| Part One Retinal hypoperfusion injury induced by bilateral common carotid artery occlusion in ratsObjective:To establish a rat model of retinal chronic hypoperfusion by bilateral common carotid artery occlusion(BCCAO)and observe changes in the retinal function and morphology.Methods:Sixty male Sprague-Dawley rats were randomly divided into the sham surgery(30 rats)and model(30 rats)groups.At the 1,2 and 4 weeks after surgery,the retinal circulation time was observed by fluorescein fundus angiography(FFA),the action potentials of retinal nerve cells were measured by electroretinogram(ERG)and the perfusion on the eyes’ surface was performed by laser speckle blood-flow imaging.After the functional examinations,the eyeballs of both groups were removed for retinal tissue sectioning.Morphologic and ultrastructural changes in the retinas were observed by hematoxylin-eosin staining and transmission electron microscopy.Results:BCCAO rats showed retinal arteriosclerosis and slight dilated retinal vein.At 14d,retina appeared small hemangioma and hemorrhage.At 28d,the vascular segmental filling and no perfusion zone were observed.The retinal arterial filling time was prolonged at the 2 and 4 weeks(P<0.05)and retinal venous filling time was significantly prolonged at the 1,2 and 4 weeks(P<0.01).The latency of a and b waves in the model group was delayed and the amplitude was decreased significantly at each time point(P<0.05).The perfusion of the eyes’surface continued to reduced(P<0.05).In the model rats,the retinal structure was loose and disorganized.The retinal ganglion cells(RGCs)presented obvious apoptosis.All of the thickness in the retinal layer,the inner nuclear layer and the outer nuclear layer were significantly thinner.Conclusions:These findings suggested that BCCAO may induced retinal hypoperfusion injury in the model rats,thus providing an ideal animal model for the study of ocular chronic ischemic.Part Two To observe the protective effect of Yiqi Wenyang Tongluo Decoction on the function and morphology of the rats retinal hypoperfusion injuryObjective:On the basis of establishing the model of rats retinal chronic hypoperfusion injury,the changes of rats retinal function and morphology of rats with Yiqi Wenyang Tongluo Decoction(YWTD)were observed.Methods:40 SD rats were randomly divided into 4groups:normal group,model group,treatment group and control group,each group had 10 rats.Rats in the model,treatment and control groups were operated by bilateral common carotid artery occlusion.The bilateral CCA in sham group rats were only separated with no occlusion.Rats in treatment group were with YWTD(14.40g/Kg/d)by gavage and control group were with prostaglandin-1(PGE1)(2.5ug/Kg/d)by intraperitoneal injection each day.The experimental period is 28d.FFA was used to observe the retinal circulation time and vascular morphological changes in rats of every groups respectively.The action potential of retinal nerve cells was observed with EGR.The changes of blood perfusion in the ocular surface of rats were observed by the laser speckle flow imaging instrument.Rats were killed through dislocated executed.The eyeballs of rats were removed and the paraffin secitons of retinal slides were made.Retinal structure was observed by optical microscope and electron microscopy.Results:(1)FFA:Compared with the model group,the arterial filling time of the treatment group and the control group was not significantly different(P>0.05),and the filling time of the retinal vein was significantly shortened at 14d and 28d(P<0.01).(2)ERG:Compared with model group,the latency of a wave was shortened,and the amplitude of a and b wave improved in the treatment group and the control group(P<0.05).(3)PSI-NR:The amount of blood perfusion on the surface of the treatment group was higher than that in the model group and control group(P<0.05).(4)HE:Compared with model group and control group rats,the treatment group rats retinal layers were arranged in order,nucleus pycnosis turned up in few of retinal ganglion cells,accidental vacuole like degeneration.The inner cluster was close to put in order,the photoreceptor cells were less vacuole-like changes of outer segments.The retinal atrophy was less than that of the model group,and the improvement was most significant(P<0.01).