Laboratory Animal Species Research Articles

Manual for maintenance of multi-host ixodid ticks in the laboratory.

Use of laboratory animals as hosts for blood-sucking arthropods remains a time-proven and the most efficient method for establishment and propagation of slowly feeding ixodid ticks, despite introduction of techniques involving artificial feeding on either animal skins or synthetic membranes. New Zealand White rabbits are usually the most accessible and most suitable hosts routinely used for establishment and maintenance of a large variety of multi-host tick species. Here we describe standard procedures for maintaining colonies of multi-host ixodid ticks by feeding all developmental stages (larvae, nymphs, and adults) upon New Zealand White rabbits. When needed, the same procedures can be easily adapted to other species of laboratory or domestic animals from mice to dogs and goats. A summary of our experience in maintaining laboratory colonies of Ixodes scapularis, Ixodes pacificus, Amblyomma americanum, Dermacentor variabilis, Dermacentor occidentalis, Haemaphysalis leporispalustris, and Rhipicephalus sanguineus with descriptions of the complete laboratory life cycles and reliable production of uninfected ticks under standardized conditions has been published by Troughton and Levin (J Med Entomol 44:732-740, 2007). Here we provide step-by-step recommendations for various procedures used in the maintenance of ixodid tick colonies based on over 20years of experience.

Treatment of endotoxaemia and septicaemia in the equine patient.

Endotoxins, constituents of the cell wall of gram-positive and gram-negative bacteria, regularly result in severe illness and death in horses. In endotoxaemia, these constituents are present in the systemic circulation; in septicaemia, whole microbes invade normally sterile parts of the body. Interaction of these endotoxins with pathogen recognition receptors leads to an inflammatory response that cannot always be sufficiently contained and hence needs direct treatment. Over the last decennia, our understanding of the pathophysiology of endotoxaemia and septicaemia has significantly increased. Based on improved understanding of the interaction between receptors and endotoxins as well as the subsequent downstream signalling pathways, new therapeutic targets have been identified in laboratory animal species and humans. Important species differences in the recognition of endotoxins and pathogens by their receptors as well as the inflammatory response to receptor activation hamper extrapolation of this information to the horse (and other species). Historically, horses with endotoxaemia and septicaemia have been treated mainly symptomatically and supportively. Based on the identified therapeutic targets, this review describes the current knowledge of the treatment for endotoxaemia and septicaemia in the horse with reference to the findings in other animal species and humans.

Applying reproductive technologies and genome resource banking to laboratory animals

The Genome Resource Bank (GRB) is a repository of frozen biological material, including semen and embryos. Cryobanking is often used in combination with modern reproductive technologies, such as rederivation, in vitro culture, and embryo transfer. Thirteen mouse and rat strains have been rederived, and 32 are kept in cryostorage at the Institute of Cytology and Genetics, Novosibirsk. Other laboratory animal species have been cryopreserved as well. Embryos of two hamster species (Djungarian and Campbell’s) of the Phodopus genus were cryopreserved, and the viability of thawed embryos was confirmed by their successful development in vitro and in vivo (by transfer to a recipient). The positive effect of the Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) was demonstrated for both of these Phodopus species. Furthermore, the sperm of Djungarian (Phodopus sungorus) and Campbell’s (Phodopus campbelli) hamsters, domestic cat (Felis catusf),leopard cat (Prionailurus bengalensis euptilurus), and bobcat (Lynx rufus) was frozen and cryopreserved. Double staining by SYBR Green/PI, followed by confocal microscopy, demonstrated that more than 40% of amur cat semen retained their viability after cryopreservation. This is the world’s first reported successful freezing of semen of these wild feline species (Prionailurus bengalensis euptilurus). The article reviews the results and discusses the prospects of using reproductive technologies for the conservation of laboratory animal species.

Comparative Renal Toxicopathology of Antisense Oligonucleotides.

