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Intraperitoneal Route of Drug Administration: Should it Be Used in Experimental Animal Studies?

• Expert Review
• Published: 23 December 2019
• Volume 37 , article number  12 , ( 2020 )

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• Abdullah Al Shoyaib 1 ,
• Sabrina Rahman Archie 2 &
• Vardan T. Karamyan   ORCID: orcid.org/0000-0003-0050-6047 1 , 3  

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Intraperitoneal (IP) route of drug administration in laboratory animals is a common practice in many in vivo studies of disease models. While this route is an easy to master, quick, suitable for chronic treatments and with low impact of stress on laboratory rodents, there is a common concern that it may not be an acceptable route for drug administration in experimental studies. The latter is likely due to sparsity of information regarding pharmacokinetics of pharmacological agents and the mechanisms through which agents get systemic exposure after IP administration. In this review, we summarize the main mechanisms involved in bioavailability of IP administered drugs and provide examples of pharmacokinetic profiles for small and large molecules in comparison to other routes of administration. We conclude with a notion that IP administration of drugs in experimental studies involving rodents is a justifiable route for pharmacological and proof-of-concept studies where the goal is to evaluate the effect(s) of target engagement rather than properties of a drug formulation and/or its pharmacokinetics for clinical translation.

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Introduction

It is well-recognized that the route of administration is a critical determinant of the final pharmacokinetics, pharmacodynamics as well as toxicity of pharmacological agents ( 1 ). Intravenous (IV), subcutaneous (SC), intraperitoneal (IP) and oral routes are the main paths of drug administration in laboratory animals, with each offering advantages and disadvantages depending on specific goal(s) of the study. One of the more commonly used routes in rodent studies is the IP route where a pharmacological agent is injected into peritoneal cavity. This easy to master technique is quick and minimally stressful for animals. It involves holding of the rodent in a supine position with its head tilted lower than the posterior part of the body and insertion of the needle in the lower quadrant of the abdomen (at ~10° angle) with care to avoid accidental penetration of the viscera ( 2 , 3 , 4 ). Large volumes of solution (up to 10 ml/kg) can be safely administered to rodents through this route ( 5 ) which may be advantageous for agents with poor solubility. This route is especially common in chronic studies involving mice for which repetitive IV access is challenging. In most cases, IP administration is also preferred over the oral route for biological agents to avoid the GI tract and potential degradation/modification of biopharmaceuticals. The main disadvantage of this route is that it is minimally used in clinic (mostly for treatment of peritoneal cancers), because of which its use in experimental studies is often questioned and discouraged. To mitigate this concern, in this review article we discuss the anatomy and physiology of the peritoneal cavity, and the mechanisms governing absorption of substances from peritoneal cavity. In addition, we provide examples and compare pharmacokinetic profiles of small and large molecules upon IP and other routes of administration in experimental animals. Based on the discussed experimental evidence, we conclude that IP administration of drugs in experimental animals is a justifiable route for pharmacological and proof-of-concept studies where the goal is to evaluate the effect(s) of target engagement rather than properties of a drug formulation and/or its pharmacokinetics for clinical translation.

Anatomy and Physiology of Peritoneal Cavity

Peritoneal cavity is a closed space within the abdomen that contains the abdominal organs and is derived from the coelomic cavity of the embryo. Peritoneal cavity is lined by the most extensive serous membrane in the body (i.e., peritoneum) which has total surface area equaling to that of the skin surface ( 6 ). Peritoneal cavity is filled with a thin film of fluid (peritoneal fluid) comprised of water, electrolytes, proteins, cells and other substances originating from the interstitial fluid of the adjacent tissues. In humans, the volume of peritoneal fluid ranges from 50 to 75 ml ( 7 ), whereas in mice its volume ranges between 0.02 and 0.1 ml ( 8 ). In addition, the peritoneal fluid contains leukocytes and antibodies to fight off infections, and plasma proteins at concentration that is about 50% of what is found in plasma ( 9 ).

Peritoneum covers most of the intra-abdomenal organs and consists of a single layer of squamous mesothelial cells ( 10 ). The mesothelial cell layer sits on a thin basement membrane and the majority of these mesothelial cells are flattered type with an approximate diameter of 25 μm. Mesothelial cells are closely connected to each other by either tight junctions, adherens junctions, gap junctions or desmosomes ( 11 ). The sub-mesothelial layer of peritoneum contains collagen, adipose tissue, lymphocytes, blood vessels as well as lymphatics ( 12 ). Fibroblasts and occasional macrophages are also present in this part of the peritoneum ( 13 ). Notably, the apical surface of mesothelial cells contain microvilli of different length, shape and density, which increase the functional surface area of the peritoneum (Fig.  1 ) ( 14 ).

Panel ( a ), parietal mesothelial cell with small number of pinocytic vesicles and a more mature basement membrane. Panel ( b ), visceral mesothelial cell with higher number of pinocytic vesicles and less mature basement membrane.

Peritoneal mesothelial cells play crucial role in maintenance of peritoneal homeostasis and transport of fluids and solutes across the membrane. Intra-abdominal organs and mesentery are supported by the visceral peritoneum, whereas the parietal peritoneum lines up the abdominal wall, pelvis, anterior surfaces of retroperitoneal organs, and inferior surface of the diaphragm. The peritoneum minimizes friction and facilitates free movement between abdominal viscera, resists or localizes infection, and stores fat, especially in the greater omentum ( 15 , 16 ). Importantly, the peritoneal mesothelial cells possess many features of epithelial cells including presence of polygonal, cobblestone morphology with surface microvilli and an ability to form a polarized monolayer that permits unidirectional transport of molecules ( 17 ). The peritoneal fluid has a pH of 7.5–8.0 and buffering capacity ( 18 ), because of which substances are rarely ionized in the peritoneal cavity after IP administration ( 19 ).

Peritoneal Blood Flow

The sub-mesothelial layer of the peritoneum supports a complex but efficient vascular network of blood and lymphatic vessels ( 20 , 21 ). Most of the circulatory vessels are blood capillaries but few arterioles and venules are also present in the sub-mesothelium ( 22 ). The density of these sub-mesothelial microvessels varies along different portions of the peritoneal cavity. For example, in the rabbit, the most vascularized area of peritoneal cavity is mesentery, which contains about 71% of peritoneal microvessels, whereas the diaphragm and parietal peritoneum contain about 18% and 11% of microvessels, respectively ( 21 , 23 ). Through these capillaries, peritoneal cavity receives 4 to 7% of the total cardiac output in the rabbit ( 24 ) and rat ( 25 , 26 ). The average peritoneal blood flow rate in the rat is 2.5–6.2 ml/min/kg ( 24 ). Notably, blood supply to visceral peritoneum and intra-peritoneal organs originates from the coeliac, superior and inferior mesenteric arteries, whereas the parietal peritoneum is irrigated form the branches of circumflex, iliac, lumbar, intercostal and epigastric arteries ( 27 , 28 ). Similarly, the venous system that drains from the visceral peritoneum empties into portal vein, whereas the veins that drain from the parietal peritoneum empty into the inferior vena cava ( 28 , 29 ). Overall the entire peritoneum is well perfused with blood capillaries and provides an excellent surface for exchange of drugs between the peritoneal cavity and plasma ( 24 ).

Peritoneal Lymphatic System

Like many other organs, peritoneum has a well-structured lymphatic network which maintains the solute and fluid balance of peritoneal tissues and prevents formation of edema ( 30 , 31 ). In the sub-mesothelial layer of the peritoneum, the terminal lymphatic capillaries are subdivided into parasternal, paravertebral, mediastinal, intercostal and retroperitoneal lymphatics. Parasternal, mediastinal and retroperitoneal lymphatics are arranged over the anterior, posterior and central part of the diaphragm, respectively. These terminal lymph capillaries unite together to form afferent collecting and prenodal lymphatic ducts which are then connected with regional lymph nodes. The parasternal, paravertebral and mediastinal lymphatics are connected with the mediastinal lymph nodes, whereas, retroperitoneal lymphatics are joined with cisternal and intestinal lymph nodes. From these lymph nodes the lymphatic system joins the venous circulatory system through thoracic duct and right lymphatic duct ( 32 ).

Absorption of Solutes from the Peritoneal Cavity, General Considerations

Peritoneal cavity is an excellent portal of entry into systemic circulation for substances after IP administration. Its large surface area, estimated about 125 cm 2 in an average rat, presence of microvilli on mesothelial cells and the vast blood supply facilitate rapid absorption of substances after IP administration ( 33 , 34 ). Additionally, lymphatic transport of substrates significantly contributes to the total transfer from the peritoneal cavity to systemic circulation ( 35 ).

To reach the vascular compartment after IP administration, a compound must cross different structures in and around the peritoneal cavity including the peritoneal fluid, mesothelium, sub-mesothelium and blood vessel wall ( 36 , 37 ). Transcellular and intercellular spaces in the visceral and parietal mesothelium are the main gateways for solutes and molecules to pass from the peritoneal cavity into surrounding tissues. Because of its structural features, visceral mesothelium is more permeable to molecules compared to parietal mesothelium. Anatomically, visceral peritoneum is composed of flat cells with large number of pinocytic vesicles which facilitate absorption of molecules. On the contrary, parietal peritoneum contains less pinocytic vesicles and has a more developed basement membrane/connective tissue which make it less permeable for molecules (Fig. 1 ) ( 38 ).

Experimental studies indicate that small to medium size molecules (MW < 5000) and fluids are predominantly absorbed from visceral peritoneum by diffusion through the splenic, inferior and superior mesenteric capillaries and drain into the portal vein ( 39 ). On the other hand, large molecules (MW > 5000), proteins, blood and immune cells are taken up by the lymphatics (Fig.  2 ) ( 40 ). It is noteworthy, that there is some amount of retrograde movement, of both small and large molecules, from capillaries to peritoneal cavity, however it minimally affects the overall absorption of pharmacological agents from peritoneal cavity to systemic circulation ( 41 , 42 ).

Schematic overview of absorption pathways for small and macromolecules from the peritoneal cavity to systemic circulation.

The physiological mechanisms of fluid and solute movement from peritoneal cavity-to-blood or blood-to-peritoneal cavity are the same, either through diffusion or convection. Rapid absorption of small molecules from the peritoneal cavity into blood capillaries are mainly governed by effective surface area (A), solute concentration gradient (ΔC) and solute permeability (Ps) ( 42 , 43 ).

In case of IP administration, the effective surface area (A) is created by the microvilli on mesothelial cells, which ensure increased absorption of IP administered substances. Because substances are efficiently carried away by capillary blood flow, a constant concentration gradient (ΔC) is available between the absorption site of the peritoneal cavity and surrounding capillaries. This concentration gradient facilitates the diffusion of administered substances from peritoneal cavity into blood capillaries. Lastly, physicochemical properties of the administered substance determine its permeability (Ps) and hence absorption from peritoneal cavity. A highly lipophilic substance has a higher distribution rate into tissues, which results in rapid removal of substance from the systemic circulation. The latter increases the concentration gradient between blood capillaries and peritoneal cavity, and leads to increased absorption rate of the substance after IP administration ( 44 ). Notably, solute permeability is the main determinant of convection rate, whereas concentration gradient is the main driving force for diffusion ( 45 ).

Capillary Absorption of Solutes after IP Administration

In the peritoneal microvascular network, majority of the solute and fluid exchange between peritoneal cavity and circulatory system occurs through capillaries. The capillary walls of the peritoneal capillaries are ‘continuous’ type, lined with single layer of continuous endothelial cells and basal lamina. These endothelial cells are very thin (0.5 μm) and highly permeable ( 46 , 47 ), plus they contain a large number of cytoplasmic vesicles ( 48 , 49 , 50 ). Intracellular clefts (6–7 nm thin channels located between adjacent endothelial cells) are also present in these capillaries which ensure rapid passage of water soluble ions and small molecules ( 21 ). Absorption of molecules with molecular size of <20,000 Da from peritoneal cavity occurs by either diffusion or convection through these capillaries ( 42 , 51 ). The rate and extent of diffusion depends on the size, charge, configuration and concentration gradient of the molecules ( 52 ). In addition, IP administration of a drug in solution increases the IP hydrostatic pressure which drives the convection of the soluble drugs along with fluid through peritoneum into the blood capillaries ( 45 ). Molecules absorbed from the visceral peritoneum, mesentery and omentum are drained into the portal vein, while molecules absorbed form the parietal peritoneal blood capillaries and lymphatics drains directly into the systemic circulation ( 53 , 54 ). Substances entering through portal circulation merge with systemic circulation after passing through liver which results in fast pass metabolism of the administered substances.

In one extensive experimental study carried out in rats, Lukas and colleagues have shown that after IP administration of small molecules (atropine, caffeine, glucose, glycine and progesterone), the primary route of absorption is through portal circulation ( 29 ). In this study the small molecules were selected to represent different physicochemical properties (basic, nonionic hydrophilic, zwitterionic and nonionic hydrophobic) and it was revealed that they all had comparable absorption pattern. In addition, the rate of absorption between IP and sub-cutaneous (SC) administrations of these compounds was compared. IP administered compounds could be detected in systemic circulation as soon as after 10 s of administration whereas it took about 60 s for SC administered compounds to reach systemic circulation. Another notable distinction between these two routs was the substantially lower degree of liver exposure and hence less biotransformation of molecules administered SC ( 29 ). Additionally, lipid solubility of IP administered compounds can affect the rate of their absorption. In general, increased lipid solubility leads to increased absorption from peritoneal cavity; e.g. only ~57.4% of barbital (lipid-water partition co-efficient of 0.001) was shown to be absorbed after IP administration in the rat, whereas absorption of thiopental (lipid-water partition co-efficient of 3.3) was shown to be 96.1% in the same study ( 55 ). Another important factor in absorption of IP-administered compounds is their ionization state at physiologic pH, which can be affected by buffering capacity of the peritoneal cavity ( 55 ). In general, absorption of IP-administered acidic substances increases as their pKa increases, e.g. benzyl penicillin with pKa of 2.8 has an absorption of 16.5%, whereas secobarbital with pKa of 7.9 has an absorption of 87% ( 55 ). On the other hand, absorption of IP-administered basic substances decreases as their pKa increases, e.g. caffeine with pKa of 0.9 has an absorption of 68.3%, whereas atropine with pKa of 9.6 has an absorption of 27% ( 55 ). Overall, unionized compounds are absorbed to a greater extent after IP administration than ionized compounds. Importantly, viscosity of the IP-administered formulations can also affect the absorption of pharmacological agents, with higher viscosity leading to decreased absorption and efficacy ( 55 ).

Lymphatic Absorption of Solutes after IP Administration

Cellular arrangement around the peritoneum and ample supply of blood and lymph vessels assures rapid absorption of not only small molecules but also proteins and cells after IP administration ( 56 , 57 ). Molecules with molecular weight larger than 30,000 Da enter the systemic circulation from the peritoneal cavity primarily via lymphatic vessels ( 41 ). Experimental studies indicate that IP administered plasma proteins are completely absorbed into systemic circulation and are distributed throughout the body similar to IV administered plasma proteins ( 58 ). Following IP administration, proteins are absorbed by lymphatic vessels, which are especially enriched around the diaphragm in most mammals including mouse, rat, dog, cat and human ( 59 , 60 ). In addition to proteins, small particles and cells are also absorbed mainly through end lymphatic vessels, which are also known as stomata ( 61 , 62 ). The peritoneal stomata are physiological openings in the peritoneum. Here, the mesothelial cells are interrupted and share a common basement membrane with lymphatic endothelium which allows a direct communication between peritoneal cavity and underlying lymphatics ( 11 ). Notably, relaxation of the diaphragm during respiration also controls the absorption of macromolecules from the peritoneal cavity ( 63 ). The diaphragm relaxes during expiration and the adjacent mesothelial cells on the border of lacunae separate from each other creating suction force and facilitating absorption of macromolecules. However, contraction during inspiration results in closing of gaps between mesothelial cells and emptying of lacunae into the efferent lymphatics. The parasternal lymphatics located in the diaphragm, especially in the right half around the liver, are mainly responsible for the transfer of peritoneal deposits form peritoneal cavity into the mediastinal nodes and then into venous circulation through the right lymphatic duct ( 32 , 62 ). A small portion of peritoneal deposits are also drained through the paravertebral and mediastinal lymphatics situated on the parietal peritoneum, mesenteric, and omentum ( 60 ). The visceral lymphatics collect solutes and fluids from the omentum as well as mesentery and drain into the complex network of visceral lymph nodes. Subsequently, these visceral lymphatics collectively drain into parietal lymph nodes and finally dump the contents into venous circulation primarily through the thoracic duct ( 64 ). Quantitatively, about 75% of the absorbed proteins are drained into the right lymph duct and about 25% into the thoracic duct, which subsequently merge with venous circulation (Fig.  3 ) ( 57 , 65 ). Notably, obstruction of the right lymphatic and thoracic ducts does not prevent systemic absorption of proteins from the peritoneal cavity because trace amounts of proteins can still be absorbed through other small lympho-venous communications and capillary walls ( 60 ).

Schematic representation of the main lymphatic absorption pathways for macromolecules after IP administration.

Pharmacokinetics of Small Molecules Administered Via IP Vs Other Routes

Recently, Durk and colleagues carried out a comprehensive study in rats to compare the pharmacokinetic parameters of IP and SC administered 9 small molecules (carbamazepine, citalopram, desmethylclozapine, diphenhydramine, gabapentine, metaclopramide, naltrexone, quinidine and risperidone) with distinct physicochemical properties ( 66 ). For all molecules, administered at 1 mg/kg dose, IP administration yielded higher C max and lower t max values in comparison to the SC route (Table I ). The authors also compared AUC 0–360 ratio of the brain interstitial fluid and plasma for all 9 molecules after IV infusion (1 mg/kg/h for 6 h) or 3 intermittent doses of IP or SC injections (2 mg/kg, every 2 h) ( 66 , 67 , 68 ). These experiments revealed that after IP administration, the brain to plasma ratios were greater than 1 for diphenhydramine and naltrexone, close to 1 for carbamazepine, metaclopramide and risperidone, and substantially less than 1 for citalopram, desmethylclozapine, gabapentin and quinidine. Notably, brain exposure of most compounds were higher after IP or SC administration in comparison to IV infusion which could be due to saturation of transporters at the blood brain barrier and rapid elimination rate after IV infusion (Table II ).

Several other studies also compared pharmacokinetic properties of small molecular drugs administered via IP and other routs (Table III ). Among them, a study carried out by Shimada and colleagues compared plasma and peritoneal concentrations of docetaxel (8 mg/kg, MW = 807.89) after IP and IV administration in mice ( 69 ). In intact mice, AUC of 4.85 and 3.37 mg h/ml were observed after IV and IP administration of the drug, respectively. Based on these data the calculated absolute bioavailability (F% = AUC IP/AUC IV × 100%) of IP administered docetaxel is 69% ( 69 ), which is much higher compared to the absolute bioavailability of docetaxel after oral administration (~2.8%) ( 70 ). Notably, the documented maximum plasma concentration (C max ) of docetaxel was also similar after IP and IV administration in this study.

In another study, the plasma profile of deramciclane (10 mg/kg, MW = 301.466) was compared after oral, IP and IV administration in rats ( 71 ). Deramciclane showed AUC 0-∞ of 106.95, 578.18 and 3127.53 ng-h/ml after oral, IP and IV administration, respectively. The absolute bioavailability of the drug was almost 6-fold higher after IP vs oral administration. Furthermore, the time required to reach maximum plasma concentration of the drug was 4 times shorter after IP vs oral administration. Importantly, in case of both oral and IP routes of administration deramciclane had poor bioavailability due to heavy first pass metabolism in the liver.

One other study compared pharmacokinetic profile of IP vs oral or IV administered 18 F-fluorodeoxyglucose (FDG, MW = 181.15 g/mol) for positron emission tomography/computed tomography (PET/CT) in mice ( 72 ). The authors concluded that the AUC and tissue uptake of FDG was greater in case of IP administration compared to the oral route. The time to reach maximum tissue concentration (t max ) was shorter after IP administration indicating a more rapid absorption of FDG after IP vs oral administration. Notably, the brain accumulation of FDG was not significantly different after IP or IV administration, however, it was lower after oral administration ( 72 ).

In another recent study, Matzneller and colleagues compared bioavailability of tariquidar (MW = 646.73 g/mol) at 15 mg/kg dose in two different formulations after oral, IP and IV administration in rats ( 73 ). For formulation A (tariquidar dissolved in 5% glucose and 2% DMSO solution), the AUC were 18.1 μg.h/ml, 23.8 μg.h/ml and 25.2 μg.h/ml after oral, IP and IV administration, respectively. Whereas, C max were 1.2 μg/ml, 1.5 μg/ml, 1.9 μg/ml and t max were 4 h, 2 h, and 0.5 h after oral, IP and IV administration, respectively. These parameters were further improved in formulation B (microemulsion of tariquidar), for which AUC were 21.9 μg.h/ml, 25.6 μg.h/ml and 25.2 μg.h/ml, C max were 1.3 μg/ml, 1.6 μg/ml, 1.9 μg/ml and t max were 3.6 h, 2 h, and 0.5 h after oral, IP and IV administration, respectively. Notably, for both formulations, the absolute bioavailability (F%) was higher after IP vs oral administration; for formulation A, F% was 71.6 and 91.4 after oral and IP administration, for formulation B, F% was 86.3 and 99.6, respectively.

In a similar study carried out in mice, it was revealed that IP administered lenalidomide (MW = 259.261 g/mol) has better bioavailability compared to oral administration ( 74 ). Administration of the drug at 10 mg/kg dose resulted in AUC of 214 μg.min/ml, 300 μg.min/ml and 284 μg.min/ml after oral, IP and IV administration, respectively. The absolute bioavailability (F%) was 75 and 105 after oral and IP administration, respectively. Whereas, the absorption rate was higher after IP vs oral administration, with an absorption constant (K a ) of 0.044 min −1 and 0.014 min −1 , respectively.

In summary, these experimental studies indicate that IP administration of small molecule pharmacological agents results in faster and more complete absorption compared to oral and SC routes. Given the fast absorption of most substances from peritoneal cavity, it is generally considrered that systemic exposure (AUC and C max ) of a IP-admnistered substance is closer to that of the IV route. However, it is difficult to state how similar the exposure profiles are for the two routes, expecially considering that IP-administered substances are subject to first pass metabolism similar to orally administered substances ( 29 , 75 ). The uncertaininty is reasoned by a close examination of the published literature indicating that a good number of pharmacokinetic studies comparing IP and other routes, did not consider the rapid absorbtion of substances from peritoneal cavity and did the first blood sampling 30 min after IP administration ( 69 , 73 , 76 ). For rapidly absorbed compounds such experimental design could substantially underestimate systemic exposure of the pharmacological agent after IP administration, and provide an indication of higher first pass metabolism. Unfortunately, analysis of the published studies does not allow to unequivocally conclude whether the first pass metabolism of IP-administered small molecules is as extensive as in case of the oral route, and future studies specifically designed to answer this question will be needed to clarify the notion.

Pharmacokinetics of Macromolecules Administered Via IP Vs Other Routes

Numerous in vivo studies conducted in various animal models of disease have shown biological effect(s) of macromolecules and recombinant proteins after IP administration indicating bioavailability of large molecules administered by this route ( 77 , 78 , 79 , 80 ). In addition, some studies focused on complete or partial pharmacokinetic characterization of macromolecules after IP administration providing more detailed information about suitability of this route for administration of macromolecules (Table IV ).

In an elegant study conducted in mice, Sumbria and colleagues investigated plasma and brain availability of IgG-TNF Decoy Receptor Fusion Protein (MW = 210 kDa) after IV, IP and SC administration at 0.7, 3 and 10 mg/kg doses ( 81 ). The observed C max values decreased in the following order IV > IP > SC for all three doses. The calculated plasma AUC for 10 mg/kg dose were 10.8 mg.min/mL, 94.521 mg.min/mL and 17.233 mg.min/mL for IV 0-1h , IP 0–24h and SC 0–24h routes, respectively, indicating a 5.5-fold higher AUC 0–24h after IP vs SC. Notably, while plasma concentrations of the noted groups were different, the brain concentrations were not different between IP and SC injections.

A recent comparative study carried out in neonatal rats evaluated the plasma and brain pharmacokinetics of recombinant human erythropoietin (rEpo, MW = 37 kDa, administered IP or SC at 5000 U/kg) ( 82 ). The calculated plasma AUC 0-∞ were 140,331 U.h/L, 117,677 U.h/L and brain AUC 0–24 were 52.5 U.h/L, 45.2 U.h/L after IP and SC administration, respectively. Plasma and brain C max were 10,015 U/L and 3.3 U/g after IP administration, and 6224 U/L and 2.8 U/g after SC administration, respectively. Plasma t max was 3 h for IP and 9 h for SC administration.

In another study, Veronese and colleagues studied bioavailability of intact superoxide dismutase (SOD, MW = 37 kDa; 4000 IU) and monomethoxypoly-ethylene glycol (MPEG) conjugated SOD (3000 IU) after IV, IP, intramuscular (IM) and SC administration in the rat ( 83 ). In case of MPEG-conjugated SOD, the calculated AUC were 71, 54 and 29% of the administered dose after IP, IM and SC administration (compared to IV route), indicating the following order of bioavailability IV > IP > IM > SC. The plasma C max values were ~300 μg/ml, 100 μg/ml, 70 μg/ml and 62 μg/ml for IV, IP, IM and SC routes, whereas absorption rates decreased in following order IP > IM > SC, and t max values were 10 h, 42 h and 40 h, respectively. In comparison to AUC of IV administered MPEG-conjugated SOD, the observed AUC of intact SOD after IV, IP, IM and SC administration were 1.20, 0.87, 0.72 and 0.59% indicating superior stability of the conjugated SOD over the intact form. The absorption rate of intact SOD was highest after IP administration, followed by IM and SC routes (IP > IM > SC), with t max values of 100 min, 150 min and 150 min, respectively.

In another study, Parker and colleagues performed pharmacokinetic evaluation of Exendin-4 (a homolog of glucagon-like peptide-1 ( 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 )amide, MW = 4186.63 Da) in rats ( 84 ). The observed AUC values were 172 ± 5 nM.h/ml, 128 ± 25 nM.h/ml and 112 ± 18 nM.h/ml following IV, IP and SC administration of 50 nmol Exendin-4. It was also revealed that IP administration results in higher C max (35.3 ± 6.1 nM) compared to SC (28 ± 4 nM) administration, whereas elimination was also more rapid after IP (t 1/2  = 157 min) compared to SC (t 1/2  = 216 min) administration.

