| Retinitis pigmentosa, is one of the most common diseases in the hereditary blindness, though many ways and multilayers of studies were carried on for the disease in various fields, so far still have no the treatment method to stop the patholgical changes progress and recover visual function. The histopathology had confirmed that RP is a primary photoreceptor degeneration and death, then secondary retinal pigment epithelium change. So, the transplantation of healthy neuroretina-RPE sheets is in order to rescue and replace degenerative photoreceptor cell, promote the resumption and regeneration of remainder retinal cells , rebuild retinal neuronal network, stop the disease progress and recover visual function. It is a method that has most the latent value for clinical application at present. Recently 20 years, the considerable researches about retinal transplantation on animals and patients which had already obtained the initial result: the safety of the surgical operation to be confirmed; the graft can be accepted by the host; The host still has the foundation of the visual function improvement. These provide the dependable laboratory basis and the applied foreground for the treatment of RP and others retinosis diseases. At present,there mainly are embryonic and fetal and adult retinal stem cell, haemopoietic stem cell, nerve stem cell , embryonic neuroretina and embryonic full-thickness retina being used for retinal transplantation ,but all of them are deficient and limited in source ;To use the animal models are most retinal natural degenerative murid models such as RCS rat,rd mouse, under looking indirectly at the dornor was transplanted into the host subretinal trans-scleral outer road method,. the surgical operation method and experience is apart from the clinical application; Currently, the contact of donor tissue and host retina can be seen by morphology and immunohistochmistry, but the study of post-transplantation function to adopt electrophysiological means still lake conviction.Then, can the donor of mature retina by a contribution to become the extensive source be carried on the transplantation? Can the neonatal retina with completed differentiaion and immature become the source path of donor? How to improve and perfect the operative method being built up though retinal transplant experiment on the large animal model ,and come true gradually the convert from animal experiment to clinical application? However,these works about the mature retina donor and large animal model are very litter in retinal transplant domain,The reports on observing morphous and measuring function to evaluate the effect of transplantation after surgery from living body not be seen yet. For this reason,it is a very important and meaningful work. to explore the application.outlook of the extensive source retinal donor and use large animal for the retinal transplant experiment. The purpose of this research of transplantation the completed differentiation and immature the full-thickness newborn piggy retinal tissue is to observe its condition of survival and growth at the subretinal cavity of adult light-induced retinal degenerative pigs, have the integration of modality and function with the host retina or not, to discuss the possibility that the retinal transplantation will be regarded as the important means of the RP clinical treatment, and to lay the foundation for the new technical application and development of transplanting retinal tissue /cells to cure seriouse blindness diseases. This experiment includes four parts:(1) Build up the model of chronic light-induced retinal degeneration in mini-pigs through imitating the process of chronic photoreceptor degeneration in human for a few months or several years, and identify the model by means of mfERG, immunohistochmistry, electron microscopy from the both sides of function and modality. To prepare for retinprepared foodtransplantation curing retinal degenerative experiment (2) To obtain neuroretina-RPE cografts by using excimer laser for microablation of newborn piggy choroidal tissue, and transplant the cografts into the subretinal cavity of light-induced adult pig retinal degeneration through vitreotomy and retinotomy, observe the survival situation and the host retinas have rejection or not by using ophthalmoscope, colour fundus photography and fundus fluorescein angiography from living body level. To develop the retinal transplant implement and set up the operative method of retinal transplant on large animals.(3) The mfERG examines the changes of the the amplitude and latency N1 wave and P1 wave after retinal transplantation so as to compare between at different time in length and breadth and meanwhile combine the results of the fundus observation and FFA , estimate syntheticly the change of retinal function after retinaltransplantation. (4) To adopt the immunohistochmistry technique for judgeing the survival ability of donor tissue under the subretinal cavity of host retina, the integrated manner of donor tissue with host retina and the relation between the survival appearance of graft and retinal function,and discuss the necessity of nueroretina and RPE co-transplant and the possible mechanism of the graft improving the host retinal function. The main results and conclusions of this research are summarized as follows: 1. Twelve adult miniature pigs aged 2 months were exposed constantly to the visible light (spectrum 400-700nm,intensity 2500lux,lighting time 12h/d )for more than 3 months, and induced slowly retinal degeneration in miniature pigs. To measure the amplitude and latency of N1,P1wave by using mfERG,and compare pre-lighting with pro-lighting itself and compare light-induced groups with normal groups, the result is that amplitude N1,P1wave lower obviously within 1-6 rings ,but latency N1,P1wave prolong obviously .In the light-induced retinosis in pigs, the photoreceptor cells were manifested