This review summarizes the current understanding of nephrotoxicity related to the administration of therapeutic oligonucleotides, particularly those with 2'-methoxy-ethyl (2'-MOE) modifications. To best understand the effects of antisense oligonucleotides (ASOs) on the kidney, the reader should have a general understanding of renal microanatomy, physiology, and general mechanisms related to toxicity, so a short review is presented. Preclinical-clinical correlates are also discussed. Collectively, the data for PS ODN and 2'-MOE-modified ASOs have shown the laboratory animal species utilized in toxicology studies generally overpredict renal effects of these agents. As such, 2'-MOE ASOs do not appear to pose as much of a risk to patients as the preclinical data would suggest. This observation has been confirmed so far in clinical investigations.

In Utero Bisphenol A Exposure Induces Abnormal Neuronal Migration in the Cerebral Cortex of Mice

Bisphenol A (BPA) has been known to have endocrine-disrupting activity to induce reproductive and behavioral abnormalities in offspring of laboratory animal species. However, morphological basis of this abnormality during brain development is largely unknown. Cerebral cortex plays a crucial role in higher brain function, and its precisely laminated structure is formed by neuronal migration. In the present study, transfecting a plasmid (pCAG-mCherry) by in utero electroporation (IUE), we visualized developing neurons and investigated the possible effects of in utero BPA exposure on neuronal migration. Pregnant mice were exposed to BPA by osmotic pump at estimated daily doses of 0, 40 (BPA-40), or 400 (BPA-400) μg/kg from embryonic day 14.5 (E14.5) to E18.5. IUE was performed at E14.5 and neuronal migration was analyzed at E18.5. Compared with the control group, neuronal migration in the cortical plate was significantly decreased in the BPA-40 group; however, there was no significant difference in the BPA-400 group. Among several neuronal migration-related genes and cortical layer-specific genes, TrkB in the BPA-400 group was found significantly upregulated. In conclusion, in utero exposure to low BPA dose was found to disrupt neuronal migration in the cerebral cortex in a dose-specific manner.

Applying reproductive technologies and genome resource banking to laboratory animals

The Genome Resource Bank (GRB) is a repository of frozen biological material, including semen and embryos. Cryo­banking is used in combination with modern reproductive technologies such as rederivation, in vitro culture and embryo transfer. Thirteen mouse and rat strains have been re-derived and 32 are kept frozen in the cryostorage at the Institute of Cytology and Genetics, Novosibirsk. Some other laboratory animal species have been cryopreserved as well. Embryos of two hamster species (Djungarian and Campbell’s) in the genus Phodopus were cryopreserved and the viability of thawed embryos was proved by their successful development in vitro and in vivo (by transfer to a recipient). A positive effect of the granulocyte-macrophage colony-stimulating factor (GM-CSF) was demonstrated with both these Phodopus species. Furthermore, semen of Djungarian (Phodopus sungorus) and Campbell’s (Phodopus campbelli) hamsters, domestic cat (Felis catus), amur cat (Prionailurus bengalensis euptilurus) and bobcat (Lynx rufus) was frozen and cryopreserved. Double staining by SYBR Green/PI and subsequent confocal microscopy demonstrated that more than 40 % of amur cat semen retained viability after cryopreservation. This is the world’s first reported successful freezing of semen of this wild felid (Prionailurus bengalensis euptilurus). This article reviews the results and discusses prospects of using reproductive technologies for conservation of laboratory species.

Application of a Micropatterned Cocultured Hepatocyte System To Predict Preclinical and Human-Specific Drug Metabolism.

