An inducible rodent glaucoma model that exhibits gradual sustained increase in intraocular pressure with distinct inner retina and optic nerve inflammation

Abstract

Glaucoma is a chronic and progressive neurodegenerative disease of the optic nerve resulting in loss of retinal ganglion cells (RGCs) and vision. The most prominent glaucoma risk factor is increased intraocular pressure (IOP), and most models focus on reproducing this aspect to study disease mechanisms and targets. Yet, current models result in IOP profiles that often do not resemble clinical glaucoma. Here we introduce a new model that results in a gradual and sustained IOP increase over time. This approach modifies a circumlimbal suture method, taking care to make the sutures 'snug' instead of tight, without inducing an initial IOP spike. This approach did not immediately affect IOPs, but generated gradual ocular hypertension (gOHT) as the sutures tighten over time, in comparison to loosely sutured control eyes (CON), resulting in an average 12.6 mmHg increase in IOP at 17 weeks (p < 0.001). Corresponding characterization revealed relevant retinal and optic nerve pathology, such as thinning of the retinal nerve fiber layer, decreased optokinetic response, RGC loss, and optic nerve head remodeling. Yet, angles remained open, with no evidence of inflammation. Corresponding biochemical profiling indicated significant increases in TGF-β2 and 3, and IL-1 family cytokines in gOHT optic nerve tissues compared to CON, with accompanying microglial reactivity, consistent with active tissue injury and repair mechanisms. Remarkably, this signature was absent from optic nerves following acute ocular hypertension (aOHT) associated with intentionally tightened sutures, although the resulting RGC loss was similar in both methods. These results suggest that the pattern of IOP change has an important impact on underlying pathophysiology.

Figure 1

The snug suturing technique results in gradual and sustained ocular hypertension. (A) Representative pictures of the procedure; IOP is measured by rebound tonometer (1), nylon 8–0 suture was passed subconjunctivally (2, 3) and tied using a slip-knot (4) to achieve a snug fit (5). One arm of a forceps could be easily passed under the suture (white arrow), demonstrating a snug fit (6) (pictures taken with a Dino-Lite Edge Polarizing Digital Microscope). (B) Timeline for the associated study indicating baseline and follow-up measurements. (C) Average IOP curves showing significant elevation in the gOHT group compared to CON (n = 12 and 8 eyes for gOHT and CON, respectively, *p < 0.001, bars are S.E.). CON; control, gOHT; gradual ocular hypertension, IOP; intraocular pressure, OCT; optical coherence tomography, RGC; retinal ganglion cell.

Figure 2

The gOHT model results in progressive RNFL thinning and decreased vision. (A) Representative fundus and corresponding cross-sectional OCT images used to measure the RNFL thickness at baseline, and at 13 and 17 weeks after surgery (scale bars represent 200 μm). The green circle represents the cross-sectional circumference and red and blue tracings outline the RNFL. (B) Quantified RNFL thickness measurements indicate progressive thinning in the gOHT group compared to CON that became highly significant after 17 weeks. ‘Snugly’ sutured eyes that showed a poor IOP response were excluded from the gOHT group (n = 8 and 12 for CON and gOHT, respectively, ****p < 0.0001, bars are SE). (C) Outer retinal thickness measured using OCT did not show statistically significant change at any time point (ns; not significant, bars are SE). (D) Comparison of visual acuity after 17 weeks (12 weeks of elevated IOP) in CON and gOHT groups shows a highly significant difference. However, there was a statistically significant decrease in vision in all groups post-suturing (p < 0.05). Notably, animals that responded poorly in terms of IOP elevation post-suturing did not show a statistically significant difference from CON (n = 4, 6 and 4 for CON, gOHT, and poor responders, respectively, ****p < 0.0001, *p < 0.05, bars are SE). RNFL, retinal nerve fiber layer.

