Assessment of Visual and Retinal Function Following In Vivo Genipin-Induced Scleral Crosslinking

Abstract

Purpose: Genipin has been proposed as a possible neuroprotective therapy in myopia and glaucoma. Here, we aim to determine the effects of prolonged genipin-induced scleral stiffening on visual function.

Methods: Eyes from Brown Norway rats were treated in vivo with either a single 15 mM genipin retrobulbar injection or sham retrobulbar injection and were compared to naïve eyes. Intraocular pressure, optomotor response, and electroretinograms were repeatedly measured over 4 weeks following retrobulbar injections to determine visual and retinal function. At 4 weeks, we quantified retinal ganglion cell axon counts. Finally, molecular changes in gene and protein expression were analyzed via real-time polymerase chain reaction (RT-PCR) and proteomics.

Results: Retrobulbar injection of genipin did not affect intraocular pressure (IOP) or retinal function, nor have a sustained impact on visual function. Although genipin-treated eyes had a small decrease in retinal ganglion cell axon counts compared to contralateral sham-treated eyes (-8,558 ± 18,646; mean ± SD), this was not statistically significant (P = 0.206, n = 9). Last, we did not observe any changes in gene or protein expression due to genipin treatment.

Conclusions: Posterior scleral stiffening with a single retrobulbar injection of 15 mM genipin causes no sustained deficits in visual or retinal function or at the molecular level in the retina and sclera. Retinal ganglion cell axon morphology appeared normal.

Translational significance: These results support future in vivo studies to determine the efficacy of genipin-induced posterior scleral stiffening to help treat ocular diseases, like myopia and glaucoma.

Keywords: crosslinking; genipin; glaucoma; myopia; scleral stiffening.

Figure 1.

Schematic of three groups of rats used in this study: Naïve/naïve rats (A) were completely naïve control rats. HBSS/naïve rats (B) received a single (unilateral) retrobulbar injection of HBSS, and genipin/HBSS rats (C) received a unilateral retrobulbar injection of genipin and a contralateral retrobulbar injection of HBSS.

Figure 2.

Ocular examination of eyes immediately and one week after retrobulbar injections show mild transient complications. In all eyes receiving a retrobulbar injection (HBSS or genipin), a bleb (A) appeared in the nasal quadrant immediately after injection. One such bleb is indicated by an arrow (OS) and can be compared to the naïve OD eye (prior to retrobulbar injection). Typically, the bleb would resolve 1 week after injection (B). In a few cases, eyes had mild conjunctival chemosis (C) or subconjunctival hemorrhage (D, arrow). All images taken 1 week after injection are oriented such that the nasal portion of the eye is on the left.

Figure 3.

Genipin-induced scleral stiffening did not affect IOP. No significant differences in IOP were found in any group at any timepoint up to 4 weeks postinjection. RM ANOVA, F(21, 133) = 0.976; P = 0.497. All data shown as mean ± SD, all n ≥ 5.

Figure 4.

Genipin treatment did not have a sustained effect on spatial frequency or contrast sensitivity. Spatial frequency (A) and contrast sensitivity (B) for HBSS/naïve and genipin/HBSS rats. Spatial frequency was not significantly decreased in any of the groups over the course of the experiment (RM ANOVA, F(15, 95) = 1.33; P = 0.201). Contrast sensitivity was transiently decreased at day 14 in genipin eyes compared with naïve eyes (P = 0.002, denoted by double asterisks) and in HBSS eyes (of genipin/HBSS rats) versus genipin eyes (P = 0.043, denoted by asterisk). All data shown as mean ± SD and analyzed by RM ANOVA, Tukey post hoc used when appropriate; all n ≥ 5.

Figure 5.

Retinal function was not altered by HBSS or genipin injections up to 4 weeks postinjection. Electroretinogram naïve responses for dark-adapted (A–D) and light-adapted (E–H) testing conditions. Plotted are representative waveforms at 1-week (A, E) and 4 weeks (B, F) postinjection for naïve (black dotted) and genipin (black solid) eyes. Mean amplitude and implicit time of all genipin (or naïve) eyes were computed at each time point and flash intensity to select waveforms that most closely matched the means to ensure proper representative waveforms. A-wave and B-wave amplitudes from the brightest dark-adapted flash (2.1 log cd s/m²) are plotted versus time in C and D, respectively. Additionally, B-wave and PhNR amplitudes from the brightest single photopic flash (1.4 log cd s/m²) are plotted versus time in G and H. All ERG data was analyzed with a 2-way RM ANOVA. No significant interactions of time and treatment were found for any flash intensity (all P > 0.05). All data shown as mean ± SD, all n ≥ 5.

Figure 6.

Genipin treatment results in a minor, non-statistically significant, loss of RGC axons. (A) Whole nerve counts from naïve (n = 7, randomly selected as OD or OS eye), HBSS (n = 9), and genipin (n = 9) eyes. Nerve counts were not different in any cohort (1-way ANOVA, F(2, 22) = 1.067, P = 0.361). (B) Contralateral optic nerve axon count differences for genipin/HBSS rats at 4 weeks postinjection. Differences are computed as whole nerve axon count in genipin eye minus whole nerve axon count in contralateral HBSS eyes. (One sample t-test, t = 1.377, df = 8, P = 0.206, black dashed lines represent SD of axon count differences from 5 naïve rats). Data shown as mean ± SD. (C, D) show representative subregions from the central region of optic nerves from a genipin/HBSS rat, with C being the HBSS eye and D being the genipin eye. Axons appear to be normal with homogenous interiors surrounded by uniform myelin sheaths.