Inflammatory Response and Defects on Myelin Integrity in the Olfactory System of K18hACE2 Mice Infected with SARS-CoV-2.

Viruses, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), use respiratory epithelial cells as an entry point for infection. Within the nasal cavity, the olfactory epithelium (OE) is particularly sensitive to infections which may lead to olfactory dysfunction. In patients suffering from coronavirus disease 2019, deficits in olfaction have been characterized as a distinctive symptom. Here, we used the K18hACE2 mice to study the spread of SARS-CoV-2 infection and inflammation in the olfactory system (OS) after 7 d of infection. In the OE, we found that SARS-CoV-2 selectively targeted the supporting/sustentacular cells (SCs) and macrophages from the lamina propria. In the brain, SARS-CoV-2 infected some microglial cells in the olfactory bulb (OB), and there was a widespread infection of projection neurons in the OB, piriform cortex (PC), and tubular striatum (TuS). Inflammation, indicated by both elevated numbers and morphologically activated IBA1+ cells (monocyte/macrophage lineages), was preferentially increased in the OE septum, while it was homogeneously distributed throughout the layers of the OB, PC, and TuS. Myelinated OS axonal tracts, the lateral olfactory tract, and the anterior commissure, exhibited decreased levels of 2',3'-cyclic-nucleotide 3'-phosphodiesterase, indicative of myelin defects. Collectively, our work supports the hypothesis that SARS-CoV-2 infected SC and macrophages in the OE and, centrally, microglia and subpopulations of OS neurons. The observed inflammation throughout the OS areas and central myelin defects may account for the long-lasting olfactory deficit.

Influence of chitosan concentration on cell viability and proliferation in vitro by changing film topography.

PURPOSE: Chitosan is a natural polysaccharide which can form gels and scaffolds that support its use as a biomaterial in various tissue engineering applications. A useful feature of chitosan polymer is that you can manipulate its properties easily. Thus, in this work we studied the effect of varying chitosan concentration in the topography and the biological properties of the chitosan films, as well as the effects in the structure of 3D gels in order to be used as nerve bridges. METHODS: Analysis of film topographies were addressed by swelling test and atomic force microscopy (AFM). In vitro biological properties were assessed through MTT viability assays on cultures of blood-brain barrier forming endothelial (bEnd5), glioma (C6) and postmitotic neuron (NGF-differentiated PC12) cell lines. The structure of tridimensional gels was studied by environmental scanning electron microscopy.
 RESULTS: Topography of 1% chitosan films showed a AFM profile with higher nano-roughness profile than that observed in 2% films, which was smoother. Moreover, swelling rate was not affected. Topography changes affected cell viability as shown by the MTT assays. Our results showed that 2% chitosan films promoted higher proliferation and viability of C6 and PC12 respectively than 1% films. Conversely, neither 1% nor 2% films promoted viability of bEnd5 cells. In order to establish the
feasibility of both type of chitosan solutions as nerve bridges, we constructed 3D gels by alkaline precipitation. Resulting gels showed that only 2% gels were rigid enough to be effectively used as nerve bridges. CONCLUSIONS: These results establish that changes in chitosan concentration affects the polymer surface topography, which has a direct effect in the growing cell behavior. Additionally, higher concentration of chitosan gels are required to be used in neural tissue engineering.

Differential adhesiveness and neurite-promoting activity for neural cells of chitosan, gelatin, and poly-L-lysine films.

Chitosan (Ch) and some of its derivatives have been proposed as good biomaterials for tissue engineering, to construct scaffolds promoting tissue regeneration. In this work we made composite films from Ch and mixtures of Ch with gelatin (G) and poly-l-lysine (PLL), and evaluated the growth on these films of PC12 and C6 lines as well as neurons and glial cells derived from cerebral tissue and dorsal root ganglia (DRG). C6 glioma cells proliferated on Ch, G, and Ch + G films, although metabolic activity was decreased by the presence of the G in the mixtures. NGF-differentiated PC12 cells, adhered preferentially on Ch and films containing PLL. Unlike NGF-treated PC12 cells, cortical and hippocampal neurons showed good adhesion to Ch and Ch + G films, where they extended neurites. Astrocytes adhered on Ch, Ch + G, and Ch + PLL mixtures, although viability decreased during the culture time. Olfactory ensheathing cells (OEC) adhered and proliferated to confluency on the wells covered with Ch + G films. Neurites from DRGs exhibited high extension on these films. These results demonstrate that Ch + G films have excellent adhesive properties for both neurons and regeneration-promoting glia (OEC). These films also promoted neurite extension from DRG, making them good candidates for tissue engineering of nerve repair.

Gelation under dynamic conditions: a strategy for in vitro cell ordering.

Ordered gelation under spin-coating conditions, as reported here, is a suitable method to order cells in biogels. Cell ordering is of great importance for functional repair of central nervous system (CNS) injuries, because therapies must include strategies to bridge chystic gaps and facilitate axon growth towards its target. Organized biocompatible and biodegradable substrates may be used for this purpose, to supply trophic support and provide directional cues for neuronal process outgrowth. Atomic force microscopy (AFM) and low temperature scanning electron microscopy (LTSEM), confirmed that fibrils in kappa-carrageenan/chitosan and fibrin hydrogels prepared under spin-coating conditions, were longitudinally arranged. The cell model was conveniently tested using rat C6 glioma cells. C6 cells were distributed regularly in fibrin gels formed under centrifugal force. The ability of ordered fibrin scaffolds to promote uniform distribution of transplanted cells, was confirmed by fluorescence microscopy.