Additionally, pharmacokinetic parameters of radiolabeled soluble interlukin-1 receptor (IL-1R, MW = 68 kDa) were studied after IV (340 ng), IP (240 ng) and SC (240 ng) administration in mice ( 85 ). Though the study did not report AUC values, the observed C max was 178 ng/mL, 32 ng/mL and 14 ng/mL, and t max was 1 min, 120 min and 240 min after IV, IP and SC administration of IL-1R, respectively. In the same study, the authors studied pharmacokinetic properties of soluble interlukin-4 receptor (IL-4R, MW = 140 kDa at 1.1 μg). For IL-4R the observed C max was 1.2 μg/mL, 0.19 μg/mL, 0.21 μg/mL, and t max was 2 min, 60 min, 60 min after IV, IP and SC, respectively.

To study the effect of dose and injection volume on pharmacokinetics of IP administered macromolecules, Barrett and colleagues injected two doses (2 and 100 μg) of IgG2 ak (IgG light chain, MW = 25 kDa) at two different injection volumes (2 and 20 ml) in rats ( 86 ). Their observations revealed that higher dose of administered IgG2 ak results in higher AUC and C max whereas, the higher volume resulted in lower AUC and C max . According to the authors and another research group ( 87 ), IP injection of higher volume induces diuresis and increased clearance of the drug leading to a lower AUC.

In another study, comparative pharmacokinetics of AD-114-PA600-6H (human single domain antibody against CXCR4, MW = 60.6 kDa) at 10 mg/kg dose was studied in mice after IV, IP and SC administration ( 88 ). The results indicated higher AUC last after IP administration compared to the SC, though the highest AUC was observed after IV administration; AUC last for IV, IP and SC were 1871 μg.h/mL, 1435 μg.h/mL and 760 μg.h/mL, respectively. The C max were 467 μg/mL, 176 μg/mL and 32.17 μg/mL with t max values of 1.8 min, 2 h and 8 h for IV, IP and SC administration of AD-114-PA600-6H, respectively.

A comprehensive pharmacokinetic study of IFN-γ (MW = 16.8 kDa, 100 μg/kg) and its PEG-ylated conjugates (with PEG-10, −20 and − 40 at 100 μg/kg dose) in rats showed higher AUC 0-∞ and C max values after IP vs SC administration for all IFN-γ forms ( 89 ). In addition, the observed t max after IP administration was 4 h, 4 h, 10 h and 2 h for PEG-10, PEG-20, PEG-40-conjugated and intact IFN-γ, respectively, whereas it exceeded 24 h for all PEG-ylated IFN-γ and was 2 h for native IFN-γ after SC administration. C max values were 160.03 ng/mL, 179.50 ng/mL, 567.29 ng/mL and 3.41 ng/mL after IP, and 15.62 ng/mL, 29.52 ng/mL, 23.47 ng/mL and 0.35 ng/mL after SC administration for PEG-10, PEG-20, PEG-40-conjugated and intact IFN-γ, respectively.

In another study, pharmacokinetics of anti-CD-20 monoclonal antibody veltuzumab (MW = 145 kDa, at 150 μg) was studied in mice after IP and SC administration ( 90 ). The calculated AUC was 106.639 nmole.h/mL after IP and 51.67 nmole.h/mL after SC administration. The observed C max were 0.195 and 0.203 nmole/mL for IP and SC administration, whereas t max was ~24 h for both routes.

In a recent study Shi and colleagues examined suitability of IP and SC routes, in comparison to IV, for administration of large plasma-derived proteins, coagulation factor VIII (phFVIII, MW = 90–200 kDa, 50 U/kg) and von Willebrand factor (phVWF, MW = 500–2000 kDa, 50 U/kg) in mice ( 91 ). Both proteins were absorbed with t max of 2 to 4 h after IP administration, with observed C max of ~250 mU/mL for phVWF and ~300 mU/mL for phFVIII, which is similar to plasma level of these proteins 2 to 4 h after IV administration. On the contrary, phVWF and phFVIII were undetectable after SC administration in mice, even though another coagulation factor with a smaller molecular size (FIX, MW = 55 kDa) was shown to be absorbed in significant amounts after SC administration in another study ( 92 ). Notably, Shi and colleagues concluded that the hindered absorption of phVWF and phFVIII after SC was likely due to their large molecular size retarding absorption through subcutaneous capillaries and lymphatics.

It is noteworthy, that not all studies documented better pharmacokinetic profile of IP vs SC administered ploypeptides or proteins. For example, in the study where Parkes and colleagues observed higher AUC of exendin-4 after IP vs SC administration, pharmacokinetics of glucagon-like peptide-1 ( 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 ) amide (also known as GLP-1) was also studied in the rat ( 84 ). Their observations revealed lower AUC for GLP-1 after IP vs SC administration, documenting AUC of 0.77 ± 0.16 nM.h/ml and 1.54 ± 0.24 nM.h/ml, respectively. Additionally, C max after IP and SC administration of 50 nmol GLP-1 were 2.64 ± 2.11 nM and 5.14 ± 1.16 nM, respectively. The authors attributed these results to potential biodegradation of GLP-1 upon absorption from peritoneal cavity ( 84 ).

Collectively, based on the above-discussed experimental studies, we can suggest that macromolecules of different molecular size get access to systemic circulation after IP administration in intact/therapeutically active form and, in majority of cases, systemic exposure of these molecules, i.e. AUC, is higher after IP than SC route of administration (summarized in Table IV ).

Bioavailability of Suspension and Nanoparticle Formulations Administered Via IP Route

In addition to solution formulations of small and macromolecules discussed above, experimental studies point out to bioavailability of suspension formulations after IP administration. Absorption of suspension preparations after IP administration is mainly through lymphatic system and is primarily affected by physicochemical properties of the pharmacological agent, dissolution rate of the suspension in the peritoneal cavity and particle size ( 93 ). For example, in a recent study Cardenas and colleagues studied systemic bioavailability of 6-methylcoumarin (water insoluble, MW = 160.17 g/mol) in rats after IP and oral administration of 200 mg/kg suspension prepared in Tween-80 and saline ( 94 ). The authors observed more rapid absorption and higher bioavailability of the suspension after IP vs oral administration, documenting t max values of 6 min and 30 min, and AUC of 2177.0 μg.min/ml and 977.2 μg.min/ml, respectively. On the other hand, systemic bioavailability of 100 mg/kg elacridar suspension (water insoluble, MW = 563.6 g/mol), prepared in hydroxypropyl-methylcellulose and Tween-80, after IP administration in mice was several fold lower compared to that of oral administration, with AUC of 90.30 μg.min/ml and 1460 μg.min/ml, respectively ( 95 ). Notably, when the same drug was given as a microemulsion (prepared with Cremophor EL, Carbiton and Captex 355) at 10 mg/kg, systemic bioavailability of the formulation after IP administration was several times higher compared to oral administration, with AUC of 962 μg.min/ml and 270 μg.min/ml, respectively ( 76 ). The authors concluded that lower bioavailability of elacridar suspension compared to emulsion was due to lower dissolution of the administered formulation in the peritoneal cavity, which contains smaller amount of fluid compared to the gastrointestinal tract. It is noteworthy, that dissolution of the suspension formulation can be improved by vehicle selection. For instance, Sofia and colleagues have compared the influence of different suspension vehicles on central effects of Δ9-tetrahydrocannabinol (water insoluble, MW = 314.45 g/mol) at 10 and 40 mg/kg doses after IP administration ( 96 ). Among four different vehicles studied (bovine serum albumin-saline (BSA), 1% Tween-80-saline, polyvinylpyrolidone-saline (PVP) and 10% propylene glycol-1% Tween 80-saline (PG)), suspension of Δ9-tetrahydrocannabinol in PG was the most effective followed by PVP suspension. In a subsequent study, the same investigators studied suspensions of Δ9-tetrahydrocannabinol in the same four vehicles after IV, IP, oral, and SC administration ( 97 ). The strongest CNS effects were observed with PG suspension and the effects descended in the following order IV > IP > SC > oral, based on the route of administration ( 97 ). Lastly, particle size of the suspension formulation can also affect the efficacy of IP administered drugs. The latter was demonstrated by Ritschel and colleagues who studied toxicity of pentobarbiturate suspension in 1% sodium-carboxymethyl cellulose at two different particle sizes (<44 μm and 297–420 μm) administered IP in mice, and observed about twice higher toxicity of the drug at small vs large particle size suspension (LD50 of 189 and 288 mg/kg, respectively) ( 98 ). Another recent study investigated pharmacokinetics of two poorly water soluble substances (at 5 μmol/kg, referred to as AC88 and BA99) after IP administration in the rat, in the form of microsuspension (prepared with hydroxylpropyl-methylcellulose) and nanosuspension (prepared with Aerosol OT, polyvinylpyrolidone and mannitol) ( 99 ). For both substances nanosuspensions showed about twice higher AUC compared to that of microsuspensions. More specifically, the observed C max , t max and AUC values were 8.24 μmol/L, 3 h and 136 kg.h/L for AC88 microsuspension (mean size 14 μm), and 21.2 μmol/L, 2 h and 233 kg.h/L for AC88 nanosuspension (mean size 219–251 nm). Similarly, for BA99 microsuspension (mean size 12 μm) the observed C max , t max and AUC values were 10.4 μmol/L, 0.7 h and 50.6 kg.h/L, whereas for BA99 nanosuspension (mean size 291 nm) the observed values were 20.1 μmol/L, 0.31 h and 85.4 kg.h/L, respectively ( 99 ). Notably, the same research group studied bioavailability of AC88 and BA99 nanosuspensions after oral and SC administration in the rat (at 5 μmol/kg) in two parallel studies, and reported ~5–8 fold lower AUC values compared to IP administration of the same nanosuspensions ( 100 , 101 ).

It is important to note, that while the small particle size of nanoformulations facilitates enhanced absorption of pharmacological agents, encapsulation of active substances in nanoformulations often results in decreased clearance from systemic circulation and ultimately higher drug exposure. In addition, depending on specifics of nanocapsulated formulation release of the drug may also be prolonged leading to higher drug exposure. For example, pharmacokinetic comparison of lipid core nanocapsule formulation of olanzapine (sparingly water soluble, MW = 312.4 g/mol) with free olanzapine after IP administration (at 10 mg/kg) indicated ~2.3 fold increased bioavailability of the nanoformulation in the rat ( 102 ). In addition, higher C max and lower clearance (CL) values were observed for olanzapine nanoformulation (3.02 μg/ml and 1.36 L/h/kg) compared to the free drug (1.12 μg/ml, and 3.12 L/h/kg). And because the observed absorption rate was comparable for both formulations (t max ~1 h) the authors concluded that the observed higher bioavailability of nanoformulation was largely due to decreased CL ( 103 ). In another study, chitosan-coated PLGA nanoparticles of docetaxel (sparingly water soluble, MW = 807.87 g/mol) were studied in the rat after IP administration (at 13 mg/kg) and ~4.7 fold higher bioavailability of the drug was observed for the nanoparticle formulation in comparison to free docetaxel suspension ( 103 ). Notably, the time to reach maximum plasma concentrations (2 and 0.5 h) as well as the elimination half-life (5.2 and 2.6 h) were higher for the nanoformulation, indicating overall longer residence time for docetaxel nanoparticle (7.4 h) in comparison to the free form (4.3 h) ( 102 ). Similarly, in a recent study Ragelle and colleagues studied bioavailability of free (at 223 mg/kg) and nanoemulsion (at 112.5 mg/kg, prepared with Miglyol® 812 N, Labrasol®, Tween-80, Lipoid® E80 and water) fisetin (sparingly water soluble, MW = 286.23 g/mol) after IP administration in the rat ( 104 ). Their observations indicate that bioavailability of fisetin nanoemulsion was about 24 fold higher compared to the free drug, and it had to do with more rapid absorption (1.97 and 5.98 h) and lower clearance (2.32 and 54.8 L/kg/h) of the nanoemulsion vs free drug, respectively ( 104 ).

In summary, these experimental studies suggest that drugs in suspension and/or nanoparticle formulations also reach systemic circulation after IP administration. Notably, the physicochemical properties of the drug, dissolution rate of the suspension in the peritoneal cavity and particle size critically affect absorption rate and bioavailability of the administered drug. In general, higher dissolution rate and smaller particle size lead to more complete and rapid absorption of such formulations from peritoneal cavity and result in higher bioavailability. From the other hand, slow dissolution rate may lead to more prolonged absorption of the drugs from such formulations and also result in longer exposure. Lastly, encapsulation of active substances in nanoformulations may prolong release of the pharmacological agent into systemic circulation, and often leads to decreased clearance of the drug.

Limitations of the IP Route

Eventhough IP administration of pharmacological agents results in faster and more complete absorption compared to oral, intramuscular and SC routes, this route as any other, has certain limitations. One limitation is the first pass metabolism, similar to what is observed with orally administered drugs, because substances absorbed from the peritoneal cavity end up in portal vein and pass through the liver. It is generally considered that, pharmacokinetics of small molecular drugs administered through the IP route resemble that of orally administered drugs in terms of metabolic fate and high rate of first pass metabolism, which leads to lower systemic exposure of IP-administered substances ( 29 , 75 ). Notably, published literature does not allow to conclusively say whether the first pass metabolism of IP-administered sustances is as expensive as in case of the oral route and future studies will likely adress this question. On the contrary, IP administered macromolecules, which reach systemic circulation through lymphatic vessels, were shown to be minimally affected by first pass metabolism. This was well-documented with a number of recombinant enzymes including neurolysin ( 80 ), angiotensin converting enzyme 2 ( 105 ) and SOD ( 83 ) which retained catalytic activity upon reaching systemic circulation after IP administration in rodents.

Two other important considerations for IP administered agents include sterility and non-irritability, because irritating compounds may cause ileus and peritoneal inflammation, which may further develop into adhesions ( 106 ). Importantly, position of IP injection can aslo affect the absorption rate of substances. Although, not studied in experimental animals, a study carried out in humans showed that the time to reach maximum plasma concentration of insulin in healthy volunteers varied about 2-fold upon administration of the agent at a position above vs below the transverse mesocolon ( 107 ). The technique of injection and its accuracy may also affect the outcome of IP administration. One common mistake associated with IP drug admnistration (~20% of cases) is puncturing the skin at a very sharp angle which results in SC administration rather than IP ( 108 ). Although occurring less frequently, inaccurate IP administration may also deposit drugs into the gastrointestinal tract, retroperitoneum or the urinary bladder ( 109 ). Another important factor is the volume of IP administered drugs because large volumes (>10 ml/kg in rodents) can lead to pain, chemical peritonitis, formation of fibrous tissue, perforation of abdominal organs, hemorrhage, and respiratory distress ( 86 , 110 , 111 ). Repeated IP administration can result in a cumulative irritant effect and needle-induced damage of the peritoneum ( 5 ). Temperature of the IP administered solutions can also affect local absorption rate ( 112 , 113 ), whereas, hypothermia and distress can be observed if a large volume of a cold substance is administered IP ( 114 , 115 ).

Concluding Remarks

Proper formulation of a drug and appropriate route of administration are crucial for clinical success at later stages of drug development. However, such questions are usually addressed after proof-of-concept studies where the goal is to evaluate the effect(s) of target engagement rather than properties of a drug formulation and/or its pharmacokinetics for clinical translation. Because of these reasons, in exploratory as well as early preclinical studies it is more practical to choose a route of drug administration that ensures bioavailability of the drug and meets the needs of a specific experiment/scientific question rather than focuses on clinically applicable administration route. In this regard, IP route of administration can be the choice for in vivo experimental studies in rodents, because it is safe for animals, ensures therapeutic bioavailability of both small and large molecules, amendable to variety of formulations, suitable for chronic treatments, robust and easy to master.

Among all routes of drug administration the IV route usually results in the highest bioavailability of a drug. However, this route is often impractical for rodent studies, because most investigational pharmacological agents are difficult to fully dissolve in water/aqueous solutions (hydrophobic nature), it requires advanced skills to practice (especially in mice, due to small vessels), and is not well-suited for chronic/repetitive treatments. To avoid these problems, researchers often prefer administration of pharmacological agents through oral, IP or SC routes, all of which have certain advantages and disadvantages (summarized in Table V ). Among these, the IP route stands out because the rate and extent of drug absorption is faster in IP followed by intramascular > SC > oral routes ( 116 ), and it is suitable for both drug solutions and suspensions/emulsions. This is primarily because IP administered pharmacological agents are exposed to a large surface area (close to that of the entire skin surface) which leads to rapid and efficient absorption. Notably, in majority of cases the effect elicited by a pharmacological agent after IV administration can be approximated more closely with IP rather than intramuscular or SC administration. Usually, the rate of absorption after IP administration is one-half to one-fourth as rapid as after IV administration ( 66 , 69 , 117 ). Following rapid absorption from the peritoneal cavity, a compound may face one of the following two pathways to reach systemic circulation: ( 1 ) it is absorbed through the visceral peritoneum, the mesentery and omentum and is drained into portal circulation, or ( 2 ) the compound gets into the systemic circulation directly bypassing liver when it is absorbed through parietal peritoneum and lymphatics. Notably, small molecular weight compounds are primarily absorbed through the first pathway, because the surface area of membranes transporting substances into the portal circulation is much larger ( 29 , 118 ). On the contrary, macromolecules access systemic circulation through the second pathway (lymphatics), which is very efficient but relatively less recognized among researchers.

Another important point of consideration is the suitability of IP route for chronic/repetitive treatments. For example, studies have shown that chronic IP administration (daily saline injection over 30 days in the same position of the abdomen) and use of different types of injection vehicles and volumes are safe and well-tolerated in laboratory animals ( 119 , 120 ).

From a technical standpoint, IP administrations is more reproducible and easy to master compared to other available routs, and upon mastering it is less stressful and safer for rodents.

Among main deficiencies of the IP route, is the metabolic fate of IP administered small molecular agents, which resembles that of the orally administered drugs ( 121 ). This may present a problem for pharmacological agents prone to extensive first-pass metabolism.

In summary, based on the knowledge discussed in this manuscript we conclude that IP administration of drugs in experimental studies involving rodents is a justifiable route for initial pharmacological and proof-of-concept studies where the goal is to evaluate the effect(s) of target engagement rather than properties of a drug formulation and/or its pharmacokinetics for clinical translation.

Turner PV, Brabb T, Pekow C, Vasbinder MA. Administration of substances to laboratory animals: routes of administration and factors to consider. J Am Assoc Lab Anim Sci. 2011;50(5):600–13.

CAS   PubMed   PubMed Central   Google Scholar  

Cunliffe-Beamer TLaL, E.P. . In The UFAW Handbook on the Care and Management of Laboratory Animals. Longman Scientific and Technical, Essex. 1987;6th:275–308.

Eldridge SF, McDonald, K.E., Renne, R.A. and Lewis, T.R. Lab Anim 11. 1982;11:50–54.

Simmons MLaB, J.O. . In The Laboratory Mouse (ed. A. Hollaender). Prentice-Hall Inc, Englewood Cliffs. 1970:127–129.

Morton DB, Jennings M, Buckwell A, Ewbank R, Godfrey C, Holgate B, Inglis I, James R, Page C, Sharman I, Verschoyle R, Westall L, Wilson AB, Joint Working Group on R. Refining procedures for the administration of substances. Report of the BVAAWF/FRAME/RSPCA/UFAW Joint Working Group on Refinement. British Veterinary Association Animal Welfare Foundation/Fund for the Replacement of Animals in Medical Experiments/Royal Society for the Prevention of Cruelty to Animals/Universities Federation for Animal Welfare. Lab Anim. 2001;35(1):1–41.

CAS   PubMed   Google Scholar  

Wegner G. Chirurgische Bemerkungen uber die peritonealhohle, mit besonderer Berrucksichtigung der Ovariotomie. Langenbecks Arch Chir. 1877;20(51):145.

Google Scholar  

Hanbidge AE, Lynch D, Wilson SR. US of the peritoneum. Radiographics. 2003;23(3):663–84 discussion 684-665.

PubMed   Google Scholar  

Hartveit F, Thunold S. Peritoneal fluid volume and the oestrus cycle in mice. Nature. 1966;210(5041):1123–5.

Aune S. Transperitoneal exchange. IV. The effect of transperitoneal fluid transport on the transfer of solutes. Scand J Gastroenterol. 1970;5(4):241–52.

Blackburn SC, Stanton MP. Anatomy and physiology of the peritoneum. Semin Pediatr Surg. 2014;23(6):326–30.

Michailova K, Wassilev W, Wedel T. Scanning and transmission electron microscopic study of visceral and parietal peritoneal regions in the rat. Ann Anat. 1999;181(3):253–60.

Albertine KH, Wiener-Kronish JP, Roos PJ, Staub NC. Structure, blood supply, and lymphatic vessels of the sheep's visceral pleura. Am J Anat. 1982;165(3):277–94.

Gotloib L, Digenis GE, Rabinovich S, Medline A, Oreopoulos DG. Ultrastructure of normal rabbit mesentery. Nephron. 1983;34(4):248–55.

Mutsaers SE, Whitaker D, Papadimitriou JM. Changes in the concentration of microvilli on the free surface of healing mesothelium are associated with alterations in surface membrane charge. J Pathol. 1996;180(3):333–9.

Whitaker D, Papadimitriou J. Mesothelial healing: morphological and kinetic investigations. J Pathol. 1985;145(2):159–75.

Shimotsuma M, Shields JW, Simpson-Morgan MW, Sakuyama A, Shirasu M, Hagiwara A, et al. Morpho-physiological function and role of omental milky spots as omentum-associated lymphoid tissue (OALT) in the peritoneal cavity. Lymphology. 1993;26(2):90–101.

Stylianou E, Jenner LA, Davies M, Coles GA, Williams JD. Isolation, culture and characterization of human peritoneal mesothelial cells. Kidney Int. 1990;37(6):1563–70.

Howard JM, Singh LM. Peritoneal fluid Ph after perforation of peptic ulcers: the myth of "acid-peritonitis". Arch Surg. 1963;87:483–4.

Moretz WH, Erickson WG. Neutralization of hydrochloric acid in the peritoneal cavity. AMA Arch Surg. 1957;75(5):834–7.

Aguirre AR, Abensur H. Physiology of fluid and solute transport across the peritoneal membrane. J Bras Nefrol. 2014;36(1):74–9.

Solass W, Horvath P, Struller F, Konigsrainer I, Beckert S, Konigsrainer A, et al. Functional vascular anatomy of the peritoneum in health and disease. Pleura Peritoneum. 2016;1(3):145–58.

PubMed   PubMed Central   Google Scholar  

Michailova KN, Usunoff KG. Serosal membranes (pleura, pericardium, peritoneum). Normal structure, development and experimental pathology. Adv Anat Embryol Cell Biol. 2006;183:i-vii, 1–144, back cover.

Gotloib L, Shustak A, Bar-Sella P, Eiali V. Heterogeneous density and ultrastructure of rabbit's peritoneal microvasculature. Int J Artif Organs. 1984;7(3):123–5.

Aune S. Transperitoneal exchange. II. Peritoneal blood flow estimated by hydrogen gas clearance. Scand J Gastroenterol. 1970;5(2):99–104.

Flessner MF. Transport of water solube solutes between the peritoneal cavity and the plasma in the rat [PhD Thesis]. Department of Chemical Engineering, University of Michigan, Ann Arbor. 1981.

Grzegorzewska AE, Moore HL, Nolph KD, Chen TW. Ultrafiltration and effective peritoneal blood flow during peritoneal dialysis in the rat. Kidney Int. 1991;39(4):608–17.

Rippe B, Rosengren BI, Venturoli D. The peritoneal microcirculation in peritoneal dialysis. Microcirculation. 2001;8(5):303–20.

Granger DN, Ulrich M, Perry MA, Kvietys PR. Peritoneal dialysis solutions and feline splanchnic blood flow. Clin Exp Pharmacol Physiol. 1984;11(5):473–81.

Lukas G, Brindle SD, Greengard P. The route of absorption of intraperitoneally administered compounds. J Pharmacol Exp Ther. 1971;178(3):562–4.

Granger HJ. Role of the interstitial matrix and lymphatic pump in regulation of transcapillary fluid balance. Microvasc Res. 1979;18(2):209–16.

Fraser PA, Smaje LH, Verrinder A. Microvascular pressures and filtration coefficients in the cat mesentery. J Physiol. 1978;283:439–56.

Olin T, Saldeen T. The lymphatic pathways from the peritoneal cavity: A Lymphangiographic study in the Rat. Cancer Res. 1964;24:1700–11.

Esperanca MJCDL. Peritoneal dialysis efficiency in relation to body weight. J Pediatr Surg. 1966;1(2):162–9.

Kuzlan M, Pawlaczyk K, Wieczorowska-Tobis K, Korybalska K, Breborowicz A, Oreopoulos DG. Peritoneal surface area and its permeability in rats. Perit Dial Int. 1997;17(3):295–300.

Zink J, Greenway CV. Control of ascites absorption in anesthetized cats: effects of intraperitoneal pressure, protein, and furosemide diuresis. Gastroenterology. 1977;73(5):1119–24.

Jacquet P, Vidal-Jove J, Zhu B, Sugarbaker P. Peritoneal carcinomatosis from gastrointestinal malignancy: natural history and new prospects for management. Acta Chir Belg. 1994;94(4):191–7.

Sugarbaker PH. Cytoreductive surgery and intraperitoneal chemotherapy with peritoneal spread of cystadenocarcinoma. Eur J Surg Suppl. 1991;561:75–82.

Fedorko ME, Hirsch JG, Fried B. Studies on transport of macromolecules and small particles across mesothelial cells of the mouse omentum. II. Kinetic features and metabolic requirements. Exp Cell Res. 1971;69(2):313–23.

Williams R, White H. The greater omentum: its applicability to cancer surgery and cancer therapy. Curr Probl Surg. 1986;23(11):789–865.

Surbone A, Myers CE. Principles and practice of intraperitoneal therapy. Antibiot Chemother (1971). 1988;40:14–25.

Flessner MF, Dedrick RL, Schultz JS. Exchange of macromolecules between peritoneal cavity and plasma. Am J Phys. 1985;248(1 Pt 2):H15–25.

CAS   Google Scholar  

Mactier RA, Khanna R. Absorption of fluid and solutes from the peritoneal cavity. Theoretic and therapeutic implications and applications. ASAIO Trans. 1989;35(2):122–31.

Gross ML, Somani P, Ribner BS, Raeader R, Freimer EH, Higgins JT Jr. Ceftizoxime elimination kinetics in continuous ambulatory peritoneal dialysis. Clin Pharmacol Ther. 1983;34(5):673–80.

Canal P, Plusquellec Y, Chatelut E, Bugat R, De Biasi J, Houin G. A pharmacokinetic model for intraperitoneal administration of drugs: application to teniposide in humans. J Pharm Sci. 1989;78(5):389–92.

Flessner MF. Peritoneal transport physiology: insights from basic research. J Am Soc Nephrol. 1991;2(2):122–35.

Chambers R, Zwiefach BW. Functional activity of the blood capillary bed, with special reference to visceral tissue. Ann N Y Acad Sci. 1946;46:683–94.

