RESUMEN
Subarachnoid hemorrhage (SAH) accounts for 5% of stroke, with women having a decreased inflammatory response compared to men; however, this mechanism has yet to be identified. One hurdle in SAH research is the lack of human brain models. Studies in murine models are helpful, but human models should be used in conjunction for improved translatability. These observations lead us to develop a 3D system to study the sex-specific microglial and neuroglial function in a novel in vitro human SAH model and compare it to our validated in vivo SAH model. Our lab has developed a 3D, membrane-based in vitro cell culture system with human astrocytes, microglia, and neurons from both sexes. The 3D cultures were incubated with male and female cerebrospinal fluid from SAH patients in the Neuro-ICU. Furthermore, microglial morphology, erythrophagocytosis, microglial inflammatory cytokine production, and neuronal apoptosis were studied and compared with our murine SAH models. The human 3D system demonstrated intercellular interactions and proportions of the three cell types similar to the adult human brain. In vitro and in vivo models of SAH showed concordance in male microglia being more inflammatory than females via morphology and flow cytometry. On the contrary, both in vitro and in vivo models revealed that female microglia were more phagocytic and less prone to damaging neurons than males. One possible explanation for the increased phagocytic ability of female microglia was the increased expression of CD206 and MerTK. Our in vitro, human, 3D cell culture SAH model showed similar results to our in vivo murine SAH model with respect to microglial morphology, inflammation, and phagocytosis when comparing the sexes. A human 3D brain model of SAH may be a useful adjunct to murine models to improve translation to SAH patients.
RESUMEN
Calorie restriction can enhance the regenerative capacity of the injured intestinal epithelium. Among other metabolic changes, calorie restriction can activate the autophagy pathway. Although independent studies have attributed the regenerative benefit of calorie restriction to downregulation of mTORC1, it is not known whether autophagy itself is required for the regenerative benefit of calorie restriction. We used mouse and organoid models with autophagy gene deletion to evaluate the contribution of autophagy to intestinal epithelial regeneration following calorie restriction. In the absence of injury, mice with intestinal epithelial-specific deletion of autophagy gene Atg7 (Atg7ΔIEC) exhibit weight loss and histological changes similar to wild-type mice following calorie restriction. Conversely, calorie-restricted Atg7ΔIEC mice displayed a significant reduction in regenerative crypt foci after irradiation compared with calorie-restricted wild-type mice. Targeted analyses of tissue metabolites in calorie-restricted mice revealed an association between calorie restriction and reduced glycocholic acid (GCA) in wild-type mice but not in Atg7ΔIEC mice. To evaluate whether GCA can directly modulate epithelial stem cell self-renewal, we performed enteroid formation assays with or without GCA. Wild-type enteroids exhibited reduced enteroid formation efficiency in response to GCA treatment, suggesting that reduced availability of GCA during calorie restriction may be one mechanism by which calorie restriction favors epithelial regeneration in a manner dependent upon epithelial autophagy. Taken together, our data support the premise that intestinal epithelial Atg7 is required for the regenerative benefit of calorie restriction, due in part to its role in modulating luminal GCA with direct effects on epithelial stem cell self-renewal.NEW & NOTEWORTHY Calorie restriction is associated with enhanced intestinal regeneration after irradiation, but the requirement of autophagy for this process is not known. Our data support the premise that intestinal epithelial autophagy is required for the regenerative benefit of calorie restriction. We also report that luminal levels of primary bile acid glycocholic acid are modulated by epithelial cell autophagy during calorie restriction with direct effects on epithelial stem cell function.
