Use Of Laboratory Animals Research Articles

Efficacy and safety of HERG channel agonists for treatment of Long QT Syndrome type 8

Abstract Background Substrate-based approach aimed at repolarization abbreviaton using mexiletine was shown to normalize the QTc interval and reduce the event rate in LQT3, and is now considered standard-of-care. Unfortunately, mexiletine is partially efficacious in other malignant forms of LQTS, such as LQT2 and LQT8. Novel compounds, such as HERG channel agonists, potentially capable of safely and efficaciously abbreviating the repolarization have not been tested in a clinically relevant model. Purpose To investigate the efficacy and safety of repolarization shortening using ICA-105574, the most potent compound belonging to the class of HERG channel agonists, in a clinically relevant, first-in-class porcine model of LQT8. Methods We used a combination of 12-lead ECG and electroanatomical mapping (EAM) to obtain data on the QTc interval, local activation time (LAT), local repolarization time (LRT) and its gradients, and reentrant vulnerability index (RVI), at baseline and after drug administration. We used LQT8 pigs of both genders weighing between 60 kg and 80 kg. All procedures were conducted following local regulations for the care and use of laboratory animals after authorization by relevant authorities. We used mixed effects linear regression model to assess the effect of ICA-105574 on the QTc interval and paired nested t-test to assess the effect of ICA-105574 on EAM-derived parameters. Results As a first step, we assessed the effects of ICA-105574 on the QTc interval. Compared to baseline, a single dose of 3 mg/kg of ICA-105574 resulted in an immediate and pronounced shortening of the QTc interval in all animals (∆QTc at 5 minutes post-injection: -28%, p<0.001). We then investigated repeated intravenous administrations of 3 mg/kg doses at 10-minute intervals for a total of 4 doses (i.e., overall drug dose of 12 mg/kg). Importantly, we did not observe excessive QTc shortening (∆QTc 6 mg/kg: -26%; ∆QTc 9 mg/kg: -26%; ∆QTc 12 mg/kg: -30%, p<0.001 for all) nor did we observe any proarrhythmic events in any of the animals studied. In a second phase of the study, we used EAM to assess the modifications in the arrhythmic substrate induced by ICA-105574. Importantly, ICA-105574 not only shortened the duration of ventricular repolarization (∆LRT: -21%, p=0.017), but also reduced the dispersion of ventricular repolarization (∆Max. LRT gradient: -69%, p=0.020). This, coupled with reduction of the activation delay as compared to baseline (∆LAT: -28%, p=0.072), had a net positive effect on the arrhythmic substrate, as evidenced by an amelioration by 30% of global RVI (∆RVIG,D: -32%, p=0.029). Conclusions In our LQT8 pig model, ICA-105574, a potent HERG channel agonist, safely and efficaciously ameliorated the arrhythmic substrate. These results suggest that HERG channel agonists, as a novel class of antiarrhythmics, could have clinical application as a novel substrate-based therapy for LQT8.Figure 1

Survey Results on the Awareness of Laboratory Animal Welfare among Medical Graduate Students

AbstractTo investigate the knowledge and attitudes of medical graduate students regarding laboratory animal welfare, a survey was conducted on 288 medical graduate students from the 2019 to 2023 cohorts through the final exam of the compulsory course ‘Medical Laboratory Animal Science.’ The survey included both closed and open-ended questions. We calculated the awareness rate from the cumulative student score/maximum score based on the questions and scoring rubrics. We found that medical graduate students showed relatively low awareness of laboratory animal welfare and limited understanding of related knowledge. There were shortcomings in theoretical knowledge and practical experience related to laboratory animals’ welfare and ethical aspects of laboratory animal care and use. Students in basic medical disciplines demonstrated slightly better awareness of laboratory animal welfare than those in other disciplines. In addition, female students exhibited a higher awareness of laboratory animal welfare than male students. In recent years, there has been a gradual increase in the awareness of the importance of laboratory animal welfare among medical graduate students, but a decrease in awareness was observed during the COVID-19 pandemic. Therefore, a need exists for strengthening education on laboratory animal welfare in medical schools to enhance students’ awareness. It is suggested that school-specific expectations with respect to the replacement, reduction, and refinement principles be established to balance the needs of medical research and laboratory animal welfare, as a way to ensure the ethical considerations and the smooth progress of medical research are both met.