Guyton AV, Hall JE. Chapter 14: Overview of the circulation; medical physics of pressure, flow, and resistance. In: Guyton AV, Hall JE, editors. Textbook of medical physiology. 11th ed. Philadelphia: Elsevier; 2006.

Karnovsky MJ. The ultrastructural basis of capillary permeability studied with peroxidase as a tracer. J Cell Biol. 1967;35(1):213–36.

Bruns RR, Palade GE. Studies on blood capillaries. II. Transport of ferritin molecules across the wall of muscle capillaries. J Cell Biol. 1968;37(2):277–99.

Di Paolo N, Sacchi G. Anatomy and physiology of the peritoneal membrane. Contrib Nephrol. 1990;84:10–26.

Bajaj G, Yeo Y. Drug delivery Systems for Intraperitoneal Therapy. Pharm Res-Dordr. 2010;27(5):735–8.

Krediet RT, Zuyderhoudt FM, Boeschoten EW, Arisz L. Alterations in the peritoneal transport of water and solutes during peritonitis in continuous ambulatory peritoneal dialysis patients. Eur J Clin Investig. 1987;17(1):43–52.

Lockhart RD, Hamilton, G. F. and Fyfe, F. W. Anatomy of the Human Body. J B Lippincott Company, Philadelphia. 1960.

Romanes GJ. Cunningham’s textbook of anatomy. London: Oxford University Press; 1964.

Torres IJ, Litterst CL, Guarino AM. Transport of model compounds across the peritoneal membrane in the rat. Pharmacology. 1978;17(6):330–40.

Allen L. On the penetrability of the lymphatics of the diaphragm. Anat Rec. 1956;124(4):639–57.

Flessner MF, Parker RJ, Sieber SM. Peritoneal lymphatic uptake of fibrinogen and erythrocytes in the rat. Am J Phys. 1983;244(1):H89–96.

Regoeczi E, Zaimi O, Chindemi PA, Charlwood PA. Absorption of plasma proteins from peritoneal cavity of normal rats. Am J Phys. 1989;256(4 Pt 1):E447–52.

Courtice FC, Steinbeck AW. The rate of absorption of heparinized plasma and of 0.9 p.c. NaCl from the peritoneal cavity of the rabbit and Guinea-pig. Aust J Exp Biol Med Sci. 1950;28(2):171–82.

Courtice FC, Steinbeck AW. The lymphatic drainage of plasma from the peritoneal cavity of the cat. Aust J Exp Biol Med Sci. 1950;28(2):161–9.

Courtice FC, Simmonds WJ. Physiological significance of lymph drainage of the serous cavities and lungs. Physiol Rev. 1954;34(3):419–48.

Raybuck HE, Allen L, Harms WS. Absorption of serum from the peritoneal cavity. Am J Phys. 1960;199:1021–4.

Allen L. Vogt E. A mechanism of lymphatic absorption from serous cavity Am J Physiol. 1937;119:776–82.

Miller FN. The peritoneal microcirculation. Peritoneal Dialysis. 1981.

Abernethy NJ, Chin W, Hay JB, Rodela H, Oreopoulos D, Johnston MG. Lymphatic drainage of the peritoneal cavity in sheep. Am J Phys. 1991;260(3 Pt 2):F353–8.

Durk MR, Deshmukh G, Valle N, Ding X, Liederer BM, Liu X. Use of subcutaneous and Intraperitoneal administration methods to facilitate cassette dosing in microdialysis studies in rats. Drug Metab Dispos. 2018;46(7):964–9.

Deshmukh G, Sun K, Liederer BM, Ding X, Liu X. Use of cassette dosing to enhance the throughput of rat brain microdialysis studies. Drug Metab Dispos. 2015;43(7):1123–8.

Liu X, Van Natta K, Yeo H, Vilenski O, Weller PE, Worboys PD, et al. Unbound drug concentration in brain homogenate and cerebral spinal fluid at steady state as a surrogate for unbound concentration in brain interstitial fluid. Drug Metab Dispos. 2009;37(4):787–93.

Shimada T, Nomura M, Yokogawa K, Endo Y, Sasaki T, Miyamoto K, et al. Pharmacokinetic advantage of intraperitoneal injection of docetaxel in the treatment for peritoneal dissemination of cancer in mice. J Pharm Pharmacol. 2005;57(2):177–81.

Hu K, Cao S, Hu F, Feng J. Enhanced oral bioavailability of docetaxel by lecithin nanoparticles: preparation, in vitro, and in vivo evaluation. Int J Nanomedicine. 2012;7:3537–45.

Nemes KB, Abermann M, Bojti E, Grezal G, Al-Behaisi S, Klebovich I. Oral, intraperitoneal and intravenous pharmacokinetics of deramciclane and its N-desmethyl metabolite in the rat. J Pharm Pharmacol. 2000;52(1):47–51.

Kim C, Kim IH, Kim SI, Kim YS, Kang SH, Moon SH, et al. Comparison of the Intraperitoneal, Retroorbital and per Oral routes for F-18 FDG administration as effective alternatives to intravenous Administration in Mouse Tumor Models Using Small Animal PET/CT studies. Nucl Med Mol Imaging. 2011;45(3):169–76.

Matzneller P, Kussmann M, Eberl S, Maier-Salamon A, Jager W, Bauer M, et al. Pharmacokinetics of the P-gp inhibitor Tariquidar in rats after intravenous, Oral, and Intraperitoneal administration. Eur J Drug Metab Pharmacokinet. 2018;43(5):599–606.

Rozewski DM, Herman SE, Towns WH 2nd, Mahoney E, Stefanovski MR, Shin JD, et al. Pharmacokinetics and tissue disposition of lenalidomide in mice. AAPS J. 2012;14(4):872–82.

Abu-Hijleh MF, Habbal OA, Moqattash ST. The role of the diaphragm in lymphatic absorption from the peritoneal cavity. J Anat. 1995;186(Pt 3):453–67.

Sane R, Mittapalli RK, Elmquist WF. Development and evaluation of a novel microemulsion formulation of elacridar to improve its bioavailability. J Pharm Sci. 2013;102(4):1343–54.

Chang R, Al Maghribi A, Vanderpoel V, Vasilevko V, Cribbs DH, Boado R, et al. Brain penetrating Bifunctional erythropoietin-transferrin receptor antibody fusion protein for Alzheimer's disease. Mol Pharm. 2018;15(11):4963–73.

Lee B, Clarke D, Al Ahmad A, Kahle M, Parham C, Auckland L, et al. Perlecan domain V is neuroprotective and proangiogenic following ischemic stroke in rodents. J Clin Invest. 2011;121(8):3005–23.

Zhang B, Dai J, Wang H, Wei H, Zhao J, Guo Y, et al. Anti-osteopontin monoclonal antibody prevents ovariectomy-induced osteoporosis in mice by promotion of osteoclast apoptosis. Biochem Biophys Res Commun. 2014;452(3):795–800.

Wangler NJ, Jayaraman S, Zhu R, Mechref Y, Abbruscato TJ, Bickel U, et al. Preparation and preliminary characterization of recombinant neurolysin for in vivo studies. J Biotechnol. 2016;234:105–15.

Sumbria RK, Zhou QH, Hui EK, Lu JZ, Boado RJ, Pardridge WM. Pharmacokinetics and brain uptake of an IgG-TNF decoy receptor fusion protein following intravenous, intraperitoneal, and subcutaneous administration in mice. Mol Pharm. 2013;10(4):1425–31.

Statler PA, McPherson RJ, Bauer LA, Kellert BA, Juul SE. Pharmacokinetics of high-dose recombinant erythropoietin in plasma and brain of neonatal rats. Pediatr Res. 2007;61(6):671–5.

Veronese FM, Caliceti P, Pastorino A, Schiavon O, Sartore L, Banci L, et al. Preparation, physico-chemical and pharmacokinetic characterization of monomethoxypoly(ethylene glycol)-derivatized superoxide dismutase. J Control Release. 1989;10(1):145–54.

Parkes D, Jodka C, Smith P, Nayak S, Rinehart L, Gingerich R, et al. Pharmacokinetic actions of Exendin-4 in the Rat: comparison with glucagon-like Peptide-1. Drug Dev Res. 2001;53:260–7.

Jacobs CA, Beckmann MP, Mohler K, Maliszewski CR, Fanslow WC, Lynch DH. Pharmacokinetic parameters and biodistribution of soluble cytokine receptors. Int Rev Exp Pathol. 1993;34 Pt B:123–135.

Barrett JS, Wagner JG, Fisher SJ, Wahl RL. Effect of intraperitoneal injection volume and antibody protein dose on the pharmacokinetics of intraperitoneally administered IgG2a kappa murine monoclonal antibody in the rat. Cancer Res. 1991;51(13):3434–44.

Koizumi K, DeNardo GL, DeNardo SJ, Hays MT, Hines HH, Scheibe PO, et al. Multicompartmental analysis of the kinetics of radioiodinated monoclonal antibody in patients with cancer. J Nucl Med. 1986;27(8):1243–54.

Griffiths K, Binder U, McDowell W, Tommasi R, Frigerio M, Darby WG, et al. Half-life extension and non-human primate pharmacokinetic safety studies of i-body AD-114 targeting human CXCR4. MAbs. 2019;11(7):1331–40.

Fam CM, Eisenberg SP, Carlson SJ, Chlipala EA, Cox GN, Rosendahl MS. PEGylation improves the pharmacokinetic properties and ability of interferon gamma to inhibit growth of a human tumor xenograft in athymic mice. J Interf Cytokine Res. 2014;34(10):759–68.

Goldenberg DM, Rossi EA, Stein R, Cardillo TM, Czuczman MS, Hernandez-Ilizaliturri FJ, et al. Properties and structure-function relationships of veltuzumab (hA20), a humanized anti-CD20 monoclonal antibody. Blood. 2009;113(5):1062–70.

Shi Q, Kuether EL, Schroeder JA, Fahs SA, Montgomery RR. Intravascular recovery of VWF and FVIII following intraperitoneal injection and differences from intravenous and subcutaneous injection in mice. Haemophilia. 2012;18(4):639–46.

Gerrard AJ, Austen DE, Brownlee GG. Subcutaneous injection of factor IX for the treatment of haemophilia B. Br J Haematol. 1992;81(4):610–3.

Claassen V. Chap: 5 - Intraperitoneal drug administration; 1994.

Cardenas PA, Kratz JM, Hernandez A, Costa GM, Ospina LF, Baena Y, Simoes CMO, Jimenez-Kairuz A, Aragon M. In vitro intestinal permeability studies, pharmacokinetics and tissue distribution of 6-methylcoumarin after oral and intraperitoneal administration in Wistar rats. Braz J Pharm Sci. 2017;53(1).

Sane R, Agarwal S, Elmquist WF. Brain distribution and bioavailability of elacridar after different routes of administration in the mouse. Drug Metab Dispos. 2012;40(8):1612–9.

Sofia RD, Kubena RK, Barry H 3rd. Comparison of four vehicles for intraperitoneal administration of -tetrahydrocannabinol. J Pharm Pharmacol. 1971;23(11):889–91.

Sofia RD, Kubena RK, Barry H 3rd. Comparison among four vehicles and four routes for administering δ9-tetrahydrocannabinol. J Pharm Pharmacol. 1974;63(6):939–41.

Ritschel WA, Siegel EG, Ring PE. Biopharmaceutical factors influencing LD50. I Viscosity Arzneimittelforschung. 1974;24(6):907–10.

Sigfridsson K, Lundqvist A, Strimfors M. Evaluation of exposure properties after injection of nanosuspensions and microsuspenions into the intraperitoneal space in rats. Drug Dev Ind Pharm. 2013;39(11):1832–9.

Sigfridsson K, Lundqvist AJ, Strimfors M. Particle size reduction and pharmacokinetic evaluation of a poorly soluble acid and a poorly soluble base during early development. Drug Dev Ind Pharm. 2011;37(3):243–51.

Sigfridsson K, Lundqvist A, Strimfors M. Subcutaneous administration of nano- and microsuspensions of poorly soluble compounds to rats. Drug Dev Ind Pharm. 2014;40(4):511–8.

Dimer FA, Pigatto MC, Boque CA, Pase CS, Roversi K, Pohlmann AR, et al. Nanoencapsulation improves relative bioavailability and antipsychotic effect of olanzapine in rats. J Biomed Nanotechnol. 2015;11(8):1482–93.

Badran MM, Alomrani AH, Harisa GI, Ashour AE, Kumar A, Yassin AE. Novel docetaxel chitosan-coated PLGA/PCL nanoparticles with magnified cytotoxicity and bioavailability. Biomed Pharmacother. 2018;106:1461–8.

Ragelle H, Crauste-Manciet S, Seguin J, Brossard D, Scherman D, Arnaud P, et al. Nanoemulsion formulation of fisetin improves bioavailability and antitumour activity in mice. Int J Pharm. 2012;427(2):452–9.

Ye M, Wysocki J, Gonzalez-Pacheco FR, Salem M, Evora K, Garcia-Halpin L, et al. Murine recombinant angiotensin-converting enzyme 2: effect on angiotensin II-dependent hypertension and distinctive angiotensin-converting enzyme 2 inhibitor characteristics on rodent and human angiotensin-converting enzyme 2. Hypertension. 2012;60(3):730–40.

Gotloib L, Wajsbrot V, Shostak A. A short review of experimental peritoneal sclerosis: from mice to men. Int J Artif Organs. 2005;28(2):97–104.

Micossi P, Cristallo M, Librenti MC, Petrella G, Galimberti G, Melandri M, et al. Free-insulin profiles after intraperitoneal, intramuscular, and subcutaneous insulin administration. Diabetes Care. 1986;9(6):575–8.

Black MC. Routes of administration for chemical agents. The laboratory fish London (UK): Academic Press. 2000.

Lewis REK, A. L.; Bell R. E. Error of intraperitoneal injections in rats. Lab Anim Care. 1966;16:505–9.

Bredberg E, Lennernas H, Paalzow L. Pharmacokinetics of levodopa and carbidopa in rats following different routes of administration. Pharm Res. 1994;11(4):549–55.

Esquis P, Consolo D, Magnin G, Pointaire P, Moretto P, Ynsa MD, et al. High intra-abdominal pressure enhances the penetration and antitumor effect of intraperitoneal cisplatin on experimental peritoneal carcinomatosis. Ann Surg. 2006;244(1):106–12.

Bendavid Y, Leblond FA, Dube P. A study of the effect of temperature on the pharmacokinetic profile of raltitrexed administered by intraperitoneal route in the rat. Med Sci Monit. 2005;11(1):BR1–5.

Jacquet P, Averbach A, Stuart OA, Chang D, Sugarbaker PH. Hyperthermic intraperitoneal doxorubicin: pharmacokinetics, metabolism, and tissue distribution in a rat model. Cancer Chemother Pharmacol. 1998;41(2):147–54.

Sharp PELR, M. C. The laboratory rat. Boca Raton (FL): CRC Press LLC; 1997.

Pekow CB, V. Common nonsurgical techniques and procedures. Handbook of laboratory animal science: Boca Raton (FL): CRC Press. 2003;2(2nd ed):351–391.

Wolfensohn SE, Lloyd MH. Aleutian disease in laboratory ferrets. Vet Rec. 1994;134(4):100.

Woodard G. In methods of animal experimentation. Academic Press, New York. 1965;1:343–59.

De Marco TJ, Levine RR. Role of the lymphatics in the intestinal absorption and distribution of drugs. J Pharmacol Exp Ther. 1969;169(1):142–51.

Davis JN, Courtney CL, Superak H, Taylor DK. Behavioral, clinical and pathological effects of multiple daily intraperitoneal injections on female mice. Lab Anim (NY). 2014;43(4):131–9.

Gad SC, Spainhour CB, Shoemake C, Pallman DR, Stricker-Krongrad A, Downing PA, et al. Tolerable levels of nonclinical vehicles and formulations used in studies by multiple routes in multiple species with notes on methods to improve utility. Int J Toxicol. 2016;35(2):95–178.

Al-Dewachi HS, Sangal BC, Zakaria MA. Synovial sarcoma of the abdominal wall: a case report and study of its fine structure. J Surg Oncol. 1981;18(4):335–44.

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ACKNOWLEDGMENTS AND DISCLOSURES

Dr. Karamyan is supported by NIH (1R01NS106879) and AHA (14BGIA20380826) grants. We apologize that the scope of this manuscript prevented citation of all published experimental studies where the IP route of drug administration was used.

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Al Shoyaib, A., Archie, S.R. & Karamyan, V.T. Intraperitoneal Route of Drug Administration: Should it Be Used in Experimental Animal Studies?. Pharm Res 37 , 12 (2020). https://doi.org/10.1007/s11095-019-2745-x

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Received : 08 January 2019

Accepted : 27 November 2019

Published : 23 December 2019

DOI : https://doi.org/10.1007/s11095-019-2745-x

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Suggested parameters and sets of instructions outlining best practices and standards for accomplishing specific animal care and use research duties.

Guidelines on Administration of Substances to Laboratory Animals

This document is designed to provide general guidelines about administration of substances to laboratory animals. All procedures must be approved by the Institutional Animal Care & Use Committee (IACUC). The route of administration, intervals between substance administration, dose range, and volume to be administered should be listed in the approved protocol specific to each study.

Responsibility

• Investigative Personnel
• Veterinary Personnel

Glossary Definitions

Administration of substances outside of the gastrointestinal tract. Routes of parenteral administration are listed below.

• Intravenous (IV):  Administration of substances into venous circulation.
• Intraperitoneal (IP):  Administration of substances into the abdominal cavity.
• Topical (epicutaneous):  The application of substances directly to the skin for topical effect.
• Transdermal (percutaneous):  The application of substances directly to the skin for systemic effect.
• Subcutaneous (SC):  Administration of substances into the subcutaneous space.
• Intradermal (ID):  Administration of substances into the dermis.
• Intramuscular (IM):  Administration of substances into the muscle
• Intranasal (IN):  Administration of substances into the nose.
• Intratracheal (IT):  Administration of substances within the trachea.
• Intracranial:  Administration of substances into the brain.
• Epidural (ED):  Administration of substances into the epidural space.
• Intrathecal (IT):  Administration of substances into the subarachnoid space (in the spinal canal but not within the spinal cord).

Administration of substances into the gastrointestinal tract. Routes of enteral administration are listed below.

• Per os (PO):  Administration of substances by mouth.
• Gavage:  Administration of substances via a tube that is passed through the nose or mouth into the esophagus or stomach.
• Rectal:  Administration of substances into the rectum.

Administration of a large volume (up to 1ml/kg) of a substance by injection.

Administration of a substance over time. Time intervals may be drug- or diluent-specific, or based on veterinary recommendation.

1. Administration of Substances

• When administering substances to laboratory animals care should be taken to select an appropriate route of administration, method of restraint, dosing interval, and dose volume.
• All personnel should be trained to safely perform the selected route of administration. Contact the ULAM Training Core or veterinary staff for assistance.

2. Parenteral Administration

• Isotonic (the same concentration of solute as the blood)
• If pH is outside of physiologic range, administer the substance through a central vessel (such as the jugular or femoral vein) or buffer the solution such that pH is appropriate.
• All substances given parenterally  must be sterile  and should be delivered aseptically.
• If the preparation is not a commercially manufactured solution, it must be mixed in a laminar flow hood or biosafety cabinet and filtered through a 0.2 micron filter.

3. Routes of Parenteral Administration

• The maximum bolus injection volume is 1 ml/kg. If larger quantities are administered, give as an infusion.
• Infusions are often administered with specific equipment (precision pumps or microdrip infusion sets).
• Rodents: Lateral tail vein, saphenous vein, or retro-orbital venous sinus
• Rabbits: Lateral ear, jugular, or cephalic vein
• Larger species: Jugular, cephalic, femoral, or saphenous vein
• Consult with veterinary staff for recommendations on refinements to improve animal comfort during repeated IV dosing.
• Injections are administered into lower abdominal quadrants. Aspirate before injecting to avoid inadvertent administration into the bladder or gastrointestinal tract.
• Repeated daily intraperitoneal dosing for up to one month is well-tolerated in rodents. Doses should be administered to alternating sides of the abdomen.
• Administration of irritating substances may cause ileus (stasis of the gastrointestinal tract) and peritonitis (inflammation of the abdominal cavity).
• Avoid application of caustic or irritating substances.
• Apply substances to skin that is unbroken and free of hair.
• Avoid application of substances to sites available for grooming by the animal.
• Transdermal dosing is typically accomplished by application of a patch impregnated with the substance of interest.
• Apply the patch so as to avoid inadvertent ingestion or removal by the animal.
• Systemic absorption is not immediate. Patches should be applied prior to the time of anticipated need according to manufacturer's instructions.
• Do not cut patches  to reduce dose size. If an appropriate dose of patch is not commercially available, consider an alternative route of administration.
• Use a small, sharp needle (25-27G).
• Tent the skin. Holding the syringe parallel to the animal, direct the needle into the dermis. Aspirate and inject.
• Inadvertent subcutaneous administration is common. Consult ULAM veterinary or training core staff for assistance.
• Tent the skin. Holding the syringe parallel to the animal, direct the needle into the subcutis. Aspirate and inject.
• The rate of absorption from the subcutis may be slower than with other parenteral routes.
• Subcutaneous infusions can be administered with the use of an oily depot or osmotic minipump. Consult veterinary staff for additional information.
• IM dosing is best used in larger species with greater muscle mass.
• In smaller animals, use the gluteal or quadriceps muscles.
• In larger animals, use the gluteal, quadriceps, biceps or epaxial muscles.
• Take care to avoid the sciatic nerve which runs along the caudal aspect of the femur. Inadvertent injection into nerves can result in paralysis and localized muscle necrosis.
• Intracranial injections require anesthesia and stereotactic equipment. Injections can be administered through a surgically implanted cerebral cannula, direct injection, or an osmotic pump catheter.
• Animals  must  be heavily sedated or anesthetized for cannula or catheter placement and for direct injections
• Epidural or intrathecal administration of substances requires highly trained personnel. Consult with veterinary staff before attempting this technique.
• Animals  must  be heavily sedated or anesthetized.
• Intranasal : Use the smallest possible volume to avoid suffocation. Systemic absorption is rapid.
• Intratracheal : This technique requires intubation or surgical access to the trachea. Consult veterinary staff before attempting this technique.

4. Enteral Administration

• Substances are typically mixed with the daily diet, flavored water, or other palatable items to encourage consumption. Care should be taken to maintain an appropriate daily caloric intake and to habituate animals to any novel food items before adding drug.
• Care should be taken to ensure animals consume all agent offered. Laboratory personnel are responsible for ensuring that food and water intake is adequate.
• Food or water containing additives should be clearly labeled and disposed of properly.
• Gavage is often used to administer an exact PO dose.
• Administration of gavage volumes greater than 5 ml/kg may cause distress in species that are unable to vomit such as mice.
• The gavage tube size should be appropriate for the species being dosed. Contact veterinary staff for assistance.
• This technique is not frequently used in laboratory animals. Substances can be administered via an enema or a suppository.

5. Recommended Dose Volumes

• If a range is provided, the first dose is ideal. If a single value is listed, it is the maximum allowable dose.
• Investigators wishing to dose greater volumes than specified must consult the veterinary staff and provide justification in the IACUC protocol.

Appendix A: Administration of substances into the skin or muscle (Turner et al., 2011)

Appendix 1.png.

Appendix B: A comparison of epidural and intrathecal injections (Turner et al., 2011)

Appendix 2.png.

• Diehl, K-H, et al. 2001. A good practice guide to the administration of substances and removal of blood including routes and volumes. J Appl. Toxicol. 21:15-23.
• Flecknell PA, Waynforth HB. 1992. Experimental and Surgical Techniques in the Rat. Second ed. Academic Press. San Diego, CA.
• Morton DB, et al. 2001. Refining procedures for the administration of substances. Laboratory Animals. 35: 1-41.
• Nebendahl, K. 2000. Routes of administration. In: Krinke GJ, ed. The Laboratory Rat. Academic Press. London. pp. 463-483.
• Turner PV, et al. 2011. Administration of substances to laboratory animals: Routes of administration and factors to consider. JAALAS. 50(5): 600-613.
• Turner PV, et al. 2011. Administration of substances to laboratory animals: Equipment considerations, vehicle selection, and solute preparation. JAALAS. 50(5): 614-627.

If you have questions or comments about this document, contact  ULAM Veterinary Staff  ( [email protected] ).

The  ULAM Training Core  ( [email protected]  or  734-763-8039 ) can be contacted to provide training in techniques at no charge.

Related Documents

Guidelines by topic / species.

GUIDANCE & OVERSIGHT Animal Care & Use Office (ACUO) 2800 Plymouth Road Ann Arbor, MI 48109 Phone: (734) 763-8028 Email: [email protected]

HUSBANDRY & VETERINARY CARE Unit for Laboratory Animal Medicine (ULAM) 2800 Plymouth Road Ann Arbor, MI 48109 Phone: (734) 764-0277 Email: [email protected]

ACUO is a unit of the U-M Office of the Vice President for Research:

ULAM is a unit of the Medical School Office of Research:

• Animal Care Services
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• Experimental Guidelines
• Surgery, Anesthesia and Analgesia
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• Animal Resource Program Home Page

Drugs and Chemical Compounds Used in Experimental Animals

Follow this link for information on specific methods and volumes for substance administration in rodents.

Research investigators are required to provide information to the IACUC on any drug or chemical compound administered to research animals as part of their experimental protocol. The  information outlined here must be included in an IACUC application.

The name of the drug/chemical, dose, administration method, volume and frequency of administration .

Information on the pharmaceutical or non-pharmaceutical grade of the drug/chemical (see below). Potential responses may include:

The drug/chemical is an FDA approved product. The drug/chemical is a USP purity grade chemical (or higher) obtained from a reputable chemical supplier because no suitable FDA approved product is available. Other (such as an experimental drug/chemical generated in a research laboratory) - An explanation must be provided.

A statement describing the potential for toxicity or adverse reactions . Potential responses may include:

The drug/chemical will be administered at a dose previously reported safe. No adverse effect/toxicity is expected.  The drug/chemical has specific toxic effects which is related to the intended purpose of use. This is an experimental compound of undetermined toxicity. (See note below.)

If toxic effects are expected to result from administration of the drug/chemical, those effects must be described in the IACUC protocol. Examples include:

DMBA used as part of a carcinogenesis model. Streptozotocin used to induce diabetes. Phenylhydrazine used to induce anemia. Certain antigen-adjuvant preparations that induce an inflammatory reaction at the site of administration.

Note:  The safety/potential toxicity of experimental drugs/chemicals must be documented prior to experimental use. This may be documented via a pilot study or by reference to published studies that confirm the drug/chemical's safety when used within the proposed dose range.

