| Optic nerve disease is characterized by the gradual death of retinal ganglion cells (RGCs). RGC death or injury can not be effectively renewable. The protection of RGC cells and the prevention of optic nerve injury have been the focus of research areas. Protein-folding reaction (UPR) is the first response after the stimulation of neuron injury and it can protect neurons or induce neurons apoptosis. Recent research suggests that UPR may be closely related to gradual death of retinal nerve cells and fibers. In this study, we have undertaken a further investigation of unfolded protein responses (UPR) in RGC cells after optical nerve crash in rats to explore the mechanism of UPR in optic nerve injury.Purpose1. To establish a reliable model of optical nerve crash in rats and perform visual electrophysiology.2. To investigate the expression of related molecules of UPR of RGC cells ( PERK, IRE-1, calnexin) and a subunit of AMPA receptor GluR2 after optical nerve crash in rats. Meanwhile, we tried to observe the apoptosis cells in RGC and analyze the interrelationship between them.MethodsPart 1: To establish a rat model of optic nerve crash and undertake visual electrophysiology.1. Establishment of a rat model of optic nerve crash: The optic nerve of SD adult male rats was exposed, the optic nerve at 2 mm behind the eye ball was crashed for 20 sec with a reverse tweezers by vertical angle.2. Visual electrophysiology: the peak latency and amplitude changes of F-VEP and ERG were recorded in normal eyes and in injured eyes at 4h, 8h, 12h, 1d, 3d and 7d.Part 2: UPR of RGC cells after optical nerve crash.1. The animals were randomly assigned into control group (4 rats) and injury groups (4h, 8h, 12h, 1d, 3d, and 7d after injury, 4 rats each). The sections of the rat retina were prepared for following examinations.2. Immunohistochemical staining: The retinal sections were prepared and stained with primary antibodies (rabbit anti-rat PERK antibody, rabbit anti-rat calnexin antibody, rabbit anti-rat IRE and AMPA antibody), and then examined with laser scanning confocal microscopy.3. Counting of ganglion cells: Four to 5 fields of each slice were selected, PERK-positive or calnexin-positive ganglion cells were counted on the retina with 10μm×10μm×10μm grid at x 40 times.Results1. After the rat model of optic nerve crash was established, the eye pupil dilated, light-reflecting decreased significantly and the retinal vascular perfusion was normal. No postoperative wound infection and retinal ischemia was found.2. The peak latency of F-VEP in normal rats was 70.19±2.14 ms, and the amplitude was 22.72±1.97μV. P100 peak latency after injury was shorter than that of normal rats and it was reduced to a minimum at 12h. The difference of peak latency was significant compared with normal and then gradually recovery after 12h. It was longer than the normal level at 7d. The amplitude was decreased significantly after injury and was lower than the normal level at different time points. It was increased shortly in 8h, 12 h and then dropped again and the difference of amplitude at 4h, 1d, 3d, 7d was significant. The latency was significantly prolonged compared to normal in the maximum response b wave at 8h, 12h and 1d. The latency was also significantly prolonged in different time points of b wave in bright adaptation and flicker response 3d compared to normal. Amplitudes were decreased significantly in the maximum response b wave at 4 h, 8 h and 12 h and the difference of amplitudes after injury compared to normal was significant. Amplitude of oscillatory potentials′s O2 wave was increased in 1d and 7d compared to normal and the difference was significant. Amplitude of photopic ERG decreased in 3 d compared to normal and the difference was significant.3. The expression of calnexin and PERK in RGC cells was significantly enhanced after optical nerve crashed detected by confocal laser scanning microscope. The number of RGC cells expressing PERK or calnexin increased 4 h after injury and reached its peak at 24 h. Compared with that of controls, expressions of PERK and calnexin were increased distinctively (PERK,15.00±0.36;calnexin,11.75±1.44,P<0.05) in RGC cells after injury. Expressions of IRE-1 and GluR2 increased 8 h after injury and the positive staining to GluR2 was obvious on the cell membrane of RGC cells. It was noted Fluoro-Jade C staining in RGC cells 72 h after injury, with co-existence with GluR2.Conclusion1. Rat model of optic nerve crash is simple, easy to operate, and it can cause the exact damage of the optic nerve.2. The amplitude of F-VEP after injury was decreased significantly while the overall composition of the ERG had little changes, suggesting that the optic nerve injury of the model was precise and retinal function was not significantly affected..2. The expression of UPR related PERK, IRE-1 and calnexin in RGC cells was enhanced after optical nerve crash and apoptosis of RGC was emerged 72h after injury, suggesting that UPR was involved in the process of protection in 24h after RGC injury. After that, UPR could induce apoptosis more easily. These findings have not been reported in literature. |
PDF Full Text Request