Abstract
Purpose :
Retinal vascular diseases present increased glial activity, inflammation, and release of inflammatory cytokines. But the signaling mechanisms that connect vascular damage to a glial response have not been elucidated. Studies in the laboratory showed that non-apoptotic activation of endothelial caspase-9 (EC-Casp9) regulated activation of astroglial caspase-6 in a mouse model of retinal vein occlusion (RVO). We investigated the effect of EC-Casp9 signaling on astroglial and microglial response in RVO.
Methods :
RVO was performed in inducible EC-Casp9 (iEC-Casp9 WT/KO) adult male mice by tail vein injection of Rose Bengal and photocoagulation of major retinal veins. After induction of RVO we collected the retinas one and two days post RVO (P-RVO). Immunohistochemistry for Iba-1, CD68, cl-Caspase-6, GFAP, and AQP-4 was done and analyzed by blinded quantification using thresholding analysis in FIJI. Data was evaluated in injured iEC-Casp9 WT (n=6-8) and iEC-Casp9 KO (n=4-9), and uninjured groups (iEC-Casp9 WT, n=3, iEC-Casp9 KO, n=3). Kruskal-Wallis followed by Dunn’s and Brown-Forsythe and Welch ANOVA tests were used for statistical analysis.
Results :
EC-Casp9 did not modify the total number of Iba-1 or CD68 microglial cells, but the latter increased in injured animals two days P-RVO (p=0.02) compared to uninjured iEC-Casp9 WT. The area of microglial CD68 particles of injured retinas was bigger P-RVO (p=0.01, p=0.001), and rescued to normal levels in injured iEC-Casp9-KO 1 day P-RVO (p=0.03). EC-Casp9 led to higher astroglial cl-caspase-6 in injured iEC-Casp9 WT at assessed time points (p=0.02, p=0.004) compared to injured iEC-Casp9 KO. However, injured retinas did not show differences in GFAP expression P-RVO, but had a downward trend in AQP-4 2 days P-RVO compared to uninjured iEC-Casp9 WT, independent of EC Casp9.
Conclusions :
EC-Casp9 expression caused increased microglial activation in a timely manner and led to higher astroglial cl-caspase-6 expression, but not GFAP or AQP-4. These data demonstrate that EC-Casp9 signaling affects glial activity in our model. Further studies will help determine whether these glial responses are detrimental to the neuroretina.
This is a 2021 ARVO Annual Meeting abstract.