Pharmaceutical versus Non-Pharmaceutical Grade

The term 'pharmaceutical grade' refers to drug products approved by the U.S. Federal Drug Administration (FDA). The FDA approves the complete product formulation including active as well as inactive ingredients and specifies the dose and route of administration. Products approved by the FDA are preferred because they have been extensively tested to verify safety, efficacy, drug absorption, metabolism, excretion and duration of action. Based on guidance documents from the U.S. National Institutes of Health Office of Laboratory Animal Welfare the following situations may justify the use of non-pharmaceutical grade drugs/chemicals in research animals.

• The compound is not available as an FDA approved drug product.
• An FDA approved product is available but not in a formulation suitable for its intended application (for example it is available in an oral dose form but not as an injectable)
• An FDA approved product is available but a significant change in formulation would be required for the intended application.
• An FDA approved product is available but contains additional ingredients that may introduce new variables.
• An FDA approved product is available but there is no equivalent vehicle control.
• An FDA approved product is available but a non-FDA approved product must be used to replicate the results of a prior study.

Drugs for Clinical Use in Animals

Pharmaceutical grade drugs must be used for clinical procedures and medical treatment of animals. This includes but is not limited to anesthetics, analgesics, and drugs used for euthanasia.There are a few exceptions commonly encountered in research:

• Avertin (2,2,2-Tribromoethanol) for anesthesia in mice.
• Pentobarbital obtained from a compounding pharmacy due to the high cost of pharmaceutical grade pentobarbital (Nembutal).
• Occasional shortages of pharmaceutical grade drugs may necessitate purchase from compounding pharmacies.

Drugs for Experimental Use in Animals

Drugs and chemical products used for experimental purposes should be pharmaceutical grade if a suitable product is available. Non-pharmaceutical grade drugs/chemicals may be used when a pharmaceutical grade product is not available or when there is a scientifically valid reason.

Preparation and Use of Drugs/Chemical Compounds Administered to Research Animals

The drug formulation must be appropriate for the route of administration and not harmful to the animal. FDA approved drugs must be used as supplied or if dilution is necessary, the vehicle will be of equivalent purity. Non-pharmaceutical grade drugs/chemicals must be prepared with a compatible vehicle of USP purity grade or higher and will be as close as possible to physiological pH and osmolality. Drugs/chemicals intended for administration via injection must be sterile and stored in a sterile injection vial. A 0.2micron filter may be used to sterilize solutions prepared from non-sterile ingredients.

USP (United States Pharmacopeia) is a non-profit organization that establishes standards and specifications for drug, chemical and food ingredient manufacture.  Products that carry the USP designation on the label were manufactured according to USP specifications. You will find the USP mark on the label of drug products approved for use in humans. Many drugs approved for use in animals do not carry the USP designation.

Chemical compounds purchased from companies such as Sigma are available in a variety of purity grades. The USP purity grade meets the USP specifications for production of the chemical and is suitable for use in the manufacture of drug and food products. However, USP grade chemicals purchased from a chemical company are not FDA approved drug products.

Storage and Stability of Drugs/Chemical Compounds

FDA approved drug products must be stored according to label instructions and discarded by the last date of the month of the expiration date. Containers/vials must be labeled with the contents and expiration date. When drugs are mixed and/or diluted, the expiration date will be that of the ingredient with the earliest expiration date . However, unless there is information available suggesting longer or shorter storage times are appropriate, the solution should be used within one month of mixing.

Example: There is evidence that mixtures of ketamine and xylazine are stable for up to six months. Example: Carprofen may be mixed with sterile water but the mixture should not be used for more than one week.

Storage conditions and stability of some drugs may be described in the literature or are available from university maintained SOPs. For example, instructions for the preparation and storage of Avertin (2,2,2-Tribromoethanol) are readily available. We recommend that the final use solution for Avertin be stored refrigerated, in the dark for up to one month.

References:

The Guide for the Care and Use of Laboratory Animals: http://grants.nih.gov/grants/olaw/guide-for-the-care-and-use-of-laboratory-animals.pdf USDA Animal Care Resources Guide, Policy Number 3: Veterinary Care: http://www.aphis.usda.gov/animal_welfare/downloads/Animal%20Care%20Policy%20Manual.pdf   OLAW Online Seminar: Regulatory Considerations for Using Pharmaceutical Seminal Products in Research Involving Laboratory Animals, June 4, 2015: Seminar Transcript and questions: http://grants.nih.gov/grants/olaw/150604_seminar_transcript.pdf                    OLAW Seminar: Use of Non-Pharmaceutical-Grade Chemicals and Other Substances in Research with Animals, March 1, 2012: Transcript: http://grants.nih.gov/grants/olaw/120301_seminar_transcript.pdf OLAW FAQ: May investigators use non-pharmaceutical grade substances in animals? http://grants.nih.gov/grants/olaw/faqs.htm#662 AAALAC FAQ: Non-Pharmaceutical Grade Compounds: http://aaalac.org/accreditation/faq_landing.cfm#B9

Use of Biohazardous Materials and Biological Products in Research Animals

The use of pathogenic organisms, radioisotopes, carcinogens or other materials that may be hazardous to animals or humans should be clearly indicated on the Animal Care Request (ACR) form. The Institutional Biosafety Committee  must approve experimental procedures involving biohazardous material prior to IACUC approval and ACR form submission.  The University Isotopes Committee must approve the use of radioisotopes prior to IACUC approval and ACR form submission. Information on working with biohazardous materials within PSU animal facilities may be found on the ARP website .

Biological products are a potential source of microorganisms, especially pathogenic viruses that may infect laboratory animals. All biological products (tumors, blood, serum or other products of animal origin) must be screened for pathogen contamination before introduction into animals. If documentation of testing of the product you wish to use is available, please submit this to ARP with the Animal Care Request form. If no or incomplete documentation is available, appropriate testing must be performed before animal experimentation can begin. Please contact ARP at least 2 weeks prior to starting any animal experimentation to allow time for testing.

Adjuvant Administration

Investigators should consult the scientific literature for alternatives to Freund's complete adjuvant (FCA). If FCA is deemed necessary, then only one injection of FCA may be administered. Freund's incomplete adjuvant (FIA) should be used for subsequent immunizations. Footpad injections are not allowed. Please see the Penn State IACUC Guideline VI for further information.

Administration Of Drugs and Experimental Compounds in Mice and Rats (IACUC)

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Introduction

Administration of compounds plays a large part in experimental design using animals. This policy addresses issues related to administration of compounds within a protocol with the goal of facilitating the procedure for the person administering the compound as well as minimizing discomfort and supporting well-being of the animal. Over 90% of animals used in research are rats ( Rattus ) and mice ( Mus ), and this policy deals only with rats and mice.

“The use of pharmaceutical-grade chemicals and other substances ensures that toxic or unwanted side effects are not introduced into studies conducted in experimental animals. They should therefore be used, when available, for all animal-related procedures.” 1

“Before administering any substance (therapeutic or experimental) to an animal subject, one must consider the pH, sterility, and chemical nature (odor, taste, mucosal irritability, osmolarity, solubility, light sensitivity, and hazard status) of the compound and make appropriate decisions on the dose to be administered, frequency of administration, volume to be administered, the solvent (if necessary), and route of administration.” 2

This policy addresses these issues plus requirements for observation and care of the animal during and after administration.

For further technical information on performing injection procedures, refer to ASC SOP VET-2 “Rodent Injection” (Kerberos log-in required).

A. Any compound to be administered must be reviewed and approved by the IACUC.

Compounds that are administered on an emergency basis as part of veterinary care do not require prior IACUC approval. These may include fluids, anesthetics, analgesics, antibiotics or other pharmaceutical compounds.

B. Parameters to be defined in the IACUC protocol include:

• Name of compound or a brief chemical description if not generally known (unless proprietary).
• Dose (mg/kg) of compound.
• Route of administration
• Volume of administration. See Tables 1 and 2 at the bottom of this page for recommended needle sizes and volumes.
• Frequency of administration, and intervals between repeated administration; for example, “once”, once daily for 6 weeks” or “continuously for two weeks via osmotic pump”.
• Information on solvent/vehicle including pH and other chemical characteristics.
• Effects of compound and/or vehicle including intended effects and side effects.
• The use of any non-pharmaceutical grade chemical or compound must be described and justified in the IACUC protocol.

Specific information for mice and rats can be found in Tables 1 and 2 at the bottom of this page.

C. Following administration of compound the animal must be monitored and findings documented

The findings must available in the animal room or in the laboratory space. A form for documenting monitoring and animal response is available online.

Frequency of monitoring depends upon the study and is determined by the Principal Investigator (PI) and the IACUC and is specified in the approved protocol.

D. Experimental compounds added to the animal’s food or water must be clearly labeled.

See IACUC Policy on Additives to Drinking Water .

II. Procedures: Routes of Administration are Designated as Enteral or Parenteral

A. enteral or per os (by mouth) routes of administration.

• Additives to the drinking water: When additives are placed in the drinking water it is the responsibility of the investigator to monitor the animal(s) and assure that adequate fluid intake occurs. See IACUC Policy on Additives to Drinking Water .
• Additives to the food: When additives are placed in the food it is the responsibility of the investigator to monitor the animal(s) and assure that adequate food intake occurs.
• Oral gavage: The investigator must be trained and experienced in this procedure prior to undertaking it. This route of administration is most ensured to deliver an exact per os (oral) dose. Contact BU ASC at [email protected] for training in this procedure.
• Rectal administration: 2 This is enteral (to the GI tract) but not per os (oral). Rectal installation of drug solution can be performed using a small-gauge soft, flexible tubing (3.5 French red rubber feeding tube). The edges of the tubing should be smooth/rounded. Attach dosing syringe to end of tubing, making sure to eliminate all air bubbles. Limit the injection volume to 0.5 ml in the mouse and 1-2 ml in the rat. 1

B. Parenteral Routes of Administration

The route chosen for administration will depend upon the species, volume and material to be injected and desired rate of absorption.

The investigator should know the physiological properties of the substance to be injected (see Characteristics of Compound and Solvent section, below) because tissue damage and discomfort can be caused by irritating vehicles or drugs. Accurate placement of the needle should always be determined prior to the actual injection by applying slight negative pressure on the syringe and observing the flashback of blood or other body fluid. 3

For specific administration volumes and needle gauges, see Tables 1 and 2 at the bottom of this page.

• Subcutaneous (SC) injections: Manually restrain the animal. The best place to inject is into the loose skin on the back of the neck. A mouse may easily be injected by one person, whereas a rat may require restraint by one person and injection by the other. Anesthesia is not required. Contact BU ASC at [email protected] for training in this procedure.
• Subcutaneous slow release formulation: If repeated or long-term SC dosing is required, and the formulation is suitable for this, slow release pellets or Silastic capsules may be implanted. See  Osmotic Pumps in Mice and Rats .
• Intraperitoneal (IP): Commonly used in rats and mice IP administration results in a faster absorption into the vasculature than SC administration and is thus akin to IV administration. Any irritating compound, such as ketamine or pentobarbital, is less irritating if administered IP. A mouse may easily be injected by one person, whereas a rat may require restraint by one person and injection by the other. Anesthesia is not required. Contact BU ASC at [email protected] for training in this procedure.
• Tail vein injections are commonly performed in mice and rats and uses the lateral tail vein located on either side of the tail. Anesthesia is not required for this procedure. The maximum volume for bolus injections is 5 ml/kg. For continuous administration the maximum IV volume is 4 ml/kg/hour. 4 Contact BU ASC at [email protected] for training in this procedure.
• In rats, the jugular vein is another site for IV administration. This requires anesthesia and aseptic preparation of the site. Catheters may also be implanted in the jugular vein for repeated infusion. Finally, continuous IV or IA administration may be achieved via an osmotic pump. See Osmotic Pumps in Mice and Rats .
• If an irritating compound designed for IV administration should extravasate (be accidentally injected outside the vein), it is recommended that it be diluted in the surrounding tissues by administration of sterile 0.9% saline to avoid tissue necrosis.
• Retro-orbital injections: Discouraged in mice and must be justified in the IACUC protocol. The retro-orbital route of administration 5 into the retro-orbital sinus may be used in mice. Anesthesia is required for this procedure. This route may be used as an alternative to tail vein administration and is acceptable provided that the operator is trained and proficient in this technique. Cell suspensions should be filtered or agitated prior to injection to prevent cell clumping. Caution is advised when administrating cell lines via this route due to the possibility of cells escaping into the surrounding tissues and introducing a retro-orbital tumor mass.
• Intramuscular (IM): Not generally recommended in mice or rats due to their small muscle mass and must be scientifically justified in the IACUC protocol. Brief anesthesia (e.g. isoflurane) is normally required for immobilization and proper placement. When done in rats, < 0.2 ml/site may be injected into the quadriceps or the gluteal muscles, which are the largest. If injecting into the gluteal muscles take care to avoid the sciatic nerve that runs along the caudal aspect of the femur. Contact BU ASC at [email protected] for training in this procedure.
• Topical: BU policy is to keep areas of skin injury or treatment to 10-20% of the total body surface of the animal and not exceed 25 % of total body surface. The body surface of a 20 g mouse is 36 cm 2 and of a 25 g mouse 42 cm 2 . The percentage of surface area to be treated must be approved in the IACUC protocol. If the skin is to be pre-treated prior to application of the test compound, such as by application of depilatory cream (e.g. Nair) or waxing, this treatment must be performed by a trained and qualified person and the pretreated area must not exceed 25% of total body surface. For depilatory creams, ensure that all of the cream is removed prior to proceeding, usually with warm water and gauze or cotton swab. After the topical treatment is applied the animals should be singly housed in order to prevent the animals from grooming the compound off each other. Topical treatments are normally placed on the dorsal surface to prevent the subject from grooming it off as well.
• Intradermal (ID): Approved only if scientifically justified in the IACUC protocol. Brief general anesthesia (e.g. isoflurane) is normally required for immobilization and proper placement. Used primarily for studies in inflammation and immunologic studies. See IACUC Policy on Adjuvants .
• Ocular: Only compounds designated for and approved for ocular administration or otherwise scientifically justified in the IACUC protocol.
• Upper Respiratory Tract – Intranasal: May require anesthesia. Maximum volume to be injected in adult mice may be as much as 62 µl but it is recommended that the volume is kept to 30 µl per nostril. 6
• Upper Respiratory Tract – Intratracheal: Maximum volume to be injected in adult mice is 50 µl. This method, requiring anesthesia, must be described in detail in the IACUC protocol. Variations on this procedure may be acceptable. In general, after being anesthetized with isoflurane, the mouse is placed in an almost vertical position, head up, the tongue is retracted and the infusate placed in the mouth. Tongue retraction helps the compound to be aspirated rather than swallowed. Alternatively, the epiglottis may be visualized for inoculation using a blunt ended syringe with the anesthetized mouse placed with incisors over a wire in ventral or sternal recumbency on an angled backboard. 9, 10 It is also possible to inoculate intratracheally either via surgical incision or percutaneously into the trachea with a needle and syringe once the area has been aseptically prepared.
• Via a surgically implanted cerebral cannula
• Via direct injection
• Via osmotic pump catheter. See Osmotic Pumps in Mice and Rats .
• Intrathoracic administration: Any compound administered percutaneously into the chest requires animal to be placed under anesthesia. This specialized procedure must be described in detail in the IACUC protocol.
• Intracardiac administration: Any compound administered percutaneously or directly via an open chest into the heart requires animal to be placed under general anesthesia. This specialized procedure must be described in detail in the IACUC protocol.
• Osmotic pumps: Used for continuous administration including SC, IP, IV and IA or to specific organs. The placement of an osmotic pump is encouraged whenever administration of a compound must continue for several days or weeks. See Osmotic Pumps in Mice and Rats .
• The higher the number (gauge) the smaller the needle.
• Always use the smallest needle (highest gauge) that will do the job. For recommended needle sizes, see Tables 1 and 2 at the bottom of this page.
• Sterile needles and syringes are necessary to administer a sterile solution. Do not reuse needles beyond the same dosing administration.
• Do not recap needles.
• Dispose of used needles and syringes in a designated sharps container.

D. Frequency of Administration and Intervals Between Administration

• Limit frequency of administration as much as possible to meet medical or experimental requirements.
• For repeat administrations of experimental compounds, consider implantation of osmotic pump.

E. Characteristics of Compound and Solvent (Vehicle)

• Buffer to pH 7 if possible
• Dilute the solution using sterile normal saline or PBS
• Do not inject a high or low pH preparation IM or SC as this will be painful and cause tissue necrosis. Recommended alternate routes of administration is IP or IV.
• Pentobarbital is a very basic pH (~11). Only administer IV or IP; never administer IM or SC.
• Ketamine has an acidic pH (~4). In rodents, ketamine is always administered IP. IM administration can cause tissue necrosis.
• Sterility: The compound and solvent must be rendered sterile either by sterilization or filtration. Investigators are expected to use pharmaceutical grade medications whenever they are available, even in acute procedures. 1 Use of non-pharmaceutical grade compounds must be justified in the IACUC protocol. See IACUC policy Use of Pharmaceutical Grade and Non-Pharmaceutical Grade Substances in Vertebrate Animals .
• Odor: An offensive odor limits voluntary intake of any compound.
• Taste: If bitter compounds are administered in the drinking water, they may be better accepted if sucrose is added. These include some common antibiotics, such as tetracycline, doxycycline and metronidazole. Adding 2.5-5.0g sucrose/l water enhances palatability. See IACUC Policy on Additives to Drinking Water .
• Mucosal irritability: PI should consider tissue compatibility when administering any compounds to mucosal surfaces, i.e. eyes, mouth, and trachea. If a compound is novel, a small pilot study may be warranted.
• Osmolarity: Compounds to be administered parenterally must be ~ iso-osmolar (280 osmoles). Note that 5% Dextrose in Lactated Ringer’s Solution (LRS), which are both iso-osmolar by themselves, may be an exception and can be administered in small amounts SC or IP since the dextrose it is metabolized to CO2 and water rendering the resultant infusion iso-osmotic. However, caution is advised in a dehydrated animal.
• Solubility: Some compounds may not be soluble and require administration as a suspension. An example is sulfamethoxazole-trimethoprim administered in the drinking water. This suspension must be shaken daily to assure proper dosage.
• Light sensitivity: Protect against light exposure either by dispensing in a colored glass or cover clear glass or plastic with foil. Examples include antibiotics, such as Septra, cephalexin, and Sulfatrim.
• Toxicity: PI to document (literature search) any new compound or vehicle to be administered to animals in order to determine limitations to use and side effects.

F. Solvents (Vehicle) Characteristics

• Water: For enteral administration only. Injectable compounds must be iso-osmotic with the possible exception of 50% dextrose. A small amount of 50% dextrose may be administered IP or IV in a hypoglycemic crisis.
• Phosphate Buffered Saline (PBS): Whenever possible use sterile normal buffered saline (PBS) as a solvent. If the compound to be administered is not soluble in PBS other possible vehicles include:
• DMSO (Dimethyl sulfoxide) 0.5% – 5%: An antioxidant, DMSO [(CH3)2SO] is a highly reactive, amphipathic molecule with a highly polar domain and two apolar groups, making it soluble in both aqueous and organic media. Due to its anti-inflammatory properties and the ability to scavenge reactive oxygen particles, DMSO has been purposed for the treatment of several diseases. Therapeutic and toxic agents that are not soluble in water are often soluble in DMSO. 7, 8 However, DMSO has been shown to cause some toxicity at low doses, especially with chronic administration. 11, 12 This should be considered in the study design process to ensure it will not confound research outcomes.
• Methylcellulose 2%: This is used as a thickener and emulsifier in various food and cosmetic products, and also as a treatment of constipation. PO or topical administration appears safe. Other routes of administration must be scientifically justified. Methylcellulose is a stable sugar with low toxicity and one of the safest and most widely used vehicles available when administered per os. Characteristics of methylcellulose affecting its viscosity include percentage and cp grade which must be considered when administered to animals.
• Corn oil: Not to be given IV. See Tables 1 and 2 for recommended volumes of administration.
• Sesame oil: Not to be given IV. See Tables 1 and 2 for recommended volumes of administration.
• Ethanol: Ethanol can be lethal if injected IV at high concentration. Therefore, if an experimental compound has been purified or dissolved in ethanol, it behooves the PI to consider concentration of EtOH, possible dilution if the concentration is high and alternate routes of administration in order to assure animal safety.
• The Guide for the Care and Use of Laboratory Animals. NCR. ILAR. Eighth Ed. 2011. P.31.
• Drug Administration. 444-454. In Ch. 13. Biomethodology and Surgical Techniques in The Mouse in Biomedical Research Vol.3. Second Ed. 2007. Academic Press. Fox, J.G. et. al. Eds.
• MIT IACUC Policies.
• Hawk, C. Terrance et.al. Formulary for Laboratory Animals. Third Ed. 2005.
• University of California at San Francisco (UCSF) Retro-orbital Injections in Mice. http://www.iacuc.ucsf.edu/Policies/awSPretroorbitalinjection.asp
• Jackson Erica L. et.al. Analysis of Lung Tumor Initiation and Progression Using Conditional Expression of Oncogenic K-ras Genes Dev. 2001, December 15 (24): 3243-3248.
• Xing, L and Remick, D.G. Mechanisms of Dimethyl Sulfoxide Augmentation of IL-1β Production. The Journal of Immunology, 2005, 174:6195-6202.
• Brayton, C.F. Dimethyl sulfoxide (DMSO): a review. Cornell Vet. 1986 Jan: 76(1):61-90.
• Revelli, D. A., Boylan, J. A., & Gherardini, F. C. (2012). A non-invasive intratracheal inoculation method for the study of pulmonary melioidosis. Frontiers in cellular and infection microbiology, 2, 164. https://doi.org/10.3389/fcimb.2012.00164
• Ortiz-Muñoz, G., & Looney, M. R. (2015). Non-invasive Intratracheal Instillation in Mice. Bio-protocol, 5(12), e1504. https://doi.org/10.21769/bioprotoc.1504
• Galvao, J., Davis, B., Tilley, M., Normando, E., Duchen, M.R. and Cordeiro, M.F. (2014), Unexpected low-dose toxicity of the universal solvent DMSO. The FASEB Journal, 28: 1317-1330. https://doi.org/10.1096/fj.13-235440
• Klaas K, Saskia ABE. Van Acker JA, Grimbergen DJ, Van Den Berg WJF, Van Der Vijgh AB. (1995) Effect of dimethyl sulfoxide (DMSO) on the electrocardiogram (ECG) in freely moving male Balb/c mice. General Pharmacology: The Vascular System, 26: 6, 1403-1407. https://doi.org/10.1016/0306-3623(94)00300-C
• Shimizu S. 2004. Ch 32: Administration of Substances. The Laboratory Mouse (Handbook of Experimental Animals). Elsevier Academic Press, Oxford UK. 527-541. http://www.usp.br/bioterio/Artigos/Procedimentos%20experimentais/Routeadministration-4.pdf

BU IACUC approved April 2009; revised January 2014, revised February 2019, Approved March 2019

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Administration of substances to laboratory animals: routes of administration and factors to consider.

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Administration of substances to laboratory animals: equipment considerations, vehicle selection, and solute preparation., current requirements for and approaches to dosing in animal studies, intraperitoneal route of drug administration: should it be used in experimental animal studies, propylene glycol and kolliphor as solvents for systemic delivery of cannabinoids via intraperitoneal and subcutaneous routes in preclinical studies: a comparative technical note, manual restraint and common compound administration routes in mice and rats., animal care and use in toxicity testing, an alternative method for oral drug administration by voluntary intake in male and female mice, review of intraperitoneal injection of sodium pentobarbital as a method of euthanasia in laboratory rodents., micropipette-guided drug administration (mda) as a non-invasive chronic oral administration method in male rats, chapter 8 – common technical procedures in rodents, 147 references, direct dosing of preweaning rodents in toxicity testing and research: deliberations of an ilsi rsi expert working group, routes of administration for chemical agents, alternative method of oral dosing for rats., the route of absorption of intraperitoneally administered compounds., intravenous drug self-administration in mice: practical considerations, a good practice guide to the administration of substances and removal of blood, including routes and volumes, a pathophysiological study of abdominal organs following intraperitoneal injections of chloral hydrate in rats: comparison between two anaesthesia protocols, biomethodology and surgical techniques, tissue response to intramuscular and intraperitoneal injections of ketamine and xylazine in rats., related papers.

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Effects of oral administration of p-chlorophenylalanine to experimental animals

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• Behavioral and biochemical effects of p-chlorophenylalanine, 3-chlorotyrosine and 3-chlorotyramine. A proposed mechanism for inhibition of self-stimulation. Stark P, Fuller RW. Stark P, et al. Neuropharmacology. 1972 Mar;11(2):261-72. doi: 10.1016/0028-3908(72)90098-6. Neuropharmacology. 1972. PMID: 4260269 No abstract available.
• Behavior and brain growth in rats treated with p-chlorophenylalanine in the first weeks of life. Hole K. Hole K. Dev Psychobiol. 1972;5(2):157-73. doi: 10.1002/dev.420050209. Dev Psychobiol. 1972. PMID: 4276421 No abstract available.
• Reduced 5-hydroxyindole synthesis reduces postnatal brain growth in rats. Hole K. Hole K. Eur J Pharmacol. 1972 May;18(3):361-6. doi: 10.1016/0014-2999(72)90037-4. Eur J Pharmacol. 1972. PMID: 4260841 No abstract available.
• Isoenzyme composition of hepatic phenylalanine hydroxylase in developing rats after treatment with cortisol, alpha-methylphenylalanine and p-chlorophenylalanine in vivo. Del Valle JA, Greengard O. Del Valle JA, et al. Biochem Med. 1978 Oct;20(2):247-55. doi: 10.1016/0006-2944(78)90071-6. Biochem Med. 1978. PMID: 153746 No abstract available.
• Mood, performance, and pain sensitivity: changes induced by food constituents. Lieberman HR, Corkin S, Spring BJ, Growdon JH, Wurtman RJ. Lieberman HR, et al. J Psychiatr Res. 1982-1983;17(2):135-45. doi: 10.1016/0022-3956(82)90015-2. J Psychiatr Res. 1982. PMID: 6764930 Review.
• Behavioral, biochemical and maturation effects of early DL-para-chlorophenylalanine treatment. Kilbey MM, Harris RT. Kilbey MM, et al. Psychopharmacologia. 1971;19(4):334-46. doi: 10.1007/BF00404378. Psychopharmacologia. 1971. PMID: 4254728 No abstract available.
• Effect of glucagon on phenylalanine metabolism and phenylalanine-degrading enzymes in the rat. Brand LM, Harper AE. Brand LM, et al. Biochem J. 1974 Aug;142(2):231-45. doi: 10.1042/bj1420231. Biochem J. 1974. PMID: 4155291 Free PMC article.
• beta-2-Thienyl-DL-alanine as an inhibitor of phenylalanine hydroxylase and phenylalanine intestinal transport. Wapnir RA, Moak GS. Wapnir RA, et al. Biochem J. 1979 Jan 1;177(1):347-52. doi: 10.1042/bj1770347. Biochem J. 1979. PMID: 570838 Free PMC article.
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Intraperitoneal Route of Drug Administration: Should it Be Used in Experimental Animal Studies?

