| Objective:Axon regeneration after central nervous system(CNS)injury is extremely limited in adult mammals.As a part of the CNS,the injury of optic nerve leads to apoptotic death of retinal ganglion cells(RGCs)and loss of vision.In recent years,accumulating evidence suggests that chitosan exerts neuroprotective effects in a variety of neurodegenerative diseases.This study intends to explore the therapeutic effects of chitosan in traumatic optic nerve injury through the classic optic nerve clamping rat model.Methods:72 adult female SD rats of SPF grade were randomly divided into sham group(n=12),TON group(n=30)and chitosan group(n=30).The right optic nerve of rats in chitosan and TON group was clamped for 10 s with a clamping force of 50 g at 2 mm behind the ball.Same volume of 300 mg/kg chitosan and 1%acetic acid solvent was infused to stomach daily starting from three days before the optic nerve crush,respectively.While in the sham group,the right optic nerve was only peeled off from the nerve sheath and not clamped.Among the model rats,18 rats(6 in sham group,6 in TON group and 6 in chitosan group)were used for flash visual evoked potentials(F-VEP)analysis at 3 days,7 days,14 days and 21 days after modeling.54 rats(6 in sham group,24 in TON group,and 24 in chitosan group)were used for immunofluorescence analysis at 3 days,7 days,14 days and 21 days after modeling.The eyeballs and optic nerve were preserved and frozen for immunofluorescence detection of the survival of RGCs,axon regeneration and the co-expression of astrocytes with IL-1β and BDNF.Results:Roland electrophysiological test showed that the sham-operation control rats exhibited a typical F-VEP waveform.Varying degrees of change of F-VEP waveform was found in TON group and chitosan treated rats.Importantly,the chitosan treatment significantly decreased the delay of NI wave and increased the N1-P1 amplitude at 21 days post-clamping,which suggested an improvement of optic nerve conduction.Immunofluorescence staining showed that the number of RGCs decreased with time after clamping the optic nerve compared with the sham group.However,the chitosan treatment improved the survival of RGCs significantly at 7 days,14 days and 21 days after optic nerve injury,respectively(P<0.05).There was no detectable expression of GAP-43 found in the optic nerve of the sham group and TON group at all time sets.However,the GAP-43 expression was detected from 14 days and 21 days post-crush in chitosan group.Notably,the expression of GAP-43 in chitosan group was significantly higher than that of in TON group(P<0.05).The resting astrocytes in the sham group were found in the retinal ganglion cell layer and the nerve fiber layer.3 days post-clamping,the astrocytes were activated and the GFAP-positive cell fibers began to extend,which further extended to the inner mesh layer at 7 days,14 days,and 21 days post-clamping.By contrast,administration of chitosan inhibited astrocytes activation and proliferation as well as the extension of GFAP-positive fibers.Immunofluorescence co-localization results showed that few astrocytes existed in retina which secreted very little IL-1β in the sham group.Astrocytes activated and proliferated,which produced IL-1β to cause inflammation at 3 days,7 days,14 days and 21 days post-clamping.In compared with TON group,chitosan treatment reduced the number of astrocytes in retina and their expression of IL-1β.Meanwhile,detectable BDNF in astrocytes was only found in chitosan group at 14 days post-clamping.Conclusion:In optic nerve clamping injury,chitosan can reduce the death of RGCs,promote the regeneration of optic nerve axons and promote the recovery of visual function F-VEP in rats.Further analysis suggested that the neuroprotective function of chitosan may rely on the inhibition of the activation of retinal astrocytes,suppression of IL-1β secretion and promoting the expression of the BDNF in astrocytes.This research would provide theoretical basis for clinical prevention and treatment of traumatic optic neuropathy with chitosan. |
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