Effect Of Dark Rearing On Local Circuit In Retina Of SD Rats After Optic Nerve Transection

Chapter I Anatomical basis of the optic nerve transection model of SD ratsObjective To provide the anatomical basis for establishing a stable and reliable optic nerve transection injury animal model of SD rats through exploring the relationship between central retinal artery and optic nerve,Method Optic nerve peripheral vessels in normal SD rats (n=6) were demonstrated by perfusion technology with red latex and disected under the stereomicroscope; According to the distance from the proximity of central retinal artery to rear pole of eye ball, optic nerves were transected intrathecally and extrathecally at different positions,2.0(n=6),5.0(n=6) and6.0mm (n=6) to eye ball respectively in different groups. The effects of the optic nerve transection at different parts on retinal blood supply was evaluated by gelatin ink perfusion technique.Result The vessels around optic nerve in normal SD rat were clearly displayed with red latex perfusion. Ophthalmic artery located at the outer side of the optic nerve. Ophthalmic artery detour the optic nerve and run forward, and gave off the branchs including central retinal artery and the inner, outer ciliary arteries, the former was the main branch of ophthalmic artery and its strating region to the posterior pole of eyeball was (5.784±0.054)mm. The central retinal artery processed front inside and underneath and cling the optic nerve sheath forwardly along the optic nerve by loose connective tissue. Four branches were issued at the posterior pole of the eyeball wall for nourishment. And the trunk of the central retinal artery penetrated the eyeball wall, giving out branches for retina nutrition.With gelatin ink perfusion, it was showed that compared to other way, best blood supply was reserved when the optic nerve was cut at6.0mm intrathecally.Conclusion The SD rat central retinal artery was paralled with the optic nerve in the optic nerve sheath. It was an ideal way to establish the optic nerve transection model of SD rat by transecting the optic nerve intrathecally at6.0mm away from the posterior pole of eyeball. Chapter II Protective effects of dark rearing on the structure of inner retina of SD rats after optic nerve transectionObjective To explore the role and influence of dark rearing on bipolar cells, ganglion cells and retinal dopaminergic amacrine cells and their synaptic connections in optic nerve transection rats.Method Animals were divided into normal group, control group and optic nerve transection groups. In each group, half of the animals were light/dark reared and half were dark reared only. The optic nerve transection model was made following the method in chapter one. Morphological structure and the number of ganglion cells were observed by Nissl staining. The expression of Recovering PKC-a in bipolar cells were measured by immunohistochemical staining. Both immunohistochemistry and Western Blot were employed to detect expression of TH in amacrine cells.Result1.It was showed by Nissl staining that in light/dark rearing group, structure disorder was observed since the3rd day after optic nerve transection and with obvious thinning of retina at the14th day. The number of retinal ganglion cell per unit area decreased at the7th day and14th day. In dark rearing group, structure disorder was observed since the5th day after optic nerve transection, which was delayed compared with the light/dark group. The number of retinal ganglion cell per unit area decreased since the14th day.2.Compared with the normal group, expression of PKC-a in light/dark rearing group was increased in bipolar cells in rat retina at the3rd day after optic nerve tansection, and then decreased at the5th day and7th day. In the14th day, the expression of PKC-a could be detected only at the synaptic sites in the inner plexiform layer and could not be seen at the soma of bipolar cells. The expression of PKC-a in the dark rearing groups was the same pattern with the normal group, except the14th day, when PKC-a expression was decreased and could only be detected at the synaptic site in the inner plexiform layer.3.The expression of Recovrerin (Rec) was increased in bipolar cells in rat retina at the1st day after optic nerve transection, and then decreased under normal level at the3rd day. At the5th day, it was up-regulated reaching normal level and down-regulated at the14th day again. In the dark rearing group, the expression of Rec was increased in bipolar cells at the1st day after optic nerve transection significantly and then regulated to normal level at the3rd day and remained this level in the5th day and7th day group. At the14th day after optic nerve transection, Rec was down-regulated under normal level. It was worth noticing that at the3rd day, Rec immune positive area in dark rearing group was significantly higher than that in the light/dark rearing group.4.1n the light/dark rearing group, the expression of TH was decreased gradually in retina since the1st day to the5th day after optic nerve transection. And it was regulated to the normal level at the7th day. In the dark rearing group, TH expression was decreased at the1st day. From then on, it was up-regulated and reached the normal level at the5th day. Western Blot results of TH expression were in accordance with those of immunohistochemistry.5.In the light/dark rearing group, axotomized synaptic connections showed by TH and double staining gradually reduced and reached the lowest level at the5th day after optic nerve transection. Then they were restored to some extend at the14th day, but far less than the normal level. At the5th day and14th day, the number of double stained synapses was higher in dark rearing group than in light/dark rearing group.Conclusion Dark rearing had a protective effect for bipolar cells, ganglion cells, DA amacrine cells and their synaptic connections in the SD rat retinal after optic nerve transection. Chapter III Possible mechanisms of dark rearing protecting the structure of inner retina after optic nerve transectionObjective To explore the possible mechanisms of the protective effect of dark rearing to retinal bipolar cells, ganglion cells, DA amacrine cells and their synaptic connections after optic nerve transection.Method Similar with Chapter Ⅱ, animals were divided into normal group, control group and optic nerve transection groups. In each group, half of the animals were light/dark reared and half were dark reared only. The optic nerve transection model was made following the method in chapter one. Expression of GAD67, BDNF and its receptor trkB in the retina were measured by immunohistochemical staining.Result1.In light/dark rearing group, the expression of GAD67increased at thelst day after optic nerve transection, reached the peak at the3rd day and then down-regulated since the5th day. In dark rearing group, the expression of GAD67at the1st and3rd day was higher than that at the same time points in light/dark rearing group.2.The expression of BDNF was decreased in the retina of the light/dark rearing group since the3rd day after optic nerve transaction, with the number of BDNF positive ganglion cells gradually reducing. While in dark rearing group, BDNF expression level wasn’t declined until the7th day.3.The expression of trkB was increased at the1st day in the light/dark rearing group after optic nerve transaction firstly, and then decreased under normal level at the3rd day. The expression of trkB was up-regulated gradually since the5th day but still below normal levels. In dark rearing group, trkB expression was up-regulated at the1st day and back to a normal level at the3rd day. From the3rd day on, at each subsequent time points, trkB expression was higher in the dark rearing group than in the light/dark rearing group.Conclusion Dark rearing may protect the structures of inner retina after optic nerve transection through increasing GABA synthesis and secretion, reducing BDNF depletion.