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Cyclophosphamide and vincristine are widely used intravenous chemotherapeutic agents in both human and veterinary oncology. Although intravenous administration of these chemotherapeutics is the gold standard in most treatment protocols, this route of administration has several disadvantages (e.g. long infusion times and risk of extravasation). Therefore, alternative routes have been explored in the past. Recently, good clinical results were achieved with intraperitoneal (i.p.) administration of cyclophosphamide and vincristine in cats. However, the bioavailability following i.p. administration of cyclophosphamide and vincristine providing proof of principle has not been investigated and is the focus of the present study. The pharmacokinetics of cyclophosphamide and vincristine after i.p. and intravenous administration was investigated in six cats in a cross-over study by analysis of plasma levels of cyclophosphamide and vincristine after simultaneously administration of 0.6 mg/m 2 vincristine and 200 mg/m 2 cyclophosphamide. The median bioavailability on i.p. administration was 76% for cyclophosphamide and 100% for vincristine.

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• Published: 08 September 2024

Microglial morphological/inflammatory phenotypes and endocannabinoid signaling in a preclinical model of periodontitis and depression

• Javier Robledo-Montaña   ORCID: orcid.org/0000-0002-6975-3452 1 , 2 ,
• César Díaz-García   ORCID: orcid.org/0009-0006-2019-8570 1 , 2 ,
• María Martínez   ORCID: orcid.org/0000-0002-6308-4243 3 , 4 ,
• Nagore Ambrosio   ORCID: orcid.org/0000-0001-9837-5393 3 , 4 ,
• Eduardo Montero   ORCID: orcid.org/0000-0003-2525-8529 3 , 4 ,
• María José Marín   ORCID: orcid.org/0000-0003-0206-4756 3 ,
• Leire Virto   ORCID: orcid.org/0000-0002-3376-5232 3 , 5 ,
• Marina Muñoz-López   ORCID: orcid.org/0000-0003-4817-9639 1 , 2 ,
• David Herrera   ORCID: orcid.org/0000-0002-5554-2777 3 , 4 ,
• Mariano Sanz   ORCID: orcid.org/0000-0002-6293-5755 3 , 4 ,
• Juan Carlos Leza   ORCID: orcid.org/0000-0002-9901-0094 1 , 2 ,
• Borja García-Bueno   ORCID: orcid.org/0000-0001-7320-1530 1 , 2 ,
• Elena Figuero   ORCID: orcid.org/0000-0002-3129-1416 3 , 4   na1 &
• David Martín-Hernández   ORCID: orcid.org/0000-0003-0195-2604 1 , 2   na1  

Journal of Neuroinflammation volume  21 , Article number:  219 ( 2024 ) Cite this article

Metrics details

Depression is a chronic psychiatric disease of multifactorial etiology, and its pathophysiology is not fully understood. Stress and other chronic inflammatory pathologies are shared risk factors for psychiatric diseases, and comorbidities are features of major depression. Epidemiological evidence suggests that periodontitis, as a source of low-grade chronic systemic inflammation, may be associated with depression, but the underlying mechanisms are not well understood.

Periodontitis (P) was induced in Wistar: Han rats through oral gavage with the pathogenic bacteria Porphyromonas gingivalis and Fusobacterium nucleatum for 12 weeks, followed by 3 weeks of chronic mild stress (CMS) to induce depressive-like behavior. The following four groups were established ( n  = 12 rats/group): periodontitis and CMS (P + CMS+), periodontitis without CMS, CMS without periodontitis, and control. The morphology and inflammatory phenotype of microglia in the frontal cortex (FC) were studied using immunofluorescence and bioinformatics tools. The endocannabinoid (EC) signaling and proteins related to synaptic plasticity were analyzed in FC samples using biochemical and immunohistochemical techniques.

Ultrastructural and fractal analyses of FC revealed a significant increase in the complexity and heterogeneity of Iba1 + parenchymal microglia in the combined experimental model (P + CMS+) and increased expression of the proinflammatory marker inducible nitric oxide synthase (iNOS), while there were no changes in the expression of cannabinoid receptor 2 (CB2). In the FC protein extracts of the P + CMS + animals, there was a decrease in the levels of the EC metabolic enzymes N-acyl phosphatidylethanolamine-specific phospholipase D (NAPE-PLD), diacylglycerol lipase (DAGL), and monoacylglycerol lipase (MAGL) compared to those in the controls, which extended to protein expression in neurons and in FC extracts of cannabinoid receptor 1 (CB1) and to the intracellular signaling molecules phosphatidylinositol-3-kinase (PI3K), protein kinase B (Akt) and extracellular signal-regulated kinase 1/2 (ERK1/2). The protein levels of brain-derived neurotrophic factor (BDNF) and synaptophysin were also lower in P + CMS + animals than in controls.

Conclusions

The combined effects on microglial morphology and inflammatory phenotype, the EC signaling, and proteins related to synaptic plasticity in P + CMS + animals may represent relevant mechanisms explaining the association between periodontitis and depression. These findings highlight potential therapeutic targets that warrant further investigation.

Graphical Abstract

Major depressive disorder (MDD) is a serious neuropsychiatric disease characterized by a depressed mood, impaired cognition, and vegetative dysfunctions [ 1 ]. Depressive disorders affect approximately 3.8% of the global population and are more prevalent in women [ 2 ]. The World Health Organization (WHO) has projected that depression will emerge as the leading cause of disability-adjusted life years (DALYs) by 2030 [ 3 ], but the aftermath of the COVID-19 pandemic may even exacerbate the importance of mental health within overall public health [ 4 ]. Indeed, the sociosanitary burden is sizable ($333.7 billion in the US in 2019 [ 5 ]), particularly for treatment-resistant patients [ 6 ], who are associated with 58.5% higher total costs [ 7 ].

Despite the evidence suggesting the involvement of biological and environmental factors in its etiology, a complete understanding of MDD pathophysiology remains elusive [ 8 ]. Notably, exposure to physical and psychological stressors that activate inflammatory mechanisms when persistent and uncontrolled may affect mood-controlling pathways in the brain at multiple levels, hampering physiological functions and affecting both function and structure [ 9 ].

In recent years, epidemiological evidence has substantiated the significant comorbidity between psychiatric disorders and chronic systemic inflammatory diseases [ 10 ]. Notably, periodontitis, arising from a dysbiotic subgingival microbiome challenging the host response, and being a source of low-grade inflammation, may be recognized as an independent potential contributor to mental health [ 11 ]. However, the nature of this association, whether causal or otherwise, and the specific mechanisms involved remain unclear. Over the last few years, our research, along with that of others, has aimed to unravel the role of the oral-brain axis in psychiatric diseases [ 11 ].

Specifically, our investigations revealed the presence of the periodontal pathogenic bacteria Fusobacterium nucleatum , neuroinflammation, alterations in the expression of key mediators regulating blood–brain barrier (BBB) permeability, and the modulation of sphingosine-1-phosphate (S1P) signaling in the frontal cortex (FC) of rats exposed to a combined model of periodontitis (oral gavage with periodontal pathogens) and depression (chronic mild stress [CMS]) [ 12 , 13 ]. In this experimental model, a notable hallmark of neuroinflammation was the increased microglial population in the FC, accompanied by qualitative changes in microglial microscopic morphology, suggesting an activated phenotype that warrants further investigation [ 12 ].

Microglia are resident cells within the central nervous system (CNS) parenchyma responsible for immune surveillance and are intimately related to the BBB through perivascular macrophages [ 14 ]. Clinical data from MDD patients and animal models have revealed that microglial activation is associated with symptoms [ 15 , 16 ]. Furthermore, microglia respond to periodontitis by adopting an activated and proinflammatory phenotype [ 17 ]. However, it is crucial to recognize the diversity of microglial populations and functions beyond mere activation states [ 18 ]. The hypothesis that a specific type or profile of microglia is associated with specific disease stages and individual heterogeneity has ignited a debate fostering the search for precision medicine pathways [ 19 ]. While classical studies have focused on M1 proinflammatory and M2 anti-inflammatory microglial phenotypes, this binary approach may oversimplify their role. Some authors have reported controversial results in various neuropathological conditions, including psychiatric disorders [ 20 , 21 , 22 , 23 ], underscoring the need for a comprehensive multilevel classification to elucidate the distinct characteristics and roles of different microglial subpopulations [ 24 ].

Beyond immune surveillance, a significant role attributed to microglia is the regulation of crucial neurophysiological processes, such as synaptic plasticity. Dysregulation of synaptic plasticity could be a pertinent factor in the physiopathology of MDD [ 25 , 26 ]. One of the extensively studied modulators of microglial actions on neuronal synaptic plasticity is the homeostatic endocannabinoid (EC) system [ 27 ], which is also altered both in MDD and periodontitis.

The association between the EC system and MDD has been extensively studied [ 28 ]. Numerous reports have documented alterations in the levels of ECs in brain and peripheral samples of MDD patients [ 29 ] and in stress-based animal models [ 30 ]. Various psychiatric drugs upregulate the EC system [ 31 ], and the EC activity appears to be critical for the mechanism of newly developed rapid-action antidepressant therapies [ 32 , 33 ]. EC signaling also plays a beneficial role directly in periodontal tissue [ 34 ]. Gingival biopsies from periodontitis patients have shown a decrease in cannabinoid receptor (CB) 2 [ 35 ], while cannabinoid-based interventions have been found to reduce gum inflammation [ 36 , 37 ], enhance periodontal regeneration [ 38 ], and promote osteogenic differentiation [ 39 ]. Interestingly, inflammation caused by the lipopolysaccharide (LPS) of Porphyromonas gingivalis in human periodontal ligament cells is attenuated after EC methanandamide treatment [ 40 ], and the EC effects on these cells are similar to those observed in microglia [ 41 ]. This evidence suggests that the anti-inflammatory properties of ECs might constitute an indirect link between periodontitis and depression.

In essence, the EC system encompasses the ECs (arachidonate-based lipids), anandamide (AEA) [ 42 ] and 2-arachidonoylglycerol (2-AG) [ 43 ]; their cannabinoid receptor CB1 [ 44 ] and CB2 [ 45 ]; their two main synthesis enzymes, N-acyl phosphatidylethanolamine phospholipase (NAPE) and diacylglycerol lipase (DAGL); and their degradation or reuptake enzymes, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) [ 46 ].

CB1 is the predominant G-protein coupled receptor (GPCR) in the mammalian brain, while CB2 is highly expressed in immune cells [ 47 ]. Their activities are primarily associated with neuroprotective and anti-inflammatory actions, emphasizing the critical role of maintaining homeostatic EC balance for overall health and disease [ 48 ]. Notably, microglial CB2 is essential for establishing long-term memory and promoting an anti-inflammatory profile [ 49 ]. Downstream signaling by CB1 and CB2 involves several second messengers, including mitogen-activated protein kinases (MAPKs), such as phosphatidylinositol-3-kinase (PI3K), protein kinase B (Akt), and extracellular signal-regulated kinase (ERK). These pathways can activate cAMP response element-binding protein (CREB), which is implicated in cellular surveillance and plasticity [ 50 ].

Alterations in synaptic plasticity driven by microglia have been postulated to be a pathophysiological mechanism underlying depression [ 51 ]. Two pivotal molecules in these processes are brain-derived neurotrophic factor (BDNF) and synaptophysin. Along with its polymorphism (Val66Met), BDNF plays a role in mediating antidepressant effects in both patients and preclinical models [ 52 , 53 ], while low levels of synaptophysin correlate with depression severity and increase in response to antidepressant treatment in animal models of depression [ 54 , 55 ]. Intriguingly, periodontitis, either independently or comorbid with other conditions such as diabetes, may impact dendritic arborization [ 56 , 57 ].

With the objective of obtaining a deeper understanding of the association between periodontitis and depression, a secondary analysis of our previously published studies [ 12 , 13 ], is presented aiming to conduct an extensive morphological and inflammatory phenotypic analysis of the microglial population, elucidating potential alterations in the expression and intracellular signaling of ECs and proteins associated with synaptic plasticity in the FC of rats exposed to a combined model of both comorbidities.

Materials & methods

This study adhered to the modified ARRIVE guidelines 2.0 for preclinical in vivo research [ 58 ] and conformed to the regulatory standards outlined by Spanish and European Union regulations (European Communities Council Directive 86/609/EEC). The in vivo experimental segment of the study was conducted at the Experimental Animal Center of the Complutense University of Madrid, following the approval of its protocol by the regional authorities (PROEX 087/18) and the Ethical Committee of Animal Experimentation.

Male Wistar Hannover rats (HsdRccHan: Wist, from Envigo, Spain) with body weights ranging from 230 to 280 g were housed in a controlled environment at a constant temperature of 24 ± 2 °C and a relative humidity of 70 ± 5%, following a 12-h light‒dark cycle (lights on at 8:00 AM). Throughout the experimental procedures, the rats had free access to fresh tap water and were provided ad libitum access to standard pellet chow (A04 SAFE, Scientific Animal Food and Engineering, Augy, France). All animals were acclimatized under constant conditions and handled daily for 7 days before the experiments.

Experimental protocol

The following four experimental groups exhibited different combinations of periodontitis (P) and chronic mild stress (CMS): (a) the control group (P-CMS-); (b) the periodontitis group (P + CMS−); (c) the CMS group (P-CMS+) and (d) the periodontitis and CMS group (P + CMS+). The induction of periodontitis preceded exposure to chronic mild stress (CMS) (Fig.  1 ). Both in vivo protocols have been previously reported and validated individually [ 59 , 60 ] and in combination [ 12 , 13 ], including the periodontal outcomes and behavioral results of the experimental groups used in this study [ 13 ].

Experimental protocol. Induction of periodontitis in Wistar male rats via experimental oral gavage with a solution of periodontal bacteria (12 weeks) and subsequent exposure to a model of chronic mild stress (3 weeks) [ 13 ]. CFU: colony-forming unit, F-IHC: fluorescence immunohistochemistry

The experimental periodontitis model consisted of 12 weeks of oral gavage (4 times per week) of inoculation with two recognized periodontal pathogens, P. gingivalis ATCC W83K1 and F. nucleatum DMSZ 20,482. These bacteria were given to the animals in a viscous solution (2% carboxymethylcellulose) that allows bacteria to adhere to the various structures of the oral cavity. Although some of the solution may potentially reach the intestine, most of it remains on the teeth and it is expelled from the mouth again, thanks to the action of the syringe (the rat cannot keep the entire volume in its mouth and swallow it directly). Furthermore, bacteria rarely colonize healthy guts, even if swallowed, due to gastrointestinal tract barriers, the presence of the resident gut microbiota, and the acidity of the stomach, especially considering the absence of antibiotic treatment previous to periodontitis induction in our experimental setting [ 11 ].

The CMS paradigm encompasses a diverse array of stressors, including [a] food deprivation, [b] water deprivation, [c] cage tilting, [d] soiled cages, [e] grouped housing after a period of water deprivation, [f] stroboscopic illumination [150 flashes/min], and [g] intermittent illumination every 2 h. These stressors were changed daily (two stressors/day) and administered unpredictably for 21 days, with an additional day to maintain stress exposure during subsequent behavioral tests [ 13 ].

Forty-eight male rats were randomly allocated to each group ( n  = 12 rats/group), underwent a 7-day acclimatization period, and subjected to the experimental procedures. Two rats died after anesthesia administration at baseline (P-CMS- group), and two died following periodontitis induction (P + CMS- and P-CMS + groups). Overall, forty-four rats completed the in vivo experimental phase. Among these, twelve were used for fluorescence immunohistochemistry (F-IHC) ( n  = 3 rats/group), while the remaining thirty-six were used for biochemical assays ( n  = 7–9 rats/group).

Tissue specimens

Samples were collected following terminal anesthesia after the last stress session utilizing sodium pentobarbital (320 mg/kg i.p.; Vetoquinol ® , Madrid, Spain). Anesthesia was consistently administered between 2:00 and 3:00 PM to mitigate potential alterations arising from circadian rhythm fluctuations. The animals were divided into two distinct sets for F-IHC and biochemical analyses.

For F-IHC, rats were perfused via the ascending aorta, initially with 200 ml of saline solution, followed by perfusion with 200 ml of 4% paraformaldehyde (PFA) in 0.1 M phosphate-buffered saline (PBS) (pH 7.4). Subsequently, the brains were collected, postfixed in 4% PFA overnight at 4 °C, cryoprotected with 30% sucrose, and frozen. Coronal sections of 30 μm from the FC were obtained using a microtome and stored at − 40 °C immersed in a cryopreserving solution.

For biochemical analyses, the brain was harvested after decapitation, and the left hemisphere FC was dissected and immediately frozen at − 80 °C. Total homogenates and nuclear extracts were prepared from FC tissue. For total homogenates, FC tissue was homogenized in 1× PBS (pH = 7) supplemented with a protease inhibitor cocktail (Complete Roche, Basel, Switzerland) using Tissue-Lyser LT (QIAGEN, Hilden, Germany) at 50/s for 4 min, followed by centrifugation at 19,083 g for 10 min. The resulting supernatant served as the FC protein extract for western blot analysis. A modified procedure based on the method of Schreiber et al. [ 61 ] was employed to obtain nuclear extracts.

Fluorescence immunohistochemistry (F-IHC)

Three slices of the anteroposterior stereotaxic coordinates from bregma 2.7, 1.7, and 1.2 mm were employed for F-IHC analyses. Antigen retrieval involved subjecting the sections to a sodium citrate solution at pH 6.0 for 40 min within a temperature range of 40 °C to 65 °C. The sections were subsequently washed with 0.02 M potassium phosphate-buffered saline (KPBS), immersed in 0.1 M glycine for 20 min to eliminate autofluorescence, washed with KPBS, and blocked for 60 min with 10% bovine serum albumin (BSA) in KPBS containing 0.1% Triton X-100. Next, the sections were incubated overnight with primary antibodies in 10%-BSA KPBS, washed again, incubated with secondary antibodies in 10%-BSA KPBS, and ultimately mounted with Fluoroshield containing 40,6-diamidino-2-phenylindole dihydrochloride (DAPI). Antibody titration to prevent nonspecific interactions and negative controls for each antibody were used to confirm the absence of nonspecific fluorescent signals. Four to six 20× confocal images per section were acquired using an FV1200 confocal Olympus microscope (Olympus, Shinjuku, Tokyo, Japan) at the CAI-UCM Flow Cytometry and Fluorescence Microscopy Unit.

Microglial morphological analysis

The primary antibody used was anti-ionized calcium-binding adapter molecule 1 (Iba1) (ab108539, Abcam, dilution 1:1000), and the secondary antibody used for microglial morphological analysis was Alexa Fluor 555-conjugated donkey anti-rabbit (A31572, Life Technologies, dilution 1:1000).

One hundred forty-four microglia/group were evaluated (4 cells/microphotography × 4 microphotography/section × 3 section/rat × 3 rat/group = 144 cell/group). The analysis was based on the protocol described by Vargas-Caraveo et al. [ 62 ] and executed using the Fiji ImageJ ® package (NIH, Bethesda, MD, USA). The threshold was adjusted to obtain binary images, and each cell was selected using the region of interest (ROI). The extra signal was removed to generate a single-cell image of microglia. The outlined and skeletonized formats of the new single-cell binary files were used for fractal and skeleton analyses, respectively.

Fractal analysis was performed on the outlined cells using the FracLac plugin, with Num G set to 4 and the metric box checked from the binary image. The outlined cells were scanned to obtain hull and circle results, selecting the span ratio, area, and circularity. The soma area was calculated using ROIs. Fractal dimension, lacunarity, and density were selected in the Box count summary, providing insights into microglial shape (elongated or round) and complexity. Cell skeletonized images were analyzed using the Analyze-Skeleton plugin, the skeleton option was selected, and the branch information box was checked. The results showed the number and length of branches.

Inducible nitric oxide synthase (iNOS) and CB2 microglial expression

For the analysis of iNOS microglial expression, the primary antibodies used were anti-iNOS (BD610329 BD Biosciences, 1:1000) and anti-Iba1, and the secondary antibodies used were Alexa Fluor 488-conjugated goat anti-mouse (A11001, Life Technologies, 1:1000) and Alexa Fluor 555-conjugated goat anti-rabbit, respectively. For the analysis of CB2 microglial expression, the primary antibodies used were anti-CB2 (sc-10076 Santa Cruz, 1:500) and anti-Iba1, and the secondary antibodies used were Alexa Fluor 488-conjugated donkey anti-goat (A11055, Life Technologies, 1:1000) and Alexa Fluor 555-conjugated goat anti-rabbit, respectively.

Thirty-six neurons/group were analyzed (4 microphotography/section × 3 section/rat × 3 rat/group = 36 microphotography/group). A threshold was applied to gray images to identify the number of Iba1 + cells. iNOS or CB2 fluorescence intensity was the ratio between the general mean value of the iNOS or CB2 signal and the number of parenchymal microglia (Iba1 + cells) in the image.

CB1 neuronal expression

For the analysis of CB1 neuronal expression, the primary antibodies used were anti-CB1 (ab23703 Abcam, 1:1000) and anti-neuronal nuclear antigen (NeuN) (ab23703 Abcam, 1:1000), and the secondary antibodies used were Alexa Fluor 488-conjugated donkey anti-rabbit (A21206, Life Technologies, 1:1000) and Alexa Fluor 555-conjugated goat anti-mouse (A21422, Life Technologies, 1:1000), respectively.

Fifty-four neurons/group were analyzed (6 microphotography/section × 3 section/rat × 3 rat/group = 54 microphotography/group). A threshold was applied to gray images to obtain the number of NeuN + cells. CB1 fluorescence intensity was the ratio between the general mean value of the CB1 signal and the number of neurons (NeuN + cells) in the image.

Western blot

Protein levels in FC homogenates were quantified using the Bradford method based on the principle of protein-dye binding. Subsequently, 15 µg of protein was mixed with Laemmli sample buffer (Bio-Rad, Hercules, CA, USA) and loaded and size separated by 8% sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (90 V). The gel contents were then transferred to nitrocellulose membranes using the Trans-Blot Turbo Transfer System (Bio-Rad, Hercules, CA, USA).

The membranes were blocked in Tris-buffered saline (TBS) containing 0.1% Tween 20 and 5% BSA for 1 h and incubated overnight at 4 °C with specific primary antibodies against PLD-NAPE (10306 Cayman Chemical, 1:1000), DAGL (sc-133307 Santa-Cruz Biotechnology 1:750, 2% BSA), FAAH (101600 Cayman Chemical 1:1000), MAGL (100035 Cayman Chemical, 1:1000), CB1 (ab23703 Abcam, 1:1000), PI3K (sc7189 Santa Cruz 1:1000, 5% BSA), phospho(p)-Akt (4060 Cell Signaling, 1:1000, BSA 2.5%), Akt (4691 Cell Signaling, 1:2000, BSA 2.5%), p-ERK (8544 Cell Signaling, 1:1000), ERK (4695 Cell Signaling, 1:1000), p-CREB (9198 Cell Signaling, 1:1000), CREB (9197 Cell Signaling, 1:1000), BDNF (ab108319 Abcam, 1:1000, BSA 2.5%), TrkB (ab18987 Abcam, 1:1000, BSA 5%), and synaptophysin (S5768 Sigma Aldrich 1:2000).

Following primary antibody incubation, the membranes were washed and incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies (anti-rabbit IgG, 7074; Cell Signaling, 1:2000; anti-mouse sc516102; Santa Cruz, 1:2000; BSA, 2.5%) for 90 min at room temperature. Finally, the membranes were developed using the ECL Prime kit following the manufacturer’s instructions (Cytiva Marlborough, MA, USA). Blots were imaged using a ChemiDoc™ (Bio-Rad ® , Hercules, CA, USA) and quantified by densitometry with the Fiji ImageJ ® package.

All densitometry data were obtained in arbitrary optical density units and are expressed as a percentage of the control group (100%). Multiple exposure times ensured the linearity of the band intensities. Beta-actin (A5441 Sigma, 1:10000) was used as the loading control for cytosolic extract samples, and GAPDH (G8795 Sigma, 1:5000) was used for nuclear extracts.

Statistical analysis

Details regarding the sample size calculations are provided in Martínez et al. [ 13 ]. In summary, a total of 48 animals (12 animals per group) were evaluated for a difference of 1.6× sigma in the protein expression of molecules associated with neuroinflammation, with a standard deviation (SD) of 25 [ 59 ]. A subset of 12 animals (3 animals per group) was subjected to immunofluorescence studies.

The animal was used as the unit of analysis. The data are presented as the mean ± standard error of the mean (SEM). The Grubbs test identified significant outliers at α = 0.05, allowing for the exclusion of one value per group. The normality of the distribution was assessed using the Shapiro–Wilk test, and variables were also checked for homogeneity of variance by the Brown–Forsythe test. When the data exhibited a Gaussian distribution and equal variances, one-way ANOVA followed by a Tukey post hoc test for multiple comparisons was applied. In cases where SDs were unequal, a Brown–Forsythe ANOVA test followed by Tamhane’s T2 post hoc test was used. For nonnormally distributed data, variables were log-transformed. If the data did not follow a Gaussian distribution after transformation, a nonparametric Kruskal–Wallis test with Dunn’s multiple comparisons was used for the original data. A p value ≤ 0.05 was considered to indicate statistical significance. The data were analyzed using GraphPad Prism 9 (GraphPad Software, San Diego, CA, USA).

Skeletal and fractal analyses provided insights into the morphological properties of microglia through skeletonized (Fig.  2 a-d) and outlined (Fig.  2 a’-d’) shapes (Fig.  2 A-D).

Skeleton analysis revealed alterations in the number and length of microglial branches (Fig.  2 E-F), revealing a significant increase in the number of microglial branches across all experimental groups compared to the control group ( p  < 0.0001) (Fig.  2 E). Notably, the group with the greatest number of branches was P + CMS+. The sum of the branch lengths exhibited a similar pattern ( p  < 0.0001) (Fig.  2 F).

A battery of parameters (cellular area, soma area, span ratio, circularity, lacunarity, fractal dimension, and density) (Fig.  2 G-M) further characterized the morphology of parenchymal microglia in the FC. The cellular area increased in every group compared to that in the control group ( p  < 0.001 vs. P + CMS-, p  < 0.0001 vs. P-CMS+, p  < 0.0001 vs. P + CMS+), with P + CMS + displaying the greatest increase (Fig.  2 G). Similarly, compared with that in the control group, the soma area in the covered surface in all the experimental groups significantly increased ( p  < 0.0001), but no differences were detected among them (Fig.  2 H).

The span ratio, a parameter that measures elongation, was significantly lower in the P-CMS + group than in the control group ( p  < 0.001) (Fig.  2 I). The circumference of microglia increased in every experimental group compared to that in the control group ( p  < 0.05 vs. P + CMS-, p  < 0.0001 vs. P-CMS+, p  < 0.001 vs. P + CMS+) (Fig.  2 J).