Use of stable isotope labeled (SIL) antibodies in cassette dosing to improve pharmacokinetics screening efficiency of ADCs with novel cytotoxic payloads

Stable isotope labelling by amino acids in cell culture (SILAC) is an established technique used in quantitative mass spectrometry (MS)-based proteomics. SILAC is also used to generate stable isotope labelled (SIL) antibodies for internal standards (IS) used in LC-MS/MS bioassays to improve quantitative robustness. Total antibody (TAb) is measured to evaluate pharmacokinetics (PK) of antibody drug conjugate (ADC) candidates measured by either ligand binding (LBA) or LC-MS/MS. Herein, we describe an application of SILAC, where multiple SIL combinations of an antibody are used for cassette dosing and PK evaluation. Our preclinical studies demonstrate SILAC-labelled ADC therapeutics did not alter antibody PK. Furthermore, with cassette dosing SIL antibodies exhibited comparable exposure to discretely administered unlabelled test articles in rats. In addition, SIL antibodies were conjugated to cytotoxic payloads to create SIL ADCs and cassette dosed in a cynomolgus monkey PK study and SIL ADCs yielded comparable PK results to discrete dosed unlabelled ADCs. In conclusion, SIL antibodies used with a cassette dosing strategy increases PK screening throughput of ADC candidates in preclinical species. Additionally, cassette dosing strategy further facilitates the responsible use of laboratory animals to achieve the three-Rs (Replacement, Reduction, and Refinement).

Artificial Intelligence-Powered Molecular Docking and Steered Molecular Dynamics for Accurate scFv Selection of Anti-CD30 Chimeric Antigen Receptors.

Chimeric antigen receptor (CAR) T cells represent a revolutionary immunotherapy that allows specific tumor recognition by a unique single-chain fragment variable (scFv) derived from monoclonal antibodies (mAbs). scFv selection is consequently a fundamental step for CAR construction, to ensure accurate and effective CAR signaling toward tumor antigen binding. However, conventional in vitro and in vivo biological approaches to compare different scFv-derived CARs are expensive and labor-intensive. With the aim to predict the finest scFv binding before CAR-T cell engineering, we performed artificial intelligence (AI)-guided molecular docking and steered molecular dynamics analysis of different anti-CD30 mAb clones. Virtual computational scFv screening showed comparable results to surface plasmon resonance (SPR) and functional CAR-T cell in vitro and in vivo assays, respectively, in terms of binding capacity and anti-tumor efficacy. The proposed fast and low-cost in silico analysis has the potential to advance the development of novel CAR constructs, with a substantial impact on reducing time, costs, and the need for laboratory animal use.

Chronic ecologically relevant stress effects on reverse-translated touchscreen assays of reward responsivity and attentional processes in male rats: Implications for depression.

Stagnation in the development of novel therapeutic strategies for treatment-resistant depression has encouraged continued interest in improving preclinical methods. One tactic prioritizes the reverse translation of behavioral tasks developed to objectively quantify depressive phenotypes in patient populations for their use in laboratory animals via touchscreen technology. After cross-species concordance in task outcomes under healthy conditions is confirmed, construct validity can be further enhanced by identifying environmental stressors that reliably produce deficits in task performance that resemble those in depressive participants. The present studies characterized in male rats the ability of two chronic ecologically relevant stressors, inescapable ice water or isolated restraint, to produce depressive-like behavioral phenotypes in the Probabilistic Reward Task (PRT) and Psychomotor Vigilance Task (PVT). These tasks previously have been reverse-translated using touchscreen technology for rodents and nonhuman primates to objectively quantify, respectively, reward responsivity (anhedonia) and attentional processes (impaired cognitive function), each of which are core features of major depressive disorder. In the PRT, both inescapable ice water and isolated restraint produced persistent anhedonic phenotypes compared to non-stressed control performance (i.e., significantly blunted response bias for the richly rewarded stimulus). In the PVT, both chronic stressors impaired attentional processing, revealed by increases in titrated reaction times; however, these deficits largely subsided by the end of the chronic condition. Taken together, these findings confirm the ability of reverse-translated touchscreen tasks to effectively generate behavioral phenotypes that exhibit expected deficits in performance outcomes following exposure to chronic ecologically relevant stress. In turn, this approach is well positioned to appraise the ability of candidate therapeutics to attenuate or reverse such behavioral deficits and, thereby, contribute to preclinical medications development for treatment-resistant depression.

