Rabbit as an Animal Model for Experimental Research (PDF)

Dental Research Journal Technical Report Rabbit as an animal model for experimental research Manjeet Mapara1, Betsy Sara Thomas2, K. M. Bhat2 Department of Periodontics, MGM Dental College and Hospital, Navi Mumbai, 2Department of Periodontics, Manipal College of Dental Sciences, 1...

Dental Research Journal Technical Report Rabbit as an animal model for experimental research Manjeet Mapara1, Betsy Sara Thomas2, K. M. Bhat2 Department of Periodontics, MGM Dental College and Hospital, Navi Mumbai, 2Department of Periodontics, Manipal College of Dental Sciences, 1 Manipal University, Manipal, India ABSTRACT Animal experimentation is carried out in consultation with the veterinary wing but it is essential Received: November 2011 that be familiar with experimental protocols of animal model to be able to design an approriate Accepted: November 2011 study. This is more so in place where the veterinary facilities are not easily available.Span Rabbits Address for correspondence: are commonly used as subjects for screening implant material. They have gained favour for their Dr. Betsy Sara Thomas, numerous advantages even though they should be ideally used prior to testing in a larger animal Department of Periodontics, model. Though experimentation on rabbits seems to be easy there are many pitfalls. Our endeavor Manipal College of Dental Sciences, Manipal in this article is to integrate all the data about maintaining rabbits as a model and to critically analyze University, Manipal, India. it on the basis of our experimentation. E-mail: betsythomas99@ gmail.com Key Words: Animal model, experimental model, rabbit model INTRODUCTION second level and rats, rabbits were at the third level. International standards established regarding the The first step in any study is to review the literature species suitable for testing implantation of materials on the model on which to perform the experiment. in bone state that dogs, sheep, goat, pigs, or rabbits When research is performed on the appropriate are suitable. Arriving at an animal model which suits animals, the information obtained should approximate all fields of application is a current goal in research what can be expected in human beings, i.e., animal and that is almost impossible. But the wide range of that is phylogenetically similar to human beings. animal species gives choice to the investigators for a The emotional attitude of some researchers, mainly most appropriate animal for their study design. in cases involving domestic animals (e.g., dogs and If there is more than one alternative, one should cats) can be a problem in long experiments where the select the least phylogenetically developed animals researcher may become emotionally attached to the available in sufficient quantity. Figure 1 represents animal. Dogs are the most widely used animal model the phylogenetic tree for vertebrates. in periodontal research, especially in the periodontal tissue regeneration studies and as periodontal disease The animals used as research models are divided into models. Despite the structural similarities in organic the following two groups: composition between human bone and canine, implant- 1. Small animal category requiring local animal associated changes evident in a canine model may not ethical committee approval: be as apparent in the human situation where there is Rats a lower rate of remodeling.[2,3] Monkeys were at a Mice Guinea pigs Access this article online Rabbit (largest animal in this group) Quick Response Code: 2. Large animal category requiring central animal Website: www.drj.ir ethical committee approval in addition to the local ethical committee are: Dogs DOI: 10.4103/1735-3327.92960 Goats Primates Dental Research Journal / Jan 2012 / Vol 9 / Issue 1 111  Mapara, et al.: Rabbit as an animal model osteons running parallel with the long axis of the bone, surrounding the medullary canal as well as the periosteal surface. The bone between these layers is comprised of dense haversian bone. The maximum mean osteon diameter described was 223.79+47.69 μm, with a mean minimum diameter of 50.79+9.71 μm. BONE COMPOSITION Although there is minimal literature on the differences between human and rabbit bone composition and density, some similarities are reported in the bone mineral density and subsequently the fracture toughness of mid-diaphyseal bone between rabbits and human beings. BONE REMODELLING In comparison with other species, such as primates and some rodents, the rabbit has faster skeletal change and bone turnover (significant intracortical, Haversian remodelling).