Animal Models in Toxicologic Research: Rabbit

1

Introduction

Throughout modern biomedical research, the rabbit has remained a core laboratory animal species. Studies using rabbits have provided important insights into toxicology, immunology, reproduction, and other fields. Interestingly, despite their common use, many key factors about rabbit biology, physiology, and disease are either poorly documented or difficult to identify in the literature. This chapter will cover some of those lesser-documented topics, as well as recommend selected resources in the literature.

2

Model Selection

3

Basic Biological Characteristics and Common Breeds

Rabbits ( Oryctolagus cuniculus , order Lagomorpha, family Leporidae) are often selected as an in vivo model system when size limitations of smaller mammals such as rodents cannot accommodate the techniques employed in a study while still representing a refinement over larger, phylogenetically higher-ranked mammalian model species (e.g., dog, pig, NHP). Refinements in rodent models have led to declining numbers of rabbits used in biomedical research ( ). Still, the Organisation for Economic Co-operation and Development and United States Environmental Protection Agency (EPA)–preferred nonrodent species in prenatal developmental toxicity studies is the rabbit (per EPA 712–C–98–207, OPPTS 870.3700), for which the New Zealand White (NZW) is the usual strain ( ). Other major applications include dermatological, cardiovascular, infectious disease, ophthalmological, orthopedic (including medical devices), cancer, reproductive biology, and regenerative/interventional biomedical research. Three outbred breeds predominate: the NZW (an albino large breed), the Dutch belted (a pigmented small breed), and the NZW x New Zealand Red (NZR) F1 cross (a pigmented large breed). Coat color genotypes are cc for the NZW, ee for the NZR, and du ^d du ^w aa for the black American Dutch ( ).

Requisite housing and husbandry conditions are stipulated by regulatory bodies. In the United States, rabbits are a United States Department of Agriculture–regulated species, elevating them above mice and rats and making them subject to standards set forth in the Guide for the Care and Use of Laboratory Animals, the Public Health Service Policy on Humane Care and Use of Laboratory Animals, and the Animal Welfare Act. One week of acclimation at a study site is generally provided prior to study recruitment. As the rabbit model is often selected for its size, adult animals are recruited within a defined body weight range. For the NZW, the minimum weight is around 2.4–2.7 kg. In the context of toxicology studies, environmental enrichment may include social noncontact enrichment and various forms of physical enrichment to enable gnawing and burrowing behavior; pen and pair housing is usually not possible for instrumented animals or those in dermatology studies ( ; ). Vendor differences have been noted to affect the incidence of background findings ( ) and the strain of a rabbit breed impacts genetic composition ( ). Commercially available, purpose-bred laboratory rabbit strains are regularly screened to be free from a defined set of pathogens, indicated by their vendor health reports. The importance of complete reporting on animal source, as part of the ARRIVE guidelines, cannot be overemphasized ( ). Rabbits are a prey species and as such have evolved with a wide visual field exceeding 330 degrees, auditory and olfactory acuity, and strong fear responses. This nervousness, combined with the high muscle mass and relatively low skeletal mass, necessitates firm and supportive handling to prevent hindleg excursions and subsequent spinal luxation or fracture.

Discussion of salient anatomic features will be limited to those relevant to test article investigation or explanation/implantation; detailed descriptions of lagomorph anatomy and physiology are well-documented in other texts ( ; ; ; ; ). Notable features of general biology ( Table 18.1 ) and reproduction ( Table 18.2 ) are provided in tabular format below.

