Prevention of Allodynia and Hyperalgesia by Cannabidiol in a Rat Model of Chemotherapy-Induced Peripheral Neuropathy

Summary

Co-administration of CBD with paclitaxel prevents the development of chemotherapy-induced peripheral neuropathy in rats. This protocol describes cannabinoid handling, inducing an allodynic phenotype in rats via chemotherapeutic administration, assessing mechanical and thermal allodynia, and using high-speed videography to distinguish allodynia and hyperalgesia.

Abstract

This study demonstrates the utility of a rat model of chemotherapy-induced peripheral neuropathy (CIPN) to assess the ability of the non-psychoactive cannabinoid cannabidiol (CBD) to modulate the development of this syndrome in vivo. The method utilizes the chemotherapeutic agent paclitaxel to generate an allodynic phenotype in the animals. This study describes how to handle and solubilize CBD, administer the chemotherapeutic agent, assess mechanical and cold sensitivity, and apply high-speed videography to measure nocifensive behavior in animals. Using the procedures outlined, the data support that CBD prevents the allodynic phenotype from developing in the treated animals. No difference was observed in the CBD-treated animals from day 0 (pre-paclitaxel baseline) to day 7 (post-sensitization) in mechanical or thermal sensitivity, while the vehicle-treated animals became significantly more sensitive. This response to treatment is durable up to the latest time point where data were collected (7 weeks). The addition of high-speed videography allows for a more granular and unbiased assessment of this behavioral phenotype (e.g., classification of analgesia and anti-allodynia). This demonstrates both the utility of this model for cannabinoid drug characterization and the potential role of CBD in mitigating neuropathic pain.

Introduction

Chemotherapy-induced peripheral neuropathy (CIPN) is a type of sensory neuropathy. It is a common side effect of chemotherapy treatment and is especially prevalent with taxane and platinum-based drugs^1,2. Some symptoms of CIPN include chronic pain, numbness, tingling, and extreme sensitivity to touch and temperature^1,2. These painful symptoms not only interfere with a patient's day-to-day life but also their cancer treatment. Some cancer patients seek a reduction in dosage or a complete termination of chemotherapy to be able to manage the decrease in quality of life associated with this syndrome³. Currently, the common clinically used treatments for CIPN are incompletely effective at symptomatic relief and include opioids, which pose significant abuse potential⁴. There is no cure for CIPN to date, therefore, finding a safe and effective alternative is essential^5,6.

Cannabidiol (CBD) has previously been shown to have efficacy at preventing the development of CIPN as modeled in mice^7,8. This demonstrates a promising pathway for avoiding the potentially long-term allodynic symptoms of CIPN for patients without the need for relief via equivocally effective drugs or potentially harmful opioids. The majority of people using CBD, especially for therapeutic purposes like managing seizures, experience mild side effects, and the drug is generally well-tolerated⁹. Most adverse effects are dose-dependent and often improve as the body adjusts.

In this widely used model of CIPN¹⁰, rats are administered paclitaxel to induce neuropathy, which recapitulates the allodynic phenotype observed in some patients after chemotherapeutic intervention, as reported previously¹¹. The goal of this induction is to have a translationally relevant platform for evaluating this type of sensory neuropathy in vivo, which is suitable for researchers investigating mechanisms or interventions for this syndrome. Mechanical sensitivity is measured using the von Frey test, and cold temperature challenges are used to measure thermal sensitivity^10,11. This allows for the quantitation of the prophylactic potential of CBD in mitigating CIPN at the two most clinically important quality-of-life endpoints for patients. Further evaluation included high-speed videography-assisted analysis of nocifensive behaviors^12,13. This expanded analysis of behavioral responses to innocuous and noxious stimuli allows for the differentiation between allodynic and hyperalgesic effects of chemotherapy administration, as well as the ability of CBD to modulate these effects.

This protocol describes how to induce the CIPN phenotype, assess and quantify mechanical and thermal hypersensitivity in rats, and apply videography for an unbiased and more granular evaluation of allodynia and hyperalgesia.

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Protocol

This protocol was approved and follows the guidelines of Temple University's Animal Care and Use Committee under approved protocols for animal research. Adult male Sprague-Dawley rats (250-300 g) were used in this study. Details regarding the reagents and equipment used are listed in the Table of Materials.

1. Preparing cannabinoids and chemotherapeutic agents for the induction of CIPN

NOTE: Figure 1 shows the process of solubilization of cannabidiol.

