Experimental Study Of Pathological Degeneration Of Optic Nerve Injury And Surgical And Drug Intervention Of Optic Nerve Injury

Objective:Optic nerve injury is serious complication of brain, facial and orbital trauma, severe visual impairment after injury, often left permanent impaired vision. For a long time, the treatment of optic nerve injury has been controversial. Existing studies suggest that the use of corticosteroids drug therapy and surgical treatment method is the most commonly used treatment, optic nerve decompression surgery has become the main treatment, is to relieve the compression directly relieve nerve swelling and vasospasm, have certain effect for optic nerve damage; dexamethasone as a corticosteroids reduce edema of optic nerve through its antioxidant effects reduce free radical damage to cell membrane structure, while blocking traumatic inflammatory mediators and produce of vasoactive substances, thereby reducing vasospasm, the severity of optic nerve contusion necrosis and microvascular spasm extent. Previous experiments biased in favor of the previous experimental animal model, Lack of clinical guidance. In this experiment, optic nerve decompression, dexamethasone and optic nerve decompression therapy combined with dexamethasone therapy for the influencing factors, Nogo receptor of the optic nerve, retinal Nogo receptor, Caspase-3as the starting point, pathophysiological observation of the rabbit optic nerve model of the retina and optic nerve receptors by light microscopy and electron microscopy. Initially prove the impact of optic nerve decompression, dexamethasone and optic nerve decompression therapy combined with dexamethasone therapy for model of the optic nerve. Provide a theoretical basis for the clinical treatment of optic nerve injury. Through the sham and control groups validate reasonabilities of the experimental design. Stable animal models for Observational study of surgery or drug therapy can obtained through temporarily clipping of optic nerve by aneurysm clips.Methods:(1) Experimental animal groups:96healthy adult rabbits, species of New Zealand white rabbits weighing2-2.5kg, from Xinjiang Medical University Animal Center. After3d adaptive feeding, both eyes inspection found that refractive clear, pupils and other large and round, light reflex sensitivity, fundus without exception; among these6is for blank control group,18for the sham group, to exclude the influence of surgical factors on optic nerve and retinal, the remaining72rabbits left eye optic nerve injury model making, were randomly divided into model group, simple optic nerve injury group, dexamethasone effects of medication group, optic nerve decompression effect group, the optic nerve decompression combined with dexamethasone therapy combined effect of group four large groups, and each group was divided into three subgroups, respectively, after1day group,7days group and21days groups,6for each sub-group.(2) animal model:Anesthetize rabbit using3%pentobarbital1ml/kg though ear vein, then the prone position fixed on the operating table; binocular microscope, set an upper eyelid lateral traction sutures, fixed and pull on the upper eyelid; cut bulbar conjunctiva, traction suture between the end of superior rectus and superior oblique muscle; pull the eye to front outer down direction of the orbital, then sharp separation of the soft tissue to expose the optic nerve to the deep part of the optic nerve about8mm under the microscope, clipping the optic nerve for20seconds at3mm behind the ball using temporary aneurysm clips (Aesculap temporary aneurysm clips FT250T, occlusal length9mm, maximum opening7mm, closing force0.88N, weight:9g.)(sham group not clipped). The eye reduction, suture bulbar conjunctiva. Saline flush eyes, dropping levofloxacin eye drops.Standard animal model:after establishing optic nerve injury animal model, to be back in the cage after awakening, check the incision healed without suppuration empyema, no bleeding exudate, observing eye injury mydriasis, direct response to light, surgery cornea transparent, non-traumatic cataract, vitreous cavity without hemorrhage, retinal blood supply to the normal bleeding and no retinal detachment, no inflammatory reaction, no bleeding and retinal vascular occlusion, no proptosis and eyelid closure, insufficiency were included in the experiment. Detected by the above evaluation reflects the success of the model. After checking the above criteria,96New Zealand white rabbits were randomly optic batches into experimental animal models. Animal feeding process, for those discharged complications experiment, and the number of experimental animals timely supplement.