Early Functional Impairment in Experimental Glaucoma Is Accompanied by Disruption of the GABAergic System and Inceptive Neuroinflammation

Abstract

Glaucoma is a leading cause of irreversible blindness worldwide, and increased intraocular pressure (IOP) is a major risk factor. We aimed to determine if early functional and molecular differences in the glaucomatous retina manifest before significant retinal ganglion cell (RGC) loss is apparent. Adenoviral vectors expressing a pathogenic form of myocilin (Ad5.MYOC) were used to induce IOP elevation in C57BL/6 mice. IOP and pattern electroretinograms (pERG) were recorded, and retinas were prepared for RNA sequencing, immunohistochemistry, or to determine RGC loss. Ocular injection of Ad5.MYOC leads to reliable IOP elevation, resulting in significant loss of RGC after nine weeks. A significant decrease in the pERG amplitude was evident in eyes three weeks after IOP elevation. Retinal gene expression analysis revealed increased expression for 291 genes related to complement cascade, inflammation, and antigen presentation in hypertensive eyes. Decreased expression was found for 378 genes associated with the γ-aminobutyric acid (GABA)ergic and glutamatergic systems and axon guidance. These data suggest that early functional changes in RGC might be due to reduced GABA_A receptor signaling and neuroinflammation that precedes RGC loss in this glaucoma model. These initial changes may offer new targets for early detection of glaucoma and the development of new interventions.

Keywords: glaucoma; myocilin; neuroinflammation; pattern electroretinography; retinal ganglion cell loss; γ-aminobutyric acid (GABA).

Figure 1

IOP and RGC density in Ad5.MYOC injected eyes. (A) Chronic IOP elevation is evident as early as one week after Ad5.MYOC vector injection into the anterior chamber of left eyes (Ad5.MYOC (OS), black squares) when compared to either normotensive contralateral eyes (Ad5.MYOC uninjected (OD), gray squares) or eyes having received Ad5.empty vector (Ad5.empty, blue circles; * p ˂ 0.001). (B) Loss of Brn3a⁺ RGC in hypertensive eyes nine weeks after intraocular Ad5.MYOC injection when compared to their contralateral uninjected eyes or to RGC density in Ad5.empty vector-injected eyes. Data in A and B are given as mean ± SD. Every point reflects counts from an individual eye. ns = not significant p > 0.05. (C) Regression analysis of RGC density in Ad5.MYOC injected eyes and Area Under Curve (AUC) IOP values demonstrate strong correlation, especially when using peripheral RGC data. (D) Representative images of Brn3a immunostaining of retinal wholemounts display reduced RGC density in the peripheral and mid-peripheral areas after IOP elevation (far right column).

Figure 2

IOP and RCG density three weeks after induction of ocular hypertension. (A) The IOP profile indicates substantial induction of ocular hypertension using intraocular Ad5.MYOC injection when compared to their contralateral uninjected eyes. * p < 0.05 (B) Analysis of RGC density at that early time point indicates no significant loss in hypertensive eyes when compared to age-matched controls. Every point reflects counts from an individual eye. (C) Analysis of the percentage RGC loss after three and nine weeks of OHT indicates a slow progressive RGC loss, which is most prominent in the peripheral part of the retina.

Figure 3

Retinal gene expression cluster analysis of hypertensive eyes using GO and KEGG pathway analysis. (A) Increased gene expression in glaucomatous retina early after induced pressure elevation is related to immune response, activation of the complement system, and increased expression of ribosomal genes related to the ubiquitin–proteasome system. (B) Decreased expression is found for genes encoding GABA_A receptor subunits involving ligand–receptor interaction. Two additional clusters are found for the decreased expression of genes associated with axonal guidance and neuron projection.

Figure 4

C1q accumulation and altered synaptic labeling in glaucomatous retinas. (A) Retinas of hypertensive eyes (Ad5.MYOC) display obvious accumulation of complement component C1 in the retinal ganglion cell layer (RGCL, arrows) when compared to naïve control retinas (ctrl). Decreased immunolabeling is evident for GABA_A receptor and PDS95, indicating synaptic loss in the inner plexiform layer (IPL, arrows). Decrease in Brn3a positive cells was not noticed after 3 weeks of chronic IOP elevation (INL = inner nuclear layer, ONL = outer nuclear layer). (B) Changes in immunohistochemical labeling are in accordance with expression changes of corresponding genes. Values of fragments per kilobase of transcript per million mapped reads (FPKM) are given as mean ± SD and p-values were calculated using Student’s t-test.

Figure 5

Pattern electroretinography (pERG) three weeks after IOP elevation. (A) Overlay of averaged traces demonstrating functional impairment in the 1 Hz pERG of hypertensive eyes (Ad5.MYOC, black squares) while age-matched naïve controls (ctrl, blue circles) and mice having received the empty Ad5 construct (Ad5.empty, gray squares) show normal pERG waveforms. (B) The P1 to N2 amplitude is significantly decreased when compared to naïve or Ad5.empty eyes. Scatter plots reflect data from individual eyes per group.