A Research On Evidences On The Development Of Unfolded Protein Responses In Optical Nerve After Its Injury

Retina and optical nerves diseases are major occoecatio diseases, their main characteristics are progressing death of the retinal ganglion cells (RGC) and their axons. Up to now, no effective therapy against Retina and optical nerves diseases can be got. The mechanism of progressing death of the retinal ganglion cells and their axons is an important problem of the research on nerves injury and regeneration, being paid attention by researchers worldwide.Endoplasmic reticulum (ER) is the place for the synthesis of proteins of secretion and membrane type and correct formation of their stereochemical structure. When the microenvironment of ER is disrupted, faulty folded proteins can be induced. If the amount of the faulty folded proteins go beyond their limits, unfolded protein response (unfolded protein response) may be triggered. Due to their characteristics, the neurons and myelinating oligodendrocyte in the central nerves system need to synthesize a great amount of membrane proteins, making them vulnerable to stress stimulus. It has been found that, UPR is the major cause of neuron loss in central nerve system degenerative diseases including Parkinson's disease and Alzheimer's disease. Recently, Ryoo found, interfering the protein folding in retina of Drosophila melanogaster to induce the UPR can induce late onset of death of optical nerve, implying UPR may be the major mechanism of progressing death of retinal RGCs and their axons. However, above evidences are all based on the known fact that degenerative proteins does exist in the cells, we still don't know if trauma induced neuron loss is related to the UPR. In previous study, we have found optical nerve clamping injury can induce UPR of RGCs, which co-exists with Fluoro-Jade C. On the basis of previous study, we associated immune electron microscopy with colloidal gold to observe UPR in RGCs and their nerves after clamping injury, aiming at research the mechanism of progressing death of RGCs and their nerves after clamping injury.Purpose1. To investigate the expression of related molecules of UPR of RGC cells (IRE-1) after optical nerve crash in rats. Meanwhile, we try to observe the neurons degeneration in RGC and analyze the interrelationship between them.2. Immune-electronic double labeling technique is adopted in making a study on RGC and UPR inside of oligodendrocyte after injury caused by jaw for optic nerve, making discussion on relation between UPR: contusion caused to optic nerve of rat by clamping and cell degeneration., aiming at exploring nerve cell on retina and finding clues to chronic death mechanism of its fiber.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.2. Visual electrophysiology: changes of F-VEP and ERG were recorded in normal eyes and in injured eyes at 4 h, 8 h, 12 h, 1d, 3 d and 7d.Part 2: The changes in morphology of retinal and optic nerve after optical nerve crash.1. The animals were randomly assigned into control group (4 rats) and injury groups (4 h, 8 h, 12 h, 1 d, 3 d, and 7d after injury, 4 rats each).the animals are reperfusioned.The sections of the rat retinal and optic nerve were prepared for following examinations.2. Changes of retinal ganglion cells (RGCs) were observed by microscopic, examination of the hemtoxylin and eosin (H&E) stained slides.3. Counting of ganglion cells: 10 to 15 fields of each specimen were selected by high power microscopic, RGCs were counted on the retina.Part 3: The cellular degeneration and UPR after optical nerve crash. Immunohistochemical staining: IRE-1 and Fluoro-Jade Cand. Part 4: Study on immune electron microscopy after optical nerve crash.1. Establishment of a rat model of optic nerve crash injury groups (12h, 1d, 3d, 5d), a total of 4 groups.2. Double labeled immunoelectron microscopic techniques were used, observed the relevant factor IRE-1 of UPR and AMPA receptor, the relevant factor IRE-1 and oligodendrocyte were double-labeled immunofluorescence. Results1. After the rat model of optic nerve crash was established, no postoperative wound infection and retinal ischemia was found.2. The peak latency of F-VEP in normal rats was 62.00±1.65 ms, and the amplitude was 19.17±2.03μV. 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 1d. 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 4 h, 1d, 3d, 7d was significant. The latency was significantly prolonged compared to normal in the maximum response b wave at 8h, 12h and 12h. The latency was also significantly prolonged in different time points of b wave in bright adaptation and flicker response 12h 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,3d and 7d compared to normal and the difference was significant. Amplitude of photopic ERG decreased in 3d compared to normal and the difference was significant.3. Morphological observation: After optic nerve of rat is contused by clamping, cell nucleus at cell layer of retinal ganglion cell is obviously sparse; while it is not visible after the contusion for 4h and 8h, this pathology is especially becoming even more obvious in later period of the contusion (1d, 3d, 7d), with loose cytoplasmic element; there are scattered nuclear chromatin margi nation and cavitating ganglion cell at ganglion cell layer; big-and-under stain nucleus decreases evidently, or even disappear; small-and-engrain nucleus reduces quite slow; the number of cell both at inner nuclear layer and outer nuclear layer decreases obviously; nuclear alignment is not compacted; inner plexiform layer turns thin clearly; the total thickness of retina is thinning, which is distinct after the contusion after 7d. Since 7d after the contusion, the extent of cell count decreasing is increased. The observation taken to HE stain for optic nerve of normal adult rat: it is visible that optic nerve fiber arranges compact and ordered, distributing oligodendrocytes shaped as particles cell in large amount. After the contusion for 4h and 8h; 12h and 1d after the contusion, obvious breakage belt caused by the contusion appears to optic nerve stem, together with bleeding. 