Effects of Valproic Acid and Mitomycin C Combination Therapy in a Rabbit Model of Minimally Invasive Glaucoma Surgery.

Purpose: This study aimed to compare the effectiveness of combination therapy consisting of low-dose mitomycin C (MMC) and valproic acid (VPA) against high-dose MMC for improving the scar phenotype in minimally invasive glaucoma surgery (MIGS). Methods: A rabbit model of MIGS incorporating the PreserFlo MicroShunt was treated with high (0.4 mg/mL) or low (0.1 mg/mL) doses of MMC or with combination therapy consisting of low-dose (0.1 mg/mL) MMC and VPA. Operated eyes were examined by live ocular imaging, histochemical evaluation, multiphoton quantitation of collagen characteristics, and molecular analyses. Results: Although high-dose MMC obliterated the vasculature, combination therapy vastly improved the postoperative tissue morphology by maintaining the vasculature without increased vascularization. Combination therapy also altered collagen morphology and reduced encapsulation of the MicroShunt distal end, which remained at risk with MMC treatment alone. Multiphoton quantitation indicated that the combination therapy significantly reduced collagen density and fiber dimensions compared with monotherapy. At the molecular level, combination therapy significantly reduced Vegfa, Vegfc, and Vegfd expression and inhibited Col1a1 upregulation from baseline levels, all of which low-dose MMC alone was unable to achieve. Notably, COL1A1 protein levels appeared more consistently suppressed by combination therapy compared with high-dose MMC alone. Conclusions: Compared with high-dose MMC, combination therapy was less toxic by sparing the vasculature and potentially more effective in reducing scarring via the regulation of collagen content and organization. Translational Relevance: VPA may be combined with low-dose MMC to replace high-dose MMC to deliver safe and effective anti-scarring outcomes.

Effect of valproic acid on functional bleb morphology in a rabbit model of minimally invasive surgery.

PURPOSE: To determine the effect of valproic acid (VPA) on bleb morphology and scar characteristics in a rabbit model of minimally invasive glaucoma surgery (MIGS). METHODS: Nine New Zealand white rabbits were subjected to MIGS with intraoperative implantation of the PreserFlo MicroShunt. Rabbits were then administered with subconjunctival injections of phosphate buffered saline (PBS) (n=4) or with VPA (n=5). Bleb morphology was examined by slit-lamp biomicroscopy and in vivo confocal microscopy. Postoperative day 28 tissues were examined by immunohistochemical evaluation and label-free multiphoton microscopy to visualise the collagen matrix, by terminal deoxynucleotidyl transferase dUTP nick-end labelling assay and immunofluorescent labelling for Ki67 expression to detect apoptosis and cell growth, and by real-time quantitative PCR to measure Col1a1, Fn, and Smad6 transcript expression. RESULTS: VPA-treated blebs were detectable on day 28, while the PBS-treated blebs were not detectable by day 14. VPA-treated blebs were diffuse, extended posteriorly with near normal conjunctival vascularity and featured a combination of reticular/blurred stromal pattern with evidence of relatively large stromal cysts. Instead of the deposition of thick, disorganised collagen fibres characteristic of the PBS bleb, the VPA bleb contained conspicuously thinner collagen fibres which were associated with similarly thinner fibronectin fibres. In corroboration, Col1a1 and Fn mRNA expression was reduced in the VPA blebs, while increased Smad6 expression implicated the disruption of the transforming growth factor beta pathway. Apoptosis and cell growth profiles appeared similar with both treatments. CONCLUSIONS: The results support the application of VPA to enhance bleb morphology associated with good bleb function in MIGS with no apparent cytotoxicity.

Positive-charge tuned gelatin hydrogel-siSPARC injectable for siRNA anti-scarring therapy in post glaucoma filtration surgery.

