Transplanted Olfactory Ensheathing Cells Reduce Retinal Degeneration In Royal College Of Surgeons Rats

Retinitis pigmentosa (RP) is a group of genetic disorders in which a progressive loss ofvision is caused by a cascade of retinal degenerative events. Currently, there is no realtreatment for RP. Among several experimental approaches for the treatment of RP, celltransplantation offers a novel therapeutic approach. However, cell-based therapeutic such asstem cell and Retinal pigment epithelial cell (RPE)transplantation have been limited todate due to donor cells source limitation, immunological rejection and ethics argument.A series of complicated morphological and function changes happen during theprocess of RP. The accumulated optic discs in subretinal space cause metabolic abnormalityof photoreceptor outer segments (POS), followed by photoreceptor death and extensiveretinal neurons' apotosis, necrosis and synapse remodeling. In normal retina, Müller cellsare involved in many retinal physiological activities including glycometabolism, bloodregulation, neurotransmitter cycling, homeostasis and control of neuronal excitability. Inearly stage of RP, the activated Müller cells proliferate dramatically, and form glial scar insubretinal space, which invest the remnant retina and seal it from access via the retinalpigmented epithelium and choroidal route, result in death of photoreceptor cells and otherneurons, and prevent regeneration of outer segment of photoreceptors. Besides, a series ofinhibiting factors expressed on the surface of Müller cells prevent the tissure repair andneuron regeneration3,20.The neuron loss and gliosis in RP is similar to that in central nervous system (CNS)injuries. Glial cells with astrocytes as main component formed glial scar at CNS injuriysites, which prevent the regeneration of neuron axons. More recent studies have shown thattransplantation of olfactory ensheathing cells (OECs) has become one of the mostpromising therapies for damage to the nervous system by protecting neurons5. They arethought to open up the glial scar, and provide a pathway for the successful axonregeneration throughout the life of adult mammals9-13, which is positively influenced by the presence of olfactory nerve fibroblasts (ONFs)14. OECs reside in the olfactory system,which supports neurogenesis throughout life6-8. There is no doubt that olfactoryensheathing cells represent the leading candidate for transplant-mediated repair of CNSinjury for supporting axon regeneration and CNS function restoration ability, especiallyautologous transplantation possibility.The possibility of OECs application in pathology and trauma of the retina and opticnerve has only recently been explored15-19. It was found that transplanted OECs ensheatheretinal ganglion cell(RGC) axons, delayed the death of axotomized RGCs and promotedregeneration of cut adult rat optic nerve axons. Until now, there is no report about theapplication of OECs in RP.Based on the all the researches above and the previous results in our lab, we make ahypothesis that OECs transplantation have the ability to reduce retinal degeneration in RPby protecting retinal neurons and inhibiting the formation of glial scar formed by Müllercells.Here we report morphological and visual function analysis of pigmented RoyalCollege of Surgeons (RCS-P+) rats transplanted with mixture of OECs,compared withunoperated and sham operated groups at the same age. We also investigate the mechanismsof OECs playing important roles in retinas. Our main results are showed as following:1. The bionomics research of OECs in vitro and in vivo. Cultured syngeneic adult ratolfactory ensheathing cells transplanted into the subretinal space of RCS-P+rats survivedand presented multiple style appearance. There was a slight increase in the total count ofcells in the3-4week group, with a decrease at6-8weeks. The transplanted cells spreadedinto the surrounding space and migrated from the subretinal space through all the overlyingretinal layers with a gradual increase at longer survivals. The transplanted OECs in normalcontrol rats didn't show obvious migration. Hints from results: OECs can live and migratein degenerated retina. Their migration ability has a close relationship with retinalenvironment.2. Immunohistochemistry and western blots showed a striking reduction in Müller cellGFAP maintained over the entire current period of survival after cell transplantation.RCS-P+rats at4,8and12weeks after OECs transplantation had an increased survival ofphotoreceptors by detecting recoverin, retinal outer segments, and PNA-positive cones. Caspase-positive apoptotic figures in the outer nuclear layer were decreased. The b-wave ofelectroretinography (ERG) was maintained. Hints: OECs inhibit the abnormal activation ofMüller cells, reduce retinal neurons' apoptosis, protect photoreceptors, improve retinalfunction and reduce the retinal degeneration in RP.3. The mechanism research of OECs'role in RP. We examined the expression ofneurotrophic factors and identified the cultured OECs mixtures expressed and secreted NGF,BDNF, and bFGF which contributed to assist survival of the photoreceptors. Furthermore,in a culture assay, we noted that OECs can phagocytose porcine POS, and the phagocyticability of OECs was even stronger than that of RPE cells. Furthermore, we explored specialproperties of OECs phagocytosis to identify the contribution of some important moleculesinvolved in phagocytic process and POS binding and internalization kinetics, includingmRNA level changes of integrinαvβ5,TLR4,CD36,pFAK,Mertk and cathepsinD. Itshowed that the phagocytosis process by OECs had similar mechanisms with RPE. Hints:The role of OECs on RP is reducted by multiple ways. The research on the mechanismmakes a basis for the application of OECs in cure of retinal diseases.Conclusions:1. The research firstly reports that OECs have ability to survive and migrate indegenerated retina, which establish basis of the clinical application of autograft;2. OECsnot only protect retinal neurons but also inhibited the over-activation of Müller cells. Themultifunction makes more contributions on the reduction of retinal degeneration process;3.OECs transplantation has a role on the rebuilding of visual function;4. OECs have abilityto secrete neurotrophins, and supported retinal neurons;5. The phagocytosis of OECs onphotoreceptor outer segments (POS) solves the problem of retinal dysbolismus resulted byRPE dysfunction, and makes great contributions on homeostasis of retina.