Lacunarity indicates morphological heterogeneity and was greater in the P + CMS + group than in the control ( p  < 0.01) and P-CMS+ ( p  < 0.01) groups (Fig.  2 K). Fractal dimension and density, defined as the foreground/hull area ratio, assess complexity (Fig.  2 L-M). The fractal dimension revealed greater complexity of microglia in all experimental groups than in the control group ( p  < 0.001 vs. P + CMS-, p  < 0.0001 vs. P-CMS+, p  < 0.0001 vs. P + CMS+) (Fig.  2 L). The density was lower in the P-CMS+ ( p  < 0.01) and P + CMS+ ( p  < 0.0001) groups than in the control group. Importantly, this parameter was even lower in the P + CMS + group than in the P-CMS + group ( p  < 0.05), emphasizing the heightened complexity of microglia in the P + CMS + group.

Analysis of microglial morphology in the FC of rats in control conditions (P-CMS-) after periodontitis induction (P + CMS-), after chronic mild stress exposure (P-CMS+), and after both protocols combined (P + CMS+). Immunofluorescence of Iba-1 in representative images of 30 mm-thick sections. (P-CMS-) ( A ), (P + CMS-) ( B ), (P-CMS+) ( C ), and (P + CMS+) ( D ) skeletonized and binaries outlined images of the microglia mentioned above. Yellow arrowheads indicate representative cells. Statistical analysis of the number of microglial branches ( E ), the sum of branch length ( F ), cell area ( G ), cellular soma area ( H ), span ratio ( I ), circularity ( J ), lacunarity ( K ), fractal dimension ( L ) and density ( M ). The data are presented as the means ± SEMs of 144 microglia per group. * p  < 0.05; ** p  < 0.01; *** p  < 0.001; **** p  < 0.0001. Scale bars = 20 μm. One-way ANOVA with Tukey’s post hoc test for the cellular soma area and the Kruskal‒Wallis test with Dunn´s post hoc test were used for the remaining parameters

Pro/antiinflammatory phenotype of parenchymal microglia

Following the comprehensive characterization of parenchymal microglial morphology, our focus shifted to their pro/antiinflammatory phenotype within our experimental groups. We investigated the expression of the proinflammatory enzyme iNOS in microglia [ 63 ] (Fig.  3 A-D). iNOS immunoreactivity was significantly greater in the P + CMS + group than in the control group ( p  < 0.001) (Fig.  3 E).

iNOS immunoreactivity in parenchymal microglia of the FC of rats in control conditions (P-CMS-), after periodontitis induction (P + CMS-), after chronic mild stress exposure (P-CMS+), and after both protocols combined (P + CMS+). Immunofluorescence of Iba1 (red), iNOS (green), and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) in nuclei (blue) was performed in representative images of 30 mm-thick sections of rat brain FC from the P-CMS- ( A ), P + CMS- ( B ), P-CMS+ ( C ), and P + CMS+ ( D ) groups. Arrowheads indicate representative cells. Quantitative analysis of iNOS expression in Iba1 + cells ( E ). The data are presented as the means ± SEMs of 34–36 microglia per group. *** p  < 0.001. Kruskal‒Wallis test with Dunn´s post hoc test. Scale bars = 20 μm

CB2 is a typical anti-inflammatory factor in microglia [ 64 ], and we studied its expression (Fig.  4 A-D). CB2 immunoreactivity decreased in the P-CMS + group compared to the control group (Fig.  4 E, p  < 0.01).

CB2 immunoreactivity in parenchymal microglia of the FC of rats in control conditions (P-CMS-), after periodontitis induction (P + CMS-), after chronic mild stress exposure (P-CMS+), and after both protocols combined (P + CMS+). Immunofluorescence of Iba1 (red), CB2 (green) and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) signals in nuclei (blue) in representative images of 30 mm-thick sections of rat brain FC from the P-CMS- ( A ), P + CMS- ( B ), P-CMS+ ( C ), and P + CMS+ ( D ) groups was performed. Arrowheads indicate representative cells. Quantitative analysis of CB2 expression in Iba1 + cells ( E ). The data are presented as the means ± SEMs of 34–36 microglia per group. ** p  < 0.01. One-way ANOVA was performed following Tukey’s post hoc test. Scale bars = 20 μm

Metabolism of ECs

The unexpected outcomes observed for CB2 in P + CMS + microglia prompted an exploration into the metabolic enzymes and signaling of ECs, as this system may contribute (at least partially) to the regulatory effects exerted by microglia on various neurophysiological processes [ 65 , 66 ], including neuroplasticity mediated through neuronal CB1 [ 67 , 68 , 69 ].

The enzyme responsible for AEA synthesis, NAPE-PLD, exhibited a greater reduction in the P + CMS + group than in the control ( p  < 0.05) and P + CMS- ( p  < 0.01) groups (Fig.  5 A). DAGL, at the helm of 2-AG synthesis, decreased in the P + CMS + group compared to the P + CMS- group ( p  < 0.01) (Fig.  5 B). The activity of the AEA degradation enzyme FAAH decreased in the P + CMS + group compared to the P + CMS- group (Fig.  5 C). MAGL is the main enzyme for 2-AG metabolism and exhibited lower protein levels in the P + CMS + group than in the P-CMS + group ( p  < 0.01) (Fig.  5 D). Overall, the protein expression of synthesis and degradation enzymes in ECs decreased in the FC of P + CMS + rats.

Endocannabinoid metabolism in the FC of rats in control conditions (P-CMS-), after periodontitis induction (P + CMS-), after chronic mild stress exposure (P-CMS+), and after both protocols combined (P + CMS+). Protein expression of N-acyl phosphatidylethanolamine-specific phospholipase D (NAPE-PLD) ( A ), diacylglycerol lipase (DAGL) ( B ), fatty acid amide hydrolase (FAAH) ( C ), and monoacylglycerol lipase (MAGL) ( D ) in FC samples by WB. The densitometric data of the band of interest were normalized to that of beta-actin (β-actin). The data are presented as the means ± SEMs of 6–9 rats per group. * p  < 0.05, ** p  < 0.01. One-way ANOVA with a Tukey post hoc test. Blots were cropped (black lines) to improve the clarity and conciseness of the presentation

Given that the CB1 receptor predominantly mediates EC functions in the CNS, including the induction of neuroplasticity, we investigated its expression in FC sections from the different experimental groups by immunofluorescence. We found predominant expression in neurons (NeuN + cells) (Fig.  6 A-D). Further analysis revealed that CB1 neural expression was lower in the P + CMS + group than in the other groups ( p  < 0.0001) (Fig.  6 E). Complementary analysis of CB1 protein expression by WB in FC samples also revealed lower levels in the P + CMS + group than in the control group ( p  < 0.05) (Fig.  6 F).

Cannabinoid receptor 1 (CB1) immunoreactivity in NeuN + cells and CB1 expression by western blot (WB) in the frontal cortex (FC) of rats in control conditions (P-CMS-), after periodontitis induction (P + CMS-) after chronic mild stress exposure (P-CMS+), and after both protocols combined (P + CMS+). Immunofluorescence of NeuN (red), CB1 (green) and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) signals in nuclei (blue) in representative images of 30 mm-thick sections of rat brain FC from the P-CMS- ( A ), P + CMS- ( B ), P-CMS+ ( C ), and P + CMS+ ( D ) groups was performed. Arrowheads indicate representative cells. Quantitative analysis of CB1 expression in NeuN + cells ( E ). The data are presented as the means ± SEMs of 54 neurons per group. **** p  < 0.0001. Scale bars = 20 μm. Protein expression of CB1 in FC samples by WB ( F ). The densitometric data of the band of interest were normalized to that of β-actin. The data are presented as the means ± SEMs of 6–9 rats per group. * p  < 0.05, ** p  < 0.01. One-way ANOVA with a Tukey post hoc test after logarithmic transformation. Blots were cropped (black lines) to improve the clarity and conciseness of the images

Intracellular signaling pathways related to CB1 modulation of neuroplasticity

PI3K/Akt and ERK1-2/CREB are cannabinoid intracellular signaling pathways that control synaptic plasticity. The protein levels of PI3K in the FC samples were significantly lower in the two groups exposed to chronic stress (P-CMS + and P + CMS+) than in the P + CMS- group ( p  < 0.05) (Fig.  7 A). The p-Akt/Akt ratio was lower in the P + CMS + group than in the control ( p  < 0.01) and P + CMS- ( p  < 0.01) groups (Fig.  7 B).

Concerning ERK1-2/CREB, the pERK/ERK ratio was lower in the P + CMS + group than in the control group ( p  < 0.05) (Fig.  7 C). However, the pCREB/CREB ratio remained unaltered across groups (Fig.  7 D).

Intracellular signaling pathways in the frontal cortex (FC) of rats in control conditions (P-CMS-), after periodontitis induction (P + CMS-), after chronic mild stress exposure (P-CMS+), and after both protocols combined (P + CMS+). Protein expression of phosphatidylinositol-3-kinase (PI3K) ( A ), phospho-protein kinase B (p-Akt)/Akt ratio ( B ), p-extracellular signal-regulated kinase (p-ERK)/ERK ratio ( C ) and phospho-cAMP response element-binding protein (p-CREB)/CREB ratio ( D ) in FC samples by WB. The densitometric data of the band of interest were normalized to that of β-actin. The data are presented as the means ± SEMs of 6–9 rats per group. * p  < 0.05, ** p  < 0.01. One-way ANOVA with a Tukey post hoc test. Blots were cropped (black lines) to improve the clarity and conciseness of the images

Synaptic plasticity proteins

BDNF and synaptophysin are pivotal proteins in synaptic plasticity. BDNF protein levels decreased in the P + CMS + group compared to those in the other groups ( p  < 0.05 vs. P-CMS-, p  < 0.01 vs. P + CMS-, p  < 0.0001 vs. P-CMS+) (Fig.  8 A). The level of the active form of the TrkB receptor, which transduces the BDNF signal, increased in the P + CMS- group compared to the control ( p  < 0.01) and P + CMS+ ( p  < 0.01) groups (Fig.  8 B). Notably, the expression pattern of synaptophysin mirrored that of BDNF, with a decrease in the P + CMS + group compared to the control ( p  < 0.01) and P-CMS+ ( p  < 0.01) groups (Fig.  8 C).

Synaptic plasticity markers in the frontal cortex (FC) of rats in control conditions (P-CMS-), after periodontitis induction (P + CMS-), after chronic mild stress exposure (P-CMS+), and after both protocols combined (P + CMS-). Protein expression of brain-derived neurotrophic factor (BDNF) ( A ), tropomyosin receptor kinase B (TrkB) ( B ) and synaptophysin ( C ) in FC samples by WB. The densitometric data of the band of interest were normalized to that of β-actin. The data are presented as the means ± SEMs of 6–9 rats per group. * p  < 0.05, ** p  < 0.01. One-way ANOVA with a Tukey post hoc test. Blots were cropped (black lines) to improve the clarity and conciseness of the images

The results from this investigation revealed a cascade of deleterious effects within the FC of rats exposed to a combined model of periodontitis and depression (Graphical Abstract). P + CMS + animals displayed notable alterations in microglial morphology, inflammatory phenotype, EC metabolism and signaling, and proteins related to synaptic plasticity. These alterations were more noticeable when both diseases were present, which may explain the behavioral impairments previously reported in the P + CMS + group [ 13 ].

The computational approach applied [ 62 , 70 ] described structural alterations in microglia across all experimental groups, characterized by a greater cell and soma size and a general increase in the number and length of branches. These changes were more pronounced in the combined P + CMS + group.

Furthermore, lacunarity and fractal dimension notably increased in the P + CMS + group. Lacunarity, which is associated with soma size, reflects the heterogeneity or rotational variability of a cell, with higher values indicating increased heterogeneity [ 71 ]. Similarly, higher fractal dimension values suggest greater structural complexity [ 71 ]. These parameters provide complementary information, allowing the discernment of differences between P-CMS + and P + CMS + that might otherwise go unnoticed [ 72 ]. Additionally, density decreased in the P + CMS + group compared to all the other groups, indicating higher levels of structural complexity. All these results imply the existence of a hyperbranched phenotype among microglia in our experimental groups [ 73 ]. This phenotype, previously observed in murine models of chronic stress [ 74 , 75 ], appears to be closely linked to depressive-like behaviors [ 76 ].

A branched microglial phenotype in rats was also evident in rats subjected solely to periodontitis (P + CMS-), aligning with previous evidence reporting mild microglial activation in the prefrontal cortex and hippocampus in models involving exposure to LPS from P. gingivalis [ 17 , 77 ]. Systemic P. gingivalis LPS can induce microglial activation at blood‒brain interfaces, such as CVOs [ 62 ]. This activation of microglia is directly associated with the emergence of depressive-like behavior [ 78 ]. Interestingly, the antidepressant imipramine inhibits LPS- P. gingivalis -induced inflammatory responses in microglia and alleviates neuronal damage associated with periodontitis [ 79 ].

The analysis of the inflammatory state of microglia within our experimental groups revealed diverse activity states of microglia. P + CMS + microglia exhibited elevated iNOS expression, which indicates an elevated proinflammatory state. Previous findings in this combined in vivo model of periodontitis and depression revealed depressive-like behavior and neuroinflammation in the FC, with increased expression of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), Toll-like receptor 4 (TLR4), nuclear factor kappa B (NF-κB), iNOS, microsomal prostaglandin E synthase (mPGES), and phospho-p38 mitogen-activated protein kinase (p-p38) [ 13 ].

Compared with the controls, all the experimental groups demonstrated decreased expression of the anti-inflammatory marker CB2, although only in the P-CMS + group there was statistically significant difference. Although specific studies examining the impact of periodontitis on CB2 expression in FC microglia are lacking, lower CB2 expression in human gingival biopsies has been reported [ 34 , 35 ]. Our CB2 results underscore the intricate nature of microglial states following consecutive immune challenges, emphasizing the necessity of employing multiple quantitative and qualitative approaches to comprehensively assess such complexity [ 80 ]. Indeed, microglial complexity and heterogeneity may not unequivocally align with a strictly pro- or anti-inflammatory phenotype and could drive other processes.

The initially unexpected result concerning neuroinflammation and CB2 microglial expression in P + CMS + animals prompted us to investigate the EC signaling in the FC due to the well-established ability of microglia to regulate other crucial cellular processes in the brain through ECs. Notably, such regulation has been documented following immune challenges with LPS and interferon-gamma (IFN-γ) [ 66 ], and both periodontitis and depression independently have demonstrated some effects on the EC system [ 28 , 34 , 41 ].

Neither periodontitis nor CMS altered the expression of EC enzymes in the FC in our experimental setting. However, the NAPE-PLD, DAGL, and MAGL were lower in the combined treatment group than in the control group. Particularly noteworthy was the significant downregulation of the synthesis enzyme NAPE-PLD without changes in the degradation enzyme FAAH, potentially leading to reduced AEA levels and the subsequent partial disruption of the homeostatic endocannabinergic tone in these animals. In line with this, previous research has reported antidepressant-like activity following FAHH inhibition in a rat model of CMS [ 81 ]. Moreover, an anti-inflammatory role for AEA has been documented in a combined model of experimental periodontitis induced by ligatures around the first inferior molars and immobilization stress for 2 h twice daily for 7 days [ 37 ]. Further research must obtain direct evidence about the AEA and 2-AG levels to confirm the indirect evidence provided by the data from EC metabolic enzymes.

CB1 predominantly governs EC actions on neurons, particularly those related to plasticity. Immunofluorescence and expression analyses of total homogenates revealed a downregulation of CB1 in the P + CMS + group. While there is no prior evidence regarding the impact of periodontitis on brain CB1, some research has indicated that periodontitis does not affect CB1 expression in human gingival biopsies [ 35 ]. In contrast, there is extensive information on the effect of chronic stress on CB1 in depression-like animal models. Chronic stress exposure can affect the EC signaling by reducing CB1 density in the hippocampus but not in the limbic forebrain [ 82 ]. Furthermore, CB1 knockout mice are more susceptible to developing depressive-like behavior after CMS exposure [ 83 ], and pharmacological modulation of CB1 prevents the effects of CMS on emotional learning and long-term potentiation [ 84 ].

The signal transduction of CB1 and other pathways related to inflammation requires the activity of PI3K/Akt and ERK1-2/CREB. Similar to the expression pattern of CB1, the levels of PI3K, Akt and ERK decreased exclusively in the P + CMS + group compared to those in the control group. However, there were no discernible alterations in the p-CREB/CREB ratio across any of the experimental groups. Previously, our group reported a reduction in PI3K and Akt in FC samples following CMS exposure [ 85 ], consistent with the outcomes observed in P-CMS + animals. Although no in vivo studies have detailed changes in these molecules in the brain during periodontitis, an in vitro study indicated that microglia stimulated with gingipains exhibited activation of protease-activated receptor 2, leading to subsequent activation of the PI3K/Akt and ERK pathways [ 86 ]. Our results are not specific to microglia and revealed no alterations in the expression of these pathways in P + CMS- animals.

CB1 signaling plays a pivotal role in regulating synaptic plasticity, and activation of the PI3K/Akt and ERK/CREB pathways has been implicated in this neuroprotective response by activating promoter 4 of the BDNF gene [ 87 , 88 ]. Consequently, genetic deletion and pharmacological inhibition of these pathways downregulate BDNF synthesis, leading to reduced cell survival and the onset of working and long-term memory alterations [ 89 ]. The P + CMS + group exhibited lower levels of BDNF, consistent with this plausible pathological scenario occurring in our combined model of periodontitis and depression. Indeed, P. gingivalis can induce depressive-like behavior by downregulating p75NTR-mediated BDNF maturation in astrocytes [ 90 ].

Controversially, BDNF increased in the P-CMS + animals in our experimental setting. Many papers have described a reduction in BDNF levels related to depression and associated its increase with antidepressant response [ 91 , 92 ]. Still, other studies from depression models have reported no changes or increased BDNF expression in different brain areas [ 93 , 94 , 95 , 96 ], with more consistent results of the BDNF reduction in the hippocampus than in the frontal cortex ( [ 97 ]. A possible explanation for the increased BDNF detected in the frontal cortex relies on a decreased turnover of BDNF leading to higher non-released tissue levels, differential stress effects between the hippocampus and the frontal cortex, or compensatory mechanisms due to cortex-hippocampal projections [ 98 ]. Interestingly, BDNF in different areas of the frontal cortex was only upregulated after antidepressant treatment and remained unchanged in MDD drug-free patients [ 99 ], suggesting a role of BDNF in antidepressant effects rather than as a pathophysiological feature of depression in this cerebral area.

Our results did not show changes in neuroplasticity molecules in the P-CMS + group. Nevertheless, we cannot ignore the temporal dynamics associated with BDNF expression [ 100 ] and the fact that we measured them at the time of sacrifice, which does not preclude that the neuroplasticity pathway was reduced any earlier time, potentially contributing to the behavioral impairments of P-CMS + animals. Importantly, the decrease in the neuroplasticity pathway detected in the P + CMS + group may aggravate the pathology [ 78 , 101 ] or affect the potential pharmacological intervention with antidepressants when periodontitis is comorbid. Undoubtedly, further research is warranted to continue delving into the mechanisms of BDNF in depression and its contribution to the disease.

TrkB transduces the BDNF signal by activating cAMP kinases, ultimately regulating the expression of synaptophysin [ 89 , 102 ]. Despite unaltered TrkB levels across all experimental groups, the level of synaptophysin decreased in the P + CMS + group. Synaptophysin plays a role in establishing and maintaining synaptic connections and regulating neurotransmitters within the synaptic space. Reduced synaptophysin expression is associated with synaptic impairment in the hippocampus [ 103 ]. Moreover, the administration of cannabidiol in murine models increased the mRNA expression levels of synaptophysin in the medial prefrontal cortex, resulting in antidepressant effects [ 104 ]. Animals solely exposed to periodontitis in our experimental setting did not exhibit changes in synaptophysin levels. However, other researchers have reported that systemic P. gingivalis infection increases the expression of IL-1β in leptomeninges while simultaneously decreasing the expression of synaptophysin in the cortex adjacent to leptomeninges in mice [ 105 ].

This investigation tackles the difficulties in addressing the combination of periodontitis and depression from a preclinical perspective by attempting to decipher the contribution of a brain cell type, microglia. Nonetheless, certain limitations warrant acknowledgment. First, the descriptive nature of our study is not sufficient to draw a consistent chronological pathway from microglial to plasticity alterations for a mechanistic explanation. Second, the analysis of the metabolic enzymes of the EC system provides only indirect evidence regarding the levels of AEA and 2-AG. Third, the findings in plasticity suggest that behavioral tests focused on cognition would have helped assess the repercussions of the observed molecular changes.

In conclusion, the results from this investigation revealed more pronounced alterations in the microglial phenotype, the EC signaling, and proteins associated with synaptic plasticity in animals exposed to periodontitis and CMS. This evidence underscores the potential of pharmacological interventions targeting these pathways in patients suffering from both conditions. In fact, a recent systematic review with meta-analysis explored the potential beneficial use of antidepressant agents in managing periodontitis [ 106 ], and other reports investigated the potential of infection control measures to treat periodontitis to ameliorate depressive-like symptoms [ 11 ]. These insights suggest promising avenues associated with microglia, the EC signaling, and plasticity for addressing the intricate interplay in the comorbidity between oral health and psychiatric disorders.

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Abbreviations

2-arachidonoylglycerol

Protein kinase B

Blood‒brain barrier

Brain-derived neurotrophic factor

Bovine serum albumin

Cannabinoid receptor

Chronic mild stress

Central nervous system

cAMP response element-binding protein

Diacylglycerol lipase

Endocannabinoid

Extracellular signal-regulated kinase

Fusobacterium nucleatum

Fatty acid amide hydrolase

Frontal cortex

Fluorescence immunohistochemistry

Glyceraldehyde-3-phosphate dehydrogenase

Anti-ionized calcium-binding adapter molecule 1

Inducible nitric oxide synthase

Potassium phosphate-buffered saline

Monoacylglycerol lipase

Mitogen-activated protein kinases

Major depressive disorder

N-acyl phosphatidylethanolamine phospholipase

Porphyromonas gingivalis

Phosphate-buffered saline

Paraformaldehyde

Phosphatidylinositol-3-kinase

Region of interest

Standard error of the mean

Tropomyosin receptor kinase B

Otte C, Gold SM, Penninx BW, Pariante CM, Etkin A, Fava M, Mohr DC, Schatzberg AF. Major depressive disorder. Nat Reviews Disease Primers. 2016;2:16065.

Article   PubMed   Google Scholar  

GBD: Global Burden of Disease Study 2019. (GBD 2019) Results tool. Seattle, United States of America: Institute for Health Metrics and Evaluation (IHME); 2021.

World Health A. Global burden of mental disorders and the need for a comprehensive, coordinated response from health and social sectors at the country level: report by the Secretariat. Geneva: World Health Organization; 2012.

Google Scholar  

Yuan K, Zheng YB, Wang YJ, Sun YK, Gong YM, Huang YT, Chen X, Liu XX, Zhong Y, Su SZ, et al. A systematic review and meta-analysis on prevalence of and risk factors associated with depression, anxiety and insomnia in infectious diseases, including COVID-19: a call to action. Mol Psychiatry. 2022;27:3214–22.

Article   CAS   PubMed   PubMed Central   Google Scholar  

Greenberg P, Chitnis A, Louie D, Suthoff E, Chen SY, Maitland J, Gagnon-Sanschagrin P, Fournier AA, Kessler RC. The Economic Burden of Adults with Major Depressive Disorder in the United States (2019). Adv Ther 2023.

Jaffe DH, Rive B, Denee TR. The humanistic and economic burden of treatment-resistant depression in Europe: a cross-sectional study. BMC Psychiatry. 2019;19:247.

Article   PubMed   PubMed Central   Google Scholar  

Pérez-Sola V, Roca M, Alonso J, Gabilondo A, Hernando T, Sicras-Mainar A, Sicras-Navarro A, Herrera B, Vieta E. Economic impact of treatment-resistant depression: a retrospective observational study. J Affect Disord. 2021;295:578–86.

Fries GR, Saldana VA, Finnstein J, Rein T. Molecular pathways of major depressive disorder converge on the synapse. Mol Psychiatry. 2023;28:284–97.

Article   CAS   PubMed   Google Scholar  

Sorrells SF, Caso JR, Munhoz CD, Sapolsky RM. The stressed CNS: when glucocorticoids aggravate inflammation. Neuron. 2009;64:33–9.

Chen X, Yao T, Cai J, Fu X, Li H, Wu J. Systemic inflammatory regulators and 7 major psychiatric disorders: a two-sample mendelian randomization study. Prog Neuropsychopharmacol Biol Psychiatry. 2022;116:110534.

Martínez M, Postolache TT, García-Bueno B, Leza JC, Figuero E, Lowry CA, Malan-Müller S. The role of the oral microbiota related to Periodontal diseases in anxiety, Mood and Trauma- and stress-related disorders. Front Psychiatry. 2022;12:814177.

Martín-Hernández D, Martínez M, Robledo-Montaña J, Muñoz-López M, Virto L, Ambrosio N, Marín MJ, Montero E, Herrera D, Sanz M et al. Neuroinflammation related to the blood-brain barrier and sphingosine-1-phosphate in a pre-clinical model of periodontal diseases and depression in rats. J Clin Periodontol 2023.

Martínez M, Martín-Hernández D, Virto L, MacDowell KS, Montero E, González-Bris Á, Marín MJ, Ambrosio N, Herrera D, Leza JC, et al. Periodontal diseases and depression: a pre-clinical in vivo study. J Clin Periodontol. 2021;48:503–27.

Serrats J, Schiltz JC, García-Bueno B, van Rooijen N, Reyes TM, Sawchenko PE. Dual roles for perivascular macrophages in immune-to-brain signaling. Neuron. 2010;65:94–106.

Li H, Sagar AP, Kéri S. Microglial markers in the frontal cortex are related to cognitive dysfunctions in major depressive disorder. J Affect Disord. 2018;241:305–10.

Brás JP, Guillot de Suduiraut I, Zanoletti O, Monari S, Meijer M, Grosse J, Barbosa MA, Santos SG, Sandi C, Almeida MI. Stress-induced depressive-like behavior in male rats is associated with microglial activation and inflammation dysregulation in the hippocampus in adulthood. Brain Behav Immun. 2022;99:397–408.

Almarhoumi R, Alvarez C, Harris T, Tognoni CM, Paster BJ, Carreras I, Dedeoglu A, Kantarci A. Microglial cell response to experimental periodontal disease. J Neuroinflammation. 2023;20:142.

Wang H, He Y, Sun Z, Ren S, Liu M, Wang G, Yang J. Microglia in depression: an overview of microglia in the pathogenesis and treatment of depression. J Neuroinflammation. 2022;19:132.