Some Alternative Methods to the Use of Laboratory Animals in Medical Research

Some Alternative Methods to the Use of Laboratory Animals in Medical Research

A-FABP and E-FABP as cutaneous biomarkers of Metabolic Syndrome

Introduction: In Metabolic syndrome (MetS), tissues such as adipose, liver, and skin with very active lipid metabolism are dysfunctional, making it diffcult to store the excess fat. Fatty acid binding proteins (FABPs) are cytosolic proteins that can reversibly bind to lipids and transport them to different cell organelles. In this work, we focus on adipocyte (A-FABP) and epidermal (E-FABP) FABPs, which have been proposed as potential biomarkers of MetS due to their different tissue expression patterns. When MetS impacts the skin, the integrity of the barrier is affected, such as changes in the structure and function of collagen fibers, changes in microcirculation and macrocirculation, damage to lymphatic vessels, diffculty in wound healing, increased production of sebum and sweat. All those changes seem to depend on the abnormal function of lipids and FABPs. In MetS, E-FABP and A-FABP increase significantly in serum and probably in the skin in order to facilitate lipid transport. For this reason, they can be considered a molecular sensors of the inflammation induced in the skin by MetS. Objective: Analyze the concentrations of A-FABP and E-FABP in the skin of a MetS model to determine their value as biomarkers. Methods: We used a Wistar rat model (N=32) of MetS with a 41% extra fat diet during 8, 18, 28, and 52 weeks. The protocol was approved by the "Internal Committee for the Care and Use of Laboratory Animals of the Faculty of Medicine of the Autonomous University of San Luis Potosí" (assigned code: BGFMUASLP -07-19). At the end of every period of time (8, 18, etc) a biopsy of the skin (1 cm2) was took and cleaned from connective and subcutaneous adipose tissue. For Western Blot (WB) experiments the protein extraction was performed using cell lysis and homogenization, and 50 μg of protein of each sample was used. The WB was performed following the supplier's specifications and the standardized protocol in our laboratory. The primary antibodies used for incubation were rat A-FABP antibody (R&D Systems, AF1443) and rat E-FABP antibody (R&D Systems, AF1476). The secondary antibody was Goat IgG HRP-conjugated Antibody (R&D Systems, HAF017). As a control gene, GAPDH reconstituted as indicated by the supplier. Bands were visualized using chemiluminescent detection, and their density in each group was analyzed using ImageJ software. Results: animals developed MetS after being 28 weeks in high fat diet showing excess of abdominal fat, hypertension, high triglycerides, and low cholesterol-HDL. At 52 weeks, we observed an increase in total cholesterol and in the severity of the blood lipids mentioned. Also, 40% of the animals showed visible skin lesions characterized by redness, eruptions, scratching marks, and wounds that do not heal, mainly in the cervical region, suggesting an inflammatory process. A-FABP expression was increased in MetS rats at 8 and 18 weeks and remained high at 28 and 52 weeks, although the increase was not significant. On the other hand E-FABP expression increased in MetS rats from week 18 and 28 but decreasing at 52. It suggests a greater presence of the protein and a probable participation of it in the inflammatory processes and skin lesions observed. Conclusions: E-FABP and A-FABP, increase significantly in the skin of MetS rats, mainly in the in the initial stages of MetS. E-FABP can considered a molecular sensor of skin inflammation induced by obesity. We also observed the presence of A-FABP in the skin, probably from subcutaneous adipose tissue. We propose the feasibility of both FABPs as early markers of MetS. This work has been supported by the National Council of Science and Technology (CONAHCYT) Project No. 320024. GE Donjuán-Loredo thanks CONAHCYT CVU 775467. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Muscle regeneration is not impaired by ablation of UCP-1-expressing cells

