Protective Effect Of RhEPO On Experimental Traumatic Optic Neuropathy

Objective: Traumatic optic neuopathy which is one of the common kinds of ocular trauma often results in sever vision deterioration even permanent blindness. After injuried,there may be no exeterior optic informance or optic nerve's informance of ophthalmoscope at first. The exact mechanism of injury and pathologic process is unclear.The research of nerve protection drugs including nerve growth factors, antioxidants, glutamate receptor,et,which curing optic injury is still in animal experimental stage.In the study,the modle of optic nerve cursh was established and given rhEPO at the early stage, the control group was established,the morphologic change of retina and optic nerve was studied by microscopy and RGC and fibers were caculated, which providing experimental and theoretical basis for clinical applications and exploring new ways for treating optic nerve injury .Methods: One hundred and twenty six healthy adult SD rats without eye diseases were divided into three groups randomly:normal control group(n=6,12 eyes),crush control group(n=60,60 eyes)and treatment group(n=48,60 eyes). According to the surviving time after crush,the groups were subdivided into 5 timepionts:1d,4d,7d ,14d and 28d.there are 12 eyes at each timepiont in each group. In addition to the normal control group, animals were injuried by left optic nerver,no damage to the right eye.Each time point and the normal control group of 12 eyes, 3 for the production of HE stained slice of RG and ON, morphological change of RG and ON was observed by light microscopy ,RGCs were counted; 3 for the production of ON electron microscope sections, which uranyl acetate and lead acid conformation double stained , ON morphological change of axons was observed by electron microscopy and counted;3 used to produce ON frozen sections were organized to the optic nerve 's GAP-43 and GAP-43mRNA qualitative and semi-quantitative detection.The animal of the crush group and treatment group received optic nerver crush in left eye by applying reverse forceps to the nerve at 2mm behind the globe for 20's.The treatment group were given rhEPO 2ul, 200ng/eye. at once. Crush control group received equal volume normal saline.All animals were sacrificed respectively at 1std,4thd,7thd,14thd and 28thd.after optic nerve crush ,and the eyes were encleated.Morphological change of RG and ON was obersved by light microscopy and eletron microscopy,GAP-43(gowth assoeiated protein-43) and its mRNA expressions were detected with immnunohistochemistry (IHC) and reversetranscription polymerase chain reaction(RT-PCR). All experimental data were inputted into computer,by SPPSS (10.0) statistical analysis package,the central tendency and dispersion tendency were descripted with means and the standard deviation, single factor more than two groups were analied with single-factor analysis of variance,comparision with each others were applied with q -test, the single factor between the two groups by t-test, test standards(α=0.05).Results: 1 the observation an calculation of RG by light microscope.HE stain indicated the gradual 1oss of cell nuclear in the ganglion cell layer and the continous atrophy of the nerve fiber layer,inner nuclear layer and outer nuclear layer in crush control group.Nuclear densifieation was observed 7day after crush and still exist 14d and 28d after crush.While the palthelogical change were remarkably alleviated after treating with rhEPO.rhEPO significantly protected the RGC(P<0.05) at the later three time -points.2 the observation of ON by light microscopye:Crush control group: The 1st day, the optic nerve fiber bundle swelled, the nerve fiber degenerated casually. The 4th day the optic nerve fiber bundle swelled seriously, nerve fibers degenerated. The 7th day the optic nerve fiber bundle swelled more seriously, nerve fiber degenerated flakly. The 14th day nerve fiber bundle swelled more, nerve fiber degenerated extensivly, the vacuole appeared. The 28th day the swelling of nerve fiber bundle reduced slightly, large flake of nerve fibers degenerated, and more large vacuoles.The 1st day there were no significant difference between treatment group and crush control group, The 4th day, 7th day, 14th day, 28th day ,contrasting to crush control group , morphologic change of treatment group reduced significantly , the swelling of the nerve fiber bundle reduced.3 the observation an calculation of ON by electron microscopy: Axons showed that the majority of low electron density, degenerated as granular structure within axons , mitochondria swelled clearly and vacuolizated in some axons, a gap apearred between the axis of some axons and myelin membrane , myelin membrane released, structure apeared disorderly, the vacuoles formed in axoplasm , microtubules and microfilaments significantly decreased even disappeared.Also can be seen in treatment group showed low electron density, and other signs of granular degeneration in axoplasm, but the swelling of mitochondria reduced significantly than crush control group, the number of mitochondria were more, microtubules and microfilaments in axoplasmic were significantly more than crush control group, myelin order were neat, demyelinating appeared lighterly.The 1st day after injury between the two groups had no significant differences in the number of axons, the 4th day, 7 th day, 14th days, 28th daythe number of axons of the treatment group were higher than crush control group at the same time, there were significant differences (p < 0.05). Axons were decreased gradually as time passed, there are significant differences (P <0.05).4 Immunohistochemistry of GAP-43 and RT-PCR of GAP-43 mRNA:Immunohistochemistry and RT-PCR results showed that at three time points of the the 1st day ,4th days, 7th days, there were no significant difference of GAP-43 and GAP-43mRNA expression between crush control group and the treatment group, at the 14th days and 28th days, GAP -43 GAP-43mRNA of treatment group expressed strongly positive, crush control group expressed weakly positive. the expression of GAP-43 and GAP-43mRNA in treatment group expressed more significantly than crush control group,by semi-quantitative analysis there there are significant differences (P <0.05).Conclusion: 1 GAP-43 is a reliable indicator which reflect the regeneration Of optic nerves.2 Introvitreal injection of rhEPO could increase the survival of the RGCs, and protect the RGCs after optic nerve injuried.3 Introvitreal injection of rhEPO could increase the survival of the optic nerve's axons.4 Introvitreal injection of rhEPO could increase the expression Of GAP-43 and GAP-43mRNA in optic nerves .It maybe play an important role of optic nerve regeneration and repair mechanisms on the optic nerve injury.