INTRODUCTION: SARS-CoV-2 is a novel virus from the coronavirus family that emerged in the Hubei province of China in December 2019 and rapidly spread throughout the world. On March 11, 2020, the World Health Organization declared a global pandemic. Infection with SARS-CoV-2 causes coronavirus disease 19 (COVID19) which can be fatal. There is an obvious and pressing need for research surrounding SARS-CoV-2 that will aid in eradication of this pandemic. OBJECTIVE: The goal of this study was to absolve the dire need for small animal models of human disease that demonstrate hallmark pathological features of infection. Due to ethical and financial obstacles, the use of animals that closely resemble human immunity, such as non-human primates, is often not a viable option. For this reason, there is a push to develop small animal models that can mimic human disease responses, particularly those in viral infections that have a narrow species tropism. To achieve this in the context of the novel coronavirus, SARS-CoV-2, we studied various mouse models and their capacity to become infected with and mount an immune response to SARS-CoV-2. Our goal was to identify a model that sufficiently mimics severe COVID19 seen in humans as well as provide molecular insight into pathways that prevent the development of severe disease. METHODS: NRG-L and HIS-NRGF-L mice were subcutaneously implanted with human fetal lung xenografts and infected with SARS-CoV-2. Tissues were stained with H&E for histopathological scoring. NRG-L and HIS-NRGF-L tissues were fluorescently labeled using 2 different multiplex immunohistochemistry panels. Slides were digitized by a Vectra Polaris™ fluorescent whole slide scanner and digital analysis was completed using HALO™. Statistical analysis was conducted using GraphPad Prism™ 9.0.1. RESULTS: Infected NRG-L mice present extensive histopathological manifestations when compared to uninfected controls. Cumulative histology scores at both 2 and 7DPI were increased when compared to uninoculated fLX. Neutrophil influx, intra-airspace necrosis, capillary fibrin thrombi, and presence of syncytial cells were the most significant independent observations that contributed to the increased cumulative score. Together these findings indicate that fLX inoculated with SARS-CoV-2 faithfully recapitulate several features of diffuse alveolar damage (DAD) described in severe COVID-19. HIS-NRGF-L mice displayed decreased influx of neutrophils, intra-airway necrosis, and syncytial cells when compared to NRG-L fLX. Hemorrhage was decreased at both 2 and 7 DPI for HIS-NRGF-L fLX compared to NRG-L fLX. Cumulative histology scores were decreased in HIS-NRGF-L fLX at 7 DPI when compared to NRG-L fLX. Taken together these findings support the hypothesis that human myeloid and lymphoid infiltrates suppress or prevent the disparate host response observed in NRGL-L fLX that manifested in pronounced diffuse alveolar damage. CONCLUSION: Using digital image analysis of multiplex immunohistochemistry paired with semi-quantitative histopathological scoring, this study characterized important hallmark lesions observed in severe COVID19 as seen in small animal models. NRG-L and HIS-NRGF-L mice that are subcutaneously implanted with human fetal lung xenografts are susceptible to infection with SARS-CoV-2 and can produce severe and moderate disease phenotypes respectively. Co-engraftment with human fetal lung tissue and human immune system components in HIS-NRGF-L mice suppresses the divergent host response that is observed in NRG-L mice. For this reason, NRG-L mice engrafted with fLX are an ideal small animal model of severe COVID19, whereas HIS-NRGF-L mice severe as a valuable and informative model for deciphering molecular mechanisms driving severe COVID-19 that will serve as targets for therapeutic development.

Damage to dura mater may occur during intracranial or spinal surgeries, which can result in cerebrospinal fluid leakage as well as other potentially fatal physiological changes. As a result, biological scaffolds derived from xenogeneic materials are typically used to repair and regenerate dura mater post intracranial or spinal surgeries. In this study we explore the mechanics, structure, and immunological capacity of xenogeneic dura mater to be considered as a replacement for human dura. A comparative analysis is done between native porcine dura and a commercially available bovine collagen-based dura graft. Native porcine dura mater was decellularized and subjected to mechanical and histological analysis. Our decellularized porcine dura was able to maintain the overall morphological/structural integrity and held an increased extensibility without sacrificing strength, which provides a solid foundation as a functional grafting material. The histological observations showed that the orientation of fibers was maintained after decellularization. We investigated the biocompatibility of native and decellularized porcine dura reseeded with fibroblast cells for in vitro study. Cell proliferation, cell viability, and mechanical properties of dural grafts were evaluated post reseeding on days 3, 7, and 14. Live-dead staining and resazurin salts quantified cell viability and cell proliferation, respectively. This in vitro study showed that the acellular porcine dural graft provided a favorable environment for rat fibroblast cell infiltration. The results of micro indentation testing show that the cell-seeded porcine dural graft provides a favorable environment for rat fibroblast cell infiltration. The mechanics and biocompatibility results provide promising insight for the potential use of porcine dura in future cranial dura mater graft applications. Lastly, a subcutaneous in vivo study of dura graft compared with the market available Lyoplant®. Grafts were evaluated for inflammation by evaluating macrophage and leukocyte invasion on 3, 7, and 14 days post implantation. Histological analysis of both implants revealed macrophage (and leukocyte infiltration, supporting reabsorption, and thus encouraging the regeneration at 14 days. Cell markers also revealed that inflammation and leukocytes decreased as the number of days increased. Future work will involve a long-term subcutaneous implantation up to 30 days and 60 days to determine the long-term immune response.