Abstract
Purpose :
Cellular senescence has been reported in human glaucoma patients and in a glaucoma mouse model. Mechanisms of senescence in the retina, and cell types implicated in this detrimental phenotype and propagation, remain incompletely understood. We aim to investigate the implication of senescence and identify the cells involved using the experimental model of retinal ischemia-reperfusion (I/R) injury in mice with the goal to prevent retinal degeneration.
Methods :
Intraocular pressure (IOP) was increased in mice for 60 minutes by doing intracameral injection of elevated saline solution. In vivo and ex vivo measurements have been performed at different time points, up to 14 days post I/R injury. Visual acuity was evaluated using optokinetic response (OKR) tracking. Structural changes were assessed using optical coherence tomography (OCT). Retinal ganglion cell (RGC) degeneration was quantified on retinal flatmounts by counting BRN3A immunostained positive cells. qPCR on retinal lysates and immunohistochemistry was utilized in order to assess spatiotemporal patterns of senescence. For all analyses, the contralateral eye has been used as control and statistics have been performed using paired t-test.
Results :
We observed a visual acuity dysfunction using OKR of the injured eye compared to control (-0.111 cycle/degree, p=0.016, n=4) at day 6. Despite being insignificant, thinning of the inner retinal layer after 14 days was noticed using OCT. RGC degeneration was measured with a loss of 59.24% at day 7 (p=0.018, n=4) and 36.28% at day 14 (p=0.030, n=6). We observed upregulation of senescence related genes in injured retinas compared to control at day 2: Cxcl1, Cdkn2b, Cdkn1a and day 7: Cxcl1, Il-1a. Using immunostaining, senescence markers were detected in injured retinas at day 2: P16INK4A and PAI1 were expressed in microglia and/or macrophages and Y-H2AX in RGCs and photoreceptors.
Conclusions :
Evidence of cellular senescence is detectable in the retina following I/R insult. Several retinal cell types appear to be involved. However, what are the specific cell types that express and/or propagate the phenotype remain unclear. Therefore, to elucidate this we are planning to do single cell RNA sequencing to address these questions. In future steps will attempt to rescue the I/R senescent phenotype to improve visual acuity, with an ultimate goal of targeting cellular senescence in glaucoma patients for neuroprotection.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.