INTRODUCTION: Recently, cells expressing uncoupling protein 1 (UCP-1) have been proposed to impact muscle physiology. First, transplant of brown adipose tissue (which has high expression of UCP-1) enhanced regeneration in the injured mouse rotator cuff. Second, isolated UCP-1+ muscle fibroadipogenic progenitors (FAPs) treated to enhance UCP-1 expression improved injured mouse rotator cuff muscle pathology. However, it remains unknown whether endogenous UCP-1-expressing cells contribute to muscle regeneration. Therefore, the objective of this study was to assess whether mice that lack UCP-1-expressing cells demonstrate impaired muscle regeneration following toxin administration. METHODS: Experiments were performed using tibialis anterior (TA) and 5th toe extensor digitorum longus (EDL) muscle of wild type C57BL/6J (WT; n=22), and uncoupling protein 1 diphtheria Toxin A (UCP1-DTA; n=18) mice, which lack all UCP-1 expressing cells. All procedures were performed in accordance with the National Institute of Health’s Guide for the Use and Care of Laboratory Animals and were approved by Washington University School of Medicine IACUC. Muscle injury was induced using intramuscular injection of 50%v/v solution of glycerol. Regeneration was assessed at “early” (7 days post-injury (dpi)), “intermediate” (14 dpi) and “late” (21 dpi) stages. Outcome measures were cell population dynamics by flow cytometry, ex-vivo physiology testing and histological scoring as appropriate for each timepoint. RESULTS: Our preliminary data in early regeneration show that UCP1-DTA mice have significantly greater percentage of FAPs compared to WT mice (29.84 ± 1.24% higher cell population, p<0.0134), however no differences in muscle satellite (stem) cells between genotypes. Physiological and histological outcomes at intermediate and late-stage regeneration find a baseline deficit in muscle mass (23.69 ± 2.3% smaller, p<0.0001), type 2B fiber cross-sectional area (32.56 ± 4.8% smaller, p<0.0001) and normalized muscle active force generation (16.2 ± 3.3% lower force, p<0.0154) in UCP1-DTA compared with WT. However, mass and contractile forces recover to a similar extent between the genotypes following glycerol injection suggesting the phenotypic drivers of the reduced mass and contractile function do not affect muscle regeneration. CONCLUSIONS: Taken together, the data from this study point to a baseline growth deficit in mice lacking UCP-1-expressing cells. However, in two separate timepoints of regeneration (14 and 21dpi), we see that UCP1-DTA mice have similar recovery of mass and normalized contractile force to baseline values suggesting regeneration can proceed normally without UCP-1 expressing cells. However, further data collection will be needed at early-stage regeneration to help discern whether mice lacking UCP-1-expressing cells have initial changes in cell population dynamics given the increased FAP population at 7dpi. ACKNOWLEDGMENTS: This work was supported by R01AR075773 and R21AR071582 to GAM. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The association between hemoglobin levels and renal function parameters during normothermic machine perfusion: A retrospective cohort study using porcine kidneys.

Ex vivo normothermic machine perfusion (NMP) is a promising tool for assessing an isolated kidney prior to transplantation. However, there is no consensus on the perfusate's optimal oxygen-carrying capacity to support renal function. To investigate the association of hemoglobin levels with renal function parameters, a retrospective analysis of isolated, normothermically, perfused porcine kidneys was performed. Between 2015 and 2021, a total of 228 kidneys underwent 4 h of NMP with perfusates that varied in hemoglobin levels. A generalized linear model was used to determine the association of hemoglobin levels with time-weighted means of renal function markers, such as fractional sodium excretion (FENa) and creatinine clearance (CrCl). Stratified by baseline hemoglobin level (<4.5, 4.5-6, or >6 mmol/L), these markers were modeled over time using a generalized linear mixed-effects model. All models were adjusted for potential confounders. Until a hemoglobin level of around 5 mmol/L was reached, increasing hemoglobin levels were associated with superior FENa and CrCl. Thereafter, this association plateaued. When hemoglobin levels were categorized, hemoglobin <4.5 mmol/L was associated with worse renal function. Hemoglobin levels were neither significantly associated with proteinuria during NMP nor with ATP levels at the end of NMP. Hemoglobin levels >6 mmol/L showed no additional benefits in renal function. In conclusion, we found an association between baseline hemoglobin levels and superior renal function parameters, but not injury, during NMP of porcine kidneys. Furthermore, we show that performing a retrospective cohort study of preclinical data is feasible and able to answer additional questions, reducing the potential use of laboratory animals.

Toxicity of minoxidil – Comprehensive in silico prediction of main toxicity endpoints: Acute toxicity, irritation of skin and eye, genetic toxicity, health effect, cardiotoxicity and endocrine system disruption