Yirmiya R. Depressive disorder-Associated Microglia as a target for a Personalized Antidepressant Approach. Biol Psychiatry. 2023;94:602–4.

Enache D, Pariante CM, Mondelli V. Markers of central inflammation in major depressive disorder: a systematic review and meta-analysis of studies examining cerebrospinal fluid, positron emission tomography and post-mortem brain tissue. Brain Behav Immun. 2019;81:24–40.

He L, Zheng Y, Huang L, Ye J, Ye Y, Luo H, Chen X, Yao W, Chen J, Zhang JC. Nrf2 regulates the arginase 1(+) microglia phenotype through the initiation of TREM2 transcription, ameliorating depression-like behavior in mice. Transl Psychiatry. 2022;12:459.

Scheepstra KWF, Mizee MR, van Scheppingen J, Adelia A, Wever DD, Mason MRJ, Dubbelaar ML, Hsiao CC, Eggen BJL, Hamann J, Huitinga I. Microglia Transcriptional profiling in Major Depressive Disorder Shows Inhibition of Cortical Gray Matter Microglia. Biol Psychiatry. 2023;94:619–29.

Setiawan E, Wilson AA, Mizrahi R, Rusjan PM, Miler L, Rajkowska G, Suridjan I, Kennedy JL, Rekkas PV, Houle S, Meyer JH. Role of Translocator Protein Density, a marker of Neuroinflammation, in the Brain during Major depressive episodes. JAMA Psychiatry. 2015;72:268–75.

Paolicelli RC, Sierra A, Stevens B, Tremblay ME, Aguzzi A, Ajami B, Amit I, Audinat E, Bechmann I, Bennett M, et al. Microglia states and nomenclature: a field at its crossroads. Neuron. 2022;110:3458–83.

Snijders G, Sneeboer MAM, Fernández-Andreu A, Udine E, Boks MP, Ormel PR, van Berlekom AB, van Mierlo HC, Bӧttcher C, Priller J, et al. Distinct non-inflammatory signature of microglia in post-mortem brain tissue of patients with major depressive disorder. Mol Psychiatry. 2021;26:3336–49.

Wohleb ES, Terwilliger R, Duman CH, Duman RS. Stress-Induced neuronal colony stimulating factor 1 provokes microglia-mediated neuronal remodeling and depressive-like Behavior. Biol Psychiatry. 2018;83:38–49.

Marcaggi P, Attwell D. Endocannabinoid signaling depends on the spatial pattern of synapse activation. Nat Neurosci. 2005;8:776–81.

Rodríguez-Muñoz M, Sánchez-Blázquez P, Callado LF, Meana JJ, Garzón-Niño J. Schizophrenia and depression, two poles of endocannabinoid system deregulation. Transl Psychiatry. 2017;7:1291.

Garani R, Watts JJ, Mizrahi R. Endocannabinoid system in psychotic and mood disorders, a review of human studies. Prog Neuropsychopharmacol Biol Psychiatry. 2021;106:110096.

Hill MN, Carrier EJ, McLaughlin RJ, Morrish AC, Meier SE, Hillard CJ, Gorzalka BB. Regional alterations in the endocannabinoid system in an animal model of depression: effects of concurrent antidepressant treatment. J Neurochem. 2008;106:2322–36.

McPartland JM, Guy GW, Di Marzo V. Care and feeding of the endocannabinoid system: a systematic review of potential clinical interventions that upregulate the endocannabinoid system. PLoS ONE. 2014;9:e89566.

Arjmand S, Landau AM, Varastehmoradi B, Andreatini R, Joca S, Wegener G. The intersection of astrocytes and the endocannabinoid system in the lateral habenula: on the fast-track to novel rapid-acting antidepressants. Mol Psychiatry. 2022;27:3138–49.

Sharafi A, Pakkhesal S, Fakhari A, Khajehnasiri N, Ahmadalipour A. Rapid treatments for depression: endocannabinoid system as a therapeutic target. Neurosci Biobehav Rev. 2022;137:104635.

Pellegrini G, Carmagnola D, Toma M, Rasperini G, Orioli M, Dellavia C. Involvement of the endocannabinoid system in current and recurrent periodontitis: a human study. J Periodontal Res. 2023;58:422–32.

Ataei A, Rahim Rezaee SA, Moeintaghavi A, Ghanbari H, Azizi M. Evaluation of cannabinoid receptors type 1–2 in periodontitis patients. Clin Exp Dent Res. 2022;8:1040–4.

Nakajima Y, Furuichi Y, Biswas KK, Hashiguchi T, Kawahara K, Yamaji K, Uchimura T, Izumi Y, Maruyama I. Endocannabinoid, anandamide in gingival tissue regulates the periodontal inflammation through NF-kappaB pathway inhibition. FEBS Lett. 2006;580:613–9.

Rettori E, De Laurentiis A, Zorrilla Zubilete M, Rettori V, Elverdin JC. Anti-inflammatory effect of the endocannabinoid anandamide in experimental periodontitis and stress in the rat. Neuroimmunomodulation. 2012;19:293–303.

Liu C, Qi X, Alhabeil J, Lu H, Zhou Z. Activation of cannabinoid receptors promote periodontal cell adhesion and migration. J Clin Periodontol. 2019;46:1264–72.

Yan W, Li L, Ge L, Zhang F, Fan Z, Hu L. The cannabinoid receptor I (CB1) enhanced the osteogenic differentiation of BMSCs by rescue impaired mitochondrial metabolism function under inflammatory condition. Stem Cell Res Ther. 2022;13:22.

Zhang F, Özdemir B, Nguyen PQ, Andrukhov O, Rausch-Fan X. Methanandamide diminish the Porphyromonas gingivalis lipopolysaccharide induced response in human periodontal ligament cells. BMC Oral Health. 2020;20:107.

Jäger A, Setiawan M, Beins E, Schmidt-Wolf I, Konermann A. Analogous modulation of inflammatory responses by the endocannabinoid system in periodontal ligament cells and microglia. Head Face Med. 2020;16:26.

Devane WA, Hanuš L, Breuer A, Pertwee RG, Stevenson LA, Griffin G, Gibson D, Mandelbaum A, Etinger A, Mechoulam R. Isolation and structure of a brain constituent that binds to the cannabinoid receptor. Science. 1992;258:1946–9.

Mechoulam R, Ben-Shabat S, Hanus L, Ligumsky M, Kaminski NE, Schatz AR, Gopher A, Almog S, Martin BR, Compton DR, et al. Identification of an endogenous 2-monoglyceride, present in canine gut, that binds to cannabinoid receptors. Biochem Pharmacol. 1995;50:83–90.

Matsuda LA, Lolait SJ, Brownstein MJ, Young AC, Bonner TI. Structure of a cannabinoid receptor and functional expression of the cloned cDNA. Nature. 1990;346:561–4.

Munro S, Thomas KL, Abu-Shaar M. Molecular characterization of a peripheral receptor for cannabinoids. Nature. 1993;365:61–5.

Maccarrone M, Di Marzo V, Gertsch J, Grether U, Howlett AC, Hua T, Makriyannis A, Piomelli D, Ueda N, van der Stelt M. Goods and bads of the Endocannabinoid System as a therapeutic target: lessons learned after 30 years. Pharmacol Rev. 2023;75:885–958.

Mackie K. Cannabinoid receptors: where they are and what they do. J Neuroendocrinol. 2008;20(Suppl 1):10–4.

Duffy SS, Hayes JP, Fiore NT, Moalem-Taylor G. The cannabinoid system and microglia in health and disease. Neuropharmacology. 2021;190:108555.

Malek N, Popiolek-Barczyk K, Mika J, Przewlocka B, Starowicz K. Anandamide, Acting via CB2 Receptors, Alleviates LPS-Induced Neuroinflammation in Rat Primary Microglial Cultures. Neural Plast 2015, 2015:130639.

Johannessen M, Delghandi MP, Moens U. What turns CREB on? Cell Signal. 2004;16:1211–27.

Innes S, Pariante CM, Borsini A. Microglial-driven changes in synaptic plasticity: a possible role in major depressive disorder. Psychoneuroendocrinology. 2019;102:236–47.

Wang T, Weng H, Zhou H, Yang Z, Tian Z, Xi B, Li Y. Esketamine alleviates postoperative depression-like behavior through anti-inflammatory actions in mouse prefrontal cortex. J Affect Disord. 2022;307:97–107.

Cheng CM, Hong CJ, Lin HC, Chu PJ, Chen MH, Tu PC, Bai YM, Chang WH, Juan CH, Lin WC, et al. Predictive roles of brain-derived neurotrophic factor Val66Met polymorphism on antidepressant efficacy of different forms of prefrontal brain stimulation monotherapy: a randomized, double-blind, sham-controlled study. J Affect Disord. 2022;297:353–9.

Kuwano N, Kato TA, Mitsuhashi M, Sato-Kasai M, Shimokawa N, Hayakawa K, Ohgidani M, Sagata N, Kubo H, Sakurai T, Kanba S. Neuron-related blood inflammatory markers as an objective evaluation tool for major depressive disorder: an exploratory pilot case-control study. J Affect Disord. 2018;240:88–98.

Varea E, Castillo-Gómez E, Gómez-Climent MA, Blasco-Ibáñez JM, Crespo C, Martínez-Guijarro FJ, Nàcher J. Chronic antidepressant treatment induces contrasting patterns of synaptophysin and PSA-NCAM expression in different regions of the adult rat telencephalon. Eur Neuropsychopharmacol. 2007;17:546–57.

Flores-Tochihuitl J, Márquez Villegas B, Peral Lemus AC, Andraca Hernández CJ, Flores G, Morales-Medina JC. Periodontitis and Diabetes reshape neuronal dendritic arborization in the thalamus and nucleus oralis in the rat. Synapse. 2021;75:e22187.

Xie C, Zhang Q, Ye X, Wu W, Cheng X, Ye X, Ruan J, Pan X. Periodontitis-induced neuroinflammation impacts dendritic spine immaturity and cognitive impairment. Oral Dis 2023.

Percie du Sert N, Ahluwalia A, Alam S, Avey MT, Baker M, Browne WJ, Clark A, Cuthill IC, Dirnagl U, Emerson M, et al. Reporting animal research: explanation and elaboration for the ARRIVE guidelines 2.0. PLoS Biol. 2020;18:e3000411.

Martín-Hernández D, Caso J, Bris Á, Maus S, Madrigal J, García-Bueno B, MacDowell K, Alou L, Gómez-Lus M, Leza J. Bacterial translocation affects intracellular neuroinflammatory pathways in a depression-like model in rats. Neuropharmacology. 2016;103:122–33.

Virto L, Cano P, Jiménez-Ortega V, Fernández-Mateos P, González J, Esquifino AI, Sanz M. Obesity and periodontitis: an experimental study to evaluate periodontal and systemic effects of comorbidity. J Periodontol. 2018;89:176–85.

Schreiber E, Matthias P, Müller MM, Schaffner W. Rapid detection of octamer binding proteins with ‘mini-extracts’, prepared from a small number of cells. Nucleic Acids Res. 1989;17:6419.

Vargas-Caraveo A, Sayd A, Robledo-Montaña J, Caso JR, Madrigal JLM, García-Bueno B, Leza JC. Toll-like receptor 4 agonist and antagonist lipopolysaccharides modify innate immune response in rat brain circumventricular organs. J Neuroinflammation. 2020;17:6.

Lisi L, Ciotti GM, Braun D, Kalinin S, Currò D, Dello Russo C, Coli A, Mangiola A, Anile C, Feinstein DL, Navarra P. Expression of iNOS, CD163 and ARG-1 taken as M1 and M2 markers of microglial polarization in human glioblastoma and the surrounding normal parenchyma. Neurosci Lett. 2017;645:106–12.

Komorowska-Müller JA, Schmöle AC. CB2 receptor in Microglia: the Guardian of Self-Control. Int J Mol Sci 2020, 22.

Morcuende A, García-Gutiérrez MS, Tambaro S, Nieto E, Manzanares J, Femenia T. Immunomodulatory Role of CB2 receptors in Emotional and Cognitive disorders. Front Psychiatry. 2022;13:866052.

Young AP, Denovan-Wright EM. The microglial endocannabinoid system is similarly regulated by lipopolysaccharide and interferon gamma. J Neuroimmunol. 2022;372:577971.

Tan H, Lauzon NM, Bishop SF, Bechard MA, Laviolette SR. Integrated cannabinoid CB1 receptor transmission within the amygdala-prefrontal cortical pathway modulates neuronal plasticity and emotional memory encoding. Cereb Cortex. 2010;20:1486–96.

Paes-Colli Y, Trindade PMP, Vitorino LC, Piscitelli F, Iannotti FA, Campos RMP, Isaac AR, de Aguiar AFL, Allodi S, de Mello FG, et al. Activation of cannabinoid type 1 receptor (CB1) modulates oligodendroglial process branching complexity in rat hippocampal cultures stimulated by olfactory ensheathing glia-conditioned medium. Front Cell Neurosci. 2023;17:1134130.

Molina-Holgado E, Vela JM, Arévalo-Martín A, Almazán G, Molina-Holgado F, Borrell J, Guaza C. Cannabinoids promote oligodendrocyte progenitor survival: involvement of cannabinoid receptors and phosphatidylinositol-3 kinase/Akt signaling. J Neurosci. 2002;22:9742–53.

Karperien AL, Jelinek HF. Fractal, multifractal, and lacunarity analysis of microglia in tissue engineering. Front Bioeng Biotechnol. 2015;3:51.

Fernández-Arjona MDM, Grondona JM, Fernández-Llebrez P, López-Ávalos MD. Microglial morphometric parameters correlate with the expression level of IL-1β, and allow identifying different activated morphotypes. Front Cell Neurosci. 2019;13:472.

Karperien A, Ahammer H, Jelinek HF. Quantitating the subtleties of microglial morphology with fractal analysis. Front Cell Neurosci. 2013;7:3.

Vidal-Itriago A, Radford RAW, Aramideh JA, Maurel C, Scherer NM, Don EK, Lee A, Chung RS, Graeber MB, Morsch M. Microglia morphophysiological diversity and its implications for the CNS. Front Immunol. 2022;13:997786.

Hinwood M, Tynan RJ, Charnley JL, Beynon SB, Day TA, Walker FR. Chronic stress induced remodeling of the prefrontal cortex: structural re-organization of microglia and the inhibitory effect of minocycline. Cereb Cortex. 2013;23:1784–97.

Walker FR, Nilsson M, Jones K. Acute and chronic stress-induced disturbances of microglial plasticity, phenotype and function. Curr Drug Targets. 2013;14:1262–76.

Hellwig S, Brioschi S, Dieni S, Frings L, Masuch A, Blank T, Biber K. Altered microglia morphology and higher resilience to stress-induced depression-like behavior in CX3CR1-deficient mice. Brain Behav Immun. 2016;55:126–37.

Mamunur R, Hashioka S, Azis IA, Jaya MA, Jerin SJF, Kimura-Kataoka K, Fujihara J, Inoue K, Inagaki M, Takeshita H. Systemic Administration of Porphyromonas Gingivalis Lipopolysaccharide induces glial activation and depressive-like Behavior in rats. J Integr Neurosci. 2023;22:120.

Li Y, Guan X, He Y, Jia X, Pan L, Wang Y, Han Y, Zhao R, Yang J, Hou T. ProBDNF signaling is involved in periodontitis-induced depression-like behavior in mouse hippocampus. Int Immunopharmacol. 2023;116:109767.

Yamawaki Y, So H, Oue K, Asano S, Furusho H, Miyauchi M, Tanimoto K, Kanematsu T. Imipramine prevents Porphyromonas gingivalis lipopolysaccharide-induced microglial neurotoxicity. Biochem Biophys Res Commun. 2022;634:92–9.

Zhang Y, Cui D. Evolving models and tools for Microglial studies in the Central Nervous System. Neurosci Bull. 2021;37:1218–33.

Bortolato M, Mangieri RA, Fu J, Kim JH, Arguello O, Duranti A, Tontini A, Mor M, Tarzia G, Piomelli D. Antidepressant-like activity of the fatty acid amide hydrolase inhibitor URB597 in a rat model of chronic mild stress. Biol Psychiatry. 2007;62:1103–10.

Hill MN, Patel S, Carrier EJ, Rademacher DJ, Ormerod BK, Hillard CJ, Gorzalka BB. Downregulation of endocannabinoid signaling in the hippocampus following chronic unpredictable stress. Neuropsychopharmacology. 2005;30:508–15.

Martin M, Ledent C, Parmentier M, Maldonado R, Valverde O. Involvement of CB1 cannabinoid receptors in emotional behaviour. Psychopharmacology. 2002;159:379–87.

Segev A, Rubin AS, Abush H, Richter-Levin G, Akirav I. Cannabinoid receptor activation prevents the effects of chronic mild stress on emotional learning and LTP in a rat model of depression. Neuropsychopharmacology. 2014;39:919–33.

Martin-Hernandez D, Bris AG, MacDowell KS, Garcia-Bueno B, Madrigal JL, Leza JC, Caso JR. Modulation of the antioxidant nuclear factor (erythroid 2-derived)-like 2 pathway by antidepressants in rats. Neuropharmacology. 2016;103:79–91.

Liu Y, Wu Z, Nakanishi Y, Ni J, Hayashi Y, Takayama F, Zhou Y, Kadowaki T, Nakanishi H. Infection of microglia with Porphyromonas gingivalis promotes cell migration and an inflammatory response through the gingipain-mediated activation of protease-activated receptor-2 in mice. Sci Rep. 2017;7:11759.

García-Gutiérrez MS, Ortega-Álvaro A, Busquets-García A, Pérez-Ortiz JM, Caltana L, Ricatti MJ, Brusco A, Maldonado R, Manzanares J. Synaptic plasticity alterations associated with memory impairment induced by deletion of CB2 cannabinoid receptors. Neuropharmacology. 2013;73:388–96.

Blázquez C, Chiarlone A, Bellocchio L, Resel E, Pruunsild P, García-Rincón D, Sendtner M, Timmusk T, Lutz B, Galve-Roperh I, Guzmán M. The CB₁ cannabinoid receptor signals striatal neuroprotection via a PI3K/Akt/mTORC1/BDNF pathway. Cell Death Differ. 2015;22:1618–29.

Bright U, Akirav I. Modulation of Endocannabinoid System Components in Depression: pre-clinical and clinical evidence. Int J Mol Sci 2022, 23.

Wang YX, Kang XN, Cao Y, Zheng DX, Lu YM, Pang CF, Wang Z, Cheng B, Peng Y. Porphyromonas gingivalis induces depression via downregulating p75NTR-mediated BDNF maturation in astrocytes. Brain Behav Immun. 2019;81:523–34.

Cavaleri D, Moretti F, Bartoccetti A, Mauro S, Crocamo C, Carrà G, Bartoli F. The role of BDNF in major depressive disorder, related clinical features, and antidepressant treatment: insight from meta-analyses. Neurosci Biobehav Rev. 2023;149:105159.

Hu X, Zhao HL, Kurban N, Qin Y, Chen X, Cui SY, Zhang YH. Reduction of BDNF Levels and Biphasic Changes in Glutamate Release in the Prefrontal Cortex Correlate with Susceptibility to Chronic Stress-Induced Anhedonia. eNeuro 2023, 10.

Allaman I, Papp M, Kraftsik R, Fiumelli H, Magistretti PJ, Martin JL. Expression of brain-derived neurotrophic factor is not modulated by chronic mild stress in the rat hippocampus and amygdala. Pharmacol Rep. 2008;60:1001–7.

CAS   PubMed   Google Scholar  

Larsen MH, Mikkelsen JD, Hay-Schmidt A, Sandi C. Regulation of brain-derived neurotrophic factor (BDNF) in the chronic unpredictable stress rat model and the effects of chronic antidepressant treatment. J Psychiatr Res. 2010;44:808–16.

Lin L, Herselman MF, Zhou XF, Bobrovskaya L. Effects of corticosterone on BDNF expression and mood behaviours in mice. Physiol Behav. 2022;247:113721.

Schulte-Herbrüggen O, Fuchs E, Abumaria N, Ziegler A, Danker-Hopfe H, Hiemke C, Hellweg R. Effects of Escitalopram on the regulation of brain-derived neurotrophic factor and nerve growth factor protein levels in a rat model of chronic stress. J Neurosci Res. 2009;87:2551–60.

Elfving B, Plougmann PH, Müller HK, Mathé AA, Rosenberg R, Wegener G. Inverse correlation of brain and blood BDNF levels in a genetic rat model of depression. Int J Neuropsychopharmacol. 2010;13:563–72.

Dionisie V, Ciobanu AM, Toma VA, Manea MC, Baldea I, Olteanu D, Sevastre-Berghian A, Clichici S, Manea M, Riga S, Filip GA. Escitalopram targets oxidative stress, Caspase-3, BDNF and MeCP2 in the Hippocampus and Frontal Cortex of a rat model of Depression Induced by Chronic unpredictable mild stress. Int J Mol Sci 2021, 22.

Sheldrick A, Camara S, Ilieva M, Riederer P, Michel TM. Brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT3) levels in post-mortem brain tissue from patients with depression compared to healthy individuals - a proof of concept study. Eur Psychiatry. 2017;46:65–71.

Caffino L, Mottarlini F, Piva A, Rizzi B, Fumagalli F, Chiamulera C. Temporal dynamics of BDNF signaling recruitment in the rat prefrontal cortex and hippocampus following a single infusion of a translational dose of ketamine. Neuropharmacology. 2024;242:109767.

Ma X, Yoo JW, Shin YJ, Park HS, Son YH, Kim DH. Alleviation of Porphyromonas gingivalis or Its Extracellular Vesicles Provoked Periodontitis and Cognitive Impairment by Lactobacillus pentosus NK357 and Bifidobacterium bifidum NK391. Nutrients 2023, 15.

Tartaglia N, Du J, Tyler WJ, Neale E, Pozzo-Miller L, Lu B. Protein synthesis-dependent and -independent regulation of hippocampal synapses by brain-derived neurotrophic factor. J Biol Chem. 2001;276:37585–93.

Zhong Q, Xu H, Qin J, Zeng LL, Hu D, Shen H. Functional parcellation of the hippocampus from resting-state dynamic functional connectivity. Brain Res. 2019;1715:165–75.

Xu C, Chang T, Du Y, Yu C, Tan X, Li X. Pharmacokinetics of oral and intravenous cannabidiol and its antidepressant-like effects in chronic mild stress mouse model. Environ Toxicol Pharmacol. 2019;70:103202.

Huang W, Zeng F, Gu Y, Jiang M, Zhang X, Yan X, Kadowaki T, Mizutani S, Kashiwazaki H, Ni J, Wu Z. Porphyromonas Gingivalis infection induces synaptic failure via increased IL-1β production in Leptomeningeal cells. J Alzheimers Dis. 2021;83:665–81.

Muniz F, Melo IM, Rösing CK, de Andrade GM, Martins RS, Moreira M, Carvalho RS. Use of antidepressive agents as a possibility in the management of periodontal diseases: a systematic review of experimental studies. J Investig Clin Dent 2018, 9.

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Acknowledgements

The authors thank the Experimental Animal Center (Centro de Asistencia a la Investigación (CAI) Animalario), Complutense University, Madrid, for the care and maintenance of the animals that were used in this study. Karina S. MacDowell is acknowledged for her support in administrative tasks.

This study was funded through a research grant from Santander-University Complutense of Madrid Projects in 2017 (PR41/17–20979; principal investigator: Elena Figuero), by MINECO-FEDER Funds (PID2019-109033RB-100 and PID2022-137932NB-I00 principal investigators: Juan Carlos Leza and Elena Figuero), and CIBERSAM/ISCIII.

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Elena Figuero and David Martín-Hernández as joint senior authors.

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Department of Pharmacology and Toxicology, School of Medicine, Faculty of Medicine, Complutense University of Madrid (UCM), Hospital 12 de Octubre Research Institute (Imas12), Neurochemistry Research Institute UCM (IUIN), Pza. Ramón y Cajal s/n, Madrid, 28040, Spain

Javier Robledo-Montaña, César Díaz-García, Marina Muñoz-López, Juan Carlos Leza, Borja García-Bueno & David Martín-Hernández

Biomedical Network Research Center of Mental Health (CIBERSAM), Institute of Health Carlos III, Madrid, Spain

ETEP (Etiology and Therapy of Periodontal and Peri-Implant Diseases) Research Group, Complutense University of Madrid, Madrid, Spain

María Martínez, Nagore Ambrosio, Eduardo Montero, María José Marín, Leire Virto, David Herrera, Mariano Sanz & Elena Figuero

Department of Dental Clinical Specialties, School of Dentistry, Faculty of Dentistry, Complutense University of Madrid, Pza. Ramón y Cajal s/n, Madrid, 28040, Spain

María Martínez, Nagore Ambrosio, Eduardo Montero, David Herrera, Mariano Sanz & Elena Figuero

Department of Anatomy and Embryology, Faculty of Optics, Complutense University of Madrid, Madrid, Spain

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All the authors conceived and planned the experiments. NA and MJM prepared the oral gavage solutions. DMH, MM, LV, and EM performed the preclinical interventions in the animals. JRM, CDG, MML, BGB, and DMH carried out the immunohistochemical and biochemical analyses. JRM performed the computational analysis of the microglial phenotype. JRM, CDG, BGB, and DMH performed the statistical analyses. DH, MS, JCL, BGB, EF, and DMH contributed to the interpretation of the results. JRM, BGB, and DMH wrote the manuscript. All authors discussed and reviewed the manuscript.

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Correspondence to Elena Figuero or David Martín-Hernández .

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This study was designed according to the modified ARRIVE guidelines 2.0 for preclinical in vivo research [ 58 ] and following Spanish and European Union regulations (European Communities Council Directive 86/609/EEC). The in vivo experimental part of the study was carried out in the Experimental Animal Center of the Complutense University of Madrid after its protocol was approved by the regional authorities (PROEX 087/18) and the Ethical Committee of Animal Experimentation.

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Robledo-Montaña, J., Díaz-García, C., Martínez, M. et al. Microglial morphological/inflammatory phenotypes and endocannabinoid signaling in a preclinical model of periodontitis and depression. J Neuroinflammation 21 , 219 (2024). https://doi.org/10.1186/s12974-024-03213-5

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Oral dosing of rodents using a palatable tablet

Sandeep s. dhawan.

1 School of Psychology and Neuroscience, University of St Andrews, St Mary’s Quad, South Street, St Andrews, Fife, KY16 9JP UK

David S. Tait

Christoffer bundgaard.

2 H. Lundbeck A/S, Discovery DMPK, 9 Ottiliavej, DK-2500 Valby, Denmark

Ellen Bowman

Verity j. brown.

Delivering orally bioavailable drugs to rodents is an important component to investigating that route of administration in novel treatments for humans. However, the traditional method of oral gavage requires training, is stressful, and can induce oesophageal damage in rodents.