[12-14] This may make it difficult to extrapolate results from studies performed in rabbits onto the likely human clinical response. However, rabbits are commonly used for screening implant materials prior to testing in a larger animal model. ADVANTAGES OF RABBIT AS A MODEL ARE AS FOLLOWS Figure 1: Phylogenetic tree for vertebrates 1. This animal is very docile and non-aggressive and Rabbits are small mammals in the family of Leporidae hence easy to handle and observe. of the order Lagomorpha found in several parts of 2. Widely bred and very economical compared with the world. Their habitats include meadows, wood, the expense of larger animals. forest, and grass lands. Although the macrostructure 3. Have short vital cycles (gestation, lactation, and and microstructure of rabbit bone are dissimilar puberty) to human bone, rabbits are commonly used for 4. It comes under the small animal category, hence screening implant material prior to testing in a larger comes under the purview of local ethical committee. animal model. Larger animals require an additional clearance from the central ethical committee clearance which is a Amongst various strains, New Zealand white strains of time-consuming process with stringent rules. rabbits are commonly being used for research activities. 5. The mandatory rearing facilities for ethical These strains are less aggressive in nature and have clearance for surgical procedures on larger animals less health problems as compared with other breeds. are very elaborate and expensive, generally only within the capacity of central animal research MICROSTRUCTURE OF RABBIT BONE facilities. Histologically, rabbit long bones have a very different Experimentation in rabbits poses a general set of microstructure from human beings.[9-10] In comparison problems, some of which are common to many with the secondary bone structure of mature human animals. bone, rabbits have a primary vascular longitudinal 1. Lack of well-equipped animal houses. tissue structure, comprising vascular canals of 2. Lack of expert handlers of the animal. 112 Dental Research Journal / Jan 2012 / Vol 9 / Issue 1  Mapara, et al.: Rabbit as an animal model 3. Lack of easy availability of post- and intraoperative The skin on the back of the rabbit’s neck is raised medicines, particularly for rabbits. upward until it is stretched enough that is comfortable 4. Scarcity of literature on the care of experimental to the rabbit. If it snaps back fast, the rabbit is healthy. utilization of rabbits. The slower it takes for the skin to return, the more is The specific disadvantages as regards to implantation the dehydration. In such a case, one can hydrate them of dental implants are as follows: by giving RGatorade—mix the Gatorade half and half 1. Being a small animal, the size of the mandible with water and feed them with a dropper. does not permit insertion of dental implants and hence loading of the implants is not possible after HANDLING implantation elsewhere in the body. 2. The rabbit is an animal of prey which has to be Daily human contact reduces stress during handling. fast and swift in nature. Thereby, they have a small Gloves should always be worn when handling rabbits. light and fragile bony structure. These animals can become nervous and inflict severe 3. The mid-femur diameter is only about 0.5 cm for bites or scratches from their powerful hind legs, if not a rabbit weighing 3 kg, making it inadequate for properly handled and restrained. Rabbits should never the larger size implants generally associated with be handled by the ears because of a high probability of human beings. causing cervical luxation and death.[17,20] They should 4. The rabbit is a vulnerable animal prone to post- be held by grasping a large fold of loose skin over antibiotic diarrhea, gastrointestinal stasis or ileus, the shoulders with one hand and either supporting or and post-implantation fractures due to a fragile grasping the rear feet with the other hand. Failing to weight-bearing femur. support or hold onto the rear feet may result in the 5. Veterinary surgeons specializing in rabbits are not animal kicking and trying to escape, which can cause many. severe spinal injury or a broken back. MAINTENANCE OF RABBITS IN THE IMPORTANT CONSIDERATIONS FOR ANIMAL HOUSE THE USE OF RABBITS FOR EXPERIMEN- TAL RESEARCH Newly arrived rabbits in the animal house should be quarantined for a minimum of two weeks and Choice of implant size examined for the most common diseases. Rabbits The implant size and length should be as small as are easily infested with scabies, which is highly possible. The recommended norm is of 2 mm diameter contagious and capable of damaging the airways and 6 mm length as there is size limitation of rabbit and predisposing to pneumonia. Quarantine also bone. Smaller size of the implant also reduces the serves as a period of adaptation to the surroundings sequencing of drills and the drilling time which is and the daily routine in the animal quarters. Adult advantageous as ketamine is a short-acting anesthetic rabbits should be kept in individual mesh cages with an induction time of half an hour during which (0.90 × 0.60 × 0.45 m) hung at a height of 0.8 cm both the legs have to be opened up and implants have from the ground so that excrement can fall out to be placed. into collecting trays. 