Experimental Studies On The Relationship Of The Retinal Ganglion Cell Damage In Mouse Glaucoma Model And CD3?

Backgrouds Glaucoma is a leading cause of irreversible blindness in the world, resulting in an initial loss of peripheral vision with central visual defects occurring much later. The progressive death of retinal ganglion cell (RGC) is a key character of glaucoma.But the pathogenesis of the death of retinal ganglion cell is still unclear now.How to reduce the RGC damage of glaucoma is a hard problem.It is very meaningful to study the decrease speed and the morphology change of RGC.Confocal scanning laser microscopy is used to observe the RGC number changes of transgenic mice with expression of fluorescent protein in RGC under some pathological conditions.Two recent studies demonstrated the possibility of visualizing RGC dendrites in vivo in monkey and mouse.However,no study has provided in vivo data to determine the morphological changes of RGC. The increased intraocular pressure does not explain glaucoma in all patients but can be considered as a risk factor of the disease. People pay more and more attention to the relationship of immune system and glaucoma. Our recent study have presented strong evidence that the immune molecule CD3?resulted in a significant change of RGC dendritic growth and elimination in developing retina.RGC dendritic density increased in mature CD3?-/-mice.RGC dendritic density increased After the expression of CD3?decreased in RGC by shRNA.CD3?affect the morphology and function of RGC dendrite under physiological condition.However,it is still unclear that whether CD3?is related to the damage of RGCPurposes1 To establish three types of glaucoma model.2 To image RGC in vivo in the selected glaucoma model.3 To construct the mCherry CD3?shRNA vector.4 To observe the effect of RNAi technique usage in retinal neuroprotection.Methods1 We set up three types of glaucoma model:1)optic nerve crush model:the optic nerve was clamped using a pair of self-closing tweezers.2)NMDA intravitreal injection model:Hamilton injector was used to inject NMDA(40nmol) into the vitreous body.3)chronic high intraocular pressure model:microbeads (10x106microbeads/ml) were injected into the anterior chamber to obstruct the angle of anterior chamber.2 A confocal scanning laser microscopy was used to image Thyl-CFP mice and Thyl-YFP mice to observe the damage of RGCs.3 The mCherry CD3?shRNA expression constructs were generated by inserting a 68 bp shRNA template sequence between BamHI and XhoI sites.4 Real-time PCR and Western blot assy were used to detect CD3?in retinal tissues of three glaucoma models.5 The mCherry CD3?shRNA was injected into the vitreous body, then set up the glaucoma models,eyes were enucleated and processed for whole mount preparation. Brn3b-positive RGCs were counted and analyzed.Results1 We set up three types of glaucoma model:1)optic nerve crush model;2)NMDA intravitreal injection model;3)chornic high intraocular pressure model.2 RGC loss in optic nerve crush model focus on the postoperative week,approximately 97.4% above control(P<0.001).NMDA induced significant loss of RGC(approximately 94.7% above control) in 24 hours(P<0.001).High IOP triggered the death of 26.7%of RGC over a 4 week period(P<0.001).3 In Thyl-CFP mice with IOP elevation,three weeks after the IOP elevation,CFP-positive RGCs were reduced by 21%±4.8%(P<0.001).CFP-positive RGCs continued to decrease in number over time and 6 weeks after IOP elevation,CFP-positive RGCs were reduced by 30%±4.7%(P<0.001).In Thyl-YFP mice with IOP elevation,the damage on RGC dendrites start several days before cell death.4 We constructed the mCherry CD3?shRNA expression vectors,CD3?shRNA knockdown the CD3?expression level by 70% in HEK293 cells.5 CD3?mRNA expression levels in retinal tissue of three glaucoma models are significantly changed compared to control(p<0.05),but CD3?protein expression levels in retinal tissue of three glaucoma models are not significantly changed compared to control.6 The damage of RGC in three glaucoma models with mCherry CD3?shRNA introvitreal injection are not significantly decreased compared to the same models without mCherry CD3?shRNA introvitreal injection.(p>0.05)Conclusions1 RGC loss in optic nerve crush model focus on the postoperative week, NMDA induced significant loss of RGC in 24 hours, high IOP trigger the progressive death of RGC over a long time.2 Confocal scanning laser microscopy is used to observe the RGC number decrease of Thy1-CFP mice and RGC dendrite loss of Thy1-YFP mice.3 CD3?mRNA expression levels in retinal tissue of three glaucoma models are significantly changed compared to control,but CD3?protein expression levels in retinal tissue of three glaucoma models are not significantly changed compared to control. The damage of RGC in three glaucoma models with mCherry CD3?shRNA introvitreai injection are not significantly decreased compared to the same models without mCherry CD3?shRNA introvitreal injection.CD3?maybe not related to the RGC damage.