This article focusses on elucidating the toxicological profile of minoxidil, a widely used pharmacological agent for alopecia, through the application of in silico methods (Percepta ACD/Labs software). This research is driven by the need to understand key toxicological endpoints: acute toxicity, skin and eye irritation, genetic toxicity, cardiotoxicity, disruption of the endocrine system, and estimation of various health effects due to the lack of experimental data for this drug. These parameters are critically evaluated to meet the stringent requirements of the pharmaceutical industry's safety assessments. The results obtained for acute toxicity (LD50 for rats and mouse) indicate that minoxidil exhibits a species-dependent acute toxicity profile e.g. 51 mg/kg bw for intravenous administration in mice. The predicted health effects indicate a 93% risk to the gastrointestinal system, 54% for the kidneys, 52% for the liver, 42% for the blood and lungs, and 39% for the cardiovascular system. The prediction of genotoxicity suggests a moderate probability (48%) of inducing a positive Ames test result. Furthermore, moderate inhibition of the hERG channel indicates potential cardiac risks of Minoxidil. Based on the information obtained, we propose subjecting minoxidil to additional toxicological assessments. The successful adoption of these in silico methodologies aligns with the 3 R s principle (replacement, reduction, and refinement) in the field of modern toxicological studies of minoxidil, all without the use of laboratory animals for the novelty of our toxicity assessment.

Tenebrio molitor as a new alternative model for the investigation of chemotherapy-induced intestinal toxicity

Alternative animal models have become increasingly necessary due to legal regulations aimed at reducing the use of laboratory animals. Invertebrates are gaining in importance and have been intensively researched in recent years due to their pathophysiological similarities with rodents. Among these organisms, Tenebrio molitor, also known as the yellow mealworm beetle, stands out. In this study, we investigated whether T. molitor could be an alternative for studying the toxicity of chemotherapeutic agents. For this purpose, T. molitor larvae were inoculated with irinotecan (IRI) or 5-fluorouracil (5-FU). Both chemotherapeutic agents increased the morbidity and mortality of T. molitor and led to an increase in total circulating cells. Glucose levels were decreased after treatment with 5-FU and alanine aminotransferase levels were decreased after IRI administration. Administration of IRI or 5-FU resulted in changes in the appearance, consistency and amount of T. molitor frass. Finally, both IRI and 5-FU promoted severe histologic damage in the midgut and increased melanin deposition in peripheral tissues. Finally, we succeeded in developing an alternative experimental model for evaluating the toxicity of IRI and 5-FU. This model exhibits significant features of toxicity and intestinal damage, making it suitable for translational research purposes. T. molitor proves to be a versatile model organism with numerous advantages for experimental studies and offers a viable alternative for acquiring and expanding knowledge in the field of toxicology and pharmacology.

Is bladder outlet obstruction rat model to induce overactive bladder (OAB) has similarity to human OAB? Research on the events in smooth muscle, collagen, interstitial cell and telocyte distribution

BackgroundCellular and cytoskeletal events of overactive bladder (OAB) have not been sufficiently explored in human bladder due to different limitations. Bladder outlet obstruction (BOO) had been induced in different animal models with different methods to induce (OAB). Similarity of the animal models of BOO to the human OAB is postulated but has not been confirmed. The interstitial cells of Cajal (ICCs), and telocytes (TCs) are an important players in smooth muscles conductivity, they had not been well investigated in the previous BOO models. Objectives are to investigate the morphological pattern of cellular, cytoskeleton and telocytes distribution in BOO rat model and to match the events in two time periods and compare it to the findings in real-world human OAB.MethodsFemale Sprague-Dawley rats (Rattus norvegicus) were randomly divided into: sham (n = 10), BOO 6 W (n = 10), BOO 8 W (n = 10). Operative procedure to Induce BOO was done under anesthesia with intraperitoneal Ketamine administration. The Effect of induction of BOO was evaluated after 6 and 8 weeks. The rats were anesthetized, and the urinary bladder was removed, while the rat was unconscious under anaesthesia it was transferred to the inhalation anaesthesia cage for euthanasia, rats were sacrificed under light anesthesia using isoflurane. Care of animals, surgical procedure, and euthanasia adhered to Guide for the Care and Use of Laboratory Animals, and AVMA Guidelines for the Euthanasia of Animals. The retrieved bladder was processed for examination with histopathology, immunohistochemistry (IHC), and transmission electron microscopy (EM).ResultsHistological examination of the bladder shows thinner urothelium, condensation of collagen between muscle bundles. IHC with c-kit shows the excess distribution of ICCs between smooth muscle bundles. EM shows frequent distribution of TCs that were situated between collagen fibers. Finings in BOO 6 W group and BOO 8 W group were comparable.ConclusionThe animal model study demonstrated increased collagen/ smooth muscle ratio, high intensity of ICCs and presence of TCs. Findings show that a minimally invasive procedure to induce BOO in rats had resulted in an OAB that has morphological changes that were stable in 6 & 8 weeks. We demonstrated the distribution of TCs and ICCs in the rat animal model and defined them. The population of TCs in the BOO rat model is described for the first time, suggests that the TCs and ICCs may contribute to the pathophysiology of OAB. Similarity of animal model to human events OAB was demonstrated. These findings warrant further study to define the role of TCs in OAB.Clinical trial registryThe study does not require a clinical trial registration; it is an experimental animal study in basic science and does not include human subjects.

