本研究来自泰山医学院秦树存教授课题组的报道，该文章昨日被眼科学杂志《Experimental Eye Research》接受并在线发表，也是该实验室发表的第3篇关于氢气的研究论文。

氢气作为抗氧化物质治疗疾病的研究越来越引起重视，本研究采用谷氨酸诱导的视网膜损伤为模型，在国际上首次采用眼球内注射氢气生理盐水的手段，证明氢气对谷氨酸诱导的视网膜损伤具有显著的保护作用。研究采用多种研究技术，例如iNOS和GRP78蛋白测定，形态学和电子显微镜观察节细胞和胶质细胞数量和改变。与对照组相比，氢气对细胞损伤和炎症反应具有显著治疗效果。

本研究如果能对比腹腔注射和眼球注射对上述指标的不同影响，可能更有价值，因为这显然是对比了两类不同的治疗方式，特别有可能就是一种剂量依赖关系。另外研究的指标非常全面，但整体上没有从逻辑上证明这些变化是氢气如何发挥影响的。当然这是氢气生物学研究最难以克服的障碍，就是具体的分子机制。

Hydrogen-rich saline protects retina against glutamate-induced excitotoxic injury in guinea pig

Lihua Wei^a, 1, Li Ge^a, 1, Shucun Qin^{b,  ,}  , Yunzhi Shi^a, Changqing Du^a, Hui Du^a, Liwei Liu^a, Yang Yu^b, Xuejun Sun^{c,  ,} 

Received 30 November 2010; revised 30 September 2011; Accepted 22 November 2011. Available online 2 December 2011.

Abstract

Molecular hydrogen (H₂) is an efficient antioxidant that can selectively reduce hydroxyl radicals and inhibit oxidative stress-induced injuries. We investigated the protective effects and mechanism of hydrogen-rich saline in a glutamate-induced retinal injury model. Retinal excitotoxicity was induced in healthy guinea pigs by injecting glutamate into the vitreous cavity. After thirty minutes, hydrogen-rich saline was injected into the vitreous cavity, the peritoneal cavity or both. Seven days later, the retinal stress response was evaluated by examining the stress biomarkers, inducible nitric-oxide synthase (iNOS) and glucose-regulated protein 78 (GRP78). The impaired glutamate uptake was assessed by the expression of the excitatory amino acid transporter 1(EAAT-1). The retinal histopathological changes were investigated, focusing on the thicknesses of the entire retina and its inner layer, the number of cells in the retinal ganglion cell layer （GCL）and the ultrastructure of the retinal ganglion cells (RGCs) and glial cells. Compared with the glutamate-induced injury group, the hydrogen-rich saline treatment reduced the loss of cells in the GCL and thinning of the retina and attenuated cellular morphological damage. These improvements were greatest in animals that received H₂ injections into both the vitreous and the peritoneal cavities. The hydrogen-rich saline also inhibited the expression of glial fibrillary acidic protein (GFAP) in Müller cells, CD11b in microglia, and iNOS and GRP78 in glial cells. Moreover, the hydrogen-rich saline increased the expression of EAAT-1. In conclusion, the administration of hydrogen-rich saline through the intravitreal or/and intraperitoneal routes could reduce the retinal excitotoxic injury and promote retinal recovery. This result likely occurs by inhibiting the activation of glial cells, decreasing the production of the iNOS and GRP78 and promoting glutamate clearance.

Highlights

► Hydrogen-rich saline treatment reduced the glutamate- induced retinal neuron injury. ► Hydrogen-rich saline treatment inhibited the activation of retinal glial cells. ► Hydrogen-rich saline treatment decreased the production of the stress biomarkers. ► Hydrogen-rich saline treatment maintained the activity of EAAT-1.

Keywords: hydrogen-rich saline; guinea pig; glutamate; vitreous cavity; peritoneal cavity; retinal excitotoxic injury

Article Outline

·             1. Introduction

·             2. Materials and methods

·      

·           2.1. Chemicals

·           2.2. Animals and protocol

·           2.3. Histopathological observation of the retina

·           2.4. Ultrastructural observation of the retina

·           2.5. Immunohistochemical staining

·           2.6. Double immunofluorescence labeling

·           2.7. Western blotting

·           2.8. Statistical analysis

·             3. Results

·      

·           3.1. Effects of hydrogen-rich saline on glutamate-induced retinal histopathological changes

·           3.2. Effects of hydrogen-rich saline on the ultrastructural changes of the retina

·           3.3. Effect of the intravitreal injection of hydrogen-rich saline on GFAP expression

·           3.4. Effect of the intravitreal injection of hydrogen-rich saline on CD11b expression

·           3.5. Effect of the intravitreal injection of hydrogen-rich saline on iNOS expression

·           3.6. Effect of the intravitreal injection of hydrogen-rich saline on GRP78 expression

·           3.7. Effect of the intravitreal injection of hydrogen-rich saline on EAAT-1 expression

·           3.8. Double immunostaining of GFAP with iNOS, GRP78 or EAAT-1

·           3.9. Western blotting analysis

·             4. Discussion

·             Acknowledgements

·             References