Dysregulation in the paraventricular nucleus of the hypothalamus (PVN) is associated with a variety of diseases including those related to obesity. Although most investigations have focused on molecular changes, structural alterations in PVN neurons can reveal underlying functional disruptions. While electron microscopy (EM) can provide nanometer resolution of brain structures, an inherent limitation of traditional transmission EM is the single field of view nature of data collection. To overcome this, we utilized large field of view high-resolution backscatter scanning electron microscopy (bSEM) of the PVN. By stitching high-resolution bSEM images, taken from normal chow and high fat diet mice, we achieved interactive, zoomable maps that allow for low magnification screening of the entire PVN and high-resolution analyses of ultrastructure at the level of the smallest cellular organelle. Using this approach, quantitative analysis across the PVN revealed marked electron dense regions within neuronal nucleoplasm following high fat diet feeding, with an increase in kurtosis, indicative of a shift away from a normal distribution. Furthermore, measures of skewness indicated a shift towards darker clustered electron dense regions, potentially indicative of heterochromatin clusters. We further demonstrate the utility to map out healthy and altered neurons throughout the PVN and the ability to remotely perform bSEM imaging in situations that require social distancing, such as the COVID-19 pandemic. Collectively, these findings present an approach that allows for the precise placement of PVN cells within an overall structural and functional map of the PVN. Moreover, they suggest that obesity may disrupt PVN neuronal chromatin structure.

Aims: The microglia activity of rat brain following exposure of the Macrovipera lebetina obtusa venom was investigated. Methods: Histochemical analysis of brain microcirculatory bed staining by Ca 2+ ATPase method for variable doses after intraperitoneal injections given for different time periods was used. The hemorrhagic activity of snake venom metalloproteinases was tested. Toxicological data were calculated using Behrens and Miller-Tainter methods. Surface, size of brain microglial cells (MGCs) and staining intensity were quantified using ImageJ software. Results: The vasodestructive action of the venom resulted in changes in ATPase activity. The intensity of staining of rat brain microcirculatory bed was venom dose-, and time-dependent. Increased activity of MGCs in hemorrhagic loci of different regions of venom affected brain was also demonstrated. Conclusion: The activation of microglia and changes of its form, size, and position strongly correlates with hemorrhage-induced cerebrovascular damage.

The paraventricular nucleus of the hypothalamus (PVN) is critical in autonomic and endocrine control. Previous work has indicated that PVN dysregulation is associated with a variety of cardiometabolic diseases including those related to obesity. However, investigations have focused primarily on molecular alterations. Importantly, changes in the anatomical structure of brain networks also leads to functional alterations. While electron microscopy (EM) can provide nanometer resolution of normal/abnormal brain structures, an inherent limitation of traditional transmission EM is the random sampling of an object within a large region of interest (i.e. PVN). To overcome the inherent limitations of EM, we introduce an approach that utilizes large field of view high‐resolution focused ion beam scanning electron microscopy (SEM) of the PVN. Six week old male C57Bl/6J mice were fed a high fat diet (HFD) or normal chow for 10 weeks (n=4/group), and brains prepared for SEM imaging. Due to the COVID‐19 pandemic, which required social distancing, SEM imaging and analysis was conducted remotely (i.e. at home) using a virtual network computing approach. We first acquired interactive, zoomable maps of the PVN at a low magnification. This image was then used to navigate precisely to the PVN and acquire a high‐resolution image encompassing ~30‐40% of the nucleus (Figure). With this approach, we could rapidly scan for any visually apparent anatomical alterations. This initial evaluation revealed more electron dense regions within PVN neuronal nuclei, potentially indicative of heterochromatin clusters, in HFD mice relative to normal chow counterparts. Thus, we performed subsequent analysis on 80‐120 neurons from each diet group by outlining neuronal nuclear envelopes and quantifying pixel distribution. This provided an indirect estimate of heterochromatin (i.e. dark pixels) and euchromatin (lighter pixels), with a shift towards darker pixels suggestive of increased chromatin “clustering”. The area and perimeter of analyzed PVN neuronal nuclei between groups was similar (e.g. area: 4.0±2.9 vs 3.7±1.4 µm2x106, normal chow vs HFD, p<0.05). However, histogram analysis of pixel intensities revealed positive kurtosis, indicating that HFD resulted in a redistribution of nuclear pixels to dark and light pixel intensities (‐0.05±0.03 vs 0.12±0.03 a.u., normal chow vs HFD, p<0.05). Measures of skew, an indicator of curve symmetry, further revealed a leftward pixel shift (i.e. more negative skew toward dark pixels) in diet‐induced obese mice (‐0.48±0.01 vs ‐0.64±0.01 a.u., normal chow vs HFD, p<0.05). Finally, we created a PVN “map” to examine where the most altered neurons were located. In short, following HFD feeding, “altered” neurons were distributed throughout the entire PVN and not located in discrete PVN sub‐nuclei. Collectively, these findings present an approach that allows for the precise placement of PVN cells within the overall region (i.e. locating a house within a city). Moreover, they suggest that HFD feeding may disrupt PVN neur...

Purpose Various treatment approaches are applied to repair damaged cartilage. However, a developing field of tissue engineering holds a realistic promise to replace injured cartilage tissue using the patient's cells. Using one type of chondrocytes to repair functionally different and far localized cartilage can be feasible from a clinical perspective. Toward this ultimate goal, we aimed to implement key protocols utilized in tissue engineering of articular (AR) and auricular (AU) cartilages. Methods The experiments were performed according to established protocols that include chondrocyte isolation, assessment of the cell proliferation rates, the degree of cell infiltration in three-dimensional scaffolds, and cartilage decellularization efficacy. ResultsThe data pointed to significant discrepancies in the size and in vitro chondrocytes proliferation rate isolated from distinct types of cartilage, with AR chondrocytes being 55% larger (p < 0.01) while having a slower rate of proliferation. Both collagenand alginate-based scaffolds showed relevant properties for cell infiltration. Lastly, we have shown that the AR and AU cartilages are decellularized to a different degree (17 ± 5.5% vs. 42 ± 8.5%, p < 0.01) while using the same SDS-based decellularization protocol. Conclusion This study will contribute to the global efforts to rebuild damaged cartilage with the help of tissue engineering.

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