1. Assemble the following materials for solubilization of CBD and dilution of paclitaxel.
   1. 100% ethanol (EtOH), Chremophor (Ethoxylated castor oil), normal saline, amber glass vials (x2), 50 ml conical tube (x2), CBD powder stock, paclitaxel clinical stock (6 mg/mL).
2. Add an appropriate volume of EtOH to each 50 mL conical tube for a final solvent preparation of 1:1:18 EtOH: ethoxylated castor oil: normal saline.
3. Mass out the appropriate amount of CBD to create a 5 mg/kg concentration for the number of animals to be tested.
4. Dissolve CBD in EtOH in one of the two prepared 50 mL conical tubes.
5. Vortex CBD-EtOH mixture at high speed until CBD is completely dissolved.
6. Add an equivalent volume of ethoxylated castor oil to each 50 mL conical tube.
7. Vortex each tube at high speed until the ethoxylated castor oil is in solution with EtOH.
8. Add an appropriate volume of normal saline to each 50 mL conical tube for a final ratio of 1:1:18.
9. Vortex until saline is in solution.
10. Transfer the vehicle control solution and CBD solution into amber glass vials.
11. Dilute stock paclitaxel solution 1:6 in normal saline to bring the final concentration to 1 mg/mL in amber vials at the appropriate volume for the number of animals to be tested.
12. Seal the amber vials with aluminum-collared rubber vial caps.
    NOTE: If prepared before initiation of the experiment, vials of vehicle, vehicle + CBD, and paclitaxel can be stored at 4-8 °C for up to 7 days. Allow refrigerated vials to equilibrate to room temperature before injection. Also, note that it is critically important to store prepared cannabinoid reagents in glass, if not for immediate use, to prevent adhesion to the vessel or precipitating out of solution.

2. Inducing allodynic phenotype in rats

1. Assemble the necessary materials for paclitaxel injection as mentioned below:
   1. Prepared 1 mg/mL paclitaxel stock solution, 1 mL disposable syringes (x n rats), 25 G syringe needles (x n rats), chemotherapeutic waste sharps container, and scale to collect animal weights.
2. Number or tag the rats with unique identifiers.
   NOTE: The animals should have acclimated to their housing for at least 1 week prior to the start of the experiment.
3. Collect the weights of each animal.
4. Prepare syringes with the appropriate volume of paclitaxel (all animals) and vehicle or CBD (by treatment group). In this case, the injection volume corresponds to 1 µL/g weight of the animals.
5. Inject paclitaxel and vehicle or paclitaxel and CBD interperitoneally.
   1. To restrain the rat for injection, grasp the rat with non-dominant from the back, with the index finger meeting the thumb around the neck and the middle finger meeting the thumb under the shoulder, using ring and pinky fingers to grip the body.
   2. Stabilize the rat's hindlegs against the body.
   3. Use caution not to apply an uncomfortable level of pressure on the animal to avoid injury.
      NOTE: There is always a risk of bite or scratch when handling rats. To mitigate this risk, the experimenter may wear additional PPE in the form of bite-resistant gloves.
   4. While manually restrained and stabilized, tilt the animal such that its abdomen is readily accessible to the syringe.
   5. Inject 1 mg/mL paclitaxel into the lower right quadrant of the animal and dispose of the syringe in the chemotherapy waste sharps container.
   6. Inject the vehicle or CBD at 1 µL/g weight into the lower left quadrant of the animal and dispose of the syringe in the regular sharps container.'
      NOTE: Alternating the sites of injection mitigates animal distress and potential irritation of the injection site.
   7. This marks experimental day 1. Repeat this process on days 2-4 to induce the allodynic phenotype. Follow institutional handling protocols for chemotherapeutic contaminated cages during this time.
   8. Take baseline mechanical and thermal allodynia measurements on day 0, as described in step 3. Include additional stimuli in baseline generation as described in step 4. Analysis of day 0 values for these stimuli will be necessary to generate pain scores.
   9. Repeat step 2.5.8 until animals demonstrate a stable, repeatable baseline to avoid heterogeneity in response caused by insufficient handling.
      NOTE: Do not interrupt the process of inducing the allodynic phenotype. If the experimenter is unable to conduct injections in 4 consecutive days, alternate dosing schedules can achieve a similar induction of the phenotype. It is also possible and perhaps recommended to combine the injection of paclitaxel and vehicle or CBD in one injection rather than 2 by adjusting the volume of the initial vehicle or CBD solution appropriately to accommodate dilution of paclitaxel in these mixtures while maintaining a 5 mg/mL final concentration of CBD and 1 mg/mL concentration of paclitaxel. Take appropriate caution while handling chemotherapy sharps to avoid accidental exposure to the experimenter.

3. Assessing mechanical and thermal sensitivity post-sensitization

NOTE: Figure 2 shows the schematic apparatus used for this study.

