Photoreceptor Degeneration And The Resultant Effect On Inner Retina In Rcs Rats

Retinitis Pigmentosa (RP) is an inherited retinal degeneration that leads to blindness. It is currently incurable. As 90% of the information people acquire from the environment are visual information, vision loss not only affect each individual's life, but cause great burden to the society. Enormous efforts have been invested to find effective treatments that can stop the progression of the disease or restore visual function of RP patients. Gene therapy, transplantation, neuroprotection and visual prosthesis have been developed. Although none of these future treatments is currently operating in humans, great experimental achievements have been achieved. Among these, retinal prosthesis has achieved extraordinary success and proven to be effective in some clinical treatments. Subretinal multielectrode implants that produce retinotopically correct patterns allow totally-blind RP patients to recognize individual letters and simple objects. These researches have brought great hope for RP patients. However, the basis of these treatments and the determinant factors whether these treatments can achieve success greatly relied on the residual morphology and function of the inner retina.Estimates of residual function of dystrophic inner retina have led to somewhat contradictory results. In some studies, it was found that survived retinal ganglion cells (RGCs) could maintain their characteristic morphology and basic physiology in late stages of retinal degeneration. In light-induced retinal degeneration rats, specifically express Channelrhodopsin-2 (CHR2) in the RGCs under the regulation of a Thy-1.2 promoter when almost all the cells in the outer nuclear layer (ONL) are absent, could restore effective vision. This further indicates that basic electrophysiological properties of these residual RGCs are preserved. Besides, studies on RP patients have also proven that lots of RGCs in dystrophic retina could maintain normal synaptic connections and function. Researchers who hold this idea strongly believe in the good preservation of the inner retina, and consider it to be the theoretical basis of many experimental therapies. However, other researches brought up the concept of'retinal remodeling'and believe that dystrophic retina have undergone great changes in both morphology and function after the gradual loss of photoreceptors. It involves loss and migration of inner neurons, the formation of miconeuroma and aberrant neural circuits, the hypertrophy of müller glia and the resultant glial seal. Retinal remodeling posseses great challenge to these experimental therapies and diminish the hope of RP patients.A series of study have been carried out in our lab concerning the morphology and function of the inner retina in Royal college surgeon (RCS) dystrophic rats. It was found that cells in the ganglion cell layers (GCL) which have the diameter larger than 12μm, including all theα-RGCs and part of theβ,γ–RGCs , were gradually decreased during retinal degeneration in RCS rats (Zhang CX, et al). In the outer plexiform layer (OPL), the expression of Met-glutamic acid receptor 6(mGluR6) stretch intoONL and synaptophysin was always found to be around it. These results indicated that the newly sprouted dendrites of these ON bipolar cells might form synaptic connection with other cells (Zhang CX, et al). Patch clamp recording of single cells revealed that the electrophysiological properties of rod bipolar cells (RB) have changed into another phenotype between RB and cone bipolar cells (CB) (Zhang CX, et al). Residual RGCs of the dystrophic RCS rats could maintain their characteristic morphology, but their discharge ability were inevitable decreased during degeneration, 26.7% of the RGCs couldn't be induced to fire action potential under depolarized stimulation at P7-8W; 63.2% of the cells lose the ability to fire action potential at P9-12W.Retina is a highly dedicated neural network. The accurate transmission of visual signal not only depend on the basic electrophysiological property of each cell, but rely greatly on the cooperation of all the cells and their special role in visual transmission. We couldn't help thinking that whether the visual transmission functions of bipolar cells and RGCs in the inner retina are well preserved during retinal degeneration in RCS rats. This might more properly reflect the function of the inner retina and could provide clues and direction for the development of new treatments.RCS rat is an inherited retinal degeneration caused by mutation of Mertk in retinal pigment epithelial cells (RPE). As cone account for 1-3% of all photoreceptors in normal rats, and cone death are secondary to rod degeneration in this dystrophic rats, early investigations in photoreceptor degeneration have neglected cone death. However cones are responsible for high-acuity and color vision, and thus very important for visual function. More than 50% of the bipolar cells are CBs, they receive synaptic inputs from cones which only account for 1-3% of all photoreceptors. This further demonstrates the crucial role of cones in visual transmission. Previous studies on RCS rats revealed that rod degeneration began shortly after eye opening around P18, rod death became obvious at about P25, until P90 almost all the rods have disappeared. We infer that cones play dominant role in affecting inner retina in RCS rats in which rods quickly degenerated.The present study investigated the characteristic of cone degeneration in RCS rats; the changes of bipolar cell synaptic connection in the OPL