Nerve Injury Model in Rabbits: Benefits and Pitfalls

Abstract

Peripheral nerve injury is widely studied through the sciatic nerve injury model. Although many animal models are used for sciatic nerve injury studies, rabbits are reported as the third most commonly used animal model. However, there is a significant gap in the literature describing common postoperative complications following sciatic nerve injury in rabbits. This chapter analyzed postoperative complications recorded from an original study that tested 40 mm sciatic nerve gap repairs in 56 rabbits. Autophagy of the toes and pressure ulcer development on the injured limb were the two most common and severe postoperative complications seen. These impairments ultimately led to 23.2% of the rabbits requiring euthanasia prior to the study endpoint. This raised the cost of the study by over $25,000. This chapter outlines the risks and benefits of using this animal model in sciatic nerve injury studies. It also proposes treatment methods for common postoperative complications that can substantially reduce future study costs. To preserve ethical animal care standards in research, we recommend alternative models be used instead of rabbits to study sciatic nerve injuries. However, if rabbits must be used, we encourage using the treatment protocol outlined below.

1. Introduction

Peripheral nerve injury is an exceedingly common injury that comprises nearly 2–3% of all patients admitted to a Level 1 trauma center [1, 2]. Despite this high incidence of patients with this injury, clinically accepted methods for treatment have yet to establish consistent success in restoring nerve function. It is reported that only 20–40% of direct nerve repairs achieve a high level of motor and sensory recovery [3]. In addition, the complex cellular and molecular pathology behind nerve recovery following injury provides an additional barrier to developing an effective treatment for traumatic peripheral neuropathies. Therefore, research endeavors have dedicated extensive efforts to developing treatment methods that address the pathophysiology of this injury to yield more remarkable functional recovery and tissue regeneration.

In pre-clinical research, animal models have massively expanded our knowledge of the biochemical mechanisms of diseases. In peripheral nerve injury research, in-vitro techniques are less commonly used due to their inability to mimic endogenous nerve regeneration complexities [4]. Conversely, in-vivo research has shown great translational potential for clinical treatments, making this method more common. The sciatic nerve injury model is often the model of choice for the exploration of this injury. The relatively large size of the sciatic nerve provides easy access for implementing and studying surgical interventions [4]. Although the sciatic nerve is the most common nerve for the study of peripheral nerve injury, there is a great deal of variance in the literature regarding the choice of animal model for experimentation.

Rats are often the animal model of choice for many researchers studying sciatic nerve injury [5, 6, 7]. In recent years, there has been a relative increase in nerve injury studies using rabbit models [8, 9, 10, 11, 12, 13]. The use of rabbits in the sciatic nerve injury model has shown to be an excellent method due to their size, docility, ease of handling, and short life span [14, 15, 16]. However, their sensitivity to surgical implants, although beneficial when determining immunologic responses, may lead to greater costs than anticipated due to experiment-related complications. Additionally, the extensive maintenance required for the welfare of these animals can lead to higher experiment costs. Cages must be made from non-toxic materials so a rabbit would not be harmed if chewed or licked. It also must be designed so that a rabbit cannot hurt itself with sharp edges and be able to be maintained easily and repeatedly [17]. Pain in rabbits can also present difficulties in the use of this animal model. Rabbits do not display explicit behaviors when in pain, which without identification, can cause rabbits to die within 36 hours from distress from the pain [14]. Additional complications such as self-mutilation and the development of pressure ulcers can also result in the early termination of the experiment.

When conducting animal research, it is vital to adhere to the “Three Rs” principle, Replacement, Reduction, and Refinement. This framework allows proper adherence to maintain the welfare and minimize the distress of animals used in experimental studies. More specifically, replacement refers to using alternative methods in an area where animals would have been used, reduction refers to lessening the number of animals used in a study, and refinement refers to improving study protocols to minimize the amount of distress experienced by the animal. Given this universal standard for conducting proper animal research and the commonality of rabbit models for the study of peripheral nerve injury, it is essential to analyze the general benefits and drawbacks of the use of rabbits in these experiments. This chapter will provide data on the unforeseen complications and proposed interventions we have documented during an original experiment testing 40 mm sciatic nerve gap repairs in rabbits [18]. We hope this chapter provides greater information on this animal model to reduce pain in rabbits in future experiments and potentially persuade others to opt for an alternative animal model when studying nerve injury.

