Microstructure of the corneal endothelial transition zone in different laboratory animals.

Purpose: To compare the microstructure of the corneal endothelial transition zone in different laboratory animals. Methods: Flat-mount corneas of rabbits, rats, and mice were stained with Alizarin Red S (ARS) and observed using scanning electron microscopy (SEM). The progenitor cell markers p75 neurotrophin receptor (p75NTR), SRY-box transcription factor 9 (SOX9), leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5), telomerase reverse transcriptase (TERT), and proliferation marker Ki-67 were examined in the flat-mounted corneas of three laboratory animals using immunofluorescence microscopy. Results: On flat mounts, proximity to the trabecular meshwork correlated with weaker ARS staining and greater polymorphism of endothelial cells in the transition zone in all animals. On SEM, distinct and smooth structures of the transition zone were negligibly detected in all animals. The endothelial cells in the transition zone had irregular shapes, with less dense, less wavy intercellular junctions, especially in murine corneas, exhibiting unique intercellular cystic spaces. In the transition zone of the rabbit cornea, progenitor cell markers p75NTR, SOX9, Lgr5, TERT, and proliferation marker Ki-67 were expressed, in contrast to those in other murine corneas. Conclusions: Although the transition zone was not identified clearly, irregular cell morphology and loss of cell-cell contact were observed in all animal corneal endothelial cells. The proliferative capacity and the presence of progenitor cells were confirmed in the transition zone, especially in the rabbit cornea.

Quantitative morphological analysis of age structural changes in prostate of experimental animals with ethanol poisoning.

OBJECTIVE: Aim: To find out the age remodeling of the structural components of the prostate gland at alcohol poisoning using quantitative morphological analysis. PATIENTS AND METHODS: Materials and Methods: The structure of the prostate gland of 4 white male rats groups were morphologically investigated. The 1 group included 30 control intact animals aged 8 months, the 2-nd group - 30 rats aged 24 months, the 3-rd group - 30 8-month-old animals with ethanol intoxication, and the 4-th group included 30 24-month-old rats with the specified simulated pathology. Ethanol intoxication was modeled by intragastric administration of 30% ethyl alcohol solution at a dose of 20 ml/kg once daily for 28 days. Rats were euthanized by bloodletting under general thiopental anesthesia 28 days after the beginning of the experiment. The area of glands, the height of glandular epithelial cells, the area of their nuclei and cytoplasm, the nuclear-cytoplasmic ratio in these cells and the stromal-parenchymal ratio in the organ were studied using light microscopy and were determined morphometrically. Morphometric parameters were processed statistically. RESULTS: Results: It was established that with age in the intact prostate of laboratory sexually mature white male rats, the area of glands, the height of glandular epitheliocytes, the area of their nuclei and cytoplasm, with the stability of nuclear-cytoplasmic ratios in the epithelial cells of the glands, significantly decreases, and the stromal-parenchymal ratio in the organ under study increases. Long-term ethanol poisoning leads to pronounced structural changes in the prostate, which is characterized by pronounced atrophy of the glandular epithelium, a decrease in the area of the glands, a decrease in the height of epithelial cells, a violation of nuclear-cytoplasmic relations in them, an increase in stromal-parenchymal ratio, and a prominent growth of the muscle-elastic stroma. The revealed structural changes of the studied components of the prostate dominated in 24-month-old experimental animals. CONCLUSION: Conclusions: Morphological analysis of the prostate gland established that morphometric and morphological changed significantly according to the age and were depend on the ethanol poisoning.

Optimizing environmental enrichment for Sprague Dawley rats: Exemplary insights into the liver proteome.

