Cannabinoid Receptor Mediated Neroprotection In N-methyl-N-nitrosourea Induced Retinal Degeneration

Part1. Morphologic characteristics of N-Nitroso-N-methylurea-induced retinal degeneration in miceObjective:To explore the morphological characteristics and mechanism of N-Nitroso-N-methylurea (MNU) induced retinal degeneration in mice.Methods:32C57/BL (5weeks) mice were randomly divided into two groups: control group and MNU group.60mg/kg of N-Nitroso-N-methylurea (MNU) was intraperitonealy (i.p.) injected in mice, while the control group was given physiological saline. Eyes were removed at1,2,3,5,7,14,21,28,90days after the injection, and processed for cryosectioning or protein extraction. Immunofluorescence displayed the morphology of damaged retinal outer segment, bipolar cell, synaptic connections, ganglion cell, as well as mitochondrial damage and oxidative stress. Protein levels were determined by Western blot. Retinal flat-mount immunofluorescence was used to observe vascular system and the number of ganglion cells. The ultrastructure of outer segment, nucleus, mitochondria and synaptic ribbon was observed with the transmission electron microscope. Ganzfeld ERG was used to test the electrophysiological changes.Results:After MNU administration, we performed immunofluorescence which showed progressively decreased thickness in the outer nuclear layer (ONL) associated with photoreceptor outer segment loss.10%sample showed differential damage of central and peripheral retina. Bipolar cell labeled with PKCademonstrated dendritic retraction. Cone pedicles and rod spherules labeled with PSD95displayed progressive lose. Reactive gliosis was confirmed by increased Glial fibrillary acidic protein (GFAP) levels. More serious damage to the central retina as opposed to the peripheral retina was found in the MNU-induced retinal degeneration model. Retinal ganglion cells (RGC) appear to be spared for at least two months after MNU administration. Following retinal vessel labelling, we observed many vascular complexes in the distal vessels, indicating retinal vessel damage. In the remnant retinal photoreceptor of the MNU-treated mouse, concentrated colouring nuclei, widening of the perinuclear space and autophagosome were detected by electron microscopy, together with the swollen mitochondria and displaced remnant synaptic ribbons in the photoreceptor. We also observed decreased mitochondrial protein levels and increased amounts of nitrosylation/nitration in the photoreceptors. Gansfield ERG showed reduced amplitude of a-waves and b-waves in mice with MNU administration.Conclusion:MNU-induced mouse retinal degeneration in the outer retina lead to photoreceptor loss and neural retinal remolding. The mechanism of MNU-induced apoptosis may result from oxidative stress or the loss of retinal blood supply. is a useful animal model for photoreceptor degeneration diseases, such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP). Part2. Cannabinoid Receptor1Antagonist SR141716A Attenuate the Photoreceptor Damage Induced by N-Nitroso-N-methylureaAbstract:To explore the neruoprotection effect of cannabinoid receptor1antagonist SR141716A (SRI) to N-Nitroso-N-methylurea (MNU) induced retinal degeneration in mice.Methods:50mg/kg of N-Nitroso-N-methylurea (MNU) was intraperitonealy (i.p.) injected in mice, while the control group was given physiological saline. Treatment group were intraperitonealy (i.p.) injected with55,212-2(WIN) or SRI. According to the drugs given to the animal,35adult C57/BL mice were randomly divided into seven groups:MNU group, WIN group, SRI group, MNU+WIN group, MNU+SR1group, MNU+WIN+SRl group and MNU+WIN+SR2group. Eyes were removed after cervical dislocation at3,5,7days after the injection, and processed for cryosectioning or RNA extraction. Immunofluorescence confirmed the localization of cannabinoid receptor1(CB1) in mouse retina. Immunofluorescence double labeling with neuron-specific markers showed colocalization CB1in several identified cells. Immunofluorescence performed in acutely dissociated cells confirmed the locating of cannabinoid receptor1in bipolar cell. Real-time PCR was used to analysis the relative gene expression of CB1and CB2. Immunofluorescence with neural markers also used the revaluate the ONL thickness, reactive gliosis. Retinal flat-mount immunofluorescence was performed to observe vascular system. Thin slice patch-clamp was used to observe the the response of ON and OFF bipolar cell to several drugs.Results:CB1was located pronimently in outer plexiform layer and inner plexiform layers of the retina. Beside, the photoreceptor cell outer segment labeled with OPN1SW, the bipolar cell labeled with PKCa and the ganglion cell labeled with Brn3a also showed colocalization with CB1. Freshly isolated rod bipolar cells labeled with PKC a showed colocalization with CB1, confirmed the expression of CB1in bipolar cell. Real-time PCR showed up-regulation of CB2after MNU administration. We observed a highter ONL/INL ratio and less gial reactivity after SRI treatment. Following retinal vessel labelling, MNU+SR1group also show less distal vascular complexes than we observed in the MNU group. MNU+WIN group, MNU+WIN+SR1group and MNU+WIN+SR1group didn’t show prominent amelioration. Thin slice patch-clamp displayed more serious damage for ON-bipolar cell than OFF-bipolar cell. The results of thin slice patch-clamp also adviced that SRI attenuated the OFF-bipolar cell damage induced by N-Nitroso-N-methylurea.Conclusion:Cannabinoid receptor1antagonist SR141716A attenuated the photoreceptor and OFF-bipolar cell damage induced by N-Nitroso-N-methylurea. The mechanism of nureoprotection may be ameliorated oxidative stress or neuron-astrocyte signaling.