Ophthalmic Combination of SurR9-C84A and Trichostatin-A Targeting Molecular Pathogenesis of Alkali Burn

Abstract

Background: Alkali burn is a frequently occurring ocular injury that resembles ocular inflammation caused by eye allergies, infection, and refractive surgeries.Methods: We investigated the synergistic regenerative potential of dominant negative survivin mutant (SurR9-C84A) and histone deacetylase (HDAC) inhibitor trichostatin-A (TSA) against alkali burn and corneal haze using human keratocytes and rabbit alkali burn model (Female New Zealand white rabbits).Results: Combination of SurR9-C84A and TSA suppressed levels of transforming growth factor (TGF)-β, alpha smooth-muscle actin (α-SMA), fibronectin and HDAC1, leading to apoptosis in myofibroblast cells and, showed the potential to clear the corneal haze. An insult with 0.5 N NaOH for 1 min led to neutrophils infiltration and formation of large vacuoles in the stroma. Treatments with TSA and SurR9-C84A for 40 min led to improvement in the conjunctival and corneal tissue integrity, marked by an increase in clathrin, and claudin expressions. An increase in TGF-β and endogenous survivin confirmed wound healing and cell proliferation in rabbit cornea. The blood analysis revealed a substantial decrease in the RBC, WBC, platelets, or the hemoglobin content post alkali burn. The cytokine array analysis revealed that NaOH induced expressions of IL-1α and MMP-9, which were found to be significantly downregulated (1.8 and 11.5 fold respectively) by the combinatorial treatment of SurR9-C84A and TSA.Conclusion: Our results confirmed that combination of SurR9-C84A with TSA worked in synergy to heal ocular injury and inflammations due to alkali burn and led to the regeneration of ocular tissue by increasing clathrin, claudin, survivin, and TGF-β and reversal of alkali burn by suppressing IL-1α and MMP-9 without inducing haze.

Topical Ophthalmic Formulation of Trichostatin A and SurR9-C84A for Quick Recovery Post-alkali Burn of Corneal Haze

“…In the present study, large vacuoles were found in the corneal stroma in eyes post-NaOH insult. In our previous study, we found that combination treatment of dominant negative survivin mutant (SurR9-C84A) and histone deacetylase inhibitor trichostatin-A (TSA) is effective for maintaining the corneal tissue’s integrity in rabbit alkaline burn model ( Roy et al, 2016 ). Moreover, including additional biomarkers of the disease pathway in the proposed rat model, will give a better understanding of haze/scarring mechanisms and will provide more information on the effectiveness of the treatment for future work in human trials.…”

“…These nanoparticles are not separable by centrifuging hence, in order to remove the unreacted STPP and unencapsulated proteins they were dialyzed using a 100 kDa dialysis tubing. The suspension was then added to 30 mg of EDC and 15 mg of NHS and stirred for 30 min ( Roy et al, 2016 ). The alpha-SMA antibody suspension (100 μg/mL in 3 mL of PBS) was added dropwise at room temperature (25 ± 5°C) under sustained magnetic stirring for 12 h. The solution was dialyzed using a 100 kDa dialysis tubing to remove unreacted antibody and EDC or NHS.…”

“…Recently, we used these formulations in an alkaline burn rabbit models ( Roy et al, 2016 ). Further to extend the published study we validated this work in another “rat model of alkali burn and haze/scarring.” In the present study, we want to use the rat model of scarring/haze to study, a number of biomarker reagents for rats are readily available than rabbits (pathologic markers, cytokine and apoptotic microarrays, primary and secondary antibodies) to study the complete mechanism which confirms/validate the pathways/molecules involved in humans.…”