Molecular Hydrogen Attenuated N-methyl-N-Nitrosourea Induced Corneal Endothelial Injury by Upregulating Anti-Apoptotic Pathway

Purpose: Previous work by our group has demonstrated the value of N-methyl-N-nitrosourea (MNU)-induced corneal endothelial decompensation in animal models. The aim of this study was to investigate the effect of molecular hydrogen (H2) on MNU-induced corneal endothelial cell (CEC) injury and the underlying mechanism. Methods: MNU-induced animal models of CEC injury were washed with hydrogen-rich saline (HRS) for 14 days. Immunofluorescence staining, immunohistochemical staining, and corneal endothelial assessment were applied to determine architectural and cellular changes on the corneal endothelium following HRS treatment. MNU-induced cell models of CEC injury were co-cultured with H2. The effect of H2 was examined using morphological and functional assays. Results: It was shown that MNU could inhibit the proliferation and specific physiological functions of CECs by increasing apoptosis and decreasing the expression of ZO-1 and Na+/K+-ATPase, whereas H2 improved the proliferation and physiological function of CECs by anti-apoptosis. Cell experiments further confirmed that H2 could reverse MNU damage to CECs by decreasing oxidative stress injury, interfering with the NF-κB/NLRP3 pathway and the FOXO3a/p53/p21 pathway. Conclusions: This study suggests that topical application of H2 could protect CECs against corneal damage factors through anti-apoptotic effect, reduce the incidence and severity of corneal endothelial decompensation, and maintain corneal transparency.

The Comparative Efficiency of Intraperitoneal and Intravitreous Injection of Hydrogen Rich Saline against N-Methyl-N-Nitrosourea Induced Retinal Degeneration: A Topographic Study

Retinitis pigmentosa (RP) comprises a heterogeneous group of inherited retinal diseases leading to blindness. The present study explored the protective effects of hydrogen rich saline (HRS) against the photoreceptor degeneration in the N-Methyl-N-nitrosourea (MNU) administrated rat, a pharmacologically induced RP model. The therapeutic effects of intraperitoneal (IP) and intravitreous (IV) injections of HRS on regional retina was quantified via topographic measurements. The MNU administrated rats received IV or IP injections of HRS, and then they were subjected to electroretinography, multi electrode array, histological and immunohistochemistry examinations. The concentrations of the retinal malondialdehyde (MDA), superoxide dismutase (SOD), as well as the mRNA levels of apoptotic-associated genes were quantified. The IP and IV delivery pathways of HRS were both effective to ameliorate MNU induced photoreceptor degeneration. Moreover, the IV acted as a more efficient delivery method than the IP in terms of therapeutic effects. Particularly, the topographic measurements suggested that the IV delivery of HRS could alleviate MNU induced photoreceptor degeneration in the posterior retina. The immunostaining experiments also verified the comparative efficiency between IV and IP delivery of HRS on regional cone photoreceptors. Focal cone photoreceptors showed different susceptibilities to HRS and exhibited as a distinct spatial disequilibrium: cone photoreceptors in the ST quadrant were preferentially rescued; meanwhile, HRS induced protection was feeblest in the IN quadrant. Furthermore, the HRS treatment increased the level of retinal SOD, while reduce the level of retinal MDA in MNU administered rats. The expression levels of sever apoptotic -associated genes were significantly altered by HRS treatment. Collectively, these findings suggest that the IV space is an excellent target for HRS delivery. The IV delivery of HRS can efficiently alleviate the photoreceptors (especially these locate at the posterior retina) from MNU toxicity and act as a candidate treatment for RP.