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

Abstract

PurposeTo determine the effect of valproic acid (VPA) on bleb morphology and scar characteristics in a rabbit model of minimally invasive glaucoma surgery (MIGS).MethodsNine 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.ResultsVPA-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.ConclusionsThe results support the application of VPA to enhance bleb morphology associated with good bleb function in MIGS with no apparent cytotoxicity.

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

“…The surgical procedure involving the Santen PreserFlo MicroShunt was performed in the rabbit as previously described. 23 MMC was applied via a surgical sponge soaked in either 0.4 mg/mL (high-dose) or 0.1 mg/mL (low-dose) solution. The sponge was placed within the conjunctival/Tenon's flap for 2 minutes before removal, then carefully and rigorously rinsed with 10 to 20 mL of saline solution, followed by insertion of the MicroShunt as per the manufacturer's instructions.…”

“…Staining with hematoxylin and eosin (H&E) or picrosirius red was performed as described previously. 23 The Masson's Trichrome Stain Kit was purchased from Electron Microscopy Sciences (Hatfield, PA) and used according to the manufacturer's instructions. Polarization microscopy for picrosirius red–stained cryosections was performed using an ECLIPSE Ti Series microscope (Nikon Instruments, Melville, NY) as described before.…”

“…Polarization microscopy for picrosirius red–stained cryosections was performed using an ECLIPSE Ti Series microscope (Nikon Instruments, Melville, NY) as described before. 23 Evaluations were made by unmasked observers.…”

“…Ltd., Singapore) as described previously. 23 The HistoIndex FibroIndex software was used to analyze the region of interest in the images ( https://www.histoindex.com/product-andservices/ ). 24 The distributions of collagen fiber morphometric traits, including density, reticulation, thickness, and length, were described by normalized quantitative fibrosis parameters.…”

“… 21 We have demonstrated that VPA was effective in reducing collagen production in a mouse model of conjunctival scarring and a rabbit model of MIGS. 22 , 23 We hypothesize that the capacity of VPA to modulate type I collagen expression via selective regulation of Smads 22 will complement the anti-metabolite activity of MMC and help provide improved reduction of fibrosis after GFS or MIGS. In this study, we compared the effectiveness of a combination therapy of VPA and low-dose MMC at 0.1 mg/mL (MMC+VPA) against that of low-dose and high-dose MMC alone to regulate postoperative scarring responses in a rabbit model of MIGS implanted with the PreserFlo MicroShunt.…”