2. Sciatic nerve injury in rabbits

Although relatively common, the use of the rabbit model in sciatic nerve injury studies still presents many gaps in the literature outlining specific techniques for success. A recent study by Merolli et al. investigated the sciatic nerve gap-injury model in rabbits and provided greater detail for nerve-gap repairs in this animal model [19]. In the clinical setting, peripheral nerve injuries may require an artificial device called a “nerve conduit” placed over the nerve to aid in the regeneration of the nerve. These devices have been most commonly studied in the rat animal model for sciatic nerve injury. However, this model provides minimal translational potential due to the relatively small size of the rat sciatic nerve restricting the length of the devices being studied. In this case, the rabbit model can be instrumental given their relatively larger sciatic nerve and general ease of handling compared to other higher species animal models. Merolli et al. successfully introduced a novel method for transecting the nerve in a uniform and timely manner [19]. Despite the higher regulatory standards for the use of rabbits in experimental studies, the authors still believe that the higher translational potential of this animal model outweighs the higher standard of care.

The rabbit sciatic nerve injury model has also been implicated in investigating potential new therapies to promote nerve regeneration following injury. Li et al. evaluated the efficacy of nerve growth factor following a sciatic nerve crush injury in rabbits. They found that high-frequency ultrasound-guided injections of nerve growth factor led to more remarkable nerve recovery outcomes [20]. The size of the rabbit sciatic nerve and the greater translational potential of this larger animal compared to smaller animal models makes their results incredibly promising. The study of nuanced treatment methods for peripheral nerve injury is becoming increasingly common in the literature. Therefore, with more studies employing this animal model, it is pertinent to develop and provide a standardized protocol for the adequate care of rabbits used to study sciatic nerve injury.

2.1 Common operative technique

The sciatic nerve injury model was first developed by Wall et al. in an effort to study the behavioral effects caused by the complete transection of this nerve in animals [21]. The sciatic nerve is the largest nerve, therefore handling and repair are comparably easier than other nerves [22]. One of the most common lesions studied is axonotmesis, or compression of the nerve [4, 23]. Compression injuries utilize a crushing mechanism that leads to damage of the axons without disrupting the integrity of the epineurium. This method of injury leads to functional damage to the sciatic nerve without the need for surgical repair. Crush experimental models are mainly used to mimic and subsequently understand the biological mechanisms behind peripheral nerve injuries caused by mechanical compression.

The other common nerve lesion studied is neurotmesis, or complete transection of the nerve. This injury requires complete disruption of the axons and epineurium. Clinically, one-third of peripheral nerve injuries are caused by a laceration from a sharp object [24, 25]. Due to the nature of this injury, lacerations often lead to neurotmesis of a peripheral nerve. Therefore, in-vivo studies investigating transection nerve lesions are more prevalent in the literature for their high translational potential into clinical nerve repair methods.

In our original experiment, we measured different repair techniques following complete transection of the sciatic nerve in rabbits. The rabbits were sedated with a mixture of Ketamine (40 mg/kg) and Xylazine (9 mg/kg) given intramuscularly once on the non-experimental leg. Anesthesia was then maintained with Isoflurane 2% at 3 mL/minute. A 5 cm incision was made longitudinally parallel to the femur using standard sterile operating procedures. The left sciatic nerve was exposed through a split-muscle technique, and 40 mm of sciatic nerve graft was harvested. In our original experiment, rabbits were subject to one of three nerve injury cohorts, cut without repair, reverse autograft, and repair with a sterile nerve conduit. For the second cohort, the sciatic nerve was cut using sharp scissors, and the harvested nerve section was reversed 180° and reattached to the proximal nerve at both ends using a 9–0 nylon suture (Ethicon, Somerville, NJ). For the third cohort, the 40 mm deficit was repaired by suturing in a nerve conduit using 9–0 Ethicon sutures (Ethicon).

