| Retinitis pigmentosa(RP) is a group of inherited neurod egenerative diseases characterized by the progressive death of photoreceptors, deterioration of visual fields and ultimate blindness. Among the general population, the prevalence is variably reported in one case for each 3000 to 7000 person. Due to the tre mendous genetic heterogeneities, the pathological mechanism of RP is not fully understood; neither any satisfactory therapeutic strategy is available. Although various genes are involved in RP, the final common pathway is apoptotic cell death of photoreceptors. Besides, reactive oxygen species(ROS) have been recognized as a critical contributor to the photoreceptor apoptosis in RP. According to the previous studies, hydrogen, a selective ROS scavenger, can exert therapeutic against to entophthalmia, catara ct and light- induced retinal damage via anti-oxidative, anti-apoptotic and anti- inflammatory activities. Therefore, we hypothesized that hydrogen exerts protective effects against RP.In order to verify our hypothese, we used N-Methyl-N-nitrosourea(MN U) induced RP rats in the study. This animal model has been widely utilized in the investigations of human RP. However, the previous studies only focused on the course feature of this model, rarely involved its spatial chareacteristics, such as the different degeneration between central and peripheral retina, nasal and temporal retina, superior and inferior retina, or ON pathway and OFF pathway.Our research is aimed at researching on the temporal topography of the MN U induced retinal degeneration and the protective effects of hydrogen against the degeneration in rats. From above research, we can put forward a new way to delay the pathological process of RP, and provide experimental basis for clinical application of hydrogen. Materials and Methods Part one: Exploring the temporal topography of the MNU induced retinal degenerationNinety Sprague-Dawley(SD) rats were randomly divided into five groups: one control group and four MNU-treated groups. The MNU-treated rats received an intraperitoneal injection of MNU at the dose of 60 mg / kg body weight, while the control rats received an intraperitoneal injection of physiological saline. Experimental examinations were performed at the time points of P1, P3, P5 or P7(day post injection).(1)To observe the temporal characteristics of the MN U treated retinal function in vivo by electroretinogram(ERG).(2) To observe the temporal topography of the MNU treated retinal function in vitro by multielectrode array(MEA).(3) To observe the temporal characteristics of the MNU treated retinal morphology in vivo by optical coherence tomography(OCT).(4) To observe the temporal topography of the MNU treated retinal morphology in vivo by hematoxylin&eosin(HE) staining.(5) To observe the temporal topography of the rod and cone of the MNU treated retinal by immunohistochemistry of retinal flat mounts.(6) The expression levels of rhodopsin and cone arrestin was determined by Western blotting at different time points.(7) The mRNA expressions of Bax, Calpain2, Caspase3 and Bcl2 were determined by q RT-PCR. Part two: The protective effects of hydrogen-rich saline(HRS) against the N-Methyl-N-nitrosourea induced photoreceptor degenerationNinety-six SD rats were randomly divided into four groups: normal control(NC), HRS control, vehicle treated(MNU + vehicle) and HRS treated(MNU + HRS). The vehicle treated and HRS treated groups received an intraperitoneal injection of MNU at the dose of 60 mg / kg body weight, while the control rats received an intraperitoneal injection of physiological saline. And a does of 10 ml / kg HRS was administered to HRS control and HRS treated rats intraperitoneally daily from 14 day prior to MNU administration until sacrifice, while the same does of physiological saline was administered to vehicle treated rats. Experimental examinations were performed at the time points of P3 and P7(day post injection of MNU).(1) To observe the effects of HRS on outer retinal function by ERG and field potentials.(2) Detect the spontaneous spikes and light induced spikes of RGCs to evaluatethe protective effects of HRS on inner retinal function.(3) HE staining was conducted to observe the effects of HRS on the retinal morphology.(4) The TUNEL assay and the level of genes expression related to apoptosis were performed to explore the mechanism of the protective effects of HRS.(5) Measure the superoxide dismutase(SOD) activity and malondialdehyde(MDA) content to explore the relationship between anti-oxidation and the protective effects of HRS. Results1. The MNU induced retinal degeneration complied with the following rules:(1) In one week after MNU administration, the retinal degeneration gradually worsened. The electrophysiology results showed a clear time-dependent deterioration of both ERG and field potentials after the MNU administration. At P7, the MNU ultimately eliminated all the responses of ERG. And the OCT and HE staining results showed a progressively decrease of ONL thickness.(2) Apoptosis of photoreceptor peaked at P3. The Bax, Calpain2 and Caspase3 expression were significantly up regulated and peaked at P3. And the maximum extent of Bcl2 downregulation occurred at P3.(3) Rod â†' Cone. The results of ERG, immunohistochemistry of retinal flat mounts and Western blotting showed that the rod degeneration was ea rlier than cone degeneration.(4) Central â†' Peripheral. The results of field potentials and HE staining indicated that the original site of MNU- induced retinal damage was the posterior pole of the central retina.(5) Inferior nasal(IN) â†' superior nasal(SN) and inferior temporal(IT) â†' superior temporal(ST). The results of field potentials, immunohistochemistry of retinal flat mounts and the expression of genes related to apoptosis conformed to the rule: IN â†' SN, IT â†'ST.(6) ON pathway â†' OFF pathway. The results of light induced firing rate of RGCs suggested that the ON pathway degenerated disproportionately to the OFF pathway, and should be considered as the main cause of the degenerated total response.2. HRS exerted protective effects against the MNU induced retinal degeneration.(1) The effects of HRS on the retinal function. The photopic ERG, scotopic ERG and field potential were effectively rescued in the HRS-treated rats. And the MEA data suggested that HRS exerts beneficial effects on both the spont aneous and the light induced response of RGCs, and thereby improve the signaling efficiency of inner retina.(2) The effects of HRS on the retinal morphology. The result of HE staining showed ONL in the HRS-treated rats retained with larger thickness.3. The protective effects showed no regional differences. The results of MEA and HE staining showed that HRS exerts effects on all the regions of retinal, central, mid-peripheral and peripheral.4. The protective effects of HRS were related to the anti-oxidation. The HRS treatment could enhance the SOD activity in the MNU- induced rats. And the MDA contents in HRS treatment rat decreased significantly.5. The protective effects of HRS were related to the anti-apoptosis. The TUNEL assay and the level of genes expression related to apoptosis indicated that the HRS- induced beneficial effects were derived from the increased photoreceptor presence.6. HRS would not affect function and morphology of normal ratina. Conclusions1. Our results suggested that the MNU ind uced retinal degeneration follow a predictable spatial- and time- dependent pattern. It is crucial for the popularization of this RP model. In the future explorations of human RP with this animol model, we should choose appropriate region of retinal to per form the examinations at an appropriate time point.2. It was found that the intraperitoneal administration of HRS could ameliorate MNU induced photoreceptor degeneration in terms of both morphology and function. And the protective effects are related to the anti-oxidative and anti-apoptotic activities of HRS.3. In the present study, no death, clinical signs or system symptoms are evident in the HRS administered animals during the experiment, providing safety profiles for future application of HRS. |
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