To demonstrate a novel administrative technique—palatable gelatine tablets—as a stress-free route of oral delivery.

Twenty-four male Lister hooded rats were sacrificed for brain tissue analysis at varying time-points after jelly administration of 30 mg/kg of the wake-promoting drug modafinil. A second group of 22 female rats were tested on locomotor activity after 30 mg/kg modafinil, or after vehicle jellies, with the locomotor data compared to the brain tissue concentrations at the corresponding times.

Modafinil was present in the brain tissue at all time-points, reducing in concentration over time. The pattern of brain tissue modafinil concentration is comparable to previously reported results following oral gavage. Modafinil-treated rats were more active than control rats, with greater activity during the later time-periods—similar to that previously reported following intraperitoneal injection of 40 mg/kg modafinil.

Conclusions

Palatable jelly tablets are an effective route of administration of thermally stable orally bioavailable compounds, eliminating the stress/discomfort and health risk of oral gavage and presenting as an alternative to previously reported palatable routes of administration where high protein and fat levels may adversely affect appetite for food reward, and uptake rate in the gastrointestinal tract.

Introduction

Administration of pharmacological agents in experimental animals, to investigate effects on the brain and behaviour, is performed via a variety of routes—e.g. intra-cerebrally (via implanted catheters in the brain); by injection (intraperitoneal—i.p.; subcutaneous—s.c.; intravenous—i.v.; or intramuscular—i.m.); transdermally (via a skin-patch; or onto a mucous membrane); by inhalation; orally (by gavage—insertion directly into the stomach; or through mixing with food or drink). The route chosen will impact the pharmacokinetics (e.g. absorption rate), and therefore influence the timing and magnitude of any behavioural effect, but there are also other practical considerations when selecting a particular route of administration. For example, there may be secondary behavioural effects of giving the drug: an interruption of ongoing behaviour might distract or arouse the animal, such that behaviour after drug administration changes irrespective of any pharmacological effect of the drug. This is likely to be a particular problem if the route of administration causes pain or discomfort, as is evident with needle-sticks and gavage. Oral gavage has been reported to induce significant increases in heart rate 2–5 h post-gavage and increase faecal corticosterone (Walker et al. 2012 ; Bonnichsen et al. 2005 ); and stress-related arousal will have behavioural consequences. Yet oral administration is a desirable route to explore, given the ultimate preference for such in treatments for human conditions, so establishing stress-free oral route for laboratory animals is a priority.

Walker et al. ( 2012 ) have shown that mice which voluntarily consumed a ‘pill’ made from Transgenic Dough Diet™ (Bioserve, Inc.) did not show a stress response compared to oral gavage. Whilst it is simple to knead drugs into the dough, there are disadvantages to using this diet: it is designed for rodents with chewing, dental or mobility impairments and therefore whilst highly palatable, is also high in protein and fat. This makes it less useful for studies that measure behaviour motivated by food and in instances where uptake may be affected by food in the gastrointestinal tract. Other low-stress palatable techniques, such as adding drugs to condensed milk (Murphy et al. 2015 ), are similarly disadvantaged by high protein, fat and sugar content; whilst training rats to drink from a syringe (e.g. Mar et al. 2017 ; Robinson 2012 ) requires a time component for both training and actual experimental dosing.

In the present study, we used a flavoured, but fat-/sugar-free, gelatine “jelly” tablet (previously described in Bowman et al. 2014 ) to orally administer modafinil (2-[(Diphenylmethyl)sulfinyl]acetamide) to rats. Modafinil is a stimulant drug used in the treatment of narcolepsy and excessive sleepiness (Bastuji and Jouvet 1988 ; Edgar and Seidel 1997 ). It has gained particular interest for its unique wake-promoting effects without exerting typical amphetamine-like side-effects, such as sleep rebound, and neither does it have abuse potential (Edgar and Seidel 1997 ; Deroche-Gamonet et al. 2002 ; Touret et al. 1995 ; Leith and Barrett 1976 ; Koob and Bloom 1988 ).

Modafinil has also been investigated for its potential cognitive-enhancing effects, where it has been linked to increased performance and accuracy on a variety of cognitive tasks in both patients with schizophrenia and healthy adults (digit span task, CANTAB-Stroop), as well as in experimental animals performing visual attentional tasks and T-maze-serial reversal learning (Minzenberg and Carter 2008 ; Turner et al. 2003 ; Randall et al. 2005 ; Morgan et al. 2007 ; Beracochea et al. 2002 ). We have recently observed effects of 30 mg/kg modafinil, administered i.p. prior to testing, on Lister hooded rat behavioural flexibility during the intradimensional/extradimensional (ID/ED) attentional set-shifting task (Chase, Tait and Brown, unpublished observations).

To determine the viability of the oral jelly method of administration, we investigated the effects of a single dose of modafinil on locomotor activity (LMA) and brain tissue concentration. Previous studies have shown that various doses of modafinil in the rat elicit either an outright increase in LMA (75–600 mg/kg; Ishizuka et al. 2008 ; Rowley et al. 2014 ), or a slowed reduction (40 mg/kg; Simon et al. 1996 ), compared to controls. Using an electroencephalogram (EEG), Edgar and Seidel ( 1997 ) observed that their (100–300 mg/kg) modafinil-induced LMA ‘increase’ derived from time spent awake—i.e. that “LMA intensity” (LMA per time spent awake) did not change. Based on our prior behavioural observations after 30 mg/kg i.p., the robust effect of 40 mg/kg i.p. on LMA (Simon et al. 1996 ), and an established pharmacokinetic profile after 32 mg/kg by oral gavage (Waters et al. 2005 ), we investigated oral jelly administration of 30 mg/kg modafinil—predicting it would induce a similar LMA profile to that observed by Simon et al. ( 1996 ): a reduction in LMA over time that was slower than that observed in controls. We also explored modafinil concentrations in brain tissue to establish a pharmacokinetic profile for the oral jelly administration route after the same dose, to allow comparison to the oral gavage route.

Forty-six (24 male; 22 female) naïve Lister hooded rats (Charles River, UK) were group-housed, but segregated by sex, and maintained on a 12 h light/dark schedule (lights-on at 7 am). They were maintained on a diet of 15–20 g of standard laboratory chow each day with water available ad libitum. The male rats weighed between 480 and 630 g and the females weighed between 185 and 250 g over the course of the experiment. All experimental procedures were carried out in accordance with the UK Animals (Scientific Procedures) Act 1986 and EU Directive 2010/63/EU.

Drug preparation and habituation

Modafinil was administered to the rats orally, suspended in a palatable gelatine (jelly) tablet as the vehicle. The jellies were made by heating a water bath to ~ 70 °C, then placing into the water bath a beaker containing 50 ml of flavoured, sugar-free, fruit juice concentrate (Robinsons Squash, Britvic PLC, UK) and adding 12 g gelatine (Dr. Oetker, UK). The mixture was stirred until the gelatine was fully dissolved. Modafinil (Sequoia Research Products Ltd., UK) doses (30 mg/kg) for individual rats were weighed out and added to the bottom of 2 ml wells in a plastic mould. The gelatine solution was then pipetted into the wells (1.5 ml/well), and the mixture carefully stirred with a small pipette tip to suspend the modafinil. Vehicle jellies were made using the same procedure, but without modafinil. The plastic mould was then placed in a fridge (3–5 °C) overnight for the jellies to cool and set. Once the jellies were set, they were removed from the moulds and stored in the fridge in airtight containers.

The rats were habituated to vehicle jellies before data collection: rats were placed individually in a large home-cage and presented with a jelly in a small ceramic pet food bowl, and left until they had fully consumed it. This was repeated once per day until rats were eating the jelly within 5 min, which was usually by the third day.

Experiment 1: the pharmacokinetic profile of orally administered modafinil

Drug administration.

On the day of the experiment, the 24 male rats were single-housed and presented with modafinil-containing jellies. The time at which a rat finished eating the jelly was recorded (typically no more than 5 min after it had started eating), and at specific time-points after that (15, 30, 45, 60, 75, 90, 120 and 150 min; n  = 3 per time-point) rats were sacrificed by decapitation. After decapitation, brains were extracted from the skull, the cerebellum was removed, and then the remainder was bisected in the sagittal plane. Each hemisphere was weighed and then rapidly frozen by immersion in isopentane (Sigma-Aldrich, UK) chilled by dry ice. The hemispheres were then wrapped in aluminium foil, individually placed in homogenisation tubes and stored at − 80 °C.

Post-mortem bioanalysis

Rat brain concentrations of modafinil were determined using ultra performance liquid chromatography (UPLC) coupled to tandem mass spectrometry (MS/MS). Brain samples were prepared by homogenising the brains 1:3 ( v / v ) with a mixture of water, 2-propanol and dimethyl sulfoxide (DMSO; 50:30:20 v / v / v ). Samples were precipitated with acetonitrile and DMSO (80:20 v / v ) containing internal standard. Following centrifugation, 10 μl was injected onto the chromatographic system consisting of an Aria TLX2 system (Thermo Fisher Scientific Inc., MA, USA) connected to a Thermo TSQ Quantum Ultra triple quadrupole mass spectrometer. Analytical separation was achieved using a Kinetex C18 column (50 × 2.1 mm, 2.6 μm particles; Phenomenex, CA, USA). The mobile phase consisted of 0.01% formic acid in acetonitrile and 0.1% formic acid in water pumped through the column using a 3-min gradient. Modafinil was detected at a parent > daughter mass to charge ratio ( m / z ) of 274.01 > 167.00. The retention time was 1.27 min. The peak area correlated linearly with the brain concentration of the analyte in the range of 40–4000 ng/g brain.

Experiment 2: the effects of orally administered modafinil on locomotion activity

Test procedure.

On the day of testing, starting 4 h after lights on, the 22 female rats were habituated to a 7 × 15 LED infrared actimeter (Hamilton-Kinder MotorMonitor ; Ponway, CA, USA) for 60 min. Following actimeter habituation, a jelly was presented in a ceramic bowl directly into the actimeter. Jellies were administered 5 h after lights-on (Circadian time 5 (CT5), where CT0 is lights-on), as it has been observed that sleep loss can impact stimulant drug efficacy (Edgar et al., 1991; Roehrs et al., 1989). CT5 (in 12 h light/dark schedule) in particular, yields the least interference from the high variability in wakefulness due to large amounts of sleep (< CT5) and the normal circadian wakefulness found closer to lights-off (> CT5) (Edgar & Seidel, 1997 ). Precisely 15 min from the point the rat started eating the jelly (with all jellies consumed within 5 min), the actimeter was reset, and testing commenced. LMA data were compiled to investigate time-periods of interest (15–30, 30–45, 45–60, 60–75, 75–90, 90–105, 105–120, 120–135, 135–150 and 150–160 min). Timings were aligned so that sacrifice time-point ‘15 min’ from experiment 1 falls within the first 5 min of the ‘15–30 min’ time-period from experiment 2 (and so on for the remaining time-points/periods—with the last two sacrifice time-points from experiment 1 falling within the last four time-periods of experiment 2).

Counterbalancing

Each rat was tested twice, with 11 rats each receiving modafinil and vehicle jellies in each test, and rats receiving modafinil and vehicle jellies once each. There was a minimum of 5 days between tests to allow for washout of modafinil.

Data compilation and analysis

The LMA data were configured as LMA/min over 10 time-periods (15–30, 30–45, 45–60, 60–75, 75–90, 90–105, 105–120, 120–135, 135–150 and 150–160 min), and analysed by ANOVA using SPSS v. 22 with the dependant variable being the total number of infrared beams crossed within the observed time-period. There were two within-subjects factors: dose (two levels: modafinil and vehicle), and time-period (ten levels: as above). A second ANOVA, with two within-subjects factors: test (test 1 and test 2) and time-period was used to confirm that rats’ performance was not affected by test order. Greenhouse-Geisser corrections were applied for sphericity violations.

Modafinil was detected in the brain tissue at all time-points (Fig.  1 ). A rapid uptake in the brain was observed with mean concentrations in the range of 300–400 ng/g during the first hour after drug intake, where after a gradual decrease in brain concentrations was observed over time.

Locomotor activity: mean ± SEM number of infrared beams crossed after oral administration of either 30 mg/kg modafinil or vehicle jellies during time-periods from 15 to 160 min after administration. The wake-promoting effects of modafinil became obvious after the first time-period (“ns” denotes the single time-period where there was no significant difference between the groups), following a greater reduction in exploratory behaviour in the control group. Pharmacokinetic profile: mean ± SEM ng/g modafinil in brain tissue collected at specific time-points (within the first 5 mins of the 15 mins LMA time-periods) after oral administration of 30 mg/kg modafinil. Concentration reduced over time, although high variability at the 30-min time-point (within the 30–45-min time-period) may mask a true peak at the 60 min time-point (within the 60–75 min time-period)

There was no effect of running the test twice: as a group, the rats were equally active in both tests (main effect of test: F (1, 21) = 1.19, not significant (ns)) and the time course of activity was also similar in each test (test by time-period interaction: F (4.72, 99.18) = 1.63, ns).

Overall, modafinil administration resulted in greater LMA compared to vehicle-treated rats (main effect of dose: F (1, 21) = 49.19, p  < 0.05; Fig. ​ Fig.1). 1 ). Furthermore, as previously observed by Simon et al. ( 1996 ), whilst the LMA of control rats decreased rapidly, that of modafinil-treated rats was less reduced (main effect of time-period ( F (4.35, 91.25) = 13.91, p  < 0.05); dose by time-period interaction ( F (6.73, 141.34) = 4.03, p  < 0.05)). Specifically, Bonferroni-corrected pairwise comparisons showed that modafinil-treated rats were significantly more active in all time-periods other than the first compared to vehicle-treated rats: there was a distinct pattern of reducing activity in the vehicle-treated rats over the course of the first few time-periods (LMA is significantly lower in all time-periods after the first, and in most after the second), with LMA stabilising at the 60–75 min time-period; whereas the modafinil-treated rats’ LMA barely reduced, differing significantly only between the first and third time-periods.

We have investigated the efficacy of a novel route of administration, in the form of a suspension in a palatable jelly tablet, for thermally stable, bio-orally available drugs. We have shown that modafinil is present in the brain up to at least 150 min after consumption of a 30 mg/kg modafinil-containing jelly, and that LMA is affected by modafinil on a similar timescale. This delivery method was presented as a reduced-stress alternative to oral gavage—a forced oral route of administration, which elicits undesirable stress responses and is known to alter an animal’s response to pharmacological agents (Brown et al. 2000 ; Roberts et al. 1995 ). The results we obtained from the pharmacokinetic profile of modafinil concentration in brain tissue show a pattern comparable to previously published data from oral administration of 32 mg/kg modafinil via gavage (Waters et al. 2005 ). The concentration levels reported by Waters et al. are, however, substantially higher at the 30–60-min time-points than those reported here—although unlike the rats in our study, which were on our standard food control regime for food-motivated behavioural testing, their rats were deprived of food overnight prior to the administration of modafinil. Despite this seeming discrepancy, both our data and that of Waters et al. demonstrate a rapid decrease in concentration after the 60-min time-point. Whilst our data shows a gradual decrease in modafinil concentration as time progresses, it is clear from Fig. ​ Fig.1 1 that our data have high variability in the early time-points, with the greatest concentration mean at 60 min—the same as reported in Waters et al. Although no statistical analysis of Waters et al.’s data was presented, the standard deviation data suggest very high variability at their two highest concentrations—30 and 60 min. Both our study and that of Waters et al. sampled three rats per time-point, and given the variability in their data at the highest concentrations reported, we do not think it reasonable to conclude that there is a substantial difference between the pharmacokinetic results. Furthermore, unpublished data from oral gavage using a dose of 64 mg/kg show a concentration of 350 ng/g modafinil in brain tissue at 60-min post-administration, although again with high variability (Bundgaard, unpublished observations)—comparing favourably to our reported 383 ng/g concentration at the same time-point.

Our data also demonstrate that rats fed modafinil in jelly form show LMA that compares favourably to data at a similar dose after i.p. administration: Simon et al. ( 1996 ) report an effect on LMA after 40 mg/kg modafinil. As in that study, we have shown that following actimeter habituation, compared to control performance, modafinil-treated rats exhibited greater LMA overall and importantly, continuously after the first time-period. Both our data and that of Simon et al. illustrate a more rapid decline in LMA in vehicle-treated than modafinil-treated rats, followed by stabilisation during later time-periods. Our observations differ, however, in that after the first time-period, modafinil-treated rats are consistently more active than vehicle-treated—whereas Simon et al. report differences only at three time-periods: 10–20, 30–40 and 70–80 min. That we used more than the twice the number of subjects, and a within-subjects design, suggests that lower variability in our sample accounts for our more robust effect—rather than, for example, a gender or strain effect.

As previously reported by Edgar and Seidel ( 1997 ), EEG recordings support the conclusion that modafinil causes an increase in LMA only in proportion to the expected time spent awake, and that LMA intensity is not affected by modafinil. In contrast, amphetamine-like stimulants not only increase LMA intensity, but also result in stereotyped behaviours such as “compulsive licking, sniffing, biting, chewing, grooming and head-waving” (Duteil et al. 1990 ). Whilst 300 mg/kg doses of modafinil do not yield increased LMA intensity in rats (Edgar and Seidel 1997 ), 600 mg/kg is reported to result in “intense chewing and sniffing… interrupted by brief bursts of locomotor activity” (Rowley et al. 2014 ).

Whilst the aim of jelly administration is to present a stress-free alternative to gavage as a route of oral administration, we recognise that our data do not show the effects of any reduction in stress—given their similarity to published data where either i.p. or oral gavage administration routes were used. It is the case that many experiments would not be sensitive to the changes between stress-free oral administration and gavage/i.p. administration. However, we consider the obvious benefits of a non-aversive means of dosing an animal to manifest when using drugs with a short profile of activity—which might require multiple dosings during a single experiment. For example, we have found that rats become reluctant to engage with a task (ID/ED attentional set-shifting) once they have learned to associate the task with an i.p. injection—i.e. when we have need to administer via i.p. in the middle of a test, on subsequent tests, rats are more reluctant to participate, as the expectation of an injection affects their interest in the task (Tait and Brown, unpublished observations). Thus, whilst we may not observe the effects of stress-reduction on the actual data, the benefits for collecting those data are obvious.

The jelly administration method presents as an alternative to other oral routes: gavage, syringe-feeding, and the ‘pill’ method described in Walker et al. ( 2012 ). Rich palatability and a capacity to manufacture a higher volume of pills per batch, makes the Transgenic Dough Diet a viable alternative to oral gavage, but the high fat and protein content is less desirable for food-motivated experiments, and where there is likely to be slower absorption of a drug because of gastrointestinal contents. Additionally, the dough diet requires the drug to be kneaded in (Walker et al. 2012 ), and therefore final concentration may be inconsistent as there may be irregular distribution within the dough unless pills are made individually. Some drugs also require a solvent to help dissolve them to aid in uniform distribution, and in some cases a thickening agent was added to the dough mixture to help finalise it for drying. The benefits, therefore, of the individualised jellies is that dosage can be customised to the weight of the rat without having to make a larger batch, thereby reducing wastage and being more cost-effective: the gelatine mixture is pipetted on top of the pre-weighed drug, which can remain in crystalline form. Furthermore, multiple rats can be habituated to/dosed with the jellies simultaneously, without the need for an experimenter to devote time to individually training/administering to the rats, as in the syringe-feeding method. As with other palatable oral methods, any aversive taste the drug may have should be masked by the palatable flavour of the jelly.

In conclusion, the current data demonstrate the efficacy of a jelly tablet as a reduced-stress alternative to oral gavage, as indexed by a pharmacokinetic profile for modafinil comparable to previous data from gavage administration, and an LMA profile comparable to previous i.p. administration of modafinil. Reducing stress-related arousal during the administration of pharmacological agents is a fundamental refinement to drug administration—and should be a goal for ethical experimentation regardless of any benefit to the data. Thus, whilst this technique should hopefully remove unwanted behavioural consequences that may mask the effects of a drug, it also promotes an overall ethical responsibility of reducing pain and distress in experimental animals.

Acknowledgements

We would like to thank Mary Latimer and the animal husbandry staff of the St Mary’s Animal Unit in the School of Psychology & Neuroscience, University of St Andrews for their help in the tissue preparation.

Compliance with ethical standards

All experimental procedures were carried out in accordance with the UK Animals (Scientific Procedures) Act 1986 and EU Directive 2010/63/EU.

The authors declare that they have no conflict of interest.

• Bastuji H, Jouvet M. Successful treatment of idiopathic hypersomnia and narcolepsy with modafinil. Prog Neuro-Psychopharmacol Biol Psychiatry. 1988; 12 (5):695–700. doi: 10.1016/0278-5846(88)90014-0. [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Beracochea D, Celerier A, Borde N, Valleau M, Peres M, Pierard C. Improvement of learning processes following chronic systemic administration of modafinil in mice. Pharmacol Biochem Behav. 2002; 73 (3):723–728. doi: 10.1016/S0091-3057(02)00877-8. [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Bonnichsen M, Dragsted N, Hansen AK. The welfare impact of gavaging laboratory rats. Anim Welf. 2005; 14 (3):223–227. [ Google Scholar ]
• Bowman EE, Xia S, Tait DS, Brown VJ (2014) Demonstrating a stress-free way to administer drugs during behavioural testing: modafinil restores attentional deficits in rats with lesions of the subthalamic nucleus. Paper presented at the Society for Neuroscience, Washington, DC,
• Brown AP, Dinger N, Levine BS. Stress produced by gavage administration in the rat. Contemp Top Lab Anim Sci. 2000; 39 (1):17–21. [ PubMed ] [ Google Scholar ]
• Deroche-Gamonet V, Darnaudery M, Bruins-Slot L, Piat F, Le Moal M, Piazza PV. Study of the addictive potential of modafinil in naive and cocaine-experienced rats. Psychopharmacology. 2002; 161 (4):387–395. doi: 10.1007/s00213-002-1080-8. [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Duteil J, Rambert FA, Pessonnier J, Hermant JF, Gombert R, Assous E. Central α1-adrenergic stimulation in relation to the behaviour stimulating effect of modafinil; studies with experimental animals. Eur J Pharmacol. 1990; 180 (1):49–58. doi: 10.1016/0014-2999(90)90591-S. [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Edgar DM, Seidel WF. Modafinil induces wakefulness without intensifying motor activity or subsequent rebound hypersomnolence in the rat. J Pharmacol Exp Ther. 1997; 283 (2):757–769. [ PubMed ] [ Google Scholar ]
• Ishizuka T, Murakami M, Yamatodani A. Involvement of central histaminergic systems in modafinil-induced but not methylphenidate-induced increases in locomotor activity in rats. Eur J Pharmacol. 2008; 578 (2–3):209–215. doi: 10.1016/j.ejphar.2007.09.009. [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Koob GF, Bloom FE. Cellular and molecular mechanisms of drug dependence. Science. 1988; 242 (4879):715–723. doi: 10.1126/science.2903550. [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Leith NJ, Barrett RJ. Amphetamine and the reward system: evidence for tolerance and post-drug depression. Psychopharmacologia. 1976; 46 (1):19–25. doi: 10.1007/BF00421544. [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Mar AC, Nilsson SRO, Gamallo-Lana B, Lei M, Dourado T, Alsio J, Saksida LM, Bussey TJ, Robbins TW. MAM-E17 rat model impairments on a novel continuous performance task: effects of potential cognitive enhancing drugs. Psychopharmacology. 2017; 234 (19):2837–2857. doi: 10.1007/s00213-017-4679-5. [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Minzenberg MJ, Carter CS. Modafinil: a review of neurochemical actions and effects on cognition. Neuropsychopharmacology. 2008; 33 (7):1477–1502. doi: 10.1038/sj.npp.1301534. [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Morgan RE, Crowley JM, Smith RH, LaRoche RB, Dopheide MM. Modafinil improves attention, inhibitory control, and reaction time in healthy, middle-aged rats. Pharmacol Biochem Behav. 2007; 86 (3):531–541. doi: 10.1016/j.pbb.2007.01.015. [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Murphy HM, Ekstrand D, Tarchick M, Wideman CH. Modafinil as a cognitive enhancer of spatial working memory in rats. Physiol Behav. 2015; 142 :126–130. doi: 10.1016/j.physbeh.2015.02.003. [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Randall DC, Shneerson JM, File SE. Cognitive effects of modafinil in student volunteers may depend on IQ. Pharmacol Biochem Behav. 2005; 82 (1):133–139. doi: 10.1016/j.pbb.2005.07.019. [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Roberts RA, Soames AR, James NH, Gill JH, Wheeldon EB. Dosing-induced stress causes hepatocyte apoptosis in rats primed by the rodent nongenotoxic hepatocarcinogen cyproterone acetate. Toxicol Appl Pharmacol. 1995; 135 (2):192–199. doi: 10.1006/taap.1995.1223. [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Robinson ES. Blockade of noradrenaline re-uptake sites improves accuracy and impulse control in rats performing a five-choice serial reaction time tasks. Psychopharmacology. 2012; 219 (2):303–312. doi: 10.1007/s00213-011-2420-3. [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Rowley HL, Kulkarni RS, Gosden J, Brammer RJ, Hackett D, Heal DJ. Differences in the neurochemical and behavioural profiles of lisdexamfetamine methylphenidate and modafinil revealed by simultaneous dual-probe microdialysis and locomotor activity measurements in freely-moving rats. J Psychopharmacol. 2014; 28 (3):254–269. doi: 10.1177/0269881113513850. [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Simon P, Hemet C, Costentin J. Analysis of stimulant locomotor effects of modafinil in various strains of mice and rats. Fundam Clin Pharmacol. 1996; 10 (5):431–435. doi: 10.1111/j.1472-8206.1996.tb00597.x. [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Touret M, Sallanon-Moulin M, Jouvet M. Awakening properties of modafinil without paradoxical sleep rebound: comparative study with amphetamine in the rat. Neurosci Lett. 1995; 189 (1):43–46. doi: 10.1016/0304-3940(95)11448-6. [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Turner DC, Robbins TW, Clark L, Aron AR, Dowson J, Sahakian BJ. Cognitive enhancing effects of modafinil in healthy volunteers. Psychopharmacology. 2003; 165 (3):260–269. doi: 10.1007/s00213-002-1250-8. [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Walker MK, Boberg JR, Walsh MT, Wolf V, Trujillo A, Duke MS, Palme R, Felton LA. A less stressful alternative to oral gavage for pharmacological and toxicological studies in mice. Toxicol Appl Pharmacol. 2012; 260 (1):65–69. doi: 10.1016/j.taap.2012.01.025. [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Waters KA, Burnham KE, O'Connor D, Dawson GR, Dias R. Assessment of modafinil on attentional processes in a five-choice serial reaction time test in the rat. J Psychopharmacol. 2005; 19 (2):149–158. doi: 10.1177/0269881105048995. [ PubMed ] [ CrossRef ] [ Google Scholar ]