12 to 14 hours of light are necessary for the colony's circadian biorhythms Rabbit size and animals should be routinely observed for food Contemporary literature survey has given little consumption and fecal characteristics.[17,18] importance to size of rabbit in experiments with implant surgery. But our experience shows that DIET size does play a crucial role. Choosing a healthy large animal more than 3 to 3.5 kg leaves one with The diet of an adult rabbit consists of fresh hay, an experimental subject with a good capacity to water, and fresh vegetables. Water should be changed withstand surgical trauma, preventing most of the every day and should be available round the clock. serious postoperative problems and leading to a better Dehydration in rabbit is a very common serious survival rate. Gender is generally not a problem, but condition that needs immediate attention. Signs of some investigators in literature have preferred female dehydration can be detected by the skin on their body. sex as it is biologically the stronger sex. Dental Research Journal / Jan 2012 / Vol 9 / Issue 1 113  Mapara, et al.: Rabbit as an animal model Position of implantation the rabbit regaining consciousness during surgery We found the ideal location for implantation to be the and flapping its legs. With ketamine alone, our first proximal femoral condyle. It has both cortical and experimental rabbit kept regaining consciousness and cancellous bone. Due to presence of cancellous bone in flapping its legs wildly and kicking off the instruments addition to cortical bone, it provides a cushioning effect from the surgical table to the ground. Most of the and prevents the cortical bone from splintering which surgery had to be performed with the rabbit being is commonly observed in other parts of the femur and physically constrained. Toward the end of the surgery, tibia. Also, the bone at this place gives adequate space i.e., after implantation, the rabbit flapped its leg and for the implantation and therefore a wider and longer broke the femur into two pieces. Subsequently, in the implant can be used. A 6 mm diameter and 8 mm long next rabbit, lignocaine was also used, which gave satisfactory results. implant can be placed with ease by this approach in comparison with the recommended norm of 2 mm But the best results were only after administration diameter and 6 mm length. of xylazine (0.6 ml Inter Muscular) which kept the animal sedated for a longer time with minimal pain. Implantation can be by the medial approach or the In several laboratory species (i.e., rodents, rabbits, lateral approach in the proximal femoral condyle. A ruminants, ferrets), xylazine has proven to be a safe lateral approach puts the following structures at risk anesthetic adjunct when coadministered with ketamine of damage: to induce short periods of surgical anesthesia. When 1. Fibular collateral ligament combined with ketamine, muscle relaxation and 2. Peroneal nerve visceral analgesia are improved, and emergence from 3. Popliteus tendon anesthesia is smoother. Xylazine has a wide margin The nerve is reflected along with the fascia with this of safety. Moreover, increasing the dose does not approach. generally increase the degree of sedation but rather the duration of effect. The medial approach puts the following structures at risk: Shaving and antisepsis 1. The infrapatellar branch of saphenous nerve. The region to be operated should always be shaved before any procedures. This can be done with a safety The distal tibial head is the next best location. Placing blade razor or electric shaver. All shaved hairs should more than one implant in one limb predisposes the be cleaned away to avoid contamination. already fragile rabbit bone to fracture and must be attempted with caution only if the implant size is Surgical procedure small, i.e., 2 to 3 mm in diameter and 6 mm or less in Lidocaine with adrenaline is administered locally. An length. We contemplate that a larger rabbit size and a incision is made using No. 22 blade. The incision is small implant can only be the way to use more than kept on the ventral side of the joint [Figure 3]. Once the one implant in the same limb safely. skin is incised, the fascia is cut with a pair of scissors until the bone is reached. Care should be taken to cut Anesthesia the fascia only and not the muscle. The periosteum is Preoperative fasting is not necessary because vomiting reflected with a periosteal elevator. First, a