Laboratory animal models are the industry standard for preclinical risk assessment of drug candidates. Thus, it is important that these species possess profiles of drug metabolites that are similar to those anticipated in human, since metabolites also could be responsible for biologic activities or unanticipated toxicity. Under most circumstances, preclinical species reflect human in vivo metabolites well; however, there have been several notable exceptions, and understanding and predicting these exceptions with an in vitro system would be very useful. Human micropatterned cocultured (MPCC) hepatocytes have been shown to recapitulate human in vivo qualitative metabolic profiles, but the same demonstration has not been performed yet for laboratory animal species. In this study, we investigated several compounds that are known to produce human-unique metabolites through CYP2C9, UGT1A4, aldehyde oxidase (AO), or N-acetyltransferase that were poorly covered or not detected at all in the selected preclinical species. To perform our investigation we used 24-well MPCC hepatocyte plates having three individual human donors and a single donor each of monkey, dog, and rat to study drug metabolism at four time points per species. Through the use of the multispecies MPCC hepatocyte system, the metabolite profiles of the selected compounds in human donors effectively captured the qualitative in vivo metabolite profile with respect to the human metabolite of interest. Human-unique metabolites that were not detected in vivo in certain preclinical species (normally dog and rat) were also not generated in the corresponding species in vitro, confirming that the MPCC hepatocytes can provide an assessment of preclinical species metabolism. From these results, we conclude that multispecies MPCC hepatocyte plates could be used as an effective in vitro tool for preclinical understanding of species metabolism relative to humans and aid in the choice of appropriate preclinical models.

Gestation age dependent transfer of human immunoglobulins across placenta in timed-pregnant guinea pigs

IntroductionWhen administered during pregnancy, antibodies and other biologic drugs that contain the Fc part of the IgG molecule can traverse the placenta. Although it is generally accepted that the FcRn receptor mediates this process, gaps remain in our understanding of underlying details in humans and in common laboratory animal species. MethodsWe expanded our previous studies in timed-pregnant guinea pigs to both measure the transport of human (h) IgG at earlier gestation ages in vivo and evaluate FcRn function in vitro using Surface Plasmon Resonance (SPR) and Madin–Darby canine kidney cells (MDCK) that express guinea pig (gp) FcRn. ResultsIn timed-pregnant guinea pigs both the average concentration of hIgG in the fetus and its ratio to maternal hIgG concentration increase exponentially with gestation age. Thus, hIgG fetal:maternal concentration ratios increase from an average of 1% to 3%, 17%, and 76% on GD ∼26, 35, 46, and 54, respectively. In vitro, gpFcRn immobilized on a solid surface can bind hIgG and gpIgG preparations in a similar manner. All engineered human Fc isotype-specific constructs were internalized by MDCK-gpFcRn cells at significant levels. While not significant, their recycling and hIgG transcytosis by this cell line also trend higher than background controls. DiscussionPregnant guinea pigs exhibit similarities with humans in the degree and timing of transplacental transfer as well as the ability of their FcRn to bind and internalize hIgG in vitro. Further studies are needed to guide building appropriate systems for the evaluation of FcRn mediated function of human immunoglobulin therapies.

Review of animal models used to study effects of bee products on wound healing: findings and applications

Abstract Non-healing wounds are associated with high morbidity and might greatly impact a patient’s well-being and economic status. For many years, scientific research has focused on developing and testing several natural and synthetic materials that enhance the rate of wound healing or eliminate healing complications. Honey has been used for thousands of years as a traditional remedy for many ailments. Recently, honey has reemerged as a promising wound care product especially for infected wounds and for wounds in diabetic patients. In addition to its proposed potent broad-spectrum antibacterial properties, honey has been claimed to promote wound healing by reducing wound hyperaemia, oedema, and exudate, and by stimulating angiogenesis, granulation tissue formation and epithelialisation. Several animal models, including large animals, dogs and cats, and different species of laboratory animals have been used to investigate the efficacy and safety of various natural and synthetic agents for wound healing enhancement. Interpreting the results obtained by these studies is, however, rather difficult and usually hampered by many limiting factors including great variation in types and origins of honey, the type of animal species used as models, the type of wounds, the number of animals, the number and type of controls, and variation in treatment protocols. In this article, we provide a comprehensive review of the most recent findings and applications of published experimental and clinical trials using honey as an agent for wound healing enhancement in different animal models.

Regulatory Forum Opinion Piece*: Dispelling Confusing Pathology Terminology: Recognition and Interpretation of Selected Rodent Renal Tubule Lesions.

