Remodeling Of Inner Retinal Neural Cells During Retinal Degeneration

BackgroundRetinitis 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.In our laboratory RCS rat, which is a kind of classical RP animal model, was adopted and found that at late stage of retinal degeneration the amount of RGCs decreased dramatically with memberane properties and action potential (AP) impaired severely. Remarkably AP could not be evoked in part of RGCs. Nevertheless the trigger mechanism of secondary degeneration is still unknown.There are a kind of special RGCs which can perceive light directly without the input of rods and cones,called mRGCs. Recent research found that mRGCs were more resistant to the pressure injury under chronic ocular hypertention, and in other diseased status mRGCs were still functional. The purpose of this study is to explore the change of RGCs and mRGCs in RP.PartOne Calcium overloading triggered retinal ganglion cells impairment following loss of photoreceptor during retinal degenerationObjectiveTo investigate the distribution, density, morphology and calcium influx of retinal ganglion cells (RGCs) in RCS rats during retinal degeneration, and to explore the influence of retinal degeneration on RGCs and the potential trigger factors of RGCs impairment.MethodsRetinal dystrophic and control RCS rats were divided into three groups according to postnatal days, P21d, P60d and P90d. Fluorogold were injected into superior colliculus and lateral geniculate body for retrograde labeling RGCs. Then retinal flat mounts were observed under fluorescence microscope for investigating the distribution, morphology and processes ramification of RGCs, and the changes of RGCs density during retinal degeneration. Fluo-4AM was used to incubate living retinal mounts or sections to investigate the calcium concentration of RGCs under laser confocal microscope.ResultsWith progress of retinal degeneration soma of RGCs varied greatly and distributed sparsely. The number of process ramifications and dendritic field decreased obviously. At late stage of retinal degeneration the number of RGCs diminished dramatically. RGCs density was5400±109/mm2at P21d,4167±134/mm2at P60d and2807±161/mm2at P90d. Only80%at P60d and58%at P90d of RGCs survived in RCS rats comparing with Control rats. At late stage of retinal degeneration calcium ion level in RGCs increased significantly and appeared calcium overloading in RCS rats. Calcium fluorescence expressed in both soma and processes, which extended to inner plexiform layer.ConclusionRGCs were secondarily impaired following the loss of photoreceptors at late stage of retinal degeneration, and the distribution, morphological properties and density of RGCs were effected dramatically. The overloading of calcium ion may be the triggering factor of degeneration of RGCs in RCS rats.Part Two Remodeling of retinal neural networks surrounding melanopsin-containing retinal ganglion cells during retinal degeneration ObjectiveMelanopsin-containing retinal ganglion cells (mRGCs) can perceive light directly and modulate the photoentrainment of circadian rhythm and control of papillary light responses. This study was to investigate the structural changes and remodeling of retinal neural networks surrounding mRGCs during retinal degeneration.MethodsRCS rats as a classical animal model of retinitis pigmentosa were sacrificed in this study. Retinal dystrophic and control RCS rats were divided into P21d, P60d and P90d groups. Retinal flat mountings and cryosections were immunofluorescence stiained with anti-melanopsin in RCS and Control rats, then observed under high power confocal microscopy. The morphology of soma, distribution and amount of mRGCs, ramification and stratify of mRGCs processes. The results were analyzed with a computer for cellular morphology, cells counting and processes contacts.ResultsWith the loss of photoreceptors the number of total retinal ganglion cells decreased dramatically, nevertheless more mRGCs survived. The morphology of soma, distribution and amount of mRGCs in RCS rat was almost the same with in Control rats during postnatal stages. However, at late stage of retinal degeneration (P60d and P90d) the expression of melanopsin on processes became more abundant and dendritic field enlarged obviously.ConclusionsWith the loss of photoreceptors in RP, mRGCs appeared more resistant to retinal degeneration and morphologicaly survived. Furthermore, the neural networks and contacts with other retinal neurons surrounding mRGC indicated the remodeling of inner retina during retinal degeneration.