Advances in Animal Models and Cutting-Edge Research in Alternatives: Proceedings of the Third International Conference on 3Rs Research and Progress, Vishakhapatnam, 2022.

Animal experimentation has been integral to drug discovery and development and safety assessment for many years, since it provides insights into the mechanisms of drug efficacy and toxicity (e.g. pharmacology, pharmacokinetics and pharmacodynamics). However, due to species differences in physiology, metabolism and sensitivity to drugs, the animal models can often fail to replicate the effects of drugs and chemicals in human patients, workers and consumers. Researchers across the globe are increasingly applying the Three Rs principles by employing innovative methods in research and testing. The Three Rs concept focuses on: the replacement of animal models (e.g. with in vitro and in silico models or human studies), on the reduction of the number of animals required to achieve research objectives, and on the refinement of existing experimental practices (e.g. eliminating distress and enhancing animal wellbeing). For the last two years, Oncoseek Bio-Acasta Health, a 3-D cell culture-based cutting-edge translational biotechnology company, has organised an annual International Conference on 3Rs Research and Progress. This series of global conferences aims to bring together researchers with diverse expertise and interests, and provides a platform where they can share and discuss their research to promote practices according to the Three Rs principles. In November 2022, the 3rd international conference, Advances in Animal Models and Cutting-Edge Research in Alternatives, took place at the GITAM University in Vishakhapatnam (AP, India) in a hybrid format (i.e. online and in-person). These conference proceedings provide details of the presentations, which were categorised under five different topic sessions. It also describes a special interactive session on in silico strategies for preclinical research in oncology, which was held at the end of the first day.

The Capability Maturity Model as a Measure of Culture of Care in Laboratory Animal Science.

Culture of care in Laboratory Animal Science (LAS) refers to a commitment toward improving animal welfare, scientific quality, staff wellbeing, and transparency for all stakeholders, ensuring that the animals and personnel involved are treated with compassion and respect. A strong culture of care can be established by the proactive implementation of the Three Rs, sharing best practices, caring for and respecting animals and colleagues, empowering staff, taking responsibility for our actions, and having a caring leadership. Culture of care, when established, should be evaluated continuously, in order to foster its progress and persistence. Even though several tools for assessing the culture of care within an institution have been proposed, an ultimate standard for measuring the concept is lacking. Here, we review the culture of care concept and propose the 'Capability Maturity Model' as a means of quantifying culture of care in the laboratory animal setting.

Advances in Animal Models and Cutting-Edge Research in Alternatives: Proceedings of the Second International Conference on 3Rs Research and Progress, Hyderabad, 2021.

The fact that animal models fail to replicate human disease faithfully is now being widely accepted by researchers across the globe. As a result, they are exploring the use of alternatives to animal models. The time has come to refine our experimental practices, reduce the numbers and eventually replace the animals used in research with human-derived and human-relevant 3-D disease models. Oncoseek Bio-Acasta Health, which is an innovative biotechnology start-up company based in Hyderabad and Vishakhapatnam, India, organises an annual International Conference on 3Rs Research and Progress. In 2021, this conference was on 'Advances in Research Animal Models and Cutting-Edge Research in Alternatives'. This annual conference is a platform that brings together eminent scientists and researchers from various parts of the world, to share recent advances from their research in the field of alternatives to animals including new approach methodologies, and to promote practices to help refine animal experiments where alternatives are not available. This report presents the proceedings of the conference, which was held in hybrid mode (i.e. virtual and in-person) in November 2021.

Gamma secretase-mediated Notch signaling worsens brain damage and functional outcome in ischemic stroke.

Mice transgenic for antisense Notch and normal mice treated with inhibitors of the Notch-activating enzyme gamma-secretase showed reduced damage to brain cells and improved functional outcome in a model of focal ischemic stroke. Notch endangers neurons by modulating pathways that increase their vulnerability to apoptosis, and by activating microglial cells and stimulating the infiltration of proinflammatory leukocytes. These findings suggest that Notch signaling may be a therapeutic target for treatment of stroke and related neurodegenerative conditions.

Resveratrol improves health and survival of mice on a high-calorie diet.

