Studies On Morphological And Functional Properties Of RBCs In RCS Rat During Retinal Degeneration

Retinitis Pigmentosa (RP) is a group of hereditary retinal degeneration characterized by progressive dysfunction of photoreceptors cells (PRC) and associated with progressive cells loss and eventually atrophy of several retinal layers. So far, although researchers have tried on retinal degenerative diseases with retinal transplantation, gene therapy, retinal prosthesis, drug application and so on, no effective treatment methods to prevent progressive retinal degeneration or to restore visual function have been established. The ultimate cause is that the pathophysiological mechanism of retinal degeneration is so complicated and inner layer neurons are also impaired following photoreceptors loss.Retinal bipolar cell(RBCs) is the glutamic acid neuro in the vertebrates retina, accept the signal importation of the photoreceptor, deliver after integrate to amacrine cell and the retinal ganglion cell, RBCs to be placed in a position of key role in the process that the retina signal transduction. In the normal mammal retina the rod system and the cone system is independent mutually in the outer plexiform layer. But in the research about the retinal degeneration animal model that the cone degeneration is the most, the cone- RBCs that lose the cone can stretch out evection to fome the ectopic synase with the remain rod, there is same result in the animal model that the rod degeneration loss much .These finding reveal that the RBCs that losing the signal from the photoreceptor have the synaptic plasticity trying to look for a new signal importation, but this will may cause a pair of changes on the Morphologgical and Functional Properties.Basis on the before researching work, we know that the all the Rod-RBCs is the ON-RBCs, however the Cone-RBCs could be divided into two kinds of RBCs , ON-RBCs and OFF-RBCs. The ino-glutamate receptor only express on the membrane of the OFF-RBCs, and the meta- glutamate receptor only express on the membrane of the ON-RBCs. These two kinds of receptor are the maintain functional receptor of the two different types of the RBCs. In the researching work of the Varela, he find that the Rod-RBCs that lose the signal from the Rod present stronger reaction to the GABA, however the reaction about the glutamat presents the trend of descending, suggesting that in the degeneration , the function of the RBCs have been changed.During the course of the retinal degeneration in the animal models that the Rod degeneration is the starting, by researching , that the mRNA of the mGluR6 in the RBCs present the tendency of the obvious ascendion in the later stage of the retinal degeneration. The trendency may be the reaction about the lossing of the glutamate receptor in the RBCs, and the reaction must influence further more about Electrophysiology functional properties of the RBCs. Marc find that , in the course of the retinal degeneration, the number of the RBCs that express the meta-glutamate receptor is less however the number of the RBCs that express the ion-glutamate receptor have been raised, and it suggesting that the main functional receptor of the RBCs have been changed in the course of the retinal degeneration, it could been conclude the ON-RBCs have the the trendency of the conversion to OFF-RBCs. For the important role of the RBCs in the the signal transduction in the retina, so the remodeling of the RBCs must influence the change of the other retina neuro, and it must be the important parts of the retinal degeneration , so it is very important for studying the remodeling of the RBCs in the retinal degeneration.In the retinal remodeling that the Rod degeneration is the start, the first influence of the second neuro is the rod-RBCs for the loss of the signal from the Rod.And the Rod-RBCs all be the ON-RBCs, the type is solo .Basis the before researching work, the RBCs that lossing the signal could have plasticity for finding new source of the signal. So we conclude the residual ON-RBCs could actively to strench the evection to find the remained photoreceptor such as the Cone, and It will change the Morphological and Functional Properties of the RBCs. Although , through the existing technique that we could not show the abnormal evection precision and integrity, to study how the abnormal evection affect the function of the RBCs, but through the research of the Morphological and Functional Properties of the RBCs, we could research the character of the ectopic evection on the another side, and we could know more about the remodeling in the retinal degeneration. So our hypothesis is that in the RCS rat that the rod degeneration primary, during the course of the degeneration, after the loss of the rod, the remodeling occur in the ON-RBCs. It may be have the ectopic synapse with the cone and resulted in the changes of the morophology and Elcetrophysiological properties of the ON-RBCs