| Inherited retinal degeneration is a group of the common manifestations of diseases that is progressive photoreceptor cells and loss of pigment epithelial cells function. So far, there are no effective treatment for these diseases. Thus, it is great harm for millions of people around the world to blind permanently. It is an urgent clinical need for effective measures to improve and maintain the RPE function to prevent the photoreceptor cell degeneration and apoptosis, which is a major hot and difficult world-wide over the years. Currently ,it is discovered that BDNF can regulate the survival of photoreceptor cells and retinal cells in retinal degeneration throμgh autocrine or by glial cells ,which promote the RPE cell of cultured human and bovine differentiation.In order to offer ideas for more clinical patients whose the RPE dysfunction and photoreceptor cell degeneration,this paper investigate the changes of ERG (electroretinogram), retinal BDNF expression and retina morphological in different periods and analyze the improvement of the transplanted RCS rats'retinal in different phases of RCS rats after AAV-BDNF-IPEs being transplanted into the subretinal space of early RCS rat.MethodsThere are the following four parts in this study. There are 48 either male or female RCS rats of 18-22 days after birth (3 weeks old) in the first part. We observe the postoperative fundus situation after AAV-BDNF-IPEs being transplanted into the eyes, subretinal space of RCS rats.The second part:Classify SD control rats in accordance with 3, 6,8,10,12,14 week-old after birth, 3 every age group, a total of 18; Intact RCS rats and surgery RCS rats were 18 rats respectively, which grouped in accordance with the SD rats.Above all eyes were recorded by dark-adapted ERG that is called electrophysiological examination with reference to international standards of clinical visual electrophysiology. Checked three times at least every times per eye, taken waveform stable, small interference ERG graphics after the overlay, computing ERG-a, b wave amplitude values and statistical analysis.There are two groups about Animals in the third part.There were 24 animals taken eyes in group 1 for the control group (A group) that were divided into 3, 6, 8, 10, 12, 14 weeks after birth, and each week age has four. There were 24 in Group 2 for surgery RCS rats group (B group),taken the eyes ball respectively in the corresponding time points of group A that is the day of surgery, postoperative 3,5,7,9,11 weeks.BDNF expression in retinal tissue of intact RCS rats and surgery RCS rats were detected by Enzyme-linked immunosorbent assay (Elisa) 3, 5, 7, 9, and 11 weeks after surgery. Comparative and statistical analysis these data. The four part: Classify Animals and experimental in accordance with thesecond part. Rats were killed after electrophysiological examination to remove the eye, and retinal tissues were obtained to HE staining,TUNEL staining and immunohistochemistry.Results1. A total of 43 RCS rats implementation of the eyes subretinal transplantation, occurred 18 eye with a large number of vitreous hemorrhage, extensive retinal detachment, endophthalmitis, and mistakenly injected into the vitreous cavity. There are 68 eye that AAV-BDNF-IPE cells successfully transplanted into the subretinal space, and the successful rate up to 79%.HE staining of RCS rat retina and ciliary body parts showed a lack of ciliary body and retinal pigment cell layer; HE staining showed the subretinal space of rats after postoperative 5 weeks brown with scattered epithelial cells in the survival when AAV-BDNF-IPE cells transplanted into the subretinal space of RCS rats, sμggesting that is post-transplant survival for the AAV-BDNF-IPE cells; no significant infiltration response of inflammatory cells and structural damage within the retina or choroid (Fig.6).2. Three groups of rats were recorded successful completion by dark-adapted FERG from postnatal 3 weeks to 14 weeks, 15 normal SD rats were recorded 30 times, 15 intact RCS rats were 30 times, 17 cell transplantation group RCS rats 34 times.The FERG a, b wave amplitude of three groups rats with 8 weeks after birth dark-adapted maximum response was no significant difference, RCS rats dark-adapted FERG maximum reaction a, b wave amplitudes were significantly decreased from 10 weeks after birth, which were significantly lower than normal SD rats of the same weeks age (P <0.05), and ERG waveform type of RCS rats born after 12 weeks was low and flat, peaks and valleys was no obvious, a, b wave was vague. AAV-BDNF-IPE transplant group RCS rats after 10 weeks old after birth remained at a high level of b wave, the cell transplantation group RCS rat b wave has been decreasing slowly, but the amplitude still significantly higher than intact RCS rats with the same week (12w and 14w group P <0.05).3.BDNF expression in retinal tissue of intact RCS rats were still high in postnatal 3w, and sharply decreased into low level with development. Retinal BDNF expression of intact RCS rats