Resveratrol (3,5,4'-trihydroxystilbene) extends the lifespan of diverse species including Saccharomyces cerevisiae, Caenorhabditis elegans and Drosophila melanogaster. In these organisms, lifespan extension is dependent on Sir2, a conserved deacetylase proposed to underlie the beneficial effects of caloric restriction. Here we show that resveratrol shifts the physiology of middle-aged mice on a high-calorie diet towards that of mice on a standard diet and significantly increases their survival. Resveratrol produces changes associated with longer lifespan, including increased insulin sensitivity, reduced insulin-like growth factor-1 (IGF-I) levels, increased AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor-gamma coactivator 1alpha (PGC-1alpha) activity, increased mitochondrial number, and improved motor function. Parametric analysis of gene set enrichment revealed that resveratrol opposed the effects of the high-calorie diet in 144 out of 153 significantly altered pathways. These data show that improving general health in mammals using small molecules is an attainable goal, and point to new approaches for treating obesity-related disorders and diseases of ageing.

Nrf2 mediates cancer protection but not prolongevity induced by caloric restriction.

Caloric restriction (CR) is the most potent intervention known to both protect against carcinogenesis and extend lifespan in laboratory animals. A variety of anticarcinogens and CR mimetics induce and activate the NF-E2-related factor 2 (Nrf2) pathway. Nrf2, in turn, induces a number of antioxidative and carcinogen-detoxifying enzymes. Thus, Nrf2 offers a promising target for anticarcinogenesis and antiaging interventions. We used Nrf2-disrupted (KO) mice to examine its role on the biological effects of CR. Here, we show that Nrf2 is responsible for most of the anticarcinogenic effects of CR, but is dispensable for increased insulin sensitivity and lifespan extension. Nrf2-deficient mice developed tumors more readily in response to carcinogen exposure than did WT mice, and CR was ineffective in suppressing tumors in the KO mice. However, CR extended lifespan and increased insulin sensitivity similarly in KO and WT mice. These findings identify a molecular pathway that dissociates the prolongevity and anticarcinogenic effects of CR.

Chronic ingestion of 2-deoxy-D-glucose induces cardiac vacuolization and increases mortality in rats.

Calorie restriction (CR), the purposeful reduction of energy intake with maintenance of adequate micronutrient intake, is well known to extend the lifespan of laboratory animals. Compounds like 2-deoxy-D-glucose (2DG) that can recapitulate the metabolic effects of CR are of great interest for their potential to extend lifespan. 2DG treatment has been shown to have potential therapeutic benefits for treating cancer and seizures. 2DG has also recapitulated some hallmarks of the CR phenotype including reduced body temperature and circulating insulin in short-term rodent trials, but one chronic feeding study in rats found toxic effects. The present studies were performed to further explore the long-term effects of 2DG in vivo. First we demonstrate that 2DG increases mortality of male Fischer-344 rats. Increased incidence of pheochromocytoma in the adrenal medulla was also noted in the 2DG treated rats. We reconfirm the cardiotoxicity of 2DG in a 6-week follow-up study evaluating male Brown Norway rats and a natural form of 2DG in addition to again examining effects in Fischer-344 rats and the original synthetic 2DG. High levels of both 2DG sources reduced weight gain secondary to reduced food intake in both strains. Histopathological analysis of the hearts revealed increasing vacuolization of cardiac myocytes with dose, and tissue staining revealed the vacuoles were free of both glycogen and lipid. We did, however, observe higher expression of both cathepsin D and LC3 in the hearts of 2DG-treated rats which indicates an increase in autophagic flux. Although a remarkable CR-like phenotype can be reproduced with 2DG treatment, the ultimate toxicity of 2DG seriously challenges 2DG as a potential CR mimetic in mammals and also raises concerns about other therapeutic applications of the compound.

AGEMAP: a gene expression database for aging in mice.

