Oromucosal as an Alternative Method for Administration of Cannabis Products in Rodents

Summary

Oral administration via gavage in rodents can induce distress associated with restraint. We explored oromucosal/buccal administration of CBD-enriched Cannabis extract, where rats were treated for 15 days with the extract at 3 mg/kg/day. Treatments were administered orally with a micropipette, using an affectionate touch technique to handle the animals.

Abstract

Oral administration of drugs in laboratory rodents such as rats is conventionally performed using the gavage technique. Despite effectiveness, gavage can induce distress associated with restraint, especially following repeated animal handling. To mitigate these adverse effects and reduce morbidity associated with traditional methods, we explored oromucosal/buccal administration of cannabidiol (CBD)-enriched Cannabis extract. In this method, male rats were treated daily for 15 days with medium-chain triglycerides (TCM) derived from coconut oil or CBD-enriched Cannabis extract. Each treatment was administered individually while animals were gently immobilized using an affectionate touch technique. The administration involved the use of a micropipette to apply the oily formulation directly into the oral mucosa. The dosage was calculated based on the CBD concentration in the Cannabis extract, standardized at 3 mg/kg/day. To ensure accuracy, animals were weighed daily, allowing for dose adjustments in accordance with weight changes over the treatment period. This method offers non-invasive and stress-reducing treatment, potentially improving animal welfare in experimental settings. The treatment with CBD-enriched Cannabis extract was safe, and the analysis of the hippocampus of these animals' showed alterations in the expression levels of GluA1 and GFAP proteins, which are directly associated with glutamatergic receptor functionality and neuroinflammation, respectively. This suggests that Cannabis extract could be applied in pathological conditions where glutamatergic excitotoxicity and astrogliosis are observed.

Introduction

The administration of medicines and substances with therapeutic potential in laboratory animals is still a critical component in the experimental design of preclinical research. Although common methods include intraperitoneal, subcutaneous injections, or oral gavage, they often have limitations in their translational capacity due to stress-induced welfare issues in animals^1,2. Additionally, these routes of administration do not always reflect the route that will be recommended for use in humans, potentially affecting the applicability of the findings to clinical settings. Rodents, particularly, play a crucial role in these investigations, serving as model organisms to simulate human clinical scenarios. To ensure the validity and reliability of these studies, it is imperative to create experimental conditions that minimize stress on research animals and mimic the human use of such substances. Furthermore, there are several drug administration protocols, and this choice requires careful consideration and planning to optimize the delivery of the substance to the animal while minimizing potential adverse experiences from the procedure³.

In this context, the goal was to create a protocol that mimics oromucosal administration^4,5 of Cannabis extract, similar to the current clinical practice^6,7,8,9, facilitating long-term treatments in rats¹⁰. Restraint by affective touch is performed in such a way that the skin behind the animal's neck is gently pulled backward, and they are raised above their cages until their tails stop touching the bedding in order to simulate how dams carry their pups. Using a micropipette, the Cannabis extract, an oily solution, is precisely deposited into their oral cavity, touching the inner lining of the cheek, followed by the return of rats to their cages. This approach not only aims to avoid stress and potentially enhance the welfare of the animals involved but also aligns preclinical methods more closely with clinical practices. The investigation demonstrated that healthy rats subjected to this oromucosal administration protocol and treated with Cannabis extract exhibited significant neurochemical modifications¹⁰. These alterations suggest a potential neuroprotective effect of the treatment. Specifically, the CBD-enriched Cannabis extracts were found to modulate the molecular architecture of glutamatergic synapses, regulate GFAP expression in astrocytes, and alter the morphology of microglial cells. Remarkably, these neurochemical and cellular adaptations occurred in the absence of observable behavioral changes, underscoring the nuanced biochemical impact of the treatment¹⁰.