Once the specific nerve injury was completed, the muscle plane was closed by layers using a 3–0 Vicryl (Ethicon) interrupted suture pattern, and the skin was closed with a 5–0 Monocryl running suture pattern (Ethicon). This operative procedure was conducted in accordance with the Guide for Care and Use of Laboratory Animals and approved by our corresponding Institutional Animal Care and Use Committee (IACUC).

2.2 Postoperative care

Postoperative care is a critical step in maintaining the well-being of the animal and avoiding any potential complications. Despite multiple regulations and rules for the overall reduction of pain and suffering of laboratory animals, complications can be inevitable. Here we outline the specific steps we took following the surgical procedures to minimize distress in the rabbits.

Immediately following surgery, rabbits were monitored by study personnel until they fully recovered and were able to be transported back to our animal facility per our IACUC protocol. For pain management, every 8 hours for the first 24 hours, 0.02–0.05 mg/kg of buprenorphine hydrochloride was administered subcutaneously or intramuscularly. After the initial 24 hours subsided, 0.1–0.3 mg/kg/24 hours of meloxicam was given as needed for 72 hours. Pain assessments were made using the rabbit grimace scale [26]. This scale evaluates facial actions such as orbital tightening, cheek flattening, nose shape, whisker position, and ear position. This scale has shown to be a reliable method for assessing pain in rabbits.

Rabbits were individually housed in Allentown rabbit cage rack systems, and a non-contact Techboard was placed underneath the racks for urine and excrement collection. Cages were changed every 2 weeks. Rabbits were fed through an automatic feeder and had free access to food and water 24 hours a day. A high-fiber diet and water were replaced every day. Additionally, the room in the animal facility that held the rabbits was set to a 12-hour light/dark cycle. Rabbits were monitored weekly by study personnel and daily by animal care staff. During the weekly inspections, any complications were recorded, and rabbits were weighed. At the end of the specified postoperative period (3, 6, or 12 weeks), the rabbits were euthanized by intravenous injection of 125 mg/kg sodium pentobarbital. For the purpose of the original study, the nerves were then harvested for either immunohistochemistry or magnetic resonance imaging [18].

3. Postoperative complications and proposed solutions

3.1 Autophagia

Injury to the sciatic nerve causes muscle weakness in the affected limb and decreased sensation [4]. This often leads to complications experienced across multiple animal models. Autophagia is often reported among these complications. Initial theories believed self-mutilation was due to the animal’s inability to sense the injured limb and consequently attack it as if it was a foreign body [23]. However, the histology behind peripheral nerve injury and known mechanisms following nerve injury support the theory that the onset of this phenomenon is linked to the axonal regeneration and subsequent generation of abnormal sensations from the injured nerve [23, 27].

In our original study, self-mutilation occurred in about 36% of the rabbits. The affected rabbits mainly chewed on the digits of the affected limb, however, there was additional chewing to the surgical site and the dorsal and web spaces of the injured foot. Our solution to this complication was to place a plastic Elizabethan collar with soft edges on the rabbits in the hope that it would deter autophagia. After this intervention’s implementation, the autophagia incidence dropped by nearly 10% in the affected rabbits (p = 0.0093). In addition, euthanasia related to self-mutilation decreased from 7.5 to 1.04% (p = 0.00164).

Initially, when autophagia was sighted, the rabbits were placed in donut collars (Figure 1). These collars were initially chosen due to their softness, lack of toxicity, and ease of application and cleaning. However, this type of collar did not prevent the rabbits from reaching their hind limb. Therefore, we were required to use plastic Elizabethan collars with soft edges. We found that due to the greater range around the rabbit’s head, these collars were more successful at preventing the rabbits from reaching and subsequently harming the toes on the injured limb. In addition, the relatively inexpensive cost of these types of collars proved to be incredibly advantageous. We purchased two sets of 10 collars, which made the treatment of autophagy expedited. It also allowed for an immediate exchange of soiled collars for clean ones.

Figure 1.

Example of an Elizabethan collar (left) and a Donut collar (right) used as a treatment for autophagy in original study.

