| Retinitis pigmentosa(RP) is a class of diseases that leads to progressive visual decline resulting from continuing loss of photoreceptor cells and outer nuclear layers of the retina. At present, this kind of degenerative retinal diseases remains incurable. The mammalian retina’s structure is organized, all light detection is performed by two visual pathways:the image forming and the non-image forming systems. In our traditional idea, there are only two kind of photoreceptors in the retina, the rod and the cone. They carry out the ?rst step in vision by capturing light and transducing it into electrical signals. But in recent years, a kind of special retinal ganglion cells were found, they express the photopigment melanopsin and are intrinsically photosensitive, so called intrinsically photosensitive ganglion cells(ip RGC). At first, ipRGCs was considered to be involved in circadian rhythm modulation of sleep and alertness, and within few years it evident that these photoreceptors mediate irradiance detection for behavioral and physiological responses to light. But whether the melanopsin be involved in the image forming system is still unclear.Experimental approaches to the treatment have advanced in recent years, including drug development, stem cell transplantation, gene therapy, light-sensitive proteins, and so on. The light-sensitive proteins, for example Channelrhodopsins(ChR) and melanopsin are the hot spots in recent years. Compared with Ch R2, melanopsin exist in human retina, We want to see if we used a viral vector to transfect melanopsin in retinal degeneration model retina, whether it can restore the visual function of them. In other words, may be modulate melanopsin function or expression is a therapeutic method for the patient who has the retinal degeneration disease.Accordingly,we use the Royal College of Surgeons rats and the rd1 mice model.RCS is the widely used retinal degeneration model which has a mutation in the Mertk gene expressed in RPE cells that results in progressive photoreceptor cell death and loss of electrophysiological response.And the rd1 is an rapidly retinal degeneration model which has a mutation in the phosphodiesterase type 6(PDE6)-beta subunit causing complete loss of rod and cone photoreceptors and lead to irreversible blindness. Some researches have reported that the mouse-melanopsin can restored the rd1 mice visual function, but no lab had used human-melanopsin, and whether the human-melanopsin can restored the visual function of retinal degeneration models is still not known.Methods:1. Overexpression of mouse melanopsin in retina whether can restores visual function in Royal College of Surgeons rats.1.1. Construction of mouse melanopsin-AV vector, and use cell patch clamp method to recorded the light response of mouse melanopsin to 480 nm light stimulus.1.2. Detection the change of ipRGC distribution and melanopsin protein expression level in the RCS rats1.3. Overexpression of mouse melanopsin in RCS rats retina: use WB method to detected the melanopsin protein level of the experimental group and the control group.1.4. Subretinal injcetion of mouse melanopsin-AV in RCS rats model: F-ERG and behavioral testing after injection..2. Transfection of human melanopsin-AAV2/8 in the retina whether can restores visual function in retinal degeneration rodent.2.1. Identification of the rd1 model: HE staining.2.2. Construction of CMV-human melanopsin-AAV2/8-YFP vector, and use cell patch clamp method to recorded the light response of mouse melanopsin to 420 nm light stimulus.2.3. Subretinal injection of CMV-human melanopsin-AAV2/8-YFP in rd1 model: immunofluorescence staining the transfecte cells, F-VEP and behavioral testing after injection.2.4. Subretinal injcetion of CMV-human melanopsin-AAV2/8-YFP in RCS rats model: immunofluorescence staining the transfecte cells, F-ERG /F-VEP and behavioral testing after injection.2.5. Vitreous injcetion of CMV-human melanopsin-AAV2/8-YFP in RCS rats model: immunofluorescence staining the transfecte cells, F-ERG /F-VEP and behavioral testing after injection.The main results of our research are as follows:Overexpression of mouse melanopsin in retina restores visual function in Royal College of Surgeons rats.Firstly,We use the method of immunohistochemisty and western blot to detected distribution and protein expression of melanopsin in RCS rat retina. The melanopsin expression was gradually depressed in retinal degeneration model. But the melanopsin positive cell numbers do not observed the similar tendency, there is no statistic difference between the three age. When we pay attention to the axons of ip RGC, we find that the length of axons is significant shorten after 30 postnatal days. These data indicated that RCS rat is a chronic degeneration model, the melanopsin expression is gradually decreased with the age. And we should ascribed this kind of diminished expression of melanopsin to decrescent length of dendritic axons of ipRGCs, but not the loss of ipRGCs number. Then, RCS rat right eye were received subretinal injection of adenovirus(AV)-OPN4-GFP, and left eye received PBS in FERG experimental group; and both eye received subretinal injection of adenovirus(AV)-OPN4-GFP in behavioral testing. The FERG(flash electroretinogram) was examined three weeks later. Behavioral test was used to evaluate the behavior scores after the injection. The results indicated that the b-wave of FERG was higher in the exprerimeatal group compared to the control group(P<0.05). And the experimental rats showed behavioral avoidance of light in an open-field test(a kind of behavioral tests). Briefly, the overexpressed of mouse melanopsin in RCS rats retina can restore visual function of retina degenerational RCS rats.Transfection of human melanopsin-AAV2/8 in the retina can restores visual function in retinal degeneration rodent.We use the method of immunohistochemisty to confirm human melanopsin were successfully transfected into the rd1 mice and the RCS rat retina. The right eye received subretinal or vitreous injection of adeno-associated virus(AAV)-h Mel-YFP, and left eye were untreated with PBS in the FERG and FVEP recording; and both eye received subretinal or vitreous injection of adeno-associated virus(AAV)-h Mel-YFP in behavioral testing. The FERG/FVEP and behavioral test were examined 30 days later. The results indicated that human melanopsin protein can successfully expressed in rd1 mice and RCS rat retina. The P1-wave of FVEP in experimental rd1 mice and RCS rat was higher compared to the control group(P<0.05). The b-wave of FERG was higher in the RCS rat exprerimental group(P<0.05),but there is no difference between the exprerimental and the control group of rd1 mice. And in the behavioral test, The AAV-human melanopsin treated rats and rd1 mice showed behavioral aversion to light,they spent longer time in the dark zone than control group.(P<0.05) and the AAV-human melanopsin treated RCS rats also peformed higher visual acuity than control group.(P<0.05).In conclusion, we draw the following conclusions:1. The melanopsin expression is gradually decreased with the age in the RCS rat, and we should ascribed this kind of diminished expression of melanopsin to decrescent length of dendritic axons of ip RGCs, but not the loss of ipRGCs number;2. Constructed mouse-melanopsin AV and overexpressed of mouse melanopsin in retina can restores the visual function in Royal College of Surgeons rats;3. Tansfection of CMV-human melanopsin-AAV2/8 can restores the visual function in rd1 mice;4. Transfection of AAV2/8-human melanopsin can preserve visual function in Royal College of Surgeons rats, and the subretinal injection method is more fit for the treatment. However, after 60 days of transfection, the human melanopsin protein was significantly decreased, we did not observe the maintenance of visual function. |
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