Small interfering RNA (siRNA) therapy is a promising epigenetic silencing strategy. However, its widespread adoption has been severely impeded by its ineffective delivery into the cellular environment. Here, a biocompatible injectable gelatin-based hydrogel with positive-charge tuned surface charge is presented as an effective platform for siRNA protection and delivery. We demonstrate a two-step synthesis of a gelatin-tyramine (Gtn-Tyr) hydrogel with simultaneous charge tunability and crosslinking ability. We discuss how different physiochemical properties of the hydrogel interact with siSPARC (siRNA for secreted protein, acidic and rich in cysteine), and study the positive-charge tuned gelatin hydrogel as an effective delivery platform for siSPARC in anti-fibrotic treatment. Through in vitro studies using mouse tenon fibroblasts, the positive-charge tuned Gtn-Tyr hydrogel shows sustained siSPARC cellular internalization and effective SPARC silencing with excellent biocompatibility. Similarly, the same hydrogel platform delivering siSPARC in an in vivo assessment employing a rabbit model shows an effective reduction in subconjunctival scarring in post glaucoma filtration surgery, and is non-cytotoxic compared to a commonly used anti-scarring agent, mitomycin-C. Overall, the current siRNA delivery strategy involving the positive-charge tuned gelatin hydrogel shows effective delivery of gene silencing siSPARC for anti-fibrotic treatment. The current charge tunable hydrogel delivery system is simple to fabricate and highly scalable. We believe this delivery platform has strong translational potential for effective siRNA delivery and epigenetic silencing therapy.

Glaucoma related retinal oximetry: a technology update.

There are two long-standing theories about the pathogenesis of glaucoma - barotrauma and the effect of vascular hypoxia. Currently, it is still unknown whether diminished blood flow is the cause or result of glaucomatous atrophy of ganglion cells and the optic nerve. Though many other imaging techniques used to directly assess ocular blood flow have been well studied, they are limited by their inability to directly assess metabolism in the ocular tissues or measure the oxygen carrying capacity in the vessels. Retinal oximetry is a relatively novel, noninvasive imaging technique that reliably measures oxygen saturation levels in the retinal vessels, offering surrogate markers for the metabolic demands of the eye. The clinical significance of these measurements has not been well established. Thus, this review gives an overview of ocular imaging and current retinal oximetry techniques, while contextualizing the important oximetry studies that have investigated the vascular theory behind glaucoma.

Retinal Oximetry in Subjects With Glaucomatous Hemifield Asymmetry.

PURPOSE: Although some studies suggest a strong link between retinal vessel oxygenation and damage to the retinal nerve fiber layer (RNFL) seen in glaucoma, it has yet to be conclusively proven. This study compares intraocular retinal oximetry in glaucomatous eyes displaying asymmetrically affected hemifields across different subgroups of glaucoma, namely primary angle closure glaucoma (PACG), primary open-angle glaucoma (POAG), and normal tension glaucoma (NTG). METHODS: In this prospective cross-sectional study, 99 patients (PACG, n=28; POAG, n=37; NTG, n=34) underwent retinal oxygenation and vessel caliber measurements using the Oxymap T1 Retinal Oximeter, Cirrus optical coherence tomography, and Humphrey visual field testing. For the comparison between different subtypes of glaucoma, an analysis of variance with Bonferroni method was performed. Intraeye differences were compared with a paired t test. Determination of the more affected and less affected hemifield was made using the Humphrey visual field pattern deviation plot. RESULTS: Considering the mean deviation and Advanced Glaucoma Interventional Study score, the visual field defects were milder in NTG as compared with POAG and PACG (P<0.05).Arteriole diameter was smaller in the more affected hemifield compared with the less affected hemifield in patients with PACG (109.30±18.07 vs. 120.57±17.92; P=0.023) and NTG (109.36±13.79 vs. 117.46±17.72; P=0.04). The more affected hemifield had a significantly thinner RNFL than the less affected hemifield in patients across all 3 groups, though this was only significant in PACG (P=0.02) and NTG patients (P<0.01). In all 3 groups, although the less affected hemifield tended to have a marginally higher arteriole and venule oxygen saturation than the more affected hemifield, no statistical significance was reached. There were no significant differences in arteriovenous difference between the more and less affected hemifield in all 3 groups. CONCLUSIONS: In our study, localized visual field losses were not associated with changes in retinal oximetry but were associated with narrower retinal arteriolar diameters in PACG and NTG. The RNFL was thinner in the more affected hemifield in these 2 groups but this was not so marked in the POAG sample, possibly limiting our ability to find a difference in arteriolar diameter there.