Renal tubule lesions often prove troublesome for toxicologic pathologists because of the diverse nature and interrelated cell types within the kidney and the presence of spontaneous lesions with overlapping morphologies similar to those induced by renal toxicants. Although there are a number of guidance documents available citing straightforward diagnostic criteria of tubule lesions for the pathologist to refer to, most are presented without further advice on the when to or to the why and the why not of diagnosing one lesion over another. Documents presenting diagnostic perspectives and recommendations derived from an author's experience are limited since guidance documents are generally based on descriptive observations. In this Regulatory Forum opinion piece, the authors attempt to dispel confusing renal tubule lesion terminology in laboratory animal species by suggesting histological advice on the recognition and interpretation of these complex entities.

Effect of sodium fluoride on male mouse fertility.

Sodium fluoride (NaF), an environmental pollutant, has been tested for its impact on fertility in several species of laboratory animals. A literature demonstrated that NaF adversely affects sperm motility, morphology, capacitation, and the acrosome reaction. However, the molecular mechanisms underlying these alterations have not yet been elucidated. Therefore, present study was designed to evaluate the regulatory pathways involved in the effect of NaF on sperm function and fertilization. In this in vitro study, mouse spermatozoa were incubated with a range of concentrations (2.5, 5, and 10 mm) of NaF for 90 min in media that support in vitro fertilization. Our results showed that NaF was associated with reduced intracellular ATP generation, motility, and motion kinematics. Likewise, short-term exposure of spermatozoa to NaF significantly reduced the intracellular calcium concentration, protein kinase-A activity, and tyrosine phosphorylation of sperm proteins, which were associated with a significant decrease in the rate of capacitation and the acrosome reaction. Finally, NaF significantly reduced the fertilization and blastocyst formation during early embryonic development. On the basis of these results, we propose that NaF reduces sperm motility, capacitation, and the acrosome reaction leading to poor fertilization and suppressed embryonic development.

Embryo and gamete cryopreservation for genetic resources conservation of laboratory animals

Article reviews the use of embryos and gametes cryopreservation for cryobanking the laboratory animal species. The special emphasis is made on the mechanisms of cryoinjury and cryoprotection during program freezing and vitrification. The species specific cryobanking problems are discussed and the prospects to overcome these problems are outlined.

Scientific and Regulatory Policy Committee Points-to-consider Paper*: Drug-induced Vascular Injury Associated with Nonsmall Molecule Therapeutics in Preclinical Development: Part I. Biotherapeutics.

Drug-induced vascular injury (DIVI) is a recurrent challenge in the development of novel pharmaceutical agents. Although DIVI in laboratory animal species has been well characterized for vasoactive small molecules, there is little available information regarding DIVI associated with biotherapeutics such as peptides/proteins or antibodies. Because of the uncertainty about whether DIVI in preclinical studies is predictive of effects in humans and the lack of robust biomarkers of DIVI, preclinical DIVI findings can cause considerable delays in or even halt development of promising new drugs. This review discusses standard terminology, characteristics, and mechanisms of DIVI associated with biotherapeutics. Guidance and points to consider for the toxicologist and pathologist facing preclinical cases of biotherapeutic-related DIVI are outlined, and examples of regulatory feedback for each of the mechanistic types of DIVI are included to provide insight into risk assessment.

Scientific and Regulatory Policy Committee Points-to-consider Paper*: Drug-induced Vascular Injury Associated with Nonsmall Molecule Therapeutics in Preclinical Development: Part 2. Antisense Oligonucleotides.