BACKGROUND: Considering the expected increase in the elderly population and the growing emphasis on aging-related biomedical research, the demand for aged laboratory animals has surged, challenging established husbandry practices. Our objective was to establish a cost-effective method for environmental enrichment, utilizing the liver as a representative organ to assess potential metabolic changes in response to differing enrichment levels. METHODS: We conducted a six-month study involving 24 male Sprague Dawley rats, randomly assigned to four environmental enrichment groups. Two groups were housed in standard cages, while the others were placed in modified rabbit cages. Half of the groups received weekly playtime in an activity focused rat housing unit. We evaluated hormone levels, playtime behavior, and subjective handling experience. Additionally, liver tissue proteomic analysis was performed. RESULTS: Initial corticosterone levels and those after 3 and 6 months showed no significant differences. Yet, testosterone levels were lower in the control group by the end of the study (p = 0.007). We observed 1871 distinct proteins in liver tissue, with 77% being common across groups. In gene ontology analysis, no specific pathways were overexpressed. In semiquantitative analysis, we observed differences in proteins associated in lipid metabolism such as Apolipoprotein A-I and Acyl-CoA 6-desaturase, which were lower in the control group (p = 0.024 and p = 0.009). Rats in the intervention groups with weekly playtime displayed the least amount of reported distress during inspection or upon room entry and were less prone to accepting treats. Removing animals from their enclosure was most effortless for those in the large cage group. Over time, there was a decrease in conflicts among rats that interacted only twice weekly during playpen time. DISCUSSION: In summary, refining husbandry practices for aging rats is both simple and budget-friendly, with no apparent adverse effects on stress levels, animal development, or relevant metabolic changes in the liver.

Impact of enriched environment on motor performance and learning in mice.

Neuroscience heavily relies on animal welfare in laboratory rodents as it can significantly affect brain development, cognitive function and memory formation. Unfortunately, laboratory animals are often raised in artificial environments devoid of physical and social stimuli, potentially leading to biased outcomes in behavioural assays. To assess this effect, we examined the impact of social and physical cage enrichment on various forms of motor coordination. Our findings indicate that while enriched-housed animals did not exhibit faster learning in eyeblink conditioning, the peak timing of their conditioned responses was slightly, but significantly, improved. Additionally, enriched-housed animals outperformed animals that were housed in standard conditions in the accelerating rotarod and ErasmusLadder test. In contrast, we found no significant effect of enrichment on the balance beam and grip strength test. Overall, our data suggest that an enriched environment can improve motor performance and motor learning under challenging and/or novel circumstances, possibly reflecting an altered state of anxiety.

Training Laboratory Rabbits to Refine Routine Husbandry Procedures.

Non-aversive handling and training techniques for laboratory animals are required to facilitate experimental and routine husbandry procedures, improving both animal welfare and scientific quality. Clicker training was utilized to develop training protocols for rabbits to refine stressful routine husbandry procedures usually associated with lifting (i.e., being picked up from the floor)/restraining (i.e., being held in the arms of a human) them. Thirteen female New Zealand White rabbits were trained over three weeks. All rabbits learned the predefined goal behaviors: they followed the target stick, jumped onto the weighing scale, entered a transport box, and reared while placing their front paws onto the trainer's hand. In addition, ten animals jumped from the floor onto the sitting trainer's lap and allowed the trainer to lift their paws off the surface while sitting on the trainer's lap. For some individuals, the protocols had to be adapted by additional interim steps. At the end of the training, the rabbits reliably showed the expected goal behaviors, even after short and long training breaks. With few exceptions, a familiar person other than the trainer could elicit the goal behaviors from the rabbits (generalization), though further sessions were required for generalization. In the voluntary approach test, the rabbits preferred interacting with the trainer in the 1st trial but spent as much time with an unfamiliar person as with the trainer in the 2nd trial. The behavioral observations suggested that picking the rabbits up with the transport box, as described in the protocol, instead of restraining them with the scruff of their neck and lifting them on the arm, was less aversive. All in all, the training protocols were feasible and can serve as a refinement strategy in laboratory animal facilities. In the interest of animal welfare, the training protocols should be applied wherever possible.

Recommendations for measuring and standardizing light for laboratory mammals to improve welfare and reproducibility in animal research.

