Histone 3 acetylation at lysine 9 (H3K9ac) is decreased in hypoosmolar-stressed astrocytes: early epigenetic changes after brain injury and edema formation

MONTEVERDE BM; VIDOS C; RAMOS AJ; VILLARREAL A

Capital Federal

Congreso; Primer Encuentro del Club de la Glia Cono Sur; 2022

Astrocytes respond to brain injury through a mechanism known as reactive astrogliosis involving transcriptional, phenotypic and functional changes. Astrocyte functional changes have high impact on brain injury outcome; however the epigenetic mechanisms regulating gene expression, such as histone modifications, remain obscure. We have recently shown that astrocytes exposed to pro-inflammatory signals increase the level of histone acetylation. However, to date, there is no available description on early epigenetic changes in injury-affected astrocytes. We hypothesize that hypo-osmolar stress, promoted by early edema, triggers epigenetic changes in astrocytes.In a model of brain cortical injury by pial disruption in adult male Wistar rats (Villarreal et al., 2011), we addressed the levels of H3K9ac in astrocyte nuclei at 1.5 and 3.5 hours post injury. We observed, using immunofluorescence, a significative higher number of astrocytes with lower levels of H3K9ac at 3.5 hours when compared to non-injured hemisphere. Also, the injury promoted an increase in GFAP and AQP4 immunoreactivity, which radially decreased at higher distances from the injury core, probably indicating astrocyte swelling in response to edema. In vitro, we exposed primary culture of astrocytes to hypotonic (20, 30 and 40% hypoosmolarity) culture medium to promote hypo-osmolar stress. We further designed a method for automated quantification by creating a macro in FIJI (ImageJ) that allows rapid and accurate analysis of all astrocyte nuclei within a microscopic field. We observed a statistically significant decrease in H3K9ac immunofluorescence after 1 and 3 hours which were restored to control values 24h after recovery in complete isotonic medium. Trichostatin A treatment prevented H3K9ac decrease after 3 h hypoosmolarity.Our results strongly suggest that astrocytes exposed to edema-like microenvironment are able to dramatically change the global levels of histone acetylation. During the recovery in histone acetylation levels, chromatin might be re-decorated but in a “reactive epigenome”. Funding: PICT2018, ISN-CAEN2019