Attending veterinarians improve the research capability and psychological well-being of researchers in animal research institutes.

The use of laboratory animals in biomedical research has significantly advanced scientific understanding, yet it raises ethical concerns about animal welfare and the mental health of researchers Recent research has highlighted the potential for stress and compassion fatigue among researchers working with distressed animals. Attending veterinarians (AVs) are crucial in mitigating the pain and stress experienced by animals and, by extension, researchers. However, the impact of AVs on researchers' psychological well-being remains understudied. This study explores how AVs contribute to researchers' research capability and psychological well-being in animal research institutions. AVs oversee animal housing, health, and welfare; their involvement is mandated or strongly recommended in developed countries. AVs enhance animal welfare by ensuring proper housing, nutrition, and social interaction. They monitor animal health, educate researchers on pain assessment, and promote compliance with post-surgical care. AVs also contribute to researchers' well-being by addressing euthanasia procedures, which can be emotionally challenging. Programs for rehoming animals after experiments offer an alternative to euthanasia and positively impact researchers' psychological well-being. Moreover, AVs promote workplace well-being by fostering positive workplace cultures, offering peer counseling, and providing social support. Programs considering animal welfare and researchers' emotions are crucial for a healthy research environment. In conclusion, AVs are essential in balancing scientific progress with animal welfare and researchers' psychological well-being. Therefore, their role should be recognized as vital in achieving social equity that considers the welfare of humans and laboratory animals.

Physiological effects of alfaxalone anesthesia on rhesus monkeys during intravenous glucose tolerance testing.

Laboratory animal research with nonhuman primates (NHPs) requires anesthesia for most procedures to ensure safety and consistency in sample collection. However, anesthesia drugs can have adverse effects on the physiological measures of interest. Alfaxalone, most notably used in dogs and cats, offers rapid onset, short duration of action, and has a high safety margin. Here, we compared our current anesthesia protocol using Telazol, to three different doses of alfaxalone during a one-hour intravenous glucose tolerance test, the standard evaluation of glucose metabolism in NHPs. Results indicate there are no differences in the rate of glucose metabolism, anesthesia depth measurements, or total duration of sedation, but induction, number of supplemental doses required, and recovery time to eating were affected by the different doses of alfaxalone. Cardiovascular measures showed variability between the four protocols in respiratory rate and systolic blood pressure rates only. These results indicate that alfaxalone can produce a reliable state of anesthesia, similar to our current protocol, and confers minimal cardiovascular or metabolic disturbance, as well as enhanced recovery characteristics. As such, alfaxalone is a promising anesthetic for use in laboratory animals and further investigation is warranted.

Drug review process advancement and required manufacturer and contract research oraganization responses.

The United States Senate passed the "FDA Modernization Act 2.0." on September 29, 2022. Although the effectiveness of this Bill, which aims to eliminate the mandatory use of laboratory animals in new drug development, is limited, it represents a significant trend that will change the shape of drug applications in the United States and other countries. However, pharmaceutical companies have not taken major steps towards the complete elimination of animal testing from the standpoint of product safety, where they prioritize patient safety. Nonetheless, society is becoming increasingly opposed to animal testing, and efforts will be made to use fewer animals and conduct fewer animal tests as a natural and reasonable response. These changes eventually alter the shape of new drug applications. Based on the assumption that fewer animal tests will be conducted or fewer animals will be used in testing, this study explored bioinformatics and new technologies as alternatives to compensate for reduced information and provide a picture of how future new drug applications may look. The authors also discuss the directions that pharmaceutical companies and nonclinical contract research organizations should adopt to promote the replacement, reduction, and refinement of animals used in research, teaching, testing, and exhibitions.

Hypoglycemic Assessment of Aqueous Leaf Extract of Moringa oleifera on Diabetic Wistar Rats.