Light enables vision and exerts widespread effects on physiology and behavior, including regulating circadian rhythms, sleep, hormone synthesis, affective state, and cognitive processes. Appropriate lighting in animal facilities may support welfare and ensure that animals enter experiments in an appropriate physiological and behavioral state. Furthermore, proper consideration of light during experimentation is important both when it is explicitly employed as an independent variable and as a general feature of the environment. This Consensus View discusses metrics to use for the quantification of light appropriate for nonhuman mammals and their application to improve animal welfare and the quality of animal research. It provides methods for measuring these metrics, practical guidance for their implementation in husbandry and experimentation, and quantitative guidance on appropriate light exposure for laboratory mammals. The guidance provided has the potential to improve data quality and contribute to reduction and refinement, helping to ensure more ethical animal use.

Differential effects of four laboratory animal control diets on gut microbiota in mice.

BACKGROUND: The gut microbiota is intricate and susceptible to multiple factors, with diet being a major contributor. The present study aimed to investigate the impact of four commonly used laboratory animal control diets, namely Keao Xieli's maintenance diet (KX), HFK's 1025 (HF), Research Diets' D12450B (RD), and Lab Diet's 5CC4 (LD), on the gut microbiota of mice. RESULTS: A total of 40 mice were randomly assigned to four groups, and each group was fed one of the four diets for a duration of 8 weeks. The assessment of gut microbiota was conducted using 16S rRNA sequencing both at the beginning of the study (week 0) and the end (week 8), which served as the baseline and endpoint samples, respectively. Following the 8-week feeding period, no significant differences were observed in physiological parameters, including body weight, visceral weight, and blood biochemical indices, across the four groups. Nonetheless, relative to the baseline, discernible alterations in the gut microbiota were observed in all groups, encompassing shifts in beta-diversity, hierarchical clustering, and key genera. Among the four diets, HF diet exhibited a significant influence on alpha-diversity, RD diet brought about notable changes in microbial composition at the phylum level, and LD diet demonstrated an interconnected co-occurrence network. Mantel analysis indicated no significant correlation between physiological parameters and gut microbiota in the four groups. CONCLUSION: Overall, our study demonstrated that the four control diets had a minimal impact on physiological parameters, while exerting a distinct influence on the gut microbiota after 8 weeks. © 2024 Society of Chemical Industry.

Immunoinformatics-guided recombinant polypeptide-based enzyme-linked immunosorbent assay for seromonitoring of laboratory animals for minute virus of mice and Kilham rat virus.

Subclinical infection of laboratory animals with one or more of several pathogens affects the results of experiments on animals. Monitoring the health of laboratory animals encompasses routine surveillance for pathogens, including several viruses. This study aimed to explore the development of an alternative assay to the existing ones for detecting infection of mice and rats with the parvoviruses minute virus of mice (MVM) and Kilham rat virus (KRV), respectively. Full-length VP2 and NS1 proteins of these parvoviruses, besides fragments containing multiple predicted epitopes stitched together, were studied for serological detection. The optimal dilution of full-length proteins and antigenic regions containing predicted epitopes for coating, test sera, and conjugate was determined using a checkerboard titration at each step. The assays were evaluated vis-à-vis commercially available ELISA kits. The results showed that an engineered fusion of fragments containing multiple predicted MVM VP2 and NS1 epitopes was better than either of the full-length proteins for detecting antibodies in 90% of the tested sera samples. For KRV ELISA, full-length VP2 was better compared to other individual recombinant protein fragments or combinations thereof for the detection of antibodies in sera. This report is the first description of an ELISA for KRV and an improved assay for MVM. Importantly, our assays could be exploited with small volumes of sera. The results also demonstrate the utility of immunoinformatics-driven polypeptide engineering in the development of diagnostic assays and the potential to develop better tests for monitoring the health status of laboratory animals.

Making sense of the costs of adversity throughout the lifespan on aging in humans and other animals.

Social adversity, particularly early in life, can cause lifelong damage to health; by now, numerous studies examine this relationship in non-human species, producing some important themes: A) Captive animals readily lack ethological validity, giving a special place to studies of natural populations; one must appreciate though, that animal studies typically benefit humans who themselves lack ecological validity, namely Westernized subjects. B) Animal studies of the links between social adversity and psychiatric maladies potentially produce anthropomorphism; however, long-term study of our closest relatives demonstrates how convincingly another primate can, for example, experience grief, rather than display "grief-like" behavior. C) Are long-term consequences of social adversity best viewed as maladaptive and pathological, or as adaptive preparation for similar adversity later in life?; the growing literature casts light on when adversity's consequences are the purview of medicine or natural history. D) Studies examining sustained adversity and aging can increasingly distinguish between aging versus diseases of aging or cohort effects, and between aging effects arising from direct physiological mechanisms or indirect behavioral ones.