(3) optic nerve decompressionIn accordance with the experimental groups, the need for the implementation of optic nerve decompression in experimental animals, according to the method of above model are maked.after clipping20s, using a1ml syringe needle isolated optic nerve tissue outside the envelope till to8mm after the ball under the microscope, then achieved optic nerve decompression purposes.(4) dexamethasone administration methodsall animals affected with Dexamethasone, beginning the day after the operation, according to a daily dose of1mg/kg, by intramuscular injection, until the death node.(5) Optic nerve injury model making:specimen collection and morphological studies of optic nerve and retinal ganglion cell layer:overdose anesthesia with3%sodium pentobarbital anesthesia, thoracotomy, through the left ventricle to the aorta cannulation, perfusion fluid discharge from opening of superior vena cava, followed by perfusion of normal saline500ml,2.5%glutaraldehyde phosphate buffer250ml, untill rabbit limb tonic convulsions, immediately removal the eye retina optic nerve specimens taken placed in fixative, and mark, specimens were fixed with4%paraformaldehyde for72h, dehydrated, embedded in paraffin for light microscopy, through the optic nerve, the eye are sliced along the vertical meridian of ball to longitudinal retinal layers, and HE staining. slices were prepared by using slicing mechanism. RGC counting method: observation under an optical microscope (40objective), each specimen slices randomly selected from the optic disc, respectively, above and below300um three regions (each region25um*25um), do statistics of total number of surviving retinal ganglion cell. Calculating the sum of retinal ganglion cells and then take the average and record experimental data, and finally the use of statistical software to analyze number of the survival of retinal ganglion cells and apoptosis law in each group at different time points, and whether there is significant difference between the groups.Specimen for electron microscope:the optic nerve specimen for electron microscope, fixed with a4%glutaraldehyde. Refrigerator thermostat kept at the electron microscope chamber4%glutaraldehyde and1%osmium tetroxide double fixation, dehydrated in acetone, Epon812epoxy resin embedding, LKB2188thin slicer slices, lead uranium electron staining, and observation under JEOL1230transmission electron microscopy.(6) observations and time points Outcome measures:observation at day1, one week, three weeks after clipping,1/histological observation of local retinal and optic nerve under light microscopy, and observation of optic nerve ultrastructure by electron microscopy;2/retinal ganglion cells (retinal ganglion cell, RGC) counts (cells in situ apoptosis detection);3/retina of Caspase-3immunohistochemistry;4/optic nerve, retinal Nogo receptor immunohistochemistry, pathology results of immunohistochemistry, correlation analysis of the effects of optic nerve decompression and nerve model for dexamethasone for the model.(7) Statistical analysis:statistical data as sets of quantitative data, if the packet or time line with normality, homogeneity of variance, analysis of variance, if not met, using the Kruskal-wallis (KW) rank test. Simultaneously at different times with different groups and each group pairwise comparison of data with sas jmp pro computer statistical software for statistical analysis. Measurement data of each group as mean±standard deviation, with P<0.05was considered statistically significant. Tables*do not meet the homogeneity of variance, using the Wilcoxon rank sum test. Results:(1) histological light microscopy, using HE staining sham group in1day,1week,3weeks after optic nerve injury, early retinal swelling, slightly thicker than normal thickness of layers, cells arranged in loose and disordered, only to see the retinal telangiectasia, a small reduction in retinal ganglion cells.HE staining of retinal tissue morphology retinal level of clarity, RGC were arranged in a single layer, neat dense nucleus clear, smooth and complete nuclear membrane in control group, pathological changes for each point time was significantly, the pathological changes progressively increased over time. Retinal damage morphological changes in day1, cell edema, the thickness is slightly thickened, ganglion cell layer slightly disordered arrangement of inner and outer layers, cell gap increases, there is a small amount of nuclear condensation, stain deepened, a large number of ganglion cell degeneration, the number of cells slightly reduced. Morphological changes in retinal injury were aggravated at one week, compared with the former group, cell edema severe ganglion stained nuclear chromatin increased, showing a large number of vacuolar degeneration of cells, outer and inner structural disorder, boundaries blurred retinal layers significantly thinner. Injury in three weeks group, retinal cell swelling has been reduced compared to the previous change, nucleoplasm stained stained layers of structural disorder, retinal thinning.