Background: Moringa oleifera leaf is used for diabetes due to its pharmacologic effects. Patients with hyperglycemia experience beta cell destruction. However, no research on risk awareness has been done to ascertain its safety. The present study describes the antidiabetic effect of Moringa oleifera leaf, such as the protection of pancreatic beta cells and the induction of glycogen synthesis, before addressing the secondary effects of diabetes, such as hepatic and renal toxicity. Methods: Forty-five Wistar rats weighed 160 ± 10 g were divided into nine groups. All animal operations complied with the National Institute of Health (NIH) guidelines for the care and use of laboratory animals as approved by the Animal Ethical Committee, University of Jos. Group I was normal control and Group II was diabetic animals induced with alloxan. Insulin and extract doses of 200, 400, and 800 mg/kg were given to diabetic Groups III-VI. Normal animals in Groups VII-IX were given extract at doses of 200, 400, and 800 mg/kg for 28 days. Tissues were retrieved for biochemical and histological investigations using standard techniques. Results: There was decrease relative body weight of diabetic animals (95.50 ± 5.50) when compared to normal control (142.75 ± 20.08) with increased levels of urea (control 6.13 ± 0.523 and diabetes 29.23 ± 1.267) and creatinine (control 0.70 ± 0.057 and diabetes 2.13 ± 0.185). Histology of the liver and pancreas also points to organ damage due to hyperglycemia. However, oral administration of extract showed antidiabetic effect with protection of pancreatic beta cells and the induction of glycogen synthesis, no glycogen was deposited in the liver, addressing the secondary effects of diabetes, such as hepatic and renal toxicity. Further discovery revealed that extract elevated antioxidant enzyme expression. Conclusion: Leaf extract from Moringa oleifera reduces blood sugar and lessens the damage caused by hyperglycemia in the pancreas and liver.

Establishing a national biosafety and biosecurity agency for the United States.

The rapid advancement of biological research and biotechnology requires a novel and robust regulatory agency to ensure uniform biosafety and biosecurity governance in the United States. The current fragmented regulatory landscape needs to be refocused to address the complexities of modern biological research, including risks associated with accidental, inadvertent, and deliberate biological incidents. An independent government agency, which we call the National Biosafety and Biosecurity Agency (NBBA), that is devoted to biosafety and biosecurity could effectively address these challenges. The NBBA would consolidate various regulatory functions, streamline processes, and enhance oversight. This oversight would encompass life sciences research in the United States, regardless of the source of funding or level of classification. The agency could also contribute to the bioeconomy by streamlining requirements to safeguard public health and the environment while fostering scientific and commercial progress. The proposed agency would govern high-risk biological pathogens, manage the Federal Select Agent Program, enforce policies related to dual use research of concern, pathogens with enhanced pandemic potential, and nucleic acid synthesis screening, administer regulations on the use and care of laboratory animals, as well as regulate other relevant biosafety and biosecurity activities. The goal would be to provide one-stop shopping for the biomedical research and biotechnology sectors subject to oversight by the Federal government. To ensure leadership in global biosafety and biosecurity, the agency's mission would include international collaboration, applied research, education, workforce development, and coordination with national security initiatives. Creating an agency like the NBBA will be politically challenging but presenting a comprehensive vision and engaging stakeholders early and frequently, and being transparent in the process, will be essential for garnering support. Creating a unified biosafety and biosecurity governance system in the United States will ensure the safe and secure advancement of biological research while sustaining innovation and maintaining international competitiveness.

Conscience of Science: Ethical Imperatives in Zoological Research

Zoological science is an intricate field which requires intimate considerations and guidelines to make sure that limits and boundaries are set for the ethical and humane conduction of research. A number of bioethical laws and international conventions have been designed by bioethical and committees and review boards that serve as moral compass steering towards responsible and humane scientific research. Animal Welfare Act (AWA) sets standards for the responsible treatment of animals in research, transport, exhibition and animal dealers, handling and research protocols, Guide for the Care and Use of Laboratory Animals that is published by National research Council of Unites States provides a framework for the proper use and optimal care of animals for in vivo research in laboratory experiments [1]. Similarly, European Union Directive 2010/63/EU is a directive which outlines the standards for the animal protection used for research purposes in the Europe. It states 3Rs of animal testing i.e., Refinement, Reduction and Replacement, and necessitates that researchers consider alternatives before resorting to the animal experiments. An elaborate directive, Declaration of Helsinki, primarily focuses on human research ethics but has principle related o animal research ethics as well [2]. Likewise, another Convention on International Trade in Endangered Species of Wild Fauna and Flora also called as CITES, deals with the trade of endangered animals ensuring that any research which involves endangered species is conducted ethically with proper permits and prevents illegal and unethical exploitation of animals [3]. Last but not the least, the ARRIVE guidelines provide a checklist of essential information which must be included while publishing the research data which describes animal experiments, promotes transparency and improve the quality of reporting in the animal research. The current global conditions necessitate ethical consciousness in zoological research. These considerations extend beyond laboratory settings and encompass wildlife conservation and the prevention of zoonotic diseases such as COVID-19. Innovations in non-invasive research methods, genetic analyses, and data-driven insights pave the way for more ethical and efficient scientific practices. Ethical consciousness aligning with the pursuit of knowledge nurtures a landscape where wonder of discovery and the ethical treatment of animals walk hand in hand.