1. Assemble the following materials for mechanical and cold sensitivity assays:
   1. Von Frey filaments (4-100 g), dry ice, 15 mL conical tube, isolation chambers, wire floor grid, ¼ inch glass panel, and stopwatch.
2. Begin assessment on experimental day 7 (e.g., 3 days post final chemotherapeutic/treatment condition injection).
3. Assemble von Frey and thermal isolation chambers.
   1. Place the isolation chambers for mechanical sensitivity (6 chambers separated by plastic partitions) on a wire floor grid 20 cm above the benchtop.
   2. Place the isolation chambers for thermal sensitivity testing (single chamber) on a 1/4-inch glass surface 20 cm above the benchtop.
4. Place the animals individually in isolation chambers for mechanical allodynia.
5. Allow acclimation to the isolation chambers for 15 min prior to the start of testing.
6. Conduct manual von Frey testing for mechanical sensitivity. This method describes manual von Frey testing, but electronic von Frey testing may be substituted for manual testing¹⁴.
   1. Start with the filament corresponding to the lowest gram of force (e.g., 4 g) and apply pressure on the mid-plantar surface of the right hind paw such that the filament is held in a c-shape for 6 s.
      NOTE: Researchers report little difference between right and left hind paws, but experimenters should be consistent between animals). Repeat 5 trials per stimulus.
   2. Repeat for each animal to be tested.
   3. Move up the filament size sequentially and repeat steps 3.5.1-3.5.2 until a pain response is observed (e.g., paw withdrawal) in at least 2 of the 5 trials.
   4. Record filament gram of force that evoked the observed paw response.
   5. Re-test the next lower filament to see if paw withdrawal is observed. If no response is observed, the recorded gram of force is final.
   6. If the animal responds at this step, record a new filament gram of force and re-test the next lower filament. Repeat until lower filaments no longer elicit a response. Representative data are shown in Figure 3A,C.
7. Conduct cold sensitivity testing.
   1. Remove the animal from the mechanical sensitivity isolation chamber and place it in a thermal sensitivity isolation chamber.
   2. Pack 15 mL conical tube with dry ice such that the final pellet extends from the top of the tube.
   3. Place the dry ice on the glass proximal to animal's right hind paw.
   4. With a stopwatch, record the time until the animal demonstrates paw withdrawal in response to temperature. Representative data are shown in Figure 3B.
   5. Re-house the rat in its home cage.
   6. Repeat all steps of 3.7 until all animals have been tested and are back in their home cage.
      NOTE: It is also important to note that animals' behavior and weights should be monitored for any signs of distress at this stage. If the animals develop baseline pain behavior or are losing bodyweight during prolonged time courses, the study should be terminated, and animals should be euthanized according to proper animal handling technique. If the animals do not display distress over the course of the experiment, the animals should be euthanized after the final testing day.