Cannabis plants are widely known for their medicinal properties, which have been explored since ancient times¹¹, but only in the last few decades their biochemical composition and pharmacological potential have been more widely investigated^12,13,14. The main phytocannabinoids, cannabidiol (CBD) and Δ⁹-tetrahydrocannabinol (Δ⁹-THC), as well as minor phytocannabinoids and terpenes, are responsible for a myriad of therapeutic effects described for several pathologies^15,16,17. In this sense, the use of Cannabis-based products in clinical practice has been approved and recommended for several clinical conditions^6,7,8,18. In addition to these clinical advancements, there has been a surge in preclinical research utilizing Cannabis-based products^9,19,20,21. Such studies are valuable for assessing physiological responses, potential adverse effects, and behavioral changes²². The route of drug administration is a critical component of studies, and in the literature, several therapeutic approaches are used to administer phytocannabinoids or Cannabis-based products, such as inhalation^23,24 (or smoke), intraperitoneal injection²⁵, minipumps^26,27,28 and gavage²⁹. These techniques vary in complexity, duration of treatment, and completion time and produce different physiological response times, affecting animal behavior. Furthermore, errors in the application of these techniques can lead to ineffective treatment, high variability in the study, and even death of the individual^{3,30,31,32,33,34}.

There has been increasing evidence showing that stimulation of mechano-afferences can be targeted to reduce stress not only in rodents but also in humans. Therefore, physical touch also modulates psychological features besides the sole impact on nerve stimulation, contributing to improving impairments caused by long-term mild stress^35,36,37. This non-invasive method was developed to facilitate long-term treatment by reducing the time of motor restriction of animals and consequent stress due to excessive handling, preserving the quality of life and survival of these animals. Furthermore, the method can be used for other rodent species and oral drugs.

Protocol

The research was conducted in accordance with ethical standards. All the procedures were approved by the Ethics Committee on the Use of Animals in Research (CEUA) of the Center of Health Sciences (CCS) at the Federal University of Rio de Janeiro (UFRJ); CEUA: 079/22 (protocol code 014/19, approved on 27 March 2019).

1. Calculation of dosage according to animal weight

1. Prior to initiating the administration protocol, divide male Wistar rats (Rattus norvegicus) at ages 45-60 days postnatal into treatment groups. One group received medium-chain triglycerides (MCT) derived from coconut oil as a control, while the other group received a CBD-enriched full-spectrum Cannabis extract. Administer both treatments daily for 15 days. Standardize the dosage of the Cannabis extract at 3 mg/kg/day, with adjustments made daily based on each animal's weight to ensure accurate dosing.
2. Weighing the animal: Place each rat individually on the scale using a small container. Wait for the weight, in g, to stabilize. Remove the animal from the scale.
3. Dosage calculation: Calculate the dose to be administered based on the animal's weight. Use the formula:
   Dose (mg) = [Weight (g) x CBD concentration (mg/kg)]/1,000
   ​NOTE: For this protocol, we used a CBD-enriched Cannabis extract with a CBD concentration of 24 mg/mL. In this case, for instance, the total volume of solution administered for a 200 g animal was 25 µL, a small volume to be placed in the inner cheek.
   1. Use the following standard formula to calculate the volume for administration
      Volume (µL) = [D (mg/Kg) x W (g)]/C (mg/mL)
      where D = dose to be administered, W = animal weight and C = solution concentration
4. Record the required dosage for each animal.

2. Animal restraining and administration of the Cannabis extract

1. Preparation: At the time of administration, take the animal, individually, from its cage group to avoid disturbances. Using a micropipette, draw the calculated dosage of the Cannabis extract for each individual animal.
   NOTE: Animals were isolated briefly, only during the administration procedure, to minimize disturbances. The rats were not kept in permanent isolation.
2. Restraining: Restrain the animal by gently pulling the skin behind its neck backward. Hold it above the cage's floor up until the tail stops touching the home bedding. Ensure that the animal's movements are adequately restricted without causing harm.
   NOTE: This step requires a firm but gentle grip to avoid distress.
   1. In case of animal agitation, perform a soft swing with the elevated animal to make it calmer.
3. Administration: Carefully place the pipette tip containing the Cannabis extract into the animal's inner cheek. Ensure the animal will swallow the Cannabis extract. This step takes about 30 s.
   1. Due to the positioning of the micropipette in the animal's oral cavity and the low volume administered, it is unlikely that the animal will spit out the solution. But if this occurs, do not administer the dose again in order to avoid an overdose.
      NOTE: The stock solution of CBD-enriched Cannabis extract used had 24 mg/mL CBD in its formulation, whose vehicle used was MCT (medium chain triglycerides - provided by coconut oil). Oils are extensively used as extract solvents of hardly water-soluble drugs such as CBD, derived cannabis plants. MCT is an efficient vehicle leading to better bioavailability and stability of these preparations^38,39,40. Such elevated concentration and high viscosity due to the oily solution allowed a very low volume to be placed in the inner cheek of the animal and a very low probability of spilling part of it out. Even though such losses may occur in some cases, besides part of its volume being directly swallowed instead of absorbed through the mucosa, it is a very reliable representation of what occurs in clinical practice, mostly with children^6,7.
4. Ensure that each set of treatments is performed by the same operator and that the operator does not use any cosmetic products with aromas.