pilot drill is during induction of anesthesia has not been reported used and then the implant site is enlarged to the final in this species. Rabbits are subject to dehydration, so diameter. Intermittent drilling with a low speed rotary it should be ascertained that they always have plenty and profuse saline irrigation is necessary while drilling. of water. These animals can be provided with water The implant is then placed and screwed into the bone until approximately 60 minutes prior to starting the [Figure 4]. The fascia is sutured with catgut and the anesthesia. Anesthetic agents are selected based on the skin is sutured with silk. The silk sutures are kept health of the animal and the duration and invasiveness long to prevent the rabbit from chewing the sutures of the procedure. off completely and opening the surgical site. Some Ketamine (50-60 mg kg Inter Venous) is the most investigators advise intracutaneous silk sutures which preferred drug and should be administered slowly do not extend above the surface of the skin. Cutaneous through the lateral auricular vein [Figure 2] after the sutures are painful if the anesthesia has started to animal is immobilized in a special chamber. Providing wear off. And this can result in rabbit kicking the legs inadequate anesthesia and analgesia will lead to vigorously. The prevention and control of pain is the 114 Dental Research Journal / Jan 2012 / Vol 9 / Issue 1  Mapara, et al.: Rabbit as an animal model Postoperative management Moderate to severe postoperative pain can represent a serious stress to the rabbit and can result in some potentially dangerous consequences. In the past, when we did not understand the full significance of pain management in rabbits, they would often survive a surgical procedure, only to die within the next 36 hours. The old cliché “the operation was a success, but the patient died” would apply to those situations. Some of those cases would most likely have survived if postsurgical pain had been managed, thereby reducing the stress on the rabbit. How can we recognize pain in rabbits? Figure 2: Administration of Ketamine through lateral auricular vein When working with rabbits, the practitioner must infer the presence of pain by observing changes from normal behavior. A normal rabbit is bright, alert, active, inquisitive, has a smooth coat and good body condition. Pain may be evident as a limp or a change in gait, withdrawal or protection of an injured part, awkward or abnormal postures, licking, rubbing or scratching at an area, or indicated by decreased food and water intake. It is important to know that rabbits evolved as a prey species, an animal that normally needs to hide any handicap in order to escape predation. Hence, Signs of pain may be subtle, such as an increase in respiration, reluctance to move, sudden aggression, persistently squinting the eyes, a loss of interest in the surroundings, or an inability to Figure 3: Incision being given on the ventral side of the joint rest or sleep normally. Animals should be carefully evaluated during the first 24 postoperative hours, checking for signs of pain, because even if they do not vocalize them, remaining quiet and immobile in the back of their cages may be a sign of distress. Excessive pain can prolong recovery time from illness or injury. It can cause a rabbit to stop eating, loading to eventual shutting down of the gastrointestinal tract and death. Rabbits in excessive pain also can go into shock and die within 24 to 48 hours, despite the fact that the illness or injury itself may not have been life-threatening. Hence, analgesics should consistently be used for all major surgeries. The most common analgesics currently used in rabbits include butorphanol, Figure 4: Implant placed at the femoral condyle buprenorphine, and nonsteroidal anti-inflammatory drugs, such as aspirin, carprofen, diclofenac, flunixin, key to the practice of anesthesia. The perioperative ibuprofen, indomethacin, ketoprofen, meloxicam, analgesic protocol has an impact on the well-being of acetaminophen, and piroxicam. the animal that often extends far beyond the immediate As an immediate postoperative management, one anesthetic period. dose of Bupivacaine (A long-acting local anesthetic Dental Research Journal / Jan 2012 / Vol 9 / Issue 1 115  Mapara, et al.: Rabbit as an animal model agent) can be given around the wound as the first Abnormal or absence of stools dose. Later Ibugesic Plus syrup can be given twice Anesthetic and analgesic agents can alter the motility or thrice daily for pain management. To administer of the gastrointestinal tract. In addition, the rabbit the syrup, take the required amount in the dropper may have eaten poorly just prior to surgery; therefore, and hold the dropper near the rabbit’s mouth. Most there may be an