Development and validation of a human bronchial epithelial spheroid model to study respiratory toxicity in vitro.

The use of laboratory animals in research has been extensively criticized. While most of the critique has been centered around the ethical aspect, also the economic and scientific aspects have been frequently mentioned as points of concern. As a result, the use of alternative methods has gradually become more enticing. The most used alternatives to laboratory animals are the 2D monolayer cell cultures. However, the limited translatability of these monolayer cell cultures to in vivo has led to the development of 3D cell cultures that are believed to better capture the in vivo physiology and pathology. Here we report on the development of a physiologically more relevant 3D cell model (spheroids) comprised of human bronchial epithelial (16HBE14o-) cells, for use in respiratory toxicity research. Culturing 16HBE14o-cells as hanging-drops led to the formation of stable spheroids which showed an increased expression of CLDN1 when compared to 2D monolayer cultured cells. In addition, cell-cycle analysis revealed an increased sub-G0 population and signs of G0/G1 arrest in spheroids. Afterwards, standard operating procedures (SOPs) were established, and existing protocols optimized, for compatibility with spheroids. Spheroids were successfully used to assess cytotoxicity, genotoxicity, apoptosis/necrosis, and oxidative stress after exposure to known cytotoxic or genotoxic compounds. The development of the bronchial epithelial spheroids and the establishment of SOPs can contribute to amore reliable toxicity assessment of chemicals and may aid in bridging the gap between in vivo and in vitro experiments.

200 The Effects of Whole Hemp Seed Supplementation on Health Status, Voluntary Physical Activity, Fecal Quality, and Fermentative End-Products in Healthy Adult Dogs

Abstract The cultivation and utilization of hemp and hemp-derived products have increased at a rapid pace since the enactment of the 2018 farm bill which provided changes in regulation related to cannabis. Although there are three hemp-derived ingredients that have been generally recognized as safe status for human consumption by the FDA, no ingredients have been approved by the FDA for consumption by companion animals or defined by AAFCO. Hemp seeds have the potential to act as a rich source of plant protein, polyunsaturated fatty-acids, and dietary fiber in companion animal nutrition. The objective of this study was to evaluate the effects of supplementing ground whole hemp seed in a baked chew on voluntary physical activity, fecal characteristics, fermentative-end products, and overall health status in healthy adult dogs. All animal procedures were approved by the University of Illinois Institutional Animal Care and Use Committee and were in accordance with the United States Public Health Service Policy on Humane Care and Use of Laboratory Animals. Thirty adult beagle dogs were used in a complete randomized design, with ten dogs assigned to each treatment. The dogs were fed the same commercially available, complete and balanced diet and were supplemented with one of three baked chews containing either a high inclusion of ground hemp seed (50%), a low inclusion of ground hemp seed (25%), or cellulose. The baked chews were fed at a level of 10% of the daily intake for 28 d and were formulated to be similar in macronutrient composition, approximately 25% fat, 20% protein, and 25% total dietary fiber. Duration and intensity of voluntary physical activity was measured using activity monitors for 4 d before supplementation and at the end of the supplementation period (d 24 to 27). Fasted blood and a fresh fecal sample were taken on d 0, 14, and 28 to monitor health status, blood variables, fecal characteristics and fermentative-end product concentrations. The baked chews were well accepted by the dogs with minimal intake refusals, and all dogs remained clinically healthy for the duration of the experimental period. No differences in fecal score or fecal pH were observed among the treatment groups (P &amp;gt; 0.05). The treatment did not have an effect on fecal concentrations of volatile fatty acids including acetate, propionate, butyrate, valerate, isovalerate, and isobutyrate (P &amp;gt; 0.05). Concentrations of fecal ammonia, phenols, and indoles were also similar among the groups (P &amp;gt; 0.05). No adverse effects were observed, and the data suggest that in a baked chew the whole ground hemp seed was comparable to the cellulose control.