Figure 3

Chronic gOHT results in inner retinal neuropathology. (A) Representative retinal images from CON and gOHT eyes after 17 weeks in the gOHT group, stained for RBPMS (green, arrows) and GFAP (red), in addition to the nuclear marker DAPI (blue) (scale bar indicates 50 μm). (B) Corresponding quantification of RGC density indicates significantly decreased survival in gOHT compared to CON (****p < 0.0001, bars are SE). (C) Representative immunostaining panels of CON and gOHT retinas in the same orientation. MMP-2 signal decreased and MMP-9 signal is increased in the gOHT inner retina compared to CON eyes (arrows). There was no notable difference in staining for CD68, CD31, F4/80, CD3e or CD4 between gOHT and CON. Note: negative controls (Neg CON) are sections stained with a secondary antibody alone. (scale bars indicate 50 μm). (D) Quantification of staining across image sets shows a significant decrease for MMP2, a significant increase for MMP9, and no change for CD68 markers (n = 8, bars are SE, ****p < 0.0001, ns; not significant). GCL, ganglion cell layer; INL, inner nuclear layer; Neg CON, Negative Control; ONL, outer nuclear layer.

Figure 4

Chronic gOHT results in optic nerve head tissue remodeling and neuroinflammation. (A) Representative phalloidin staining (red) highlights an intact pseudolaminar region in control (CON) tissue (arrows), which was disrupted in gOHT eyes (scale bar indicates 50 μm). (B) Representative immunostaining panels of CON and gOHT in sections of optic nerve head tissue. For orientation of all images, the white arrow indicates DAPI stained retinal layers on either side, * indicates the vitreous cavity adjacent to optic nerve head, and # indicates the optic nerve). There was mildly increased staining for MMP-9, and strongly increased CD68 in gOHT compared to CON tissues. There were no notable differences for any other marker (scale bar indicates 50 μm).

Figure 5

Eye growth does not contribute to increased IOP in the gOHT model. (A) Average corneal diameter was measured from 3 weeks of age until it plateaued at 12–14 weeks. Note the range of measured values were too narrow to display error bars (n = 8). (B) IOP measurements from eyes sutured at 14 weeks (n = 8) show the same increased IOP trend as eyes sutured at 6 weeks (n = 12, bars are SE).

Figure 6

There is no notable change in angle appearance or pathology between CON and gOHT eyes. (A) Representative OCT images of the anterior chamber angle show the same eye at the area between the peripheral cornea (arrows) and iris (arrowheads). The angle remained open until the last follow-up after 17 weeks post-suturing. (B) Line graphs indicate that the anterior chamber angle did not significantly change over time in either CON or gOHT groups. (C) A panel of relevant immunofluorescent markers indicates no significant differences in staining between CON and gOHT angles for MMP-2, MMP-9, CD68, CD31 or F4/80 staining (scale bars indicate 50 μm, all images are shown in the same orientation; the white asterisk indicates the ora serrata, blue arrow points to the ciliary processes, orange arrow to the iris and white arrows to Schlemm’s canal.

Figure 7

Retinal neuroinflammatory signaling differs in acute and gradual ocular hypertension models. (A) Experiment timeline showing weekly IOP measurements after suturing (CON, aOHT and gOHT groups), as well as pathology and cytokine profiling endpoints after 10 weeks post-suturing (pictures taken with a Dino-Lite Edge Polarizing Digital Microscope). (B) Recorded IOP curves show minimal change in IOP for the control (CON) group, for the aOHT group there was a marked transient spike in IOP at induction followed by tapering values, and for the gOHT group there was a gradual increase in IOP that remained consistently elevated above 20 mmHg from 3 weeks after suturing (n = 4, 6 and 6 for CON, aOHT and gOHT, respectively; bars are SE). (C) Representative retinal staining and RGC survival analysis shows significant and comparable loss in the aOHT and gOHT groups compared to CON (scale bar indicates 50 μm, ***p < 0.001, bars are SE, ns; not significant). (D) Multiplex cytokine analysis of retinal tissues revealed significantly elevated IL-17A in the aOHT group compared to gOHT. IL-12p70 levels were significantly elevated in the aOHT group compared to control and gOHT. In contrast, VEGF was significantly elevated in the gOHT group compared to control and aOHT. (*p < 0.05, **p < 0.01, bars are SE). (E) Multiplex cytokine analysis of the optic nerve identified significant elevation of TGF-β2, TGF-β3, IL-1α, IL-18 and LIX in the gOHT group, but no changes in the aOHT group (*p < 0.05, **p < 0.01, ***p < 0.001, bars are SE). (F) CD68 staining was significantly higher in the gOHT group compared to CON and aOHT groups (**p < 0.01, bars are SE). aOHT, acute ocular hypertension; IL, interleukin; LIX, TGF, transforming growth factor; TNF, tumor necrosis factor; VEGF, vascular endothelial growth factor.