Sex Differences in Mouse Hippocampal Astrocytes after <em>In-Vitro</em> Ischemia

Abstract

Astrogliosis following hypoxia/ischemia (HI)-related brain injury plays a role in increased morbidity and mortality in neonates. Recent clinical studies indicate that the severity of brain injury appear to be sex dependent, and that the male neonates are more susceptible to the effects of HI-related brain injury, resulting in more severe neurological outcomes as compared to females with comparable brain injuries. The development of reliable methods to isolate and maintain highly enriched populations of sexed hippocampal astrocytes is essential to understand the cellular basis of sex differences in the pathological consequences of neonatal HI. In this study, we describe a method for creating sex specific hippocampal astrocyte cultures that are subjected to a model of in-vitro ischemia, oxygen-glucose deprivation, followed by reoxygenation. Subsequent reactive astrogliosis was examined by immunostaining for the Glial Fibrillary Acidic Protein (GFAP) and S100B. This method provides a useful tool to study the role of male and female hippocampal astrocytes following neonatal HI, separately. Video LinkThe video component of this article can be found at

Aggravated brain injury after neonatal hypoxic ischemia in microglia-depleted mice

“…Taken together, our findings indicate that microglial depletion aggravates neuronal death due to a lack of anti-inflammatory cytokines. However, other sex-related mechanisms may inhibit neuronal damage in females, including the mitochondrial respiratory capacity of astrocytes, as well as the different contribution of TGF-β by resident microglia [55,65,66]. Consistent with our findings, Jin reported that microglial depletion increased ischemia-induced astrocytic inflammatory response and neural injury [26].…”

Technical Feasibility and Physiological Relevance of Hypoxic Cell Culture Models

“…Notwithstanding, the absence of pericellular O 2 measurements makes it virtually impossible to ascertain whether there is a higher concentration of oxygen in the control group and by what margin. This makes any interpretation of the results complicated, especially because the cell cultures used in these studies for their sensitivity to ischemic hypoxic injury, such as cardiomyocytes (132), neurons (137), kidney (138), and endothelial cells (139), tend to have relatively high oxygen consumption rates (85). Moreover, these studies generally do not include information about the height of the medium overlay above the adherent cell culture, which introduces yet another unknown variable that possibly affects the results and reproducibility (17,18).…”