The gastrointestinal tract of rabbits has many unique features. The rabbit is a hindgut fermenting herbivore with commensurate large intestinal microbiota and distinctive anatomical features. Compared to other species, adult rabbits have a lower gastric pH (1.9) and higher bile flow rate (130 mL/d/kg) ( ). The ileum adjoins the cecum at the sacculus rotundus, a small, bulbous structure heavily laden with lymphoid tissue. An elongated appendix forms the blind end of the cecum and the cecum adjoins the colon at the ampulla cecalis coli, another small outpouching opposite the sacculus rotundus. The cecum is large, holding approximately 40% of total ingesta ( ). Located between the transverse and descending colon, the nonhaustrated fusus coli serves to segregate fibrous ingesta and has intestinal pacemaker function ( ). Gut-associated lymphoid tissue is extensive ( ) comprising about 50% of the total body lymphoid mass ( ). Expression of rabbit intestinal CYP3A, canalicular multispecific organic anion transporter, and p-glycoprotein is reportedly similar to that in humans ( ). Cecotrophy, the ingestion of “night feces,” facilitates reprocessing of protein and vitamin-rich fecal matter.

Rabbits are lissencephalic, lacking gyri and sulci on the brain surface. Rabbits are obligate nasal breathers. The olfactory ecosystem is important in rabbit behavior and physiology and includes secretion of 2-phenoxyethanol, produced from the submandibular skin gland (“chin gland”) secretion from dominant rabbits ( ). The skin is thin and delicate. Ample subcutaneous space in the cervical and interscapular regions makes this a convenient injection site. The pinnae are highly vascular and provide thermoregulation since they represent approximately 12% total body surface area in the NZW ( ). In the laboratory setting, this facilitates measurement of pulse oximetry and blood pressure, as well as blood collection/injection via the marginal ear vein. Rabbits are prone to fatal cardiac arrhythmias following prolonged exposure to halothane anesthesia ( ).

Rabbits exhibit similar clinical pathology features as other nonclinical species with some exceptions. Lymphocytes are the predominant circulating leukocyte in the baseline state with heterophils (equivalent to neutrophils in other species) superseding in periods of inflammation or infection. Basophils are more prevalent than in most veterinary species, with upward of 30% being within normal limits ( ; ). Heterophils should not be mistaken for eosinophils ( ). The cervicofacial lymph node topography of the NZW is reportedly similar to humans, numbering 12–18 in total ( ). The fractional urinary excretion of calcium for rabbits is 45%–60% as compared to less than 2% in most mammals ( ). Crystalluria is normal, consisting of calcium carbonate and ammonium magnesium phosphate ( ).

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Regulatory Aspects and Examples of Use of Rabbits in Biomedical Research

Many guidelines and regulations that apply broadly to use of animals in biomedical research are also relevant to studies with rabbits. There are also specific documents for more specialized studies (e.g., medical devices, regenerative medicine) for which rabbits are a disproportionally popular animal model (reviewed recently by ). Key resources are summarized in Table 18.3 .

While rabbits have been used extensively in studies of biopharmaceutical, industrial/environmental chemicals, and medical device products, their use in cosmetics research may be the most widely recognized use outside of research groups. Cosmetic testing involving rabbits has influenced how some regulations/guidances have developed. For example, the seventh Amendment to the European Union (EU) Cosmetics Directive states that cosmetic products and/or their ingredients are not permitted to be marketed in the EU if they underwent testing in animals including sensitization and irritation evaluations commonly still performed in rabbits in the United States. While rabbit use is being replaced with in vitro assays for specific study types (e.g., dermal irritation), these replacement assays may not be accepted by individual regulatory groups.

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Pharmacokinetic and Toxicity Studies

Many standard nonclinical studies (e.g., pharmacokinetic and general toxicity studies) are not commonly performed in rabbits in part due to limitations with rabbits compared to rodents including relatively limited historical control data, larger amounts of compound needed, and increased costs. Rabbits are more commonly used as a secondary species for toxicity studies or the primary species for specialized studies such as those for the development of vaccines, medical devices, and regenerative medicine modalities (see Vaccines, Stem Cells and Regenerative Medicine, and Biomedical Materials and Devices , Vol 2 , Chap 9, 10, and 11). Specific areas of focus are discussed in more detail below. For other general references on rabbits in basic research and safety studies, the reader is directed to recent references ( ; ).