Our main results are showed as following:1. The study of the morophological properties of the RBCs in RCS rats at different development stageImmunofluorescence of the retinal frozen section, and Lucifer Yellow staining solo ON-RBCs.Results:1) Total amount of the ON-RBCs is less during the retinal degeneration, P60d and P90d significantly less than control rat(P<0.05 or P<0.01).2)The amount of the ON-RBCs that is in the posterior retina that the Cone Concentrated district is increased in the retinal degeneration, much higer than the control group in P60d and P90d. 3) The amount of the ON-RBCs that is in the perimeter pole of the retina that the Rod Concentrated district is significantly decreased during the retinal degeneration, much less than the control group in P60d and P90d(P<0.01). 4)The dendrites of the ON-RBCs is underdevelopment and the terminal branch of axon decrease in the retinal degeneration, the cell is immature appearance. At the same time ,ON-RBCs strench out the evection that express mGluR6 in different direction . In the late period of the degeneration ,those ectopic evection could be entwined togather and it is seems have been the evidence that it have the functionally synapse activity.2. Elcetrophysiological properties of ON-RBCs during retinal dengeration. Methods: patch clamp recording of the ON-RBCs cells in the retinal slice. Results:1)With the progress of RD, RMP of ON-RBCs cells from RCS rats retina depolarized dramatically, and IR, Cm increased significantly higher than control (P<0.05) .2) outward currents were recorded when giving hyper- and depolarized voltage steps, which could be blocked by TEA, implying a predominant distribution of K+ channels on ON-RBCs cells . 3)during the course of the degeneration of RCS rats retinal degeneration, the outward currents increased, and were significantly higher than control, which is considered to eliminate the extracelluar K+ produced in the RD,. 4)At P60d the K+ currents density of ON-RBCs cells in RCS rats was significantly higher (P<0.05) than control; The increased of K+ current density implied that the number of K+ channels might increased during retinal degeneration. 5)The I-V curves of RCS rats’ON-RBCs cells showed, at P60d, with the increase of voltage impulse, the current amplitude of RCS rats ON-RBCs cells increased more rapidly than control. 6)In the P60d and P90d of the RCS rat, the amplitude and density of the outward K+ current and density of the current of the ON-RBCs in the RCS are significant different with the ON-RBCs and OFF-RBCs in the control Rat. electrophysiological characteristics of the ON-RBCs in the RCS is different with any kinds of RBCs in the control ratBased on the results, we concluded:1. In the course of the retinal degeneration of the RCS, the total number of the ON-RBCs cells is descent. It suggest that the ON-RBCs have been loss after the signal from the photoreceptor loss.2. In the retinal degeneration of the RCS, For the signal loss, the ON-RBCs that in the position of the ambitus-retina which have much more rod have been severely loss. It suggest that the loss of the signal from the photoreceptor induce the severely loss of the ON-RBCs.3. In the retinal degeneration of the RCS, the ON-RBCs that in the position of the posterior retina which have much more cone have no loss, It suggest that reason for the ON-RBCs suriving in the district of the retina is that the signal from the cone existed.4. In the retinal remodeling of the RCS, the morphology of the dendrites of the ON-RBCs is immature, the branch of the axon is less than normal. At the same time, ON-RBCs stretch out the ectopic evection in all direction that positive express the mGluR6. And in the late stage of the retinal degeneration, those ectopic evection tangle together, and the proof of the functional activity of the synapse could been seen. It conclude that, in the retina degeneration, the ON-RBCs have the synaptic plasticity for searching for new signal.5. The action of the ion channel in the ON-RBCs of the RCS rat is outward K+ current in the depolarization. In the retinal degeneration of the RCS, the amplitude of the outward current is significant higher than the control. It reveal the function of the ON-RBCs have been changed in the course of the retinal degeneration, and maybe the ON-RBCs lower the K+ retention in the cell by the way, and the change of the function of the transduction of the ON-RBCs6. In the P60d and P90d of the retinal degeneration in RCS, the electrophysiological characteristics, amplitude of the outward K+ current and density of the current of the ON-RBCs in the RCS are significant different with the ON-RBCs and OFF-RBCs in the control Rat. It suggest that in the retinal remodeling the type of and the function of the K+ ion channel have been modified, and the change result in electrophysiological characteristics of the ON-RBCs in the RCS is different with any kinds of RBCs in the control rat.