Hepatic glucuronidation of tetrabromobisphenol A and tetrachlorobisphenol A: interspecies differences in humans and laboratory animals and responsible UDP-glucuronosyltransferase isoforms in humans.

Tetrabromobisphenol A (TBBPA) and tetrachlorobisphenol A (TCBPA), bisphenol A (BPA) analogs, are endocrine-disrupting chemicals predominantly metabolized into glucuronides by UDP-glucuronosyltransferase (UGT) enzymes in humans and rats. In the present study, TBBPA and TCBPA glucuronidation by the liver microsomes of humans and laboratory animals (monkeys, dogs, minipigs, rats, mice, and hamsters) and recombinant human hepatic UGTs (10 isoforms) were examined. TBBPA glucuronidation by the liver microsomes followed the Michaelis-Menten model kinetics in humans, rats, and hamsters and the biphasic model in monkeys, dogs, minipigs, and mice. The CLint values based on the Eadie-Hofstee plots were mice (147) > monkeys (122) > minipigs (108) > humans (100) and rats (98) > dogs (81) > hamsters (47). TCBPA glucuronidation kinetics by the liver microsomes followed the biphasic model in all species except for minipigs, which followed the Michaelis-Menten model. The CLint values were monkeys (172) > rats (151) > mice (134) > minipigs (104), dogs (102), and humans (100) > hamsters (88). Among recombinant human UGTs examined, UGT1A1 and UGT1A9 showed higher TBBPA and TCBPA glucuronidation abilities. The kinetics of TBBPA and TCBPA glucuronidation followed the substrate inhibition model in UGT1A1 and the Michaelis-Menten model in UGT1A9. The CLint values were UGT1A1 (100) > UGT1A9 (42) for TBBPA glucuronidation and UGT1A1 (100) > UGT1A9 (53) for TCBPA glucuronidation, and the activities at high substrate concentration ranges were higher in UGT1A9 than in UGT1A1 for both TBBPA and TCBPA. These results suggest that the glucuronidation abilities toward TBBPA and TCBPA in the liver differ extensively across species, and that UGT1A1 and UGT1A9 expressed in the liver mainly contribute to the metabolism and detoxification of TBBPA and TCBPA in humans.

Craniofacial and olfactory sensory changes after long-term unilateral nasal obstruction-an animal study using MMP-3-LUC transgenic rats.

Nasal obstruction exerts considerable physiological effects on the respiratory system and craniofacial morphology during the developmental stage. This study used MMP-3-LUC transgenic rats for in vivo tracking of long-term expression in the rat nasal region after unilateral nasal obstruction. Skeletal changes of the craniofacial, nasal, and sinus regions were measured through micro-computed tomography examination and analysis with 3D image processing and calculation. Matrix metalloproteinase-3 and olfactory marker protein expression were also investigated through immunohistochemistry (IHC). Unilateral nasal obstruction significantly reduced the MMP-3 signal in the nasal region of MMP-3-LUC transgenic rats, which was mainly expressed in the respiratory epithelium. Long-term obstruction also caused morphological changes of the craniofacial hard tissue, such as nasal septal deviation, longer inter-jaw distance, and increased maxillary molar dental height. It also caused compensatory growth in olfactory nerve bundles and the olfactory epithelium, as confirmed by IHC. In our study, long-term unilateral nasal obstruction caused nasal septal deviation toward the unobstructed side, hyper divergent facial development including longer molar dental height, and reduced MMP-3 production. However, further investigation is necessary to explore the mechanism in depth.

Fabrication and characterization of a hydrocolloid wound dressing functionalized with human placental derived extracellular matrix for management of skin wounds: An animal study.