as shortening of outer segments and reduction of cells in the outer nuclear layer (ONL), and the thicknesses of ONL are reduced to 20.98%; TUNEL showed immunohistochmically the positive apoptosis in the ONL of the posterior polar retina by light microscopy. The mitochondria of photoreceper cells swell obviously to follow the rupture of crista and the reduction of quantity by means of the electron microscopy in light damaged pig retinas. The model of light-induced retinal degeneration in the miniature pigs were identified by function and the modality both sides: light harm part was in photoreceper cells, simulated the process of mankind slow photoreceper degeneration in a few months or several years, the model was established successfully, whith had prepared well for the experimental study of the retinal transplantation treating light-induced pig retinal degeneration by vitreo-retinal surgery. 2. Retinas from stages PN1-6 (postnatal days 1-6) normal GuangXin Bama pigs were used as donor tissue. To ablate a 6.5mm area by using PTK mode of excimer laser for microablation of choroidal tissue, the ablated depth from 45μm to 75μm.the choroid was drived out but the RPE layer was kept intactly. Two normal and eight lighting adult pigs retinal transplantations through vitreotomy and using the developed instrument by ourselves were operated successfully on the nineteen eyes, among of lighting pigs, the false surgical operation on one eye. The grafts were transplanted into the subretinal in 16 eyes; the operative success rate is 84.2%. Perioperative complications included limited lens injury,hemorrhage and shallow detachment of the host neuroretina. Postoperative complication was mainly vitreoretinal proliferation. The neuroretina-RPE cografts harvestried were identified that the choroids were wipped off but RPE was kept well by histopathology, the cografts were obtained successfully; the implement for the experimental surgery of retinal transplantation was be expoldered; Through the foundation of the surgery method of retinal transplantation by the vitreo-retinal surgery on large animals, explore the invert path from animals to clinical application. 3. To observe the operated eyes for the normal and lighting pigs by using the ophthalmoscope, colour fundus photography on days 5-7, months 1,2,3,4,5 after retinal transplantation. In host retina the gray-black graft inside transplantation bed can be seen clearly after post-transplant 1-2 months, but there was not the graft in the false operated eye. After post-transplant 1-2 months, the FFA checkd the transplant leaked fluorescence. Retinal transplant months 1-5 after surgery in the lighting pigs, the amplitude and lantency of N1,P1wave were examined by mfERG .The vertical comparison between different period showed the amplitude of N1,P1 wave heightened with the extension of the survival time, and the area of active responsion were in ring 2 and ring 3;But the lantency of N1,P1 wave were shorten markedly in each ring than pre-operation especially in the survive later period. The horizontal comparison to the same lighting pig in different period notified the amplitude of N1,P1 wave were higher greatly than months 2 and months 3,and that the lantency of N1,P1 wave shorten markedly. Using the mfERG to compare the variety of light-induced pig visual function after transplantation from function side, observe the fundus synchronously by ophthalmoscope, FFA from modality, evaluate totally the effect of the visual function improvement, thus confirmed completed differentiation new-born pig retinal transplantation really improved the retinal function of light-induced pigs. 4. After the retinal transplantation, the morphology study found most immature neonatal pig retinas can develop continually,the grafs and host retinas took place to fuse partly, the manner of fusion was a neuronal integration between layers of retina close together , almost all types retinal cells participate the integration .The müller cells can be seen between the graft and host in a fibrillary meshwork, but the fiber of rod bipolar cells and amactine cells have not been discovered yet both of them.The limited integration of transplants and host retinas and the role supporter of the müller cells, that may be thereconstituted foundation for conduction of the retinal information. The neuroretinas of the grafts can most show a laminated appearance with a correct polar in the host subretinal space ,when it lost RPE ,it can form a rosettes and other shape,whith suggested the existence of RPE affect the appearance structure. In addition, we also found the retinal function can be improved on the operative eye if the graft had a opposite polor or unlaminated appearance ,the findings revelated the improvement of retinal function had nothing to do with the survival appearance of the graft . The completed differentiation alloggrafts can survive but did not be rejected in the host eyes at least 5 months , the safety of the host can be confirmed. This research observed that the retinas with completed differentiation and immature of the newborn pigs can develop completely, survive safely, extend to the host in search for the fit target and form the limited integration with the host. The mfERG had confirmed really retinal transplantation to improve the retinal function of the light–induced pigs after surgery. Therefore, we can consider the postnatal retina as a kind of source for retinal transplant donor. The retinal transplant instrument was developed by ourselves ,and the operative method was established through the retinal transplantation experiment on the large animals, explore the operative path of the retinal transplantation treating light-induced retinal degeneration by the vitreo-retinal surgery and the possibility from experiment to the clinical, and we are full of hope for the treatment of retinal degeneration in future.
|
PDF Full Text Request