We present the AGEMAP (Atlas of Gene Expression in Mouse Aging Project) gene expression database, which is a resource that catalogs changes in gene expression as a function of age in mice. The AGEMAP database includes expression changes for 8,932 genes in 16 tissues as a function of age. We found great heterogeneity in the amount of transcriptional changes with age in different tissues. Some tissues displayed large transcriptional differences in old mice, suggesting that these tissues may contribute strongly to organismal decline. Other tissues showed few or no changes in expression with age, indicating strong levels of homeostasis throughout life. Based on the pattern of age-related transcriptional changes, we found that tissues could be classified into one of three aging processes: (1) a pattern common to neural tissues, (2) a pattern for vascular tissues, and (3) a pattern for steroid-responsive tissues. We observed that different tissues age in a coordinated fashion in individual mice, such that certain mice exhibit rapid aging, whereas others exhibit slow aging for multiple tissues. Finally, we compared the transcriptional profiles for aging in mice to those from humans, flies, and worms. We found that genes involved in the electron transport chain show common age regulation in all four species, indicating that these genes may be exceptionally good markers of aging. However, we saw no overall correlation of age regulation between mice and humans, suggesting that aging processes in mice and humans may be fundamentally different.

Effects of aging and calorie restriction on the global gene expression profiles of mouse testis and ovary.

BACKGROUND: The aging of reproductive organs is not only a major social issue, but of special interest in aging research. A long-standing view of 'immortal germ line versus mortal soma' poses an important question of whether the reproductive tissues age in similar ways to the somatic tissues. As a first step to understand this phenomenon, we examine global changes in gene expression patterns by DNA microarrays in ovaries and testes of C57BL/6 mice at 1, 6, 16, and 24 months of age. In addition, we compared a group of mice on ad libitum (AL) feeding with a group on lifespan-extending 40% calorie restriction (CR). RESULTS: We found that gene expression changes occurred in aging gonads, but were generally different from those in somatic organs during aging. For example, only two functional categories of genes previously associated with aging in muscle, kidney, and brain were confirmed in ovary: genes associated with complement activation were upregulated, and genes associated with mitochondrial electron transport were downregulated. The bulk of the changes in gonads were mostly related to gonad-specific functions. Ovaries showed extensive gene expression changes with age, especially in the period when ovulation ceases (from 6 to 16 months), whereas testes showed only limited age-related changes. The same trend was seen for the effects of CR: CR-mediated reversal of age-associated gene expression changes, reported in somatic organs previously, was limited to a small number of genes in gonads. Instead, in both ovary and testis, CR caused small and mostly gonad-specific effects: suppression of ovulation in ovary and activation of testis-specific genes in testis. CONCLUSION: Overall, the results are consistent with unique modes of aging and its modification by CR in testis and ovary.

Cardioprotective and survival benefits of long-term combined therapy with beta2 adrenoreceptor (AR) agonist and beta1 AR blocker in dilated cardiomyopathy postmyocardial infarction.

We have reported therapeutic effectiveness of pharmacological stimulation of beta2 adrenoreceptors (ARs) to attenuate the cardiac remodeling and myocardial infarction (MI) expansion in a rat model of dilated cardiomyopathy (DCM) post-MI. Furthermore, the combination of beta2 AR stimulation with beta1 AR blockade exceeded the therapeutic effectiveness of beta1 AR blockade. However, these studies were relatively short (6 weeks). In this study, in the same experimental model, we compared different effects, including survival benefit, of combined therapy with the beta1 AR blocker, metoprolol, plus the beta2 AR agonist, fenoterol (beta1-beta2+), and either therapy alone (beta1- or beta2+) during the 1-year study. Therapy was started 2 weeks after permanent ligation of the left coronary artery. Cardiac remodeling, MI expansion, and left ventricular function were assessed by serial echocardiography and compared with untreated animals (nT). Sixty-seven percent mortality in nT was reduced to 33% in the beta1-beta2+ (p < 0.01). Progressive cardiac remodeling observed in nT and beta1- was significantly attenuated in beta1-beta2+ during the first 6 months of treatment. In beta1-beta2+, MI expansion was completely prevented, and functional decline was significantly attenuated during the entire year. Myocardial apoptosis was significantly reduced in both beta1-beta2+ and beta1-. A reduction of cardiac beta1 AR density and decreases in chronotropic and contractile responses to beta2 AR-specific stimulation in the absence of a reduction of beta2 AR density in nT were precluded in rats receiving combined therapy. The results demonstrate the cardioprotective and survival benefit of long-term combination therapy of beta2 AR agonists and beta1 AR blockers in a model of DCM.

Improvement of Vivarium Biodecontamination through Data-acquisition Systems and Automation.