A critical aspect of this treatment to note if used is that often our study personnel would find the collars covered in waste. Therefore, to maintain sanitary conditions and prevent eye infections in the rabbits, the collars had to be changed regularly and checked daily. Additionally, with these collars, if moisture collects where it is attached to the neck of the animal, fungal rashes can occur. Thus, it is also essential to check the necks of the rabbits to ensure there is no excessive moisture. Lastly, we found that the rabbits would often chew at the rims of the collars, so we had to provide non-toxic coverings, such as hypoallergenic surgical tape, to replace the soft sides of the collar. Despite these minor issues, this simple solution was shown to be successful in reducing this complication in our rabbit model.

3.2 Pressure ulcers

An additional complication we encountered was pressure ulcers on the plantar aspect of the injured foot. Pressure ulcers can cause excruciating pain and adequate steps must be taken to prevent the development of this ailment in the animals. Ulcers, specifically on animals’ heels, are incredibly common in sciatic nerve injury models. Rabbits are often more prone to this type of injury due to the overall lack of sub-dermal padding in the heel of their hind limbs [28]. Additionally, following nerve injury, dysesthesia can occur in the injured limb leading to dragging of the foot, ultimately contributing to the overall poor condition of the injured foot [29].

In our original study, we reported that about 23% of the rabbits developed pressure ulcers. This was the second most frequent postoperative complication experienced. These ulcers often developed into severe morbidities where the base of the ulcer reached the calcaneus. To prevent and lessen animal suffering, we developed an “ulcer cushion” and utilized this device to treat the ulcers. Our “ulcer cushion” consisted of a foam sheet custom-fitted to the bottom of the rabbits’ foot (Figure 2). Two personnel were used to apply the cushion, one to safely hold the rabbit and another to place the foam onto the affected foot. After the foam was placed onto the foot, it was wrapped in cast padding, and the entire foot was wrapped in veterinary bandaging. When the bandaging was placed onto the foot, appropriate pressure was applied so the cushion would stay in place but not affect the circulation of the foot.

Figure 2.

Illustration of our developed “ulcer cushion” utilized in our original experiment to treat and prevent severe pressure ulcers.

This treatment method was based on the clinical standard for treating this condition in humans. Unfortunately, there is yet to be a commercially made device to treat this ailment in animals, which led to the development of our own manufactured device. We believe this method eased pressure from the foot and even acted as a disease prevention mechanism for autophagia. Additionally, besides routine changing of the bandages, there were no other issues found with this treatment compared to the collars mentioned previously. Therefore, applying this cushion to the injured foot following sciatic nerve injury in rabbits is strongly recommended as a preventative tool for pressure ulcers.

4. Cost analysis

Compared to larger animal models, rabbits are a cost-effective option for sciatic nerve injury experimentation [4]. Yet, rabbits can often experience many complications that ultimately lead to increased costs. When the animal experiences complications beyond remedy, euthanasia must occur, and additional rabbits and their corresponding supplies will have to be ordered to replace the lost animals. This goes against the principle of reduction, a pillar in the universal framework to promote humane animal research. Our treatment methods outlined above completely subsided the need and cost for additional rabbits. In our original study, the total cost for the purchase and care of the rabbits required to complete the experiment should have been around $108,000. The added cost of acquiring additional rabbits to replace the ones that were lost was $25,414.94. Therefore, complications alone increased the cost of the study by 23.54%.

The cost of our interventions proved to be substantially inexpensive. When the cost of all the materials used for the treatment of the autophagy and pressure ulcers were summed, our treatments were around 66 times less expensive than ordering more rabbits ($376.95 vs. $25,038.44). These intervention methods proved to have the ability to reduce the number of animals required for the completion of the study, which can save thousands of dollars in experimental costs.

5. Discussion

The use of rabbits for sciatic nerve injury models can provide greater acuity for the investigation of peripheral nerve injury repair methods compared to smaller rodent animal models. In addition, their ease of handling and biocompatibility make rabbits an excellent choice for nerve injury experimentation. Although this model is less costly than larger animal models, rabbits can experience many complications that can almost negate this difference. In our original experiment, where we utilized rabbits to test sciatic nerve repairs, we recorded all postoperative complications experienced by the subjects. We found autophagy and ulcers of the heel to be the most common complications experienced by the rabbits. In this chapter, we outline how we treated and managed these complications in the hope that future studies can use these techniques to prevent these morbidities from occurring.