3d after the contusion, bleeding and breakage belt is magnifying, microglia infiltration occurred, and injure is turning sub-acute. 7d after the contusion, bleeding stops with cell death in quantity. The number of RGCs is reducing obviously after optic nerve is damaged; it is comparatively fast within 7d after the contusion. The number of RGCs was reduced significantly after optic nerve injury, quicker within 7d.4. Immunohistochemistry:In each time group: 1d, 3d and 5d after the contusion caused by clamping for optic nerve, Fluoro-Jade C stain, its cell nucleus at RGC layer is aligning loosely and disorderly; clear boundary for every layer of retina with expression (red mark) at RGC layer made by IRE-1 and degeneration neuron are visible; it is especially that strengthened expression occurs in tissue of ganglion cell on retina by IRE-1 in 1d Time Group after the contusion, and appearing stain coexistence (yellow mark) of IRE-1 strong expression cell and dye FJC of degeneration cell to stain central nerve in Time Group 3d after the contusion, it is becoming more distinct after 5d of the contusion, which is widely distributed at RGC layer and outer grained layer. In Time Groups 1d, 3d and 5d separately after the contusion, expression (green mark) at RGC layer by damaged degeneration cell of FJC stain; FJC under stain in Time Group 1d after the contusion; FJC engrain in Time Group 3d after the contusion. Fluoro-Jade C positive reaction is appeared to individual bipolar cell/ amacrine cell at inner nuclear layer. In Time Group 5d and In Time Group after the contusion, double-labeling percentage increases to 39.47±12.13%.49.36%,P < 0. 01,which has clear difference.5. TEM observation: Normal ultra-structure and ultra-structure of oligodendrocyte after contusion by clamping observed by electron microscope: structure and shape of membrane for the former is normal with clear layers and boundaries; while mitochondria of the latter is swelled with broken crystal; degeneration is caused for vacuolar; inclusion bulk sample material exists in endoplasmic reticulum (ER); protein sis appears; loose structure for medullar sheath; vacuolation resulted with hyperplasia becomes heavier, all of these are performances of typical b on morphology. The normal medullary sheath is compacted and smooth with ordered alignment. Optic nerve after injury caused by clamping, change is taken to medullary sheath: board layer of medullary sheath after being damaged, alignment becomes loose; medullary sheath is twisted and degeneration is occurred to vacuolation. At the same time, it is visible that ER of cell for ganglion is enlarged and mitochondrion is swelled. Gold grain can be seen in RGC, distributing as round single grain scattered or several grains gathered with sizes are uniform; the outline of cell is heavy in electron-density, compacted and clear. Small amount of gold grain is existed in endochylema, most of them distributing on membrane surface of ER and its neighboring areas. Apart from this, gold grain can be found on oligodendrocyte; consequently oligodendrocyte is complete in structure. The places with negative control have no gold grain. Immune reaction product of peroxydase for DAB reaction of heavy electron-density is distributed on oligodendrocyte like fuzz; cellular organs such as round & limpid synaptic vesicle and mitochondria are visible.6. IRE-1 distribution in RGC: After optic nerve of rat, colloidal gold labeling of IRE1 in the distribution of RGC increased obviously, control group: 18.4±5.1, in time group 12h: 30.4±7.2, in time group 1d: 35.1±6.4, in time group 3d: 48.5±9.7, in time group 5d: 46.5±9.3, 12h compared to control group increased obviously(p<0.05), 1d,3d,5d compared to control group and the difference was significant(p<0.01).on the shortest linear distance between every colloidal gold and nearest endoplasmic reticulum, control group: -1.1±2.2 nm, in time group 12h: 8.8±2.7 nm, in time group 1d: 18.8±2.0 nm, in time group 3d: 19.4±5.7nm, in time group 5d: 18.6±3.0nm, 1d,3d,5d compared to control group and the difference was significan(tp<0.01).IRE-1 distribution in oligodendrocyte: IRE-1 showed labeling gold, after optic nerve of rat, the cell marked CNPase (oligodendrocyte identity) ,the number of colloidal gold increased obviously ,control group: 12.3±3.5, in time group 12h: 17.4±3.2, in time group 1d: 35.1±6.4, in time group 3d: 39.0±13.3, in time group 5d: 38.1±10.6, there was an increased tredancy in 12h group, but no significant difference was observed compared with control group(p>0.05), 1d,3d,5d compared to control group and the difference was significant(p<0.01). on the shortest linear distance between every colloidal gold and nearest endoplasmic reticulum, control group: 0.3±1.6 nm ,in time group 12h: 8.5±2.4 nm, in time group 1d: 14.8±4.1 nm, in time group 3d: 15.3±5.1nm, in time group 5d: 5d组17.3±5.1nm, 1d,3d,5d compared to control group and the difference was significant(p<0.01).Conclusion1. The reduction of RGCs number after optic nerve injury is the reason of electrophysiological changes of visual.2. Traumatic injury caused to optic nerve could result oligodendrocyte and RGC in producing UPR, while UPR is earlier than RGC losing; in addition, RGC generating UPR is the one under degeneration.3. Jaw for optic nerve can cause oligodendrocyte on optic nerve stem, abnormity on distributed part of IRE-1 in RGC and increasing marked number of colloidal gold, it shows that jaw for optic nerve is available for these cells to trigger UPR. This may be the causes for RGC after jaw injury for optic nerve and chronic death of its fiber.