Bleb related infections: clinical characteristics, risk factors, and outcomes in an Asian population.

PURPOSE: Comparison of the demographic, ocular, systemic and microbiological characteristics of eyes with bleb related infection (BRI) and bleb related endophthalmitis (BRE). METHODS: Retrospective chart review of patients with BRI from January 1996-July 2013. Identification done via the center's longstanding endophthalmitis audit, BRI audit and laboratory database identifying all conjunctival swabs from blebs. Blebitis was defined as anterior segment inflammation with mucopurulent material in or around the bleb, with anterior chamber cells but no hypopyon. BRE was defined by the presence of hypopyon or vitreous inflammation. RESULTS: Twenty-nine patients with blebitis and 10 with BRE were identified. Mean age of subjects (n=39) was 68.4 (±13.3) with a preponderance of men (74.4%) and Chinese ethnicity (74.4%). BRE patients were 10.7 years older than blebitis patients (P=0.026). 28 (71.8%) subjects had primary open angle glaucoma. The presenting intraocular pressure (IOP) dropped in blebitis but almost doubled in BRE (P=0.011) compared to average preinfective IOP. Two weeks after treatment, IOPs in both groups returned to close to preinfective levels. Subjects with blebitis more often had an avascular bleb (88.0%) while those with BRE trended toward a moderately vascular bleb (50%). The distribution of causative microorganisms between the groups was similar. CONCLUSION: Our study indicates that risk factors are similar in both groups even though the visual outcome and clinical course, in the form of IOP findings and bleb vascularity, can diverge significantly. The decreased IOP in blebitis subjects represents objective evidence of subclinical leaks or bleb sweating.

Inter-eye comparison of retinal oximetry and vessel caliber between eyes with asymmetrical glaucoma severity in different glaucoma subtypes.

BACKGROUND: To compare retinal vessel oxygenation and vessel caliber in primary angle-closure glaucoma (PACG), primary open-angle glaucoma (POAG), normal-tension glaucoma (NTG), and normal controls, as well as between eyes of asymmetrical glaucoma severity. METHODS: This was a prospective, cross-sectional study. The 159 subjects (PACG, n=39; POAG, n=41; NTG, n=41; normal controls, n=38) underwent retinal oxygen saturation measurements using the Oxymap T1 Retinal Oximeter, optical coherence tomography, and Humphrey visual field testing. Retinal oxygen saturation and vessel diameter were compared between the glaucoma groups and normal controls, as well as between eyes of asymmetrical glaucoma severity. Kruskal-Wallis test was performed for comparison among different subtypes of glaucoma. Wilcoxon signed-rank test was used to compare the inter-eye differences. RESULTS: Compared to normal controls, arteriolar oxygen saturation was increased in PACG eyes (P=0.048) but not in POAG or NTG eyes. There were no significant differences in oxygen saturation in venules or arteriovenous (AV) difference in all three glaucoma groups. Venular diameter was significantly reduced in all glaucoma groups compared to normal controls (P<0.001), but no such change was observed in arteriolar diameter (P=0.10). When comparing between eyes of asymmetrical glaucoma severity, arteriolar oxygen saturation (P=0.03) and AV difference (P=0.04) were significantly higher, while arteriolar diameter was significantly lower (P=0.001) in the worse eye in PACG group. There were no significant differences in oximetric parameters or vessel calibers between the worse and the better eyes in POAG and NTG groups. CONCLUSION: Eyes with PACG showed increased arteriolar oxygen saturation and increased AV difference. This was not observed in POAG and NTG eyes. Arteriolar diameter in PACG and venular diameter in all three glaucoma groups were reduced. The difference observed in PACG eyes may be due to an increased metabolic demand in the disease process compared to open-angle glaucoma.