(5)Electron microscope:compared with sham group rats,The RGCs of the model rats were seriously damaged,with karyopyknosis,chromatic agglutination and decreased or swelling organelles.The outer nuclear layer cells were of varied in size,the nuclear membranes were shrunken and invaginated,and the chromatin was uneven density.Compared with model group and control group rats,the treatment group rats intercellular spaces of retinal ganglion cells dilatation but in good order,nucleus pycnosis and chromatin margination and small glial cells showed up.The outer layer of the core layer was close to put in order,the electron density in nucleus were homogeneous.Mitochondria swelled and vacuolation were hard to find.Conclusions:YWTD may through increasing the ocular blood perfusion and promoting the blood circulation to reduce the retinal chronic hypoperfusion injury of rats,and then to protect the morphology and improving the excitability of retinal nerve cells.Part Three The effect mechanism of the YWTD on the rats retinal chronic hypoperfusion injuryObjective:On the basis of establishing the model of rats retinal chronic hypoperfusion injury,the effect mechanism of the YWTD was researched by molecular biology technique.Methods:40 SD rats were randomly divided into 4groups:normal group,model group,treatment group and control group,each group had 10 rats.Rats in the model,treatment and control groups were operated by bilateral common carotid artery occlusion.The bilateral common carotid arterys in sham group rats were only separated with no occlusion.Rats in treatment group were with YWTD(14.40g/Kg/d)by gavage and control group were with PGE1(2.5ug/Kg/d)by intraperitoneal injection each day.The experimental period is 28d.10%chloral hydrate anesthesia executed rats and they were killed through dislocated executed.One side of eyeballs of rats were removed and the paraffin secitons of retinal slides were made.Retinal structure was observed by optical microscope and electron microscopy.The expression of iNOS,NOX4,Bax and Bcl-2 in the retina was detected by immunohistochemical staining.The color intensity of immunohistochemistry was measured by image analysis software and quantitative analysis was conducted.The expression of AQP4 and GFAP were detected by immunofluorescence double-standard technique.On the other side,the retina was removed on the ice through the same methods.The content of malondialdehyde(MDA)and peroxide dismutase(SOD)was detected.The expression of iNOS,NOX4,Bax,Bcl-2 and AQP4 was analyzed by Western Blot.Results:(1)MDA and SOD content changes:compared with the sham group,SOD content in the retina of the model group decreased and MDA content increased(P<0.01),Compared with the model group and control group,SOD level of treatment group increased and MDA level decreased(P<0.05).(2)Immunohistochemical:① NOX4 and iNOS expression:compared with the sham group,the positive expression of iNOS and NOX4 of model group rats retina were significantly higher(P<0.01).Compared with model group,the expression of NOX4 and iNOS in the treatment group rats after the intervention were reduced(P<0.05).The positive expression was in the ganglion cell layer,nerve fiber layer,inner layer,outer layer and the photoreceptor cells,such as a tan or brown granules.② Bax and Bcl-2 expression:compared with the sham group,the positive expression of Bax in the retina of the model group was significantly increased(P<0.01),and the expression of Bcl-2 was significantly decreased(P<0.01).Compared with the model group,the expression of Bax in the retina of the treatment group was decreased(P<0.05),and the expression of bcl-2 was significantly increased(P<0.01).(3)Immunofluorescence:AQP4 and GFAP were expressed in the Muller cells.The expression of AQP4 and GFAP in the rat retina of the model group increased,and the expression area involved the retinal layers.The positive expression of AQP4 and GFAP in rats in the treatment group decreased.(4)Western-Blot:① The relative expression levels of iNOS and NOX4 proteins:compared with the sham group,the relative expression levels of the retinal iNOS and NOX4 proteins in the model group were significantly increased(P<0.01).Compared with the model group,the relative expression of the retinal iNOS and NOX4 in the treatment group was significantly reduced(P<0.01,P<0.05).② Bax and Bcl-2 protein relative expression:compared with the sham group,the relative expression of the retinal Bax protein in the model group rats increased significantly(P<0.01).The relative expression of the retinal Bcl-2 protein decreased significantly(P<0.01).Compared with the model group,the relative expression of the retinal Bax protein in the treatment group rats decreased(P<0.05),and the relative expression of the retinal Bcl-2 protein increased significantly(P<0.01).③The relative expression of AQP4 and GFAP protein:ther was a small amount of AQP4 and GFAP protein in the rats retina of the sham group.The relative expression of AQP4 and GFAP protein in the rats retina of the model group was significantly increased(P<0.01).Compared with the model group,the relative expression of the retinal AQP4 protein in the treatment group rats decreased(P<0.01),but the GFAP showed no significant difference(P>0.05).Conclusions:1.The retina chronic hypoperfusion injury in the BCCAO rats may beclosely related to the mechanism of oxygen-radical damage,cell apoptosis and glial cell activation and proliferation.2.The YWTD may improve the retinal chronic hypoperfusion injury through regulating the anti-oxidation and anti-apoptosis and inhibiting the proliferation of glial cells. |
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