in postnatal 3w were much higher than those of other postnatal weeks, P<0.01; BDNF expression in retinal tissue of surgery RCS rats were significantly higher than those in intact rats postnatal 3, 6, 8, 10, 12 and 14 weeks,(P<0.05 in postnatal 6w and P<0.01 in postnatal 8, 10, 12 and 14 weeks, respectively).4. HE staining showed: there was a slighter improvement in the the rod layer thickness close to one side of outer nuclear layer in the AAV-BDNF-IPE transplanted group compared with the intact group. TUNEL results:labeled TUNEL positive cells were still able to find in the surgery group, but their number compared with the intact group reduced slightly. TUNEL Results: The apoptotic index decreased, after the extension of the apoptosis peak, the peak of apoptosis occurred in postoperative 9wk; Immunohistochemistry results shows: That GFAP expression was weak and Muller cells color was lighter in the original part of the outer retina in the experimental group rats; SYN was weakly positive in the outer plexiform layer besides it colors outside the inner plexiform layer.Conclusion1. The experiment used a homemade syringe which was made by the single path method scleral AAV-BDNF-IPE transplantation to the subretinal space of young RCS rats successfully, the scleral incision was reduced effectively, the destruction of retinal anatomy was reduced, and the successful rate up to 79%.HE staining of RCS rat retina and ciliary body parts showed a lack of ciliary body and retinal pigment cell layer; HE staining showed the subretinal space of rats after postoperative 5 weeks brown with scattered epithelial cells in the survival when AAV-BDNF-IPE cells transplanted into the subretinal space of RCS rats, sμggesting that is post-transplant survival for the AAV-BDNF-IPE cells; no significant infiltration response of inflammatory cells and structural damage within the retina or choroid.we can not judge the transplantation of retinal cells and their host organizations to establish the correct anatomical contact, but our experimental results show that the way throμgh the improved scleral road that transplanted AAV-BDNF-IPE cells into the subretinal space is safe and reliable and the transplanted cells not only survived,but also do not occur immune rejection.2.Normal control SD rats, RCS rats and AAV-BDNF-IPE cells transplanted RCS rat was discovered in this study that the FERG a, b wave amplitude of three groups rats with 8 weeks after birth dark-adapted maximum response was no significant difference, RCS rats dark-adapted FERG maximum reaction a, b wave amplitudes were significantly decreased from 10 weeks after birth, which were significantly lower than normal SD rats of the same weeks age (P <0.05), and ERG waveform type of RCS rats born after 12 weeks was low and flat, peaks and valleys was no obvious, a, b wave was vague. These results confirm that the progress of retinal pigment degeneration of RCS rats, progressive loss of retinal function. The ERG changes of RCS rats lag behind the changes in retinal morphology.AAV-BDNF-IPEs were transplanted into the subretinal space of RCS rats, the FERG b wave amplitude can be significantly improved, with postnatal weeks prolonged, but RCS rats, b wave has been decreasing slowly, AAV-BDNF-IPE transplant group RCS rats after 10 weeks old after birth remained at a high level of b wave, and the amplitude still significantly higher than intact RCS rats with the same week (12w and 14w group P <0.05), points that the AAV-BDNF-IPE cells transplantation slow the progress of retinal pigment degeneration of RCS rats and improve the part of the retinal function to a certain extent.3. BDNF expression in retinal tissue of intact RCS rats were still high in postnatal 3w, and sharply decreased into a low level with development. BDNF expression in retinal tissue of surgery RCS rats were significantly higher than those in intact rats from 6 weeks after birth to 14 weeks of age during the different periods. These results sμggest that retinal tissue can sustain and stable express high level BDNF after AAV-BDNF-IPEs being transplanted into the subretinal space of RCS rats, which is the important cause that AAV-BDNF-IPE transplantation can improve host retinal function .4.The transplanted group of AAV-BDNF-IPE cells is a little better than the intact group: there was a slight improvement in the side of the thickness of rod layer near the outer nuclear layer, which is consistent with previous studies; TUNEL assays confirmed that the loss of visual cells is a apoptosis phenomenon,and that also sμggest that AAV-BDNF-IPE treatment may inhibit apoptosis and play a role; Immunohistochemical results sμggest that there may be a compensatory response to reduce the proliferation of glial fibers and may also be demonstrated in order to restore the characteristics of synaptic ultrastructure changes. In summary, the experiment sμggest that this method may save the degeneration of photoreceptors and regain function.
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