Animal models for COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the aetiological agent of coronavirus disease 2019 (COVID-19), an emerging respiratory infection caused by the introduction of a novel coronavirus into humans late in 2019 (first detected in Hubei province, China). As of 18 September 2020, SARS-CoV-2 has spread to 215 countries, has infected more than 30 million people and has caused more than 950,000 deaths. As humans do not have pre-existing immunity to SARS-CoV-2, there is an urgent need to develop therapeutic agents and vaccines to mitigate the current pandemic and to prevent the re-emergence of COVID-19. In February 2020, the World Health Organization (WHO) assembled an international panel to develop animal models for COVID-19 to accelerate the testing of vaccines and therapeutic agents. Here we summarize the findings to date and provides relevant information for preclinical testing of vaccine candidates and therapeutic agents for COVID-19.

Replacement, Refinement, and Reduction in Animal Studies With Biohazardous Agents.

Animal models are critical to the advancement of our knowledge of infectious disease pathogenesis, diagnostics, therapeutics, and prevention strategies. The use of animal models requires thoughtful consideration for their well-being, as infections can significantly impact the general health of an animal and impair their welfare. Application of the 3Rs-replacement, refinement, and reduction-to animal models using biohazardous agents can improve the scientific merit and animal welfare. Replacement of animal models can use in vitro techniques such as cell culture systems, mathematical models, and engineered tissues or invertebrate animal hosts such as amoeba, worms, fruit flies, and cockroaches. Refinements can use a variety of techniques to more closely monitor the course of disease. These include the use of biomarkers, body temperature, behavioral observations, and clinical scoring systems. Reduction is possible using advanced technologies such as in vivo telemetry and imaging, allowing longitudinal assessment of animals during the course of disease. While there is no single method to universally replace, refine, or reduce animal models, the alternatives and techniques discussed are broadly applicable and they should be considered when infectious disease animal models are developed.

Use of ferrets for electrophysiologic monitoring of ion transport.

Limited success achieved in translating basic science discoveries into clinical applications for chronic airway diseases is attributed to differences in respiratory anatomy and physiology, poor approximation of pathologic processes, and lack of correlative clinical endpoints between humans and laboratory animal models. Here, we discuss advantages of using ferrets (Mustela putorus furo) as a model for improved understanding of human airway physiology and demonstrate assays for quantifying airway epithelial ion transport in vivo and ex vivo, and establish air-liquid interface cultures of ferret airway epithelial cells as a complementary in vitro model for mechanistic studies. We present data here that establishes the feasibility of measuring these human disease endpoints in ferrets. Briefly, potential difference across the nasal and the lower airway epithelium in ferrets could be consistently assessed, were highly reproducible, and responsive to experimental interventions. Additionally, ferret airway epithelial cells were amenable to primary cell culture methods for in vitro experiments as was the use of ferret tracheal explants as an ex vivo system for assessing ion transport. The feasibility of conducting multiple assessments of disease outcomes supports the adoption of ferrets as a highly relevant model for research in obstructive airway diseases.

A ferret model of COPD-related chronic bronchitis.

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death in the US. The majority of COPD patients have symptoms of chronic bronchitis, which lacks specific therapies. A major impediment to therapeutic development has been the absence of animal models that recapitulate key clinical and pathologic features of human disease. Ferrets are well suited for the investigation of the significance of respiratory diseases, given prior data indicating similarities to human airway physiology and submucosal gland distribution. Here, we exposed ferrets to chronic cigarette smoke and found them to approximate complex clinical features of human COPD. Unlike mice, which develop solely emphysema, smoke-exposed ferrets exhibited markedly higher numbers of early-morning spontaneous coughs and sporadic infectious exacerbations as well as a higher level of airway obstruction accompanied by goblet cell metaplasia/hyperplasia and increased mucus expression in small airways, indicative of chronic bronchitis and bronchiolitis. Overall, we demonstrate the first COPD animal model exhibiting clinical and pathologic features of chronic bronchitis to our knowledge, providing a key advance that will greatly facilitate the preclinical development of novel treatments for this disease.

Second-hand eating? Maternal perception of the food environment affects reproductive investment in mice.

OBJECTIVE: Little information exists on how perception of the food (or "energetic") environment affects body composition and reproductive investment. The hypothesis was tested that female mice, who are themselves consuming standard chow diets but who are exposed to conspecifics eating a rich "cafeteria diet," will exhibit altered weight gain and reproductive investment. METHODS: Female C57BL/6 mice were raised on a cafeteria diet. At maturity, subjects were switched to a standard chow diet, and their cage-mates were assigned to consume either a cafeteria diet (treatment, n = 20) or standard chow (control, n = 20). Subjects were mated and pups raised to weaning. Subjects and pups were analyzed for body composition. RESULTS: Treatment had no discernable effect on dam body weight or composition but caused pups to have lower body weight (P = 0.036) and less fat mass (P = 0.041). A nearly significant treatment effect on "time to successful reproduction" (avg. 55 versus 44 days), likely due to increased failed first pregnancies, (14/19 versus 8/19, P = 0.099) was found. CONCLUSIONS: These data indicate that perceived food environment (independent of the diet actually consumed) can produce small pups with less body fat and possibly induce difficulties in pregnancy for dams. Replication and mechanistic studies should follow.

Modeled microgravity and hindlimb unloading sensitize osteoclast precursors to RANKL-mediated osteoclastogenesis.

Mechanical forces are essential to maintain skeletal integrity, and microgravity exposure leads to bone loss. The underlying molecular mechanisms leading to the changes in osteoblasts and osteoclast differentiation and function remain to be fully elucidated. Because of the infrequency of spaceflights and payload constraints, establishing in vitro and in vivo systems that mimic microgravity conditions becomes necessary. We have established a simulated microgravity (modeled microgravity, MMG) system to study the changes induced in osteoclast precursors. We observed that MMG, on its own, was unable to induce osteoclastogenesis of osteoclast precursors; however, 24 h of MMG activates osteoclastogenesis-related signaling molecules ERK, p38, PLCγ2, and NFATc1. Receptor activator of NFkB ligand (RANKL) (with or without M-CSF) stimulation for 3-4 days in gravity of cells that had been exposed to MMG for 24 h enhanced the formation of very large tartrate-resistant acid phosphatase (TRAP)-positive multinucleated (>30 nuclei) osteoclasts accompanied by an upregulation of the osteoclast marker genes TRAP and cathepsin K. To validate the in vitro system, we studied the hindlimb unloading (HLU) system using BALB/c mice and observed a decrease in BMD of femurs and a loss of 3D microstructure of both cortical and trabecular bone as determined by micro-CT. There was a marked stimulation of osteoclastogenesis as determined by the total number of TRAP-positive multinucleated osteoclasts formed and also an increase in RANKL-stimulated osteoclastogenesis from precursors removed from the tibias of mice after 28 days of HLU. In contrast to earlier reported findings, we did not observe any histomorphometric changes in the bone formation parameters. Thus, the foregoing observations indicate that microgravity sensitizes osteoclast precursors for increased differentiation. The in vitro model system described here is potentially a valid system for testing drugs for preventing microgravity-induced bone loss by targeting the molecular events occurring in microgravity-induced enhanced osteoclastogenesis.