Astrogliosis in the GFAP-Cre^ERT2:Rosa26^iDTR mouse induces retinal microglia activation and Müller cell gliosis in absence of pathological insult

Abstract

Purpose : Vision loss due to diabetic retinopathy (DR) affects over 140 million people globally. DR is characterized by persistent inflammation, neuronal and vascular damage, and hypoxia leading to vision loss. Despite our understanding of the role of many retinal cells in the pathology of DR, the role of astrocytes and how signals of inflammation and hypoxia drive their contribution to retinal pathology is still unclear.

Methods : We utilize of a model of hypoxia-induced retinopathy to model the ischemia and neovascularization of DR. The GFAP-Cre^ERT2:Rosa26^iDTR mouse line renders astrocytes sensitive to diphtheria toxin (DTx) following tamoxifen administration and induces astrocyte hypertrophy. To test for astrocyte responses, retinas were collected immediately or after a 30-day recovery following DTx treatment. To assess the effect of astrocyte activation on retinopathy animals were exposed to 7.5% O₂ for 7-days following DTx recovery. Glial, neuronal and vascular changes were quantified using IF with antibodies against GFAP, vimentin, IBA-1, NeuN, fibrinogen, and CD31. In addition, protein assays from CNS tissue and serum were utilized to measure cytokine levels.

Results : DTx treatment fails to deplete astrocytes but induces reactive astrogliosis visualized by increased GFAP immunoreactivity 30-days following DTx administration. Astrocyte reactivity persisted under hypoxic, but not normoxic conditions. Vimentin expression increased in only DTx-treated groups. We observed evidence of microglia reactivity in PBS-treated hypoxic and DTx-treated retinas. However, the combination of DTx and hypoxia did not induce an exacerbated microglia response. When examining the protein response to DTx and hypoxia, we found that there was no statistically significant change in IL-1b, IL2, IL-4, IL-5, IL-6, IL-10, GM-CSF, TNF-α, and IFN-γ expression.

Conclusions : DTx fails to deplete astrocytes but induces astrocyte reactivity that activates Müller glia and microglia. The lack of vascular pathology and that microglia reactivity is not exacerbated by hypoxia suggests that astrocyte activation may induce an anti-inflammatory response. This is corroborated by not observing an increase in inflammatory mediators in CNS tissues or serum. To further investigate the mechanisms driving pathology, we seek to quantify the additional markers such as CCL17, TGFB1, IL-15, IL-18 and LIF.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.