(2) ultrastructure Observation of retina and optic nerve under electronic microscopy day1, Figure3-1shows that slight deformation of nerve fibers and myelin lamellar structure mild separation, partial visible vesicular structures visible spherical protruding into the axonal and myelin lamellar structure separated axonal neurofibrillary loosely arranged in crude fiber-based, vascular endothelial cell swelling, luminal incomplete obstruction, nerve fibers crushing, collapse and other acute traumatic change myelin malalignment few minor swelling mitochondria, day7, Figure3-2shows that lysis of myelin and myelin degeneration in general, onion-like body formation, glial proliferation significantly, there is still residual edema soften the cell thickness increases, the plate separation layer multi-layer structure, the individual performance of demyelination. Slight swelling of glial cells, mitochondria vacuolation, some nerve axonal vacuolation. day21, Figure3-3shows that nerve degeneration, irregular structure of nerve fibers and myelin structural disorder apparent dissociation visible sheath bubbly, some pulp fiber axis vacuolation, edema disappeared malacia by glial replaced each treatment group no significant change.(3) retinal ganglion cells:in the model of acute optic nerve injury after optic canal decompression therapy, dexamethasone and decompression in the early therapy (1d) to prevent apoptosis of optic nerve injury RGC layer and inner nuclear layer cell, while in7days21days time point of acute optic nerve and retina after optic nerve injury was no significant difference in effect,(P>0.05), and no statistically significant.(4) immunohistochemical detection of Caspase-3of retinaOptic nerve decompression and decompression dexamethasone dexamethasone three treatment groups with simple injury time points in one day there are differences (P<0.05), and possessed statistically significant. With simple injury in7days,21days time points were no differences (P>0.05), and no statistical significance.(5) immunohistochemistry of optic retina Nogo receptorNogo. A are widely distributed in normal adult rat retina and optic nerve. Acute optic nerve damage can promote Nogo. A upregulation. there are differences (P<0.05) between Optic nerve decompression and decompression dexamethasone, dexamethasone three treatment groups with simple injury group time points in one day and possessed statistically significant. And there were no differences (P>0.05) in7days,21days time points and no statistical significance. Conclusion:(1) This study create a stable, repeatable, easy to operate for optic nerve surgery or medication observational studies in animal models through temporary clipping of optic nerve by temporary aneurysm, sham group through the optic nerve and retinal ganglion cells and statistical analysis of the expression of special Molecular Biology, sham and control groups validate the experimental design is reasonable, and may be better use of experimental animal model of optic nerve damage.(2) the results of this study show:RGC apoptosis after acute optic nerve injury is an apoptosis that sustained over time.(3) acute optic nerve damage can promote caspase3-positive expression, which indicates that caspase3expression in the optic nerve nerve regeneration process plays an important role. Nogo-A are widely distributed in normal adult rat optic nerve and retina, acute optic nerve damage can promote Nogo-A upregulation, indicating that Nogo-A plays an important role in optic nerve regeneration after optic nerve injury.(4) in the model of acute optic nerve injury after optic canal decompression therapy, dexamethasone and decompression in the early therapy (1d) to prevent apoptosis of optic nerve injury RGC layer and inner nuclear layer cell, while in7days21days time point of acute optic nerve and retina after optic nerve injury was no significant difference in effect, P>0.05, and no statistically significant, protective effect of three treatment for acute optic nerve injury RGC layer and inner nuclear layer cell is uncertaint.(5) the results of this study shows that the therapeutic effect of optic canal decompression may equal to decompression dexamethasone group and dexamethasone group, the experimental results explains the three treatment methods reduce the number of apoptotic cells of optic nerve retinal ganglion after injury, but the exact mechanism requires further study. The experimental results need further analysis. But existing differences between the three method still need further study.