Evaluation of Spatially Targeted Scleral Stiffening on Neuroprotection in a Rat Model of Glaucoma

Abstract

Purpose: Scleral stiffening may protect against glaucomatous retinal ganglion cell (RGC) loss or dysfunction associated with ocular hypertension. Here, we assess the potential neuroprotective effects of two treatments designed to stiffen either the entire posterior sclera or only the sclera adjacent to the peripapillary sclera in an experimental model of glaucoma.

Methods: Rat sclerae were stiffened in vivo using either genipin (crosslinking the entire posterior sclera) or a regionally selective photosensitizer, methylene blue (stiffening only the juxtaperipapillary region surrounding the optic nerve). Ocular hypertension was induced using magnetic microbeads delivered to the anterior chamber. Morphological and functional outcomes, including optic nerve axon count and appearance, retinal thickness measured by optical coherence tomography, optomotor response, and electroretinography traces, were assessed.

Results: Both local (juxtaperipapillary) and global (whole posterior) scleral stiffening treatments were successful at increasing scleral stiffness, but neither provided demonstrable neuroprotection in hypertensive eyes as assessed by RGC axon counts and appearance, optomotor response, or electroretinography. There was a weak indication that scleral crosslinking protected against retinal thinning as assessed by optical coherence tomography.

Conclusions: Scleral stiffening was not demonstrated to be neuroprotective in ocular hypertensive rats. We hypothesize that the absence of benefit may in part be due to RGC loss associated with the scleral stiffening agents themselves (mild in the case of genipin, and moderate in the case of methylene blue), negating any potential benefit of scleral stiffening.

Translational relevance: The development of scleral stiffening as a neuroprotective treatment will require the identification of better tolerated stiffening protocols and further preclinical testing.

Figure 1.

Experimental design of the study. (A) An overview of the experimental approach, showing scleral crosslinked eyes receiving one of three treatments by retrobulbar injection: HBSS (sham vehicle), GP, or MB. Those in the MB group also received 30 minutes of localized red light (660 nm) to selectively stiffen the juxtaperipapillary (but not peripheral) sclera. (B) Timeline of experiments. Seven days after scleral stiffening treatment, the treated (“experimental”) eye received a microbead injection to induce OHT. Taking the date of OHT induction as day 0, rats were sacrificed at day 14. (C) Timing of experiments. IOP measurements were taken at days –7, 0, 1, 3, 4, 7, 10, and 14. OMR measurements were taken at days 0, 7, and 14. ERG measurements were taken at days –7, 7, and 14, and OCT images were acquired at days –7 and 14. DIC and axon count measurements were taken by collecting the sclerae and optic nerves on day 14 immediately after euthanasia.

Figure 2.

GP and MB significantly increased scleral stiffness in OHT eyes. (A) Representation of the posterior eye showing the juxtaperipapillary sclera, here defined as the region enclosed by a 2-mm diameter circle centered at the ON. The peripheral sclera was defined as the sclera outside this region. (B) Whole globe inflation testing showed that posterior scleral stiffening with GP and targeted juxtaperipapillary sclera stiffening with MB were effective. The reported values are the mean strains over the juxtaperipapillary and peripheral scleral regions, a quantity that is inversely proportional to scleral stiffness. (C) IOP burden did not differ significantly between treatment groups. (D) IOP increased after induction of OHT at day 0 in microbead-injected eyes compared with normotensive control eyes. Statistical significance is indicated as: *P < 0.05, **P < 0.01, **P < 0.001, and ****P < 0.0001. Data shown as mean ± standard deviation. DIC = digital image correlation.

Figure 3.

