The Influence Of Inner Retinal Neurons And Glial Cells In Retinitis Pigmentosa Rats

In vertebrates, retinal pigment epithelial (RPE) cells locate between the retina nerve layer and choroid. They are irregular polygon and closely arranged in a single layer. There apical microvilli extensivily contact with the light-sensitive outer segments of photoreceptor cells, which make information to be transmitted between RPE cells and photoreceptors. The main functions of RPE include exchanging photopigments with photoreceptors, phagocytizing outer segments of photoreceptors, uptakeng metabolic wastes from photorecepters, transporting nutrition to photorecepters and secreting some growth factors. Therefore, RPE plays an important role in normal function of photorecepters and the dynamic balance of visual pathways. The destruction of RPE structural or funtional integrity is the basal pathology in many retinal diseases, such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD). Additionly, various retinal diseases are thought to be connected with RPE degeneration such as Stargardt disease, retinitis pigmentosa and retinal dystrophies.The previours studise of RPE degenerated animal models, specifically the acute degeneration, were mostly focus on RPE and photoreceptors. Few studies reported the changes of inner retinal neurons. We must realize the importance of integral inner retinal neurons to the therapy of RPE degenerated diseases such as retinal transplantation and prosthesis. Only enough healthy inner retinal neurons present, visaul signal can be transmitted in from repaired outer retina and visaul function can be restored effectively. Thus we need to research what morphological or functional changes happen to the inner retinal neurons and whether these changes are in a direct way or secondary to the injurey of photoreceptors in the RPE degenerated diseases and what the mechanisms are.In addition, M(?)ller cells are the main glial cells in retina and have many important physiologic functions include structural support, regulation of osmotic balance, neurons nutrient transport and metabolism and Blood-retina barrier and so on. Recent researches show that Miiller cells have the characteristic of retinal progenitor cells in both structure and function and are considered to be an endogenous retinal stem cells. In some lower vertebrate, Miiller cells can dedifferentiate into retinal progenitor cells and reenter the cell cycle under specific conditions. Furthermore these dedifferentiated M(?)ller cells will trandifferentiate into retinal neurons, which restore the visual function completely. However, in mammals, the activation of Miiller cells after retinal injuries is very difficult and the number of dedifferentiated M(?)ller cells is small. Then M(?)ller cells form glial scar in the subretinal space soon and accelerate the process of lesions. Thus we want to know whether Miiller cells have the characteristic of retinal progenitor cells in RPE degenerated diseases and what the mechanisms are contained. Further we hope to find the key that starts M(?)ller cells dedifferentiate into retinal progenitor cells and prevents them form glial scar simultaneously, and then promotes Miiller cells trandifferentiate into retinal neurons to repair the damaged retina.Based on the researches above, this study adopts the acute retinitis pigmentosa model: Sodium iodate (NaIO3)-induced retinal injury model and the chronic retinitis pigmentosa model:Royal college of surgeonrat (RCS) rat to explore the changes of inner retinal neurons during acute retinitis pigmentosa and the changes of Miiller cells during chronic retinitis pigmentosa. The main results are as follows:The influence of inner retinal neurons in acute retinitis pigmentosa rats.We found the inner retinal neurons could be injuried by NaIO3 at the early stage of retinal degeneration. The dentritic fields of dopaminergic amacrine cells (DA-ACs), melanopsin-expressing retinal ganglion cells (mRGCs) and horizontal cells were all decreased. At the same time, NaI03 also had damage to the ventral tegmental area (VTA) and substantia Nigra Pars Compacta (SNpc), which are the regions content abundant dopaminergic neurons in midbrain. This suggests that there is aiso a direct mechanism in the injury of DA-ACs in retina. In addition, we observed that the expression of microRNA 133b was significantly upregulated in DA-ACs after NaIO3 administration, which was thought to be specificlly expressed in midbrain dopaminergic neurons. The overexpressed mir-133b decreased the expression level of pitx3, which was an important transcription factor in the dopaminergic system, and then downregulated the expression of tyrosine hydroxylase (TH) and D2 recepter, and the production of dopamine (DA). This may be the main cause lead to the DA-ACs, mRGCs and horizontal cells injury. When mir-133b level was inhibited by mir-133b/RNAi, those markers reduced above were rescued and the dentritic fields of DA-ACs and mRGCs were restored. It was worth noting that the the amplitude of b-wave and summed OPs of Electroretinogram (ERG) were both increased significantly, suggesting the visaul funtion was improved to some extent.The influence of M(?)ller cells in chrinic retinitis pigmentosa rats.We confirmed M(?)ller cells could express the retinal progenitor cell markers transitorily at the early stage of retinal dengneration in RCS rats, but they soon formed glial scar and aggravated photoreceptor apoptosis. Our primary research shown the let-7 family moleculars, let-7e and let-7i were upregulated in M(?)ller cells during the retinal degeneration. A RNA binding protein, Lin28, is considered to be the post-transcriptionally regulated factor of let-7 family moleculars. Our results demonstrated the downregulation of Lin28B was the key point responsible for overexpression of let-7e and let-7i. When Lin28B was upregulated in retina in vivo or in vitro, let-7e and let-7i expression would be reduced in M(?)ller cells, Thereby M(?)ller cells were activated to dedifferentiate, proliferate and transdifferentiate into retinal neurons. Simultaneously the glial fate acquisition of Miiller cells was inhibited. ERG recording shown the amplitude of a-wave and b-wave were increased significantly after Lin28B ectopic expression. These data indicate that Lin28B/let-7 signaling pathways may play a very important role in the retinal regeneration and repaire in RCS rats.In summeray, we draw the following conclusions:1. Inner retinal neurons are damaged in different degrees in NaIO3-reduced acute retinitis pigmentosa rats, which is not only caused by loss of input message from photoreceptors but also the diretct influence from pathological factors.2. The structure and function of DA-ACs are severely affected in NaIO3-reduced acute retinitis pigmentosa rats, which leads to the secondary degeneration of mRGCs.3. Overexpressin of mir-133b and downregulation of its downstream factor pitx3 is responsible for the degeneration of DA-ACs and mRGCs in NaI03-reduced acute retinitis pigmentosa rats.4. M(?)ller cells have the characteristic of retinal progenitor cells at the early stage of retinal degeneration in RCS rats, but they form glial scar soon with the development of lesion and aggravated photoreceptor apoptosis.5. Downregulation of Lin28B and upregulation of let-7e and let-7i are the mechanism impeted M(?)ller cells dedifferentiate into retinal progenitor cells in RCS rats.6. Overexpressin of Lin28B can prevent M(?)ller cells gliosis and promote the dedifferentiated MUller cells transdifferentiate into retinal neurons in RCS rats.