4. Mechanical sensitivity assessment via high-speed videography

1. Assemble the following materials as mentioned below for mechanical sensitivity assay and videography:
   1. Isolation chambers, Wire floor grid, Cotton swab, Makeup brush, Pins, high-speed camera, infrared lights, laptop with compatible analysis software.
2. Assess in conjunction with previously described mechanical sensitivity testing¹¹or as a standalone assay as described below.
3. Assemble the isolation chambers for mechanical sensitivity (single chamber) by placing them on a wire floor grid 20 cm above the benchtop.
4. Place the camera on a tripod at roughly a 45-degree angle between 1 and 2 feet away from the isolation chamber.
5. Position the infrared lights on tripods to illuminate the chamber for experimentation and subsequent scoring.
6. Place the animals individually in the isolation chamber for mechanical stimulus assessment.
7. Allow acclimation to isolation chambers for 15 m prior to the start of testing.
8. Conduct somatosensory testing for mechanical sensitivity with different stimuli (Figure 4).
   1. Use a laptop with Photron FastCAM Analysis software to record the isolation chamber at 2000 frames/s for each stimulus (press the corresponding button to start recording).
   2. Use the cotton swab to make gentle contact between the cotton tip and the hind paw of the rat while recording, and stop after completing the recording.
   3. Use the makeup brush to wipe across the hind paw from back to front and stop after completing.
   4. Use the pin to conduct low- and high-pressure pin pricks on the hind paw of the rat. For low-pressure pricks, gently touch the pin to the hind paw, withdraw after contact while recording, and stop after completion.
   5. For high-pressure pin pricks, sharply press the pin into the hind paw and, withdraw after contact while recording and stop after completion.
   6. Return the animal to the home cage.
   7. Repeat for each animal to be tested.
9. Manually assess nocifensive behavior recorded via high-speed videography using videos processed with FastCAM software¹⁵, as demonstrated in Figure 5A,B.
   1. Measure the paw height (in cm) from the mesh floor to its highest point after paw stimulation. To do this in the software, navigate to Dimensions > Measurements and select Two points.
      1. Play the video through the entire paw movement to determine the moment when the paw reaches its peak during the initial upward motion. Select a clearly identifiable region of the paw (e.g., the center of the plantar surface, the intersection of a digit with the paw, heel, etc.) and set it as the initial point.
      2. Rewind the video to locate the position of that region before stimulation and mark the second point at that location along the y-axis. Ensure that the measurement line remains parallel to the y-axis and not angled. The height will be displayed in the top left corner of the screen15.
   2. Determine the paw speed by calculating the distance (in cm) from the initial lift to the highest point, divided by the time (in seconds) between these two points. This measurement can be performed in the software by selecting Dimensions > Measurements and choosing Two points.
   3. Play the video through the entire paw movement to identify the frames where the paw initiates its first upward motion. Measure paw velocity by selecting two points at different frames within this movement and calculating the distance divided by the number of frames or the time elapsed between the start and end frames¹⁵.
   4. Evaluate the videos for orbital tightening, paw shake, paw guard, and jumping behaviors. For instance, if an animal were to display one of these behaviors during stimulus application, their nocifensive behavior score would be 1.
      NOTE: Orbital tightening was scored when eyes went from fully open to partially or fully closed during stimulus application. Paw shaking was scored when high-frequency paw flinching was observed during stimulus application. Jumping was scored when three or more paws were off the wire grid floor during stimulus application.
   5. Use baseline data from the stimuli to generate distribution and error estimates for this cohort of animals as previously described¹⁵. Briefly, record videos of each animal being used in the studies in response to static touch, dynamic touch, light pinprick, and heavy pinprick at pre-treatment baseline to capture the full range of possible behavioral features.
      1. Calculate the average and standard deviation of each behavioral feature (velocity, height, pain score) for the group across all stimuli (static touch, cotton swab, light pinprick, heavy pinprick).
   6. Generate z-scores from raw data observed in each video using variables generated in step 4.9.4 and the procedures previously described¹⁶.
   7. Combine all z-scores into a one-dimensional eigenvalue using principal component analysis using statistical analysis software¹⁷. This allows for the combination of transformed z-scores from the three-dimensional dataset to be computed as a single weighted total value¹⁶.
   8. Plot PCA-generated pain scores encompassing these multiple behavioral dimensions to separate behaviors associated with noxious and innocuous stimuli (Figure 5C) using statistical analysis software.
      NOTE: Videography-assisted mechanical testing iterates on von Frey testing by incorporating increased dimensionality. In the context of CIPN, it allows for the description of and distinction between antiallodynia and analgesia. Video scoring can also be conducted by parties independent of the experimenter who conducted the sensory testing. This is a powerful tool that can both reduce experimenter bias and increase the depth of information gathered. It is important to note that this method is reliant on specialized hardware, software, and statistical techniques. It serves as a supplement to the more accessible method described in previous sections. Furthermore, it should be noted that data collection is time-consuming due to the size of the files generated by the camera and that file storage space can be a limiting factor. These factors should be considered at the outset of the experiment.

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Results

The behavioral results elicited by this CIPN model in rats are highly reproducible and consistent. Figure 3A shows baseline and post-paclitaxel sensitization mechanical sensitivity results in control and CBD-treated animals. At baseline, Sprague-Dawley rats generally start to exhibit paw withdrawal at 26-60 g of force as applied by von Frey filaments. This is observable in both treatment groups on day zero. After paclitaxel is administered, vehicle-treated animals become sensitive to the low...

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Discussion

The rat model of chemotherapy-induced peripheral neuropathy recapitulates two of the most reported quality-of-life issues for patients receiving certain types of chemotherapy in mechanical and thermal sensitivity. With this model, relatively inexpensive and efficient methods can quantify these sensitivities via von Frey filament testing and thermal sensitivity through dry ice exposure. This method also provides an additional method for more specialized testing via high-speed videography, which can be us...

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Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cannabidiol	Caymen Chemical	90080	Ann Arbor, MI
Chremophor	Millipore-Sigma	238470	St. Lous, MO
Ethanol	Millipore-Sigma	E7023	St. Lous, MO
FastCAM Analysis software	Photon		https://photron.com, open-sourced
High-speed camera	Photron	AX 50
Infrared lights	CMVision	IP65
Normal Saline	Millipore-Sigma	S0817	St. Lous, MO
Paclitaxel	Temple University Hospital Pharmacy	N/A	Philadelphia, PA
Sprague-Dawley rats (250–300 g)	Taconic Laboratories, Cranbury, NJ