| Objective: Along with the development of science and technology,the continuous updating of the traffic tools,the traumatic optic nerve injury is becoming more and more common in the Department of Ophthalmology.The clinical therapy for optic nerve injury generally give drug treatment,if there are indications for surgery of optic nerve canal decompression,but most of the patients in the treatment effect is unsatisfactory,for the current visual nerve injury disease control and intervention,there is no specific drugs or methods so far.Now it is clear that it is necessary to put forward a new strategy to reduce the number of neuron degeneration and protection of the survival of retinal ganglion cells(retinal ganglion cells,RGCs),on the vision damage of optic nerve diseases,including glaucoma,affects more than 65 million people in the world,is an irreversible blindness.Thus,optic nerve injury is one of common blinding factor in eye disease,patients recovery and prognosis are low expectations,which gave a heavy blow to the treatment of patients with confidence.The degeneration of retinal ganglion cells and their axons is the cause of visual impairment in patients.How to reduce the complications of optic nerve injury,reduce the disease rate of blindness,becoming the biggest challenge we are facing.The latest research shows that epithelium-derived factor pigment(PEDF)has a powerful neuroprotective effect.In this study,we established the animal model of optic nerve injury,and the PEDF was injected into the glass,and to observe and explore the protective mechanism of PEDF on the optic nerve.Method: Ninety healthy female SD rats(provided by the experimental animal research center of Hebei Medical University)were selected,the body weight is 200-250 g.All of them had no disease of eyes according to examination.Adaptive feeding 1w,they were randomly divided into three groups,normal group with 30 rats(30 eyes,the normal group had no treatment),control group(ONC+PBS group)with 30 rats and treatment group(ONC+PEDF group)with 30 rats.There was no significant difference in body weight between the three groups.In both ONC+PBS group and ONC+PEDF group,the left eye of every rat was made to the incomplete injury model of optic nerve.No treatment of right eye.Rats in ONC+PBS group and ONC+PEDF group intraperitoneal injection of 10% chloral hydrate(0.35ml/100g)anesthesia after fasting for 12 hours.Along the corneoscleral limbus annular cut temporal bulbar conjunctiva,blunt separation of exposing the optic nerve of intraorbital segment,calibrated with the power of the forceps in the ball 2 mm optic nerve crush,causing injury time is 20 s and the l0-0 nylon thread Korfball conjunctiva with 2 needles.In the ONC+PBS group,after the optic nerve injury model was made successfully,then injected balanced salt solution 5μl into the vitreous cavity immediately,and the first week and second week injected balanced salt solution 5μl into the vitreous cavity respectively.In the ONC+PEDF group,after the optic nerve injury model was made successfully,then injected PEDF5μl into the vitreous cavity immediately,and the first week and second week injected PEDF 5μl into the vitreous cavity respectively.Conjunctival sac coated ofloxacin eye ointment.All rats of 3 groups were anesthetized with 10% chloral hydrate of(0.35ml/100g)at third weeks,and the left eye(20 eyes)were randomly selected from the control group(20 eyes),and 20 rats(20 eyes)were both taken from the ONC+PBS group and the ONC+PEDF group.Cut off the wall of the eyeball after the serrated edge 0.5mm,removing the anterior segment and vitreous body.With the volume fraction of 4% paraformaldehyde fixation,4 ℃ refrigerator overnight,the whole layer of paraffin embedding,preparation of 6μm retinal slice,microscope image acquisition and synchronization.Morphological changes of retina were observed by routine HE staining,and count the survival rate of RGCs.Application of image analysis system(*400)to analyze the results(each section 4 view,at a distance of papillary1.5mm retina were beginning to take a view on all sides of the 1 eyeballs,each2 sections).Caspase-3 was detected by immunohistochemical method and computer image analysis.The average optical density(AOD)was obtained by analyzing the positive reaction site in the retina.The expression of the antigen was expressed by AOD,and the higher the expression of AOD was higher.In the three groups,10 rats(10 eyes)were randomly selected in each group.Ultrastructural changes of retinal tissue were observed under electron microscope.The experimental data was calculated by using ` X ± S(mean ±standard deviation).The difference between the groups indicated by two sample t test,P<0.01 thought there was a statistically significant difference.Results:1 RGCs morphological changes under light microscopeBlank control group: from inside to outside the retinal ganglion cell layer,bipolar cell layer and photoreceptor cell layer.The cells in the ganglion cell layer are arranged in a single layer,the cells are in order,and the cell nucleus is clear.The bipolar cell layer and the photoreceptor cell layer are arranged in a multilayer arrangement.ONC+PBS group: retinal nerve cell layer cell nuclei arranged in a loose,arranged disorder,bipolar cell layer and photoreceptor cell layer can reduce the number of cells.Group ONC+PEDF: the cell nuclei of the retinal nerve cells were closely arranged in the ONC+PBS group.2 Quantity changes of RGCs under light microscopeThe number of retinal ganglion cells in normal control group was29.53±2.05.The average number of retinal ganglion cells in the ONC+PBS group was 8.5±1.59.The average number of retinal ganglion cells was14.03±1.66,and the difference was statistically significant(P < 0.01).3 Ultrastructural changes of RGCs under electron microscopeNormal rat RGCs nucleus was oval,nuclear,homogenous nucleoplasm,see ultrastructure of Golgi body,rough endoplasmic reticulum,mitochondria,ribosomes.Some retinal ganglion cell chromatin showed a homogeneous shape,nuclear membrane integrity or unclear,cytoplasmic organelles reduction,dissolution and disappearance,showed the characteristics of necrosis.In addition,still visible other some cells staining obvious aggregation,concentrated,with cytoplasmic atrophy,increased electron density,edge set on karyotheca,uneven thickness,nuclear membrane and cell membrane integrity,showed characteristic features of apoptosis.All ONC+PEDF group and ONC+PBS group of retinal ganglion cells reduced visible rough endoplasmic reticulum,Golgi bodies,ribosomes,mitochondria cristae,dissolved or disappeared,also visible ganglion cells staining obvious aggregation,concentration,with cytoplasmic atrophy,increased electron density,edge set on karyotheca,uneven thickness,cell degeneration,dissolution,reduce.In ONC+PEDF group,the retinal layers were clear and the membrane discs were arranged in order,the membrane structure was normal,and the morphology of the cells in the retina was normal.In ONC+PEDF group,ganglion cell chromatin showed a homogeneous shape,nuclear membrane integrity or unclear,cytoplasmic organelles reduction,dissolution and disappearance,another section stained cells were aggregated and concentrated,with cytoplasmic atrophy,increased electron density,edge set on karyotheca,uneven thickness.4 caspase-3 expression in the retinaThe expression of Caspase-3 in the blank control group was very weak or even no expression;The expression of Caspase-3 in ONC+PEDF group was weak,and the cells were stained with a little light,and the number of cells was less;The expression of Caspase-3 in ONC+PBS group was significantly increased,and the cells were stained with a number of cells.Conclusion:1 PEDF support in the survival and recovery of RGCs after optic nerve injury.2 PEDF can protect RGCs by reducing the expression of caspase 3 in retinal,inhibiting apoptotic cascade reaction. |
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