and the resultant changes in output signal of ON/OFF bipolar cells; at last light stimulation patch clamp tech were applied to investigate the light response properties ofα-RGCs follow photoreceptor degeneration and output signal change of bipolar cells. The study was divided into three parts.Part 1. Temporal and spatial characteristics of cone degeneration in RCS rats during retinal degeneration.Peanut agglutinin (PNA) was used as biomarker for cone. Both retinal slices and whole mounts were used in PNA histochemistry to demonstrate cone degeneration in RCS rats. It was found that:1. Cone degeneration began around P30 in RCS rats during retinal degeneration. Early signs of cone degeneration were shorten of outer segments (OSs), swollen of inner segments (ISs) and then the cell died. Cone density in P45 RCS rats further decreased, and all the OSs of cones disappeared, ISs became more swollen. By P60, OSs and ISs of cones in the central retina totally disappeared, leaving only enlarged cone pedicles. The number of enlarged cone pedicles was reduced in P90 RCS rats. In RCS rats, rods degenerate immediately after eye open and progress very quickly. Cones degenerate secondary to rods and become the major source of visual function in dystrophic retina.2. In RCS rats, cone degeneration began in the central retina and gradually spread to the periphery. In the same dystrophic retina, residual cones in the peripheral retina posses comparatively better morphology than central retina, and cone density in the peripheral is greater than that in the central retina. Among the four quadrant of the retina, the rate of IS density decline was slowest in the inferior and greatest in the superior. During retina degeneration in RCS rats, follow the path of rod degeneration, cone degeneration progress from the central to the peripheral, and the degeneration rate differs in each quadrant.Part 2. Changes in bipolar cells after photoreceptor degeneration in RCS rats. PKCa immunohistochemistry and double labeling tech were used to investigate the changes in the dendrites of RB in the OPL. Recoverin immunohistiochemistry were used to label both type II and type VIII CB. The axon terminals of ON/OFF bipolar cells were evaluated by statistical analysis of axon terminals of RB, VIII CB and II CB. It was found that:1. With the degeneration of rods, RBs withdrew their dendrites around P30; all the dendrites had lost until P45. By P60, RBs in the central retina sprouted abnormal process into the subretinal space fulfilled with debris of photoreceptors. The amount of abnormal process increased at P90, and this phenomenon was very rare in the peripheral retina. In RCS rats, as the output signal from rods diminishes, the dendrites of RBs were gradually lost. In the late stages of retinal degeneration, to seek more signals to maintain their survival, RBs sprout many new abnormal processes.2. The relationship between the abnormal processes of RB and the residual cone pedicles became closer during retinal degeneration in RCS rats. At P60, abnormal processes of RBs were found to embrace the enlarged cone pedicles. By P90, they overlap each other, indicating that synaptic connections might be formed between RBs and cones in dystrophic retina. 3. Axon terminals of ON pathway represented by of RB and VIII CB began to decrease around P30 in RCS rats, while minor decrease in the axon terminals of OFF pathway represented by II CB was seen until P60. In addition, the number of VIII CB began to decrease around P30, while the proportion of II CB increased with the progression of the disease. In RCS rats, as the differential degeneration of rods and cones, and the changed microenvironment in dystrophic retina, OFF pathway were comparatively well preserved than ON pathway at the level of bipolar cell. As all the all the input signals of OFF pathway come from cones, these results further demonstrate that, with the progress of retinal degeneration, cones and cone signal pathway gradually become the major source of visual function in dystrophic retina.Part 3. Light response properties ofα-RGCs during retinal degeneration in RCS rats.Light stimulation platform were built on the basis of patch clamp recording system. And light stimulation patch clamp tech were applied to investigate the light response properties ofα-RGCs in RCS rats. Besides, the response of light irresponsiveα-RGCs to deplorize pulse and direct bipolar electrical stimulation were recorded. It was found that:1. Based on the patch clamp recording systems in the lab, we successfully built the light stimulation platform. It consisted of light control software, light deliver screen, concave lens, cold mirror, photodiode circuit board, 1440 A/D digital converter. The use of cold mirror ensured that the stimulation pattern could be projected onto the retina. And the use of photodiode circuit board and the connected 1440 A/D digital converter ensured the synchronism of light stimulation and light response. We successfully recorded classic ON and OFF light responses in normal adult rat retina. Light stimulation platform and light stimulation patch clamp tech make it possible to record the light response of single cell in the retina, which could more directly reflect visual transmission function of retinal cells.2. In RCS rats, at P30, 66.7% of theα-RGCs possessed light response ability, and among these responsive cells, ON/OFF ratio was 7:11. At P45, only 47.6% of theα-RGCs remain light responsive, ON/OFF ratio decreased to 1:4. By P60 and P90, the proportion of light responsive cells further decreased to 21.4% and 12.5% respectively, ON/OFF