BACKGROUND: Functionalization of wound dressing is one of the main approaches for promoting wound healing in skin wound management. In this study, our aim is to fabricate a bio-functionalized hydrocolloid wound dressing. METHODS: The extracellular matrix (ECM) was extracted from human placental tissue. A hydrocolloid film was fabricated using Na-CMC, pectin, gelatin, styrene-isoprene-styrene adhesive, glycerol, and 0.5%-2.5% powdered ECM. A polyurethane film and a release liner were used in the hydrocolloid/ECM films. The mechanical, adhesion, swelling rate, and integrity of the films were investigated. Cell proliferation, adhesion, and migration assays, as well as, SEM and FTIR spectroscopy were also conducted. Macroscopic and microscopic evaluations of wound healing process and formation of blood vessels were conducted in mouse animal models. RESULTS: We successfully fabricated a three-layered ECM-functionalized hydrocolloid dressing with a water vapor transmission rate of 371 g/m2 /day and an adhesion peel strength of 176 KPa. Cellular adhesion, proliferation and migration were promoted by ECM. In the animal tests, ECM-functionalized hydrocolloids significantly improved wound closure and re-epithelialization at days 14 and 21. Also, ECM-functionalized hydrocolloids promoted the formation of hair follicles. CONCLUSIONS: Our findings suggest that ECM could enhance the wound healing properties of hydrocolloid wound dressings. This wound dressing could be considered for application in hard-to-heal acute wounds.

The Numbers of Animals Used in Mexico for Scientific and Educational Purposes.

In Mexico, there are no official public and reliably reported data on the total number and species of non-human animals used for scientific purposes. The aim of the current study was to calculate the total numbers of animals used for scientific and educational purposes in Mexico, from January 2015 to October 2021, based on data requested from the National Institute of Transparency, Access to Information and Protection of Personal Data (INAI, in Spanish). In this period, authorised laboratory animal facilities reported the use of 5,437,263 animals for scientific and educational purposes. However, these data should be viewed with caution, since there is no official register of all Mexican institutions that use animals for these purposes. The use of various species of different taxonomic groups was reported, including mammals, birds, reptiles, amphibians, fish and invertebrates. The main scientific purposes of this animal use were: technological development; innovation; laboratory testing; production of biologicals; quality control; diagnostic purposes; basic and applied research; and education. A robust system for the licensing and approval of animal use, as well as a means to ensure compliance with the relevant regulations, are both urgently required. In addition, in order to regulate animal use, monitor animal care and protect their welfare, the creation of a publicly accessible national database that records the number and species of the animals used is imperative.

Neuroactive steroids and Parkinson's disease: Review of human and animal studies.

The greater prevalence and incidence of Parkinson's disease (PD) in men suggest a beneficial effect of sex hormones. Neuroactive steroids have neuroprotective activities thus offering interesting option for disease-modifying therapy for PD. Neuroactive steroids are also neuromodulators of neurotransmitter systems and may thus help to control PD symptoms and side effect of dopamine medication. Here, we review the effect on sex hormones (estrogen, androgen, progesterone and its metabolites) as well as androstenediol, pregnenolone and dehydroepiandrosterone) in human studies and in animal models of PD. The effect of neuroactive steroids is reviewed by considering sex and hormonal status to help identify specifically for women and men with PD what might be a preventive approach or a symptomatic treatment. PD is a complex disease and the pathogenesis likely involves multiple cellular processes. Thus it might be useful to target different cellular mechanisms that contribute to neuronal loss and neuroactive steroids provide therapeutics options as they have multiple mechanisms of action.

Microbiological assessment of Suncus murinus bred and managed as laboratory animals.

Suncus murinus is gaining prominence as a laboratory animal; however, there is no generally accepted method for microbiological monitoring. This study aimed to apply non-serological microbiological monitoring of laboratory mice for S. murinus and identify the subdominant species obtained by culture methods for microbial assessment. Culture and PCR were used to test S. murinus for the laboratory mice test panels including 10 bacterial species and orthohantaviruses, all of which were negative. The species that grew sub-dominantly in rectal feces were identified as Aeromonas hydrophila, which is pathogenic to mammals. These results indicate that microbiological monitoring should be used to detect pathogens directly from S. murinus, not from sentinel animals, due to the host-specific microbial environment.