3. Post-administration care and analysis

1. Gently release the animal and return it to its group.
2. Following administration, monitor animals briefly (approximately 5-10 min) for any immediate adverse reactions. This includes close observation for changes in behavior, specifically signs related to the cannabinoid tetrad (catalepsy, decreased locomotor activity, ptosis, and hypothermia), which could indicate an unexpected or excessive response to the Cannabis extract. Additionally, monitor the animals for signs of respiratory distress (labored breathing, wheezing) and oral irritation (inflammation, excessive salivation).
3. Pay particular attention to signs of gastrointestinal distress, such as vomiting or diarrhea, as these could compromise animal welfare and potentially interfere with treatment⁴¹. It is important to note that no adverse reactions were observed in this study.
4. To further assess the impact of the 15-day treatment, perform a comprehensive set of analyses. These include behavioral assessments using the open field test to evaluate locomotor activity and the novel object recognition test to assess short-term memory.
5. Euthanize animals and collect hippocampal tissue for Western blotting to quantify protein levels related to synaptic plasticity and glial activity. Perform immunohistochemistry to examine microglial morphology and phagocytic activity within the hippocampus, providing a detailed understanding of the cellular and molecular changes induced by long-term exposure to the CBD-enriched Cannabis extract¹⁰.

4. Open field test protocol

1. Place the animal in the center of the square arena individually. Start recording with the camera connected to the computer. Ensure the camera is placed above the square arena so that you can view the entire area of the arena.
2. Set a stopwatch for 5 min. Each rat has to freely explore the arena. After the time period elapses, pause the stopwatch and the recording.
3. Remove the animal from the square arena.

Results

The methodology has been previously used by our group. This study investigates the long-term effects of cannabidiol-enriched Cannabis extract, focusing on growth, behavior, and synaptic changes. Oromucosal administration of a CBD-enriched Cannabis extract at a dose of 3 mg/kg/day did not adversely affect animal growth from adolescence to early adulthood. Key physiological parameters, including urine output, food and water intake, and weight gain, were meticulously assessed. A two-way ANOVA revealed a significant effect o...

Discussion

The protocol described offers a refined approach to the oromucosal administration of Cannabis extracts in rats, presenting a notable advantage of being comfortable for both the operator and, most importantly, the animal, providing well-being, safety, and precise control over the administered dose for effective absorption. The oromucosal route is economical and relatively safe, and some animals can be trained to cooperate voluntarily⁴, depending on the compound being administered^3...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
CBD-enriched Cannabis extract	Farmacannabis project - Federal University of Rio de Janeiro (UFRJ)	x	Cannabinoid concentrations: CBD = 24.0 mg/mL; ?9-THC = 1.0 mg/mL; CBDA = 0.5 mg/mL; THCA and CBN were not detected.
Gloves	NUGARD	CA 41.485	Synthetic rubber material (nitrile)
Micropipette	Labmate pro/LabMate+	LMP-200	20-200ul
Oily solution of medium chain triglycerides (MCT)	MCT; Brain TCM, Puravida	7907981000102	Use as a vehicle solution
Scale	123 util	1783912	The minimum and maximum sensitivity is 1 g and 10 kg, respectively