absence of stools for a period up to of the rabbits will suck the syrup out of the dropper. three days after anesthesia/surgery. Watery diarrhea For the ones which do not, insert your fingers should not be present and is cause for immediate behind the incisors and the jaw of the rabbit, so concern. that its mouth is forced open. The syrup is forced Sutures into the rabbit’s mouth using a dropper until the The rabbits have a tendency to bite off their sutures rabbit swallows the sweet syrup by reflex. The with their sharp teeth. This can lead to gaping of the intramuscular and subcutaneous route for the pain wound. This can be overcome with a help of RHealex killer and antibiotics should be avoided for a small spray which forms a polymer layer over the wound. It animal like rabbit as the trauma of the injections will add substantially to the shock of surgery and should never be applied when the rabbit is conscious the rabbit may go into gastrointestinal ileus. The as the burning sensation from the spray can make antibiotic of choice is enrofloxacin which is given the rabbit run wild leading to fracture of the already orally with the help of a dropper, for 5 to 7 days. weakened legs. Amoxicillin should not be given as it leads to fatal Death diarrhea. The most likely cause of death reported in rabbit model is gastrointestinal stasis or ileus. This is caused RABBIT BEHAVIOR POST-ANESTHESIA/ due to reduction in peristalsis which is caused due to SURGERY factors like: 1. Fractures There are specific signs to be aware of in rabbits after 2. Loss of mate an anesthetic/surgical procedure. They are as follows: 3. New environment (can lower their immunity Quiet behavior making them more prone to illness) Your rabbit may want to sleep and stay quietly in one 4. Stress area. The lack of activity can be caused by residual In gastrointestinal stasis due to stoppage of peristalsis, anesthetic in the body, pain, or medication. Some the toxins produced by Clostridium accumulate and analgesics have sedative properties and may add to may cause death in 12 hours. General sign of this is, the lethargy. Check with your veterinarian about what the rabbit stops eating and dies eventually. This can you should expect with any drugs you are giving be avoided by giving prokinetics (Cisapride 0.2 mg/ to your rabbit. If your pet is extremely lethargic, kg orally, Simethicone, Metoclopramide). crying out, or appears unable to move normally and sits hunched in one spot, you should contact your As a caregiver, you can do a number of things to veterinarian immediately. minimize your rabbit's discomfort, such as careful handling of the sick rabbit, prompt communication Poor appetite with your veterinarian, gentle nursing care to improve The rabbit may not eat or drink at all for the first 24 your rabbit's comfort, access to food and water, and a hours after the surgery. Analgesics may improve this palatable diet to keep the rabbit eating. It is important to situation, but not completely eliminate it. You can prevent changes in gastrointestinal motility, especially syringe feed your pet with thin slurry blenderized when the rabbit is already stressed by disease. The fresh vegetables mixed with fruit juice one to two faster the rabbit starts eating, the faster the recovery. times during this initial 24-hour period. A food supplement called Fibreplex is available which helps Euthanasia maintain good gut motility and provides probiotics Euthanasia can be accomplished easily, painlessly, and prebiotics to encourage normal digestive function, and without prior stress. The technique preferred by which is vital for their recovery. If your rabbit still veterinarians involves sedation followed by injection refuses to eat after 24 hours, you should contact your of a barbiturate. Sodium pentobarbital is the drug of veterinarian immediately. choice because it acts fast and effectively. 116 Dental Research Journal / Jan 2012 / Vol 9 / Issue 1  Mapara, et al.: Rabbit as an animal model Our experience because certain of the research areas obviously cannot One of the main complications of implant surgery be done on human beings for practical and ethical in rabbits which we observed was the fracture reasons. And, rabbit being an easily available and of the femur. This can happen while drilling or less aggressive animal is a promising model if the postoperatively. The hallucinating effect while guidelines described above are followed. coming out of the short-acting anesthesia can make them jump around violently and can result in usually REFERENCES fracture of the femur. Despite a Spica cast (used for femur fractures and to hold the hip or thigh muscles 1. Liebschner MA. Biomechanical considerations of animal models used in tissue engineering of bone. 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