Drug-induced vascular injury (DIVI) is a recurrent challenge in the development of novel pharmaceutical agents. In recent years, DIVI has been occasionally observed in nonhuman primates given RNA-targeting therapeutics such as antisense oligonucleotide therapies (ASOs) during chronic toxicity studies. While DIVI in laboratory animal species has been well characterized for vasoactive small molecules, and immune-mediated responses against large molecule biotherapeutics have been well described, there is little published information regarding DIVI induced by ASOs to date. Preclinical DIVI findings in monkeys have caused considerable delays in development of promising new ASO therapies, because of the uncertainty about whether DIVI in preclinical studies is predictive of effects in humans, and the lack of robust biomarkers of DIVI. This review of DIVI discusses clinical and microscopic features of vasculitis in monkeys, their pathogenic mechanisms, and points to consider for the toxicologist and pathologist when confronted with ASO-related DIVI. Relevant examples of regulatory feedback are included to provide insight into risk assessment of ASO therapies.

Gained in Translation

Atherosclerotic renovascular disease (ARVD) is more commonly identified than ever before, particularly among the elderly population. By some accounts, >10% of new patients entering dialysis programs in the United States have ARVD as a major contributor to their end-stage renal disease. ARVD is also associated with accelerated renal injury, hypertension, and cardiovascular complications. Renovascular hypertension (RVH) is an independent cardiovascular risk factor and induces greater decline in cardiac function and structure than essential hypertension.1 However, clinical trials show consistently that revascularization of the stenotic renal artery using percutaneous transluminal renal angioplasty (PTRA) results in limited and inconsistent recovery of renal function.2 The reasons for the failure of revascularization to resolve renal dysfunction are complex and may include procedural complications, as well as pre-existing parenchymal injury, including endothelial dysfunction, inflammation, fibrosis, and microvascular remodeling in the poststenotic kidney.3 Atherogenic comorbidities constitute important catalysts of renal damage in ARVD. In fact, atherosclerotic nephropathy can produce renal damage even with hemodynamically insignificant stenoses,4 suggesting a direct effect of atherosclerosis on the kidney. Furthermore, unlike patients with fibromuscular dysplasia, in patients with ARVD a decrease in stenotic-kidney perfusion does not correlate with the angiographic degree of stenosis,5 and cortical perfusion is reduced even with mild stenoses, underscoring the contribution of additional pathogenic factors to renal damage. Therefore, the quest for underlying mechanisms and promising interventions mandates development of experimental platforms that not only involve renal artery stenosis but also emulate the atherosclerotic milieu. Useful models of renal artery stenosis have been achieved in many laboratory animal species, including mice, rats, hamsters, rabbits, dogs, and monkeys, but usually without application of comorbidities.6 Our studies focused on developing a swine model with gradually developing renal artery stenosis secondary to unilateral renal arterial implantation of a local irritant coil. The advantages …

Deep subconjunctival injection of gentamicin for the treatment of bacterial conjunctivitis in macaques (Macaca mulatta and Macaca fascicularis).

Infectious conjunctivitis occurs in a number of domestic and laboratory animal species and is usually treated topically with eye drops or eye ointments, which must be administered several times a day and sometimes for a prolonged period of time. In aggressive nonhuman primates or other large laboratory animal species, this may require the use of anesthesia or physical restraint before each treatment, which can be stressful to the animals and demanding for personnel. The authors describe a technique for administering deep subconjunctival injections of an antibiotic to laboratory macaques for the treatment of conjunctivitis. Three cases of recurrent conjunctivitis in macaques that responded poorly to other treatment approaches were effectively treated using this technique. This approach is recommended for the treatment of conjunctivitis in macaques and other large animal species.

A jump persistent turning walker to model zebrafish locomotion.

Zebrafish are gaining momentum as a laboratory animal species for the investigation of several functional and dysfunctional biological processes. Mathematical models of zebrafish behaviour are expected to considerably aid in the design of hypothesis-driven studies by enabling preliminary in silico tests that can be used to infer possible experimental outcomes without the use of zebrafish. This study is motivated by observations of sudden, drastic changes in zebrafish locomotion in the form of large deviations in turn rate. We demonstrate that such deviations can be captured through a stochastic mean reverting jump diffusion model, a process that is commonly used in financial engineering to describe large changes in the price of an asset. The jump process-based model is validated on trajectory data of adult subjects swimming in a shallow circular tank obtained from an overhead camera. Through statistical comparison of the empirical distribution of the turn rate against theoretical predictions, we demonstrate the feasibility of describing zebrafish as a jump persistent turning walker. The critical role of the jump term is assessed through comparison with a simplified mean reversion diffusion model, which does not allow for describing the heavy-tailed distributions observed in the fish turn rate.