Backcrossing to Generate a Congenic Mouse Strain.

Genetic background can have subtle or profound effects on mutant phenotypes, providing additional information regarding the function of the gene. If your mutation is maintained on one genetic background but you wish to analyze it on another, it is a simple matter to transfer the mutation to a recipient strain background by repeated backcrossing (introgression) as detailed in this protocol. The resulting strain is called a congenic strain, defined as a strain carrying the mutation within a segment of chromosome from the donor strain with the remainder of the genome from the recipient strain.

The effectiveness and safety of low-intensity transcranial ultrasound stimulation: A systematic review of human and animal studies.

Low-intensity transcranial ultrasound stimulation (LITUS) is a novel non-invasive neuromodulation technique. We conducted a systematic review to evaluate current evidence on the efficacy and safety of LITUS neuromodulation. Five databases were searched from inception to May 31, 2023. Randomized controlled human trials and controlled animal studies were included. The neuromodulation effects of LITUS on clinical or pre-clinical, neurophysiological, neuroimaging, histological and biochemical outcomes, and adverse events were summarized. In total, 11 human studies and 44 animal studies were identified. LITUS demonstrated therapeutic efficacy in neurological disorders, psychiatric disorders, pain, sleep disorders and hypertension. LITUS-related changes in neuronal structure and cortical activity were found. From histological and biochemical perspectives, prominent findings included suppressing the inflammatory response and facilitating neurogenesis. No adverse effects were reported in controlled animal studies included in our review, while reversible headache, nausea, and vomiting were reported in a few human subjects. Overall, LITUS alleviates various symptoms and modulates associated brain circuits without major side effects. Future research needs to establish a solid therapeutic framework for LITUS.

SARS-CoV-2 inactivation in laboratory animal tissues with 4% formaldehyde or 5% glutaraldehyde for transfer to biosafety level 1 laboratories.

The SARS-CoV-2 pandemic required the immediate need to transfer inactivated tissue from biosafety level (BSL)-3 to BSL-1 areas to enable downstream analytical methods. No validated SARS-CoV-2 inactivation protocols were available for either formaldehyde (FA)-fixed or glutaraldehyde (GA)-fixed tissues. Therefore, representative tissue from ferrets and hamsters was spiked with 2.2 × 106 tissue culture infectious dose 50% per ml (TCID50/ml) SARS-CoV-2 or were obtained from mice experimentally infected with SARS-CoV-2. SARS-CoV-2 inactivation was demonstrated with 4% FA or 5% GA at room temperature for 72 hours by a titer reduction of up to 103.8 TCID50/ml in different animal tissues with a maximum protein content of 100 µg/mg and a thickness of up to 10 mm for FA and 8 mm for GA. Our protocols can be easily adapted for validating the inactivation of other pathogens to allow for the transfer of biological samples from BSL-3 areas to BSL-1 laboratories.

A Commentary on Fasting of Nonclinical Research Animals.

This commentary discusses the implementation of fasting in nonclinical animal experimental subjects. The short-term removal of food from cages of experimental animals is in all respects innocuous. The term "stress" is ill-defined and the statutes and regulations governing animal research laboratories that exert their authority in the performance of their operations do so without substantive grounds to base compliance. The legislative and administrative history of the implementation of the Animal Welfare Act (AWA) has evolved into the development of laboratory management strategies that focus on the reduction of the biological cost of stress to the animals and the determination of when subclinical stress (eustress) becomes distress. Animal welfare is based on the tenet that in laboratories conducting animal research in compliance with Good Laboratory Practices (Title 21 USC, Chapter 13,§58), it is the study protocol and the study director that establish procedures and processes that are approved by each Institutional Animal Care and Use Committee to ensure the humane care and use of animals in research, teaching, and testing and to ensure compliance with guidelines and regulations. This approval process establishes the justification of eustress in the environment that do not rise to the threshold of distress under the AWA.