OHT-induced globe enlargement. (A) Anterior chamber depth, (B) equatorial width, and (C) axial length were quantified versus IOP burden. It is interesting and perhaps unexpected that crosslinked eyes showed axial elongation (Fig. 3C); we speculate that this was due to corneal remodeling and perhaps some scleral remodeling in cross-linked sclerae. In this and subsequent graphs, the left side shows a plot of the outcome measure versus IOP burden at day 14, with raw data and linear regressions for each scleral stiffening treatment group shown. Dashed horizontal lines and grey bands represent the mean values and corresponding standard deviations from normotensive control eyes. The dashed red vertical line shows the mean IOP burden as reported in Supplementary Table S3. The right shows data corrected to this mean IOP burden by the statistical analysis. Statistical significance is judged by differences at the mean IOP burden as reported in Supplementary Table S3. Significance of slopes and comparisons with each group are listed in Supplementary Table S4. Data shown as mean ± standard deviation. Ctrl = control; Exp = experimental; ns = not significant.

Figure 4.

The number of RGCs and TRT did not demonstrably benefit from scleral stiffening treatments. (A) Whole nerve axon counts for hypertensive experimental eyes. (B) Nerve gradings (on a scale of 1 to 5; 1 being a healthy nerve and 5 being a highly damaged nerve) for hypertensive experimental ONs. Both analyses reported in (A) and (B) showed no significant protective effects of crosslinking. (C) TRT measured 0.5 mm from the ONH versus IOP burden. Retinal thickness in hypertensive GP eyes was not different than in GP normotensive controls, while thickness in hypertensive HBSS eyes was significantly less than in HBSS normotensive controls. Thickness in hypertensive MB eyes was less than in hypertensive GP and HBSS eyes (* indicates P = 0.027, *** indicates P = 0.0002). (D) TRT measured 1.2 mm from the ONH. Thicknesses in both GP- and MB-treated hypertensive eyes were not significantly different from their respective normotensive contralateral control eye thicknesses, whereas thickness in hypertensive HBSS-treated eyes was significantly less than in HBSS normotensive control eyes. Thickness in hypertensive GP eyes was significantly greater than in hypertensive HBSS-treated eyes (*P = 0.01). Significance of slopes and comparisons with each group are listed in Supplementary Table S4. See legend for Fig. 3 for interpretation of graphs. ns = not significant. Data shown as mean ± standard deviation.

Figure 5.

Visual function was unchanged by scleral stiffening treatment. (A) Spatial frequency thresholds and (B) CS thresholds at days 7 and 14 for hypertensive experimental eyes. All plotted quantities are corrected to the mean IOP burden at either day 7 or 14, as appropriate, as described in Fig. 3. In this figure, the dashed lines and grey bands represent mean values and their corresponding standard deviations from baseline hypertensive experimental eyes (owing to hyperacuity of control eyes, Supplementary Fig. S3). Significance of comparisons are listed in Supplementary Table S4. ns = not significant. Data shown as mean ± standard deviation.

Figure 6.

Retinal function is diminished in eyes with OHT and unaltered by scleral stiffening treatments. ERG functional outcomes at days 7 and 14 post-microbead injection for positive scotopic threshold (A) and negative scotopic threshold (B) were measured at –6.0 log cd s/m², b-wave (C) was measured at a dim flash of –3.0 log cd s/m², and OP3 (D) was measured at a bright flash of 2.1 log cd s/m². Dashed line represents mean values from normotensive controls and their corresponding standard deviations (grey band). Refer to caption of Fig. 5 for further interpretation of graphs. Significance of comparisons are listed in Supplementary Table S4. Data shown as mean ± standard deviation. ns = not significant.

Figure 7.

Overview of outcome measured for each scleral stiffening treatment group. Outcome measures in hypertensive rat eyes were organized into mechanical, morphological, and functional categories for each crosslinking treatment, and cross-correlations were evaluated. The plotted quantity is Pearson's correlation coefficient (r), a measure of the strength of association. Mechanical parameters included IOP burden, scleral strain, and eye dimensions. Morphological parameters include optic nerve cross-sectional area, axon count/density, and retinal thickness. Functional parameters include OMR and ERG data. (A) HBSS hypertensive experimental eye matrix. (B) GP hypertensive experimental eye matrix. (C) MB hypertensive experimental eye matrix. Statistical significance was calculated for null hypothesis of zero correlation (*P < 0.05, Bonferroni-corrected level). nSTR = negative scotopic threshold; pSTR = positive scotopic threshold.