Effects of orally administered galacto-oligosaccharides on immunological parameters in foals: a pilot study.

BackgroundIn the first phase of life, in which the immune system is primed and the bacterial colonization of epithelial surfaces takes place, foals are highly susceptible to bacterial infections. Next to strategies to optimize maternally acquired immunity in individual foals, current research explores other options to modulate immune responses in foals. During the past decades, oligosaccharide supplements were developed to mimic beneficial properties of the oligosaccharides, which are present in colostrum and milk. In human infants and laboratory animal species, dietary supplementation with galacto-oligosaccharides (GOS) has been shown to result in prebiotic and immunomodulating effects, with long-term beneficial consequences for both defensive and allergic immune responses. As yet, no studies are published concerning the in vivo effects of GOS in horses. The current study was designed as a pilot study to investigate the effects of an orally applied, commercially available GOS product in a group of pony foals. The treatment and the control group consisted of six and four foals, respectively. Foals were treated during the first four weeks of life and subsequently followed up for another ten weeks.ResultsIn peripheral blood mononuclear cells (PBMCs) derived from GOS-treated foals at day 28, a standardized lipopolysaccharide challenge resulted in significantly lower relative mRNA expression levels of the pro-inflammatory cytokines interferon-γ and interleukin-6 compared with PBMCs of control foals. In the 98-day period of investigation, no significant effects of the GOS supplement were observed on clinical and blood parameters for immunity and general health in these foals.ConclusionsBased on these first results, we can conclude that this dose regimen of GOS was well accepted by the foals and did not result in any detectable undesirable side effects. More clinical trials are required to confirm the attenuating effects of GOS treatment on equine pro-inflammatory immune responses and to implement this into practice.Electronic supplementary materialThe online version of this article (doi:10.1186/s12917-014-0278-4) contains supplementary material, which is available to authorized users.

Dermal toxicity studies: factors impacting study interpretation and outcome.

The field of dermal toxicity continues to evolve in order to accurately predict dermal (and systemic) responses in humans to topically applied chemicals. Although the testing methods have undergone extensive refinements, idiosyncrasies and unexpected issues during the conduct of these studies are not unusual due to the plethora of new vehicles available for formulating test substances, changing regulatory requirements, and introducting new strain and/or species of laboratory animals as no single species or method seems to suffice for evaluating skin toxicity. The objective of this article is to illustrate some pragmatic issues that should be considered during the conduct as well as interpretation of dermal toxicity studies. Routine procedure-related issues such as hair clipping, tape stripping, and wrapping the animal's torso to prevent oral ingestion can influence the interpretation. Excipients used in dermal toxicity studies may be nontoxic when used alone but complex dermal formulations can result in unexpected irritation and toxicity. In conclusion, interpretation and risk assessment of dermal toxicity studies should be done in a comprehensive manner, taking into account procedure-related impact on study results, unique species susceptibility, limitation of gross visual (naked eye) observation for evidence of toxicity, and normal anatomical variation.

A Method for Immunohistochemical Detection of Cholinergic Neurons in the Central Nervous System of Laboratory Animals

A protocol for the immunohistochemical detection of choline acetyltransferase (CAT) – a key enzyme in acetylcholine synthesis – on paraffin brain sections from laboratory animals is presented. The method is simple, gives good reproducibility, and allows CAT to be detected in neurons, fibers, and terminals on brain preparations in at least three species of laboratory animals: rats, rabbits, and cats. Different fixatives were suitable for immunohistochemical detection of CAT – 10% formalin, 4% paraformaldehyde, and zinc-ethanol-formaldehyde, though the best results were obtained with zinc-ethanol-formaldehyde.