Autophagia or self-mutilation of the toes of the injured limb is a common complication across multiple animal models. With sciatic nerve injury, dysesthesia is common and is suspected to be the cause of this response in rabbits [22]. Amputation or euthanasia before the intended study endpoint is often required when this complication becomes severe. However, these solutions lead to greater distress for the rabbit and increased costs. Our proposed solution for this complication includes placing an Elizabethan collar on the rabbit, making it more challenging to bother the injured limb. With this inexpensive and simple method, we were able to reduce the number of animals with autophagia by nearly 10% and reduce the number of animals requiring euthanasia due to severe self-mutilation by 6%.

The second most common complication we encountered was the development of ulcers on the heel of the injured foot. Currently, there is no standard method to treat this complication in animal models. These ulcers can become extremely severe if left untreated, often developing so deep into the foot that the bone of the heel can be visible. Therefore, to maintain the well-being of the rabbits, it is vital to develop a standardized method of care when this morbidity occurs. Our solution was the development of an “ulcer cushion.” This essentially was a custom-fitted piece of foam placed under the injured foot and then secured with cast padding and veterinary tape. Although we did not perform a statistical analysis on the effectiveness of this method, we believe this solution alleviated the pressure placed on the injured foot and even worked indirectly to prevent autophagia.

These developed treatments drastically decreased the overall cost of the experiment as well. The price of replacing rabbits that had to be euthanized prior to the intended study endpoint can lead to substantial unbudgeted costs for the experiment. In our original study, the price for additional rabbits and the subsequent housing and care required, led to an additional cost of $25,414.94. This raised the cost of the entire experiment by nearly 24%. However, our proposed treatments dramatically reduce this cost to merely $377. Therefore, these developed treatment methods can save thousands of dollars, increase the welfare of the animals, and lessen the number of animals needed to complete the experiment.

One aspect of both treatments to note is that they require high levels of maintenance. The collars had to be changed often to maintain sanitation and avoid bacterial infections. Study personnel often returned to see the collars covered in feces. Additionally, moisture can be easily trapped around the neck of the rabbit where the collar is placed, leading to a high risk of developing fungal dermatitis. Similarly, the ulcer cushions had to be replaced and monitored often throughout the study. Another aspect future researchers should consider if utilizing these proposed treatments is how behavioral assessments may be affected. Moreso, with the placement of the cushion on the injured foot, the rabbit’s mobility will be disturbed and subsequently interfere with any walking assessments. Ambulatory assessments are the most used postoperative measurements in nerve repair studies [30, 31, 32, 33]. Therefore, with a cumbersome cast placed on the rabbit’s foot, one can anticipate the animal to drag their foot and even have an exaggerated movement of the protected foot when raising it, compared to the unaffected limb. These details should be strongly considered when implementing these treatments in an experiment that requires postoperative behavioral assessments. However, if these additional features of the treatments cannot be handled accordingly, we encourage using alternative animal models that allow for large sciatic nerve injury modeling but may not be as sensitive as the rabbit model.

6. Conclusions

In this chapter, we have outlined our experience working with rabbits to study repair methods for sciatic nerve injury. This animal model can provide great benefits for pre-clinical experimentation of peripheral nerve injuries. However, rabbits tend to have greater sensitivity to this particular impairment. With the lack of literature outlining common post-operative complications experienced in this animal model, it was imperative to discuss our findings and potential solutions for future research. Therefore, if an experiment cannot meet the suggested treatment protocols for maintaining the animals’ well-being, we suggest opting for an alternative, more resilient model for studying sciatic nerve injury. If a rabbit animal model must be used, applying the treatment methods outlined in this chapter immediately postoperatively can drastically reduce the amount of animal suffering and allow the experiment to progress successfully.

Conflict of interest

The authors declare no conflict of interest.