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Objective: Traumatic optic neuropathy (traumatic optic neuropathy, TON) is the external orbital bone conduction through the optic nerve (optic nerve, ON) indirect damage. Injury can have no external or initial endoscopic optic fundus damage the performance, but often result in temporary or permanent visual impairment. The exact mechanism of injury and pathological process is unclear. Currently there is no conclusive clinical treatment programs, poor efficacy. Currently nerve growth factor, antioxidants, glutamate receptor antagonists neuroprotective agent in the treatment of optic nerve applications are still in the present only in animal experiments. In this study, through the establishment of traumatic optic nerve injury in animal models, early application of recombinant human erythropoietin (recombanant human erythropoietin, rhEPO), a control group, rat retina (retinal ganglion RG) and ON morphology, growth associated protein detection - 43 (growth associated protein-43, GAP-43) and the expression of GAP-43mRNA for the clinical application of theoretical and experimental basis, to explore new ways to treat nerve damage. METHODS: SD rats of clean grade 126 healthy adult, female, weighing 230g-250g, external eye and fundus examination was normal. Randomly divided into normal control group (6 12), injury control group (60 60) that the optic nerve clamping ten saline group, EPO treatment group (60 60) that the optic nerve clamping ten EPO group three groups. Damage control group and EPO treatment group survival time after injury and then were randomly divided into one day, 4 days, 7 days, 14 days, 28 days 5 groups. Injury group and treatment group at each time point of the 12 eyes. In addition to the normal control group, each animal are left ON injury, the right eye is not damaged. Each time point and the normal control group, 12 eyes, 3 for the production of RG and ON sections were stained with HE RG morphological changes under light microscope observation and counting and ON morphological observation, three sections for the production of electron microscopy ON for uranyl acetate and lead acid construct rafters double row, as the electron microscope after staining morphology and axon counts ON, six frozen sections for the production of ON and ON total RNA extraction were performed optic nerve tissue GAP-43 and GAP-43mRNA qualitative and Semi-quantitative detection. Pressure constant reverse tweezers, holding the ball at the optic nerve after 2mm 20s, making ON injury animal model. Treated immediately after injury after disinfection 10μL micro thrusters absorb drugs to intravitreal injection rhEPO 2μL, ie 200ng / eye intravitreal injection of saline control group 2μL. Animals in each group after reaching the expected survival time, the complete extraction eyeballs and ON, and ON RG prepared slices were examined by light microscopy and electron microscopy and counting, and the use of immunohistochemistry and reverse transcription-polymerase chain reaction ( RT-PCR) and protein levels from the molecular level detection of different disposal methods in different recovery time points after the ON axons and GAP-43 mRNA expression and extent. All data entry computer, using SPSS (10.0) software package for statistical analysis, central tendency and dispersion tendency described by mean and standard deviation describe univariate comparison between groups using ANOVA, single factor between the two groups compared with t test, test level (α = 0.05). Results: 1 RG stained slices morphology and retinal ganglion cells (retinalganglion cell, RGC) Count: ON 1,4 days after injury, three pathological groups and no significant change in the number of RGCs. 7,14,28 days, the injured group, the treatment group compared with the control group, showing part of the nucleus RGCs layer shrinkage, staining deepened, Presentation of the condensation forms, reducing the number compared with the normal group, showing increased trend. 7, 14 and 28 days treatment group compared with pathological changes RG injury time point was significantly reduced. 1 day, 4 days, three groups of RGCs was no significant difference in the number (p gt; 0.05); treatment group seven days, 14 days and 28 days the number of RGCs were higher than the corresponding time points injury group, the difference was statistically significant (p lt; 0.05). 4 days, 7 days, 14 days and 28 days the number of groups RGCs were decreasing over time, the difference was statistically significant (P lt; 0.05). 2 HE stained sections morphology ON: injury group: l day ON swelling of fiber bundles, scattered in the nerve fiber degeneration. 4 days fiber bundles extra swelling, nerve fiber degeneration. 7 days swelling of fiber bundles, sheets nerve fiber degeneration. 14 days fibers swelling, vacuolar degeneration of nerve fiber tracts, large vacuoles. 28 days fiber bundles slightly reduce swelling, vacuolar degeneration of nerve fiber tracts, many large vacuoles. 1 day after injury injury lesions were treated group compared with no significant difference in 4 days, 7 days, 14 days, 28 days treatment group than injury lesions were significantly reduced, reducing nerve fiber degeneration, mild swelling of the fiber bundle. 3 ON ultrastructural morphological observation and counting 3.1 Ultrastructural: injury group: Visible Most axons exhibit low electron density, axonal degeneration within the granular structure, some axons were seen in mitochondrial swelling and vacuolization obvious, part of the axon shaft there is a gap between the membrane and the formation of myelin and myelin release, structural disorder, axonal vacuolization, microtubules, microfilaments, mitochondria significantly reduced or disappeared. Treatment groups: You can also see Axoplasmic presents a low electron density, granular degeneration and other signs, but the degree of swelling of mitochondria in axonal injury group was significantly reduced compared with mitochondria, microtubules, microfilaments injury group increased significantly compared with myelin arranged in neat rows, level of clarity, demyelination lighter. 3.2 Micro observed one day after injury count number of axons three groups no significant difference. 4 days, 7 days, 14 days, 28 days treatment group were higher than the same number of axonal injury time point group, the difference was significant (p lt; 0.05); groups axon decreasing over time, the difference was statistically significant ( P lt; 0.05). 4 GAP-43 mRNA and the qualitative and semi-quantitative detection by immunohistochemistry and RT-PCR results showed that the three groups after injury, one day no expression; 4,7 days of injury and treatment groups GAP-43 and GAP-43mRNA were positive, but the difference is not obvious; 14 days, 28 days, the treatment group GAP-43 and GAP-43mRNA expression was strongly positive, weakly positive expression injury group, the treatment group GAP-43 expression and GAP-43mRNA stronger than injury group, semi-quantitative analysis the difference was statistically significant (P lt; 0.05). Conclusions: 1 GAP-43 is a reliable indicator reflects ON regeneration. 2 RhEPO intravitreal injection can promote the survival of RGCs after optic nerve injury, play a protective effect on RGCs. 3 ON incomplete injury intravitreal rhEPO, ON can promote axonal repair and regeneration. 4 ON incomplete injury intravitreal rhEPO, can promote nerve GAP-43mRNA, GAP-43 protein expression, possibly in the ON optic nerve regeneration and repair mechanisms play an important role.
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