ratio continue to decrease with the progression of the disease. Until P90, all the three recorded light responsive cells were OFFα-RGCs. Statistical analysis of the signal-noise ratio and light-evoked action potential frequency of ON/OFF cells revealed that S-N ratio and light-evoked frequency of both type greatly decreased since P30, and the decrease rate of ONα-RGCs is greater than OFFα-RGCs. The change in ON/OFF ratio and the differential decrease in S-N ratio and light-evoked frequency indicated that light response of OFFα-RGCs were comparatively well preserved than ONα-RGCs in RCS rats during retinal degeneration. The light response of OFFα-RGCs relied on the input signal from cone and OFF CB. The survival of cone and better preservation of OFF pathway in dystrophic retina ensured the well preservation of OFFα-RGCs light response. In late stages of retinal degeneration in RCS rats, all the recorded light responsive cells were OFFα-RGCs further confirmed that cone and OFF pathway gradually became the dominant factors in affecting the function of dystrophic retina.In addition, it was found that the spontaneous spike frequency of ON and OFFα-RGCs increased first and then decreased; and the change in spike frequency of ONα-RGCs was larger than that of OFF cells. Spike frequency of irresponsiveα-RGCs gradually increased with age in RCS rats. The decrease in spike frequency of ON/OFFα-RGCs and the increase in spike frequency of irresponsiveα-RGCs were indications of injury. And the cause and significance for the changes in ON/OFF spike frequency and the difference between them needs further investigation.3. In P30 RCS rats, all the light irresponsiveα-RGCs could be induced to fire action potential under depolarize pulse. In P45 and P60 RCS rats, more than 80% of the irresponsive cells respond to depolarize pulse. By P90, only 53.8% of the irresponsive cells maintain this property. Statistical analysis demonstrated that induced spike frequency under 20pA current gradually decreased with age in RCS rats, suggesting that the function of these irresponsiveα-RGCs were partially compromised. Besides, direct bipolar stimulation of the irresponsiveα-RGCs in P90 RCS rats could elicit action potential, and the fire pattern changed with the alternation of the stimulation parameters. When the stimulation parameter were adjusted to 1 volt, 1ms, and 10s separation, the fire pattern to each stimulation were almost the same. In RCS rats, the ability to fire action potential under depolarize pulse were higher than under light stimulation indicates that the loss of input signal from photoreceptors is the major reason whyα-RGCs lost response to light stimulation; if appropriate and efficient signal input is provided, irresponsiveα-RGCs in dystrophic retina could encode this stimulation into action potential.We came to the conclusion that:1. During retinal degeneration in RCS rats, cones degenerate secondary to rods and progress from the central to the peripheral. Survived cones gradually become the major source of visual function in dystrophic retina.2. In RCS rats, with the degeneration of photoreceptors, at early stages RBs withdraw their normal dendrites because gradual loss of input signals from rods. At late stages of degeneration, to maintain its survival, RBs in the central retina sprouted many abnormal processes. And some of these abnormal processes of RBs might make synaptic connection with the residual cone pedicles.3. In RCS rats, as the differential degeneration of rods and cones, axon terminals of OFF bipolar cells diminish later and slower than ON bipolar cells, and the proportion of II CB increases with the progress of retinal degeneration. These results indicate that OFF pathway are comparatively better preserved than ON pathway at the level of bipolar cell. As all the input signal of OFF pathway come from cones, these results further demonstrate that, with the progress of retinal degeneration, cones and cone signal pathway gradually become the major source of visual function in dystrophic retina.4. Increasing numbers ofα-RGCs lost response to light during retinal degeneration in RCS rats. Among the light responsive cells, the proportion of OFFα-RGCs increased gradually, and the signal-noise ratio and light-evoked spike frequency of OFFα-RGCs were better preserved. Light response of OFFα-RGCs depends on the cone drive OFF pathway. It's just the well preservation of cones and OFF cone pathway that ensures the resultant well preservation of OFFα-RGCs, and makes it the dominant factor affecting visual function of dystrophic retina.5. Lots of the light irresponsiveα-RGCs still posses the ability to respond to depolarize pulse indicating that the main reason whyα-RGCs lost light response is the defect in input signal from photoreceptors. However, the decrease in induced spike frequency with age suggests that the electrophysilogical function of these irresponsiveα-RGCs is partially compromised. Besides, direct bipolar stimulation of the irresponsiveα-RGCs in P90 RCS rats could elicit action potential. This suggested that the light irresponsiveα-RGCs could fire action potential under appropriate and efficient signal input. However, the following problems still need to be addressed before further investigation. What kind of electrical stimulation could be used in vivo; whether the direction stimulation results in permanent cell damage or elicits abnormal vision; how the electrical parameter reflect the visual information from the environment; whether the action potential elicited by electrical stimulation could be transmitted to visual center.