The Role And Mechanism Of Aquaporins In Retina Injuries After Acute Intraocular Pressure Elevated

PARTâ… THE EFFECT OF INTRAOCULAR PRESSURE ALTERATIONS ON EXPRESSION OF AQPS IN RAT RETINAObjective:The aim of this study was to investigate the alteration of AQP1,3,4,5,9 expression in rat retina under different acute intraocular increase(AOI),so as to explore the mechanism of some diseases related to the abnormal intraocular pressure.Methods:(1)in vivo experiment Acute intraocular hypertension model was induced in one eye of adult Wistar rats,while the other eye remained untreated or sham-treated,and served as control.The animals were peritoniumlly anesthesed with ketamine/xylazine.The anterior chamber of the treated eye was cannulated from the pars plana with a 27-gauge infusion needle connected to a bag containing normal saline.The intraocular pressure was increased to 75 cm WC,125 cm WC or 150 cm WC for 60 min by elevating the saline bag.The sham-treated eyes underwent similar procedures but without elevation of the saline bag.Eyes were enucleated at 3 h,12 h,24 h,48 h,72 h or 5 days after reperfusion and kept immersed in fixative solution containing 4%paraformaldehyde for 3 h. After washing in saline,the tissue was embedded in saline containing 3% agarose(w/v),and 12μm-thick slices were cut by using a vibratome. Subsequently,the morphological changes were observed with HE staining and transmission electron microscope(TEM).Retina water content(RWC) was measured by using wet-dry weighing method and the changes of vascular permeability in rat retina was detected by Mozhi-perfusing test. The mRNA and protein expression of AQP1,3,4,5,9 in the rat retina were detected with in situ hybridization(ISH),reverse transcription polymerase chain reaction(RT-PCR),immunohistochemistry(IHC)and double immunofluorescent staining.(2)in vitro experiment Rat Müller cells were prepared from newborn pups after born 7 days.Müller cells purification was assessed by staining with glial fibrillary acidic protein(GFAP)antibody and Vimentin(the GFAP-positive and Vimentin-positive cells were more than 98%).The cultured cells were divided into two groups at random,control group without anly trearmentand experimental group.In the experimental group, the cells were cultured under pressure of 0.15 MPa,0.30 MPa or 0.60 MPa for 2 h additional incubation.Each group was examined at interval times of 3 h,6 h,12 h,24 h and 48 h,respectively.The morphology of the cells in both of the control group and the experimental group was observed by inverse phase microscope and electron microscope,the viability of the cells was measured by MTT colorimetric method and trypan blue staining.The alterations of AQP1,AQP 3,AQP 4,AQP 5,AQP 9 and its mRNA expression of the cultured cells were studied by the methods of in situ hybridization,immunocytochemistry,double immunofluorescent stainings and imaging analysis.Results:In ivo there were no significantly changes of the morphological phenotype and RWC in the control group,and the content of AQP 1,AQP 3,AQP 4,AQP 5,AQP 9 and its mRNA expression was in low levels.While in the experimental groups,The RWC was increased signicantly after enhancing perfusion stopped,and reached its first peak at 12 h,then decresed slightly but still higher than the normal levels; however,at 48 h,RWC increased quickly and reached to a second peak;at 72 h,RWC decreased to the normal levels.The thickness changes of rat retina inner layer was in accordence with the BWC changes.The morphological changes of the rat retina was including the enlargement of intracellular space and the decreased cells density was observed,and the permeability of rat retina vascular in experimental group was only slightly increased.The expression of AQP4 and AQP9 in the rat retina was strongly increased after AOI.The expression of AQP4 and AQP9 was significantly correlated with its mRNA during the retinal edema formation.The expression of AQP4 and AQP9 was correlated with the RWC and the increase in rat retina inner layer thickness after AOI.The other AQPs (AQP1,AQP3,AQP5)expression was only slightly altered as compared with the control.In vitro the GFAP expression of cells in experimental group was increased after cultured under different pressure incubation. Compared with the control grooup,the cells in experimental group was swelling and the volume of the cells increased after AOI,and their viability was decreased.The alterations of AQP 4,AQP 9 and its mRNA expression of the cells in experimental group was incresed significantly,and the increase of AQP4 and AQP9 expression was co-relative with the levels of pressure.Conclusions:The RWC and the thickness of inner layer in rat retina was gradually improved and the cells in retina tissue were swelling after AOI,indicating that the retina edema appeared.The expression of AQP4, AQP9 and their mRNA up-regulated in rat retina along with the increasing of IOP.In vitro there was an co-relationship between the increase of AQP4 or AQP9 expression and the Müller cell injuries.These results indicate that AQP4 and AQP9 may play an important role in the retinal edema after AOI, and may act as pressure sensor and water channel in the process of water transportation balance in the retina. Objective:To determine the effect of intraocular pressure elevation on the AQP4,Kir4.1,laminin andα-syntrophin expression in rat retina,so as to explore the effect of abnormality anchoring mechanism of AQP4 and Kir4.1 in visual dysfunction.Methods:(1)In vivo rat AOI model was induced by acute anterior chamber pressured perfusing in one eye of adult Wistar rats,while another eye remained untreated or sham-treated,and served as control. Electroretinograms(ERG)were recorded over a 10⁵-fold range of flash intensities in dark-adapted rat and analyzed for a- and b-wave amplitude and latency.The morphological changes of rat retina in experimental group were observed with HE staining and transmission electron microscope (TEM).Retina water content(RWC)was measured by using wet-dry weighing method.The mRNA and protein expression of AQP4,Kir4.1,α-syntrophin and lamininin the rat retina were detected with in reverse transcription polymerase chain reaction(RT-PCR),immunohistochemistry (IHC),double immunofluorescent stainings.(2)In vitro experiment Rat Müller cells were prepared from newborn pups after born 7 days.Müller cells purification was assessed by staining with glial fibrillary acidic protein(GFAP)antibody and Vimentin(the GFAP-positive and Vimentin-positive cells were more than 98%).The cultured cells were divided into two groups at random,control group without and experimental group.In the experimental group,the cells were cultured under pressure of 0.15 MPa,0.30 MPa or 0.60 MPa for 2 h additional incubation.The cells in both of the control group and experimental group were sub-divided into 3 sub-groups at random,one was control group cultured without anly trearment,the other two groups cultured in an DMEM contained 7 nM^-1or 15nM^-1content laminin-1.The cells in each of the group were examined at interval times of 3h,6h,12h, 24 h and 48 h,respectively.The morphology of the cells in both of the control group and the experimental group was observed by inverse phase microscope and electron microscope,the viability of the cells was measured by MTT colorimetric method and trypan blue staining.The alterations of AQP4与Kir4.1及α-syntrophin,laminin and its mRNA expression of the cultured cells were studied by the methods of in situ hybridization,immunocytochemistry,immunofluorescent staining and imaging analysis.The double immunofluorescent staining and confocal scope were used to detect the changes of AQP4 and Kir4.1 co-expression in rat retina Müller cell after AOI.Results:In ivo there were no significantly changes of the morphological phenotype and the ERG b-wave abnormal in the control group,and the expression of AQP4,Kir4.1,α-syntrophin,lamininin and their mRNA was in low levels.While in the experimental groups,The RWC was increased signicantly and