Biodecontamination is important for eliminating pathogens at research animal facilities, thereby preventing contamination within barrier systems. We enhanced our facility's standard biodecontamination method to replace the traditional foggers, and the new system was used effectively after creating bypass ducts in HVAC units so that individual rooms could be isolated. The entire system was controlled by inhouse-developed supervisory control and data-acquisition software that supported multiple cycles of decontamination by equipment, which had different decontamination capacities, operated in parallel, and used different agents, including H2O2 vapor and ClO2 gas. The process was validated according to facility mapping, and effectiveness was assessed by using biologic (Geobacillus stearothermophilus) and chemical indicator strips, which were positioned before decontamination, and by sampling contact plates after the completion of each cycle. The results of biologic indicators showed 6-log reduction in microbial counts after successful decontamination cycles for both agents and found to be compatible with clean-room panels including commonly used materials in vivarium such as racks, cages, trolleys, cage changing stations, biosafety cabinets, refrigerators and other equipment in both procedure and animal rooms. In conclusion, the automated process enabled users to perform effective decontamination through multiple cycles with realtime documentation and provided additional capability to deal with potential outbreaks. Enabling software integration of automation improved quality-control systems in our vivarium.

Laws, Regulations, Guidelines and Standards for Animal Care and Use for Scientific Purposes in the Countries of Singapore, Thailand, Indonesia, Malaysia, and India.

The laws, regulations, guidelines, and standards on animal care and use for scientific purposes in the countries of Singapore, Thailand, Indonesia, and Malaysia, and India are described in this manuscript. For each of these five countries, a brief introduction is provided on the history of how the need for animal welfare in research, education, training, and testing came to being. This is followed by some background information leading to the current status of regulations and guidelines in each of the five countries. There is also a description of the responsibilities and functions of institutional animal welfare and ethics oversight bodies, enforcement agencies, penalties, and organizations supporting the industry. Finally, a conclusion with insights into the future of laboratory animal welfare and science in each of these five countries in Asia is provided.

A synthetic uric acid analog accelerates cutaneous wound healing in mice.

Wound healing is a complex process involving intrinsic dermal and epidermal cells, and infiltrating macrophages and leukocytes. Excessive oxidative stress and associated inflammatory processes can impair wound healing, and antioxidants have been reported to improve wound healing in animal models and human subjects. Uric acid (UA) is an efficient free radical scavenger, but has a very low solubility and poor tissue penetrability. We recently developed novel UA analogs with increased solubility and excellent free radical-scavenging properties and demonstrated their ability to protect neural cells against oxidative damage. Here we show that the uric acid analog (6, 8 dithio-UA, but not equimolar concentrations of UA or 1, 7 dimethyl-UA) modified the behaviors of cultured vascular endothelial cells, keratinocytes and fibroblasts in ways consistent with enhancement of the wound healing functions of all three cell types. We further show that 6, 8 dithio-UA significantly accelerates the wound healing process when applied topically (once daily) to full-thickness wounds in mice. Levels of Cu/Zn superoxide dismutase were increased in wound tissue from mice treated with 6, 8 dithio-UA compared to vehicle-treated mice, suggesting that the UA analog enhances endogenous cellular antioxidant defenses. These results support an adverse role for oxidative stress in wound healing and tissue repair, and provide a rationale for the development of UA analogs in the treatment of wounds and for modulation of angiogenesis in other pathological conditions.

Age-related changes of claudin expression in mouse liver, kidney, and pancreas.

Tight junctions (TJs) play crucial roles in tissue homeostasis and inflammation through their roles in the control of paracellular transport and barrier function. There is evidence that these functions are compromised in older organisms, but the exact mechanisms leading to TJ deterioration are not well understood. Claudin proteins are a family of membrane proteins that constitute the structural barrier elements of TJs and therefore play a major role in their formation and function. Using immunohistochemistry and immunoblotting, we have studied the expression of six different claudin proteins (claudin-1, -2, -3, -4, -5, and -7) in three tissues (liver, kidney, and pancreas) of aging male and female mice. In general, we find an age-dependent decrease in the expression of several claudin proteins in all three tissues observed, although the exact changes are tissue specific. Our findings provide a possible basis for the decrease in tissue barrier function in older organisms.

Transcriptome analysis of age-, gender- and diet-associated changes in murine thymus.

The loss of thymic function with age may be due to diminished numbers of T-cell progenitors and the loss of critical mediators within the thymic microenvironment. To assess the molecular changes associated with this loss, we examined transcriptomes of progressively aging mouse thymi, of different sexes and on caloric-restricted (CR) vs. ad libitum (AL) diets. Genes involved in various biological and molecular processes including transcriptional regulators, stress response, inflammation and immune function significantly changed during thymic aging. These differences depended on variables such as sex and diet. Interestingly, many changes associated with thymic aging are either muted or almost completely reversed in mice on caloric-restricted diets. These studies provide valuable insight into the molecular mechanisms associated with thymic aging and emphasize the need to account for biological variables such as sex and diet when elucidating the genomic correlates that influence the molecular pathways responsible for thymic involution.

Involvement of notch signaling in wound healing.

The Notch signaling pathway is critically involved in cell fate decisions during development of many tissues and organs. In the present study we employed in vivo and cell culture models to elucidate the role of Notch signaling in wound healing. The healing of full-thickness dermal wounds was significantly delayed in Notch antisense transgenic mice and in normal mice treated with gamma-secretase inhibitors that block proteolytic cleavage and activation of Notch. In contrast, mice treated with a Notch ligand Jagged peptide showed significantly enhanced wound healing compared to controls. Activation or inhibition of Notch signaling altered the behaviors of cultured vascular endothelial cells, keratinocytes and fibroblasts in a scratch wound healing model in ways consistent with roles for Notch signaling in wound healing functions all three cell types. These results suggest that Notch signaling plays important roles in wound healing and tissue repair, and that targeting the Notch pathway might provide a novel strategy for treatment of wounds and for modulation of angiogenesis in other pathological conditions.

Gene expression atlas of the mouse central nervous system: impact and interactions of age, energy intake and gender.

BACKGROUND: The structural and functional complexity of the mammalian central nervous system (CNS) is organized and modified by complicated molecular signaling processes that are poorly understood. RESULTS: We measured transcripts of 16,896 genes in 5 CNS regions from cohorts of young, middle-aged and old male and female mice that had been maintained on either a control diet or a low energy diet known to retard aging. Each CNS region (cerebral cortex, hippocampus, striatum, cerebellum and spinal cord) possessed its own unique transcriptome fingerprint that was independent of age, gender and energy intake. Less than 10% of genes were significantly affected by age, diet or gender, with most of these changes occurring between middle and old age. The transcriptome of the spinal cord was the most responsive to age, diet and gender, while the striatal transcriptome was the least responsive. Gender and energy restriction had particularly robust influences on the hippocampal transcriptome of middle-aged mice. Prominent functional groups of age- and energy-sensitive genes were those encoding proteins involved in DNA damage responses (Werner and telomere-associated proteins), mitochondrial and proteasome functions, cell fate determination (Wnt and Notch signaling) and synaptic vesicle trafficking. CONCLUSION: Mouse CNS transcriptomes responded to age, energy intake and gender in a regionally distinctive manner. The systematic transcriptome dataset also provides a window into mechanisms of age-, diet- and sex-related CNS plasticity and vulnerability.

Red cell interactions with amyloid-beta(1-40) fibrils in a murine model.

Vascular amyloidosis in Alzheimer's disease (AD) results in the exposure of red blood cells to beta-amyloid fibrils (A beta). The potential in vivo ramifications of this exposure have been investigated by injecting A beta(1-40) alone or A beta-bound mouse red blood cells into the circulation of C57BL/6 mice. Results indicate that when A beta(1-40) is injected alone, a transient uptake of the fibrils by red blood cells occurs in vivo. When A beta-bound red blood cells were injected, beta-amyloid is rapidly removed from these cells in vivo. Double-labeling experiments indicate that while some of the red blood cells bound to A beta(1-40) are removed from circulation, a major fraction of these cells remain in circulation even after A beta is removed. Immunohistochemistry of murine tissue samples obtained after sacrificing the animals suggests that within 1 h after injection of A beta(1-40) or A beta-bound red blood cells, A beta is found in spleen phagocytes and liver Kupffer cells. Heme staining further indicates that the binding of A beta(1-40) to red blood cells enhances red cell phagocytosis by the spleen.