Abstract
Purpose :
Early pathological changes in the human retina and optic nerve head due to elevated intraocular pressure (IOP) remain elusive due to the unique biomechanical properties of human eye in the context of glaucoma. We generated a new model of acute IOP elevation based on a recently characterized organotypic whole-globe human eye perfusion approach. This system enables us to study the initial response of ex vivo human retina and lamina cribrosa (LC) to pathologically relevant pressure insult.
Methods :
Human eyes received from the Eyebank of Canada within 24 hours of enucleation were immersed in Neurobasal-A Medium with supplements. Physiological fluid convection was restored in the eyes by infusion of synthetic aqueous humor into the anterior chamber over 6 hours. For each pair of eye, one was perfused to maintain a physiological IOP of 18-21mmHg, and the contralateral eye was maintained at an elevated IOP of 30-50 mmHg. Following perfusion eyes were fixed in 4% paraformaldehyde and sectioned for confocal microscopy. RBPMS staining and TUNEL assay were quantified to assess retinal ganglion cell (RGC) survival. GFAP and Iba1 staining were quantified to assess astrocyte reactivity and microglial activation in the retina and LC. Two-tailed paired student’s t-test was used for statistical analysis.
Results :
Whole-globe human eyes maintained a sustained physiological or elevated IOP for 6 hours, for at least three pairs. The retina and LC ctyoarchitecture were well preserved in all samples. There was minimal loss in the number of RBPMS-positive RGC (p=0.50) after IOP elevation. TUNEL assay also showed no apoptosis in the ganglion cell layer in both normal or elevated IOP eyes. IOP elevation led to significant activation of retinal microglia (p=0.015), indicated by changes in their distribution. IOP elevation showed a trend of increased GFAP intensity in the retina and LC, indicating potential early astrocyte reactivity.
Conclusions :
Our model produces sustained, elevated IOP in ex-vivo whole globe human eyes. The pressure insult induces rapid microglial activation that precedes visible neuronal injury. These changes replicate findings in other early-stage glaucoma models, and provide insight into pathological events relevant to early human glaucoma. With further optimization, this model will be used to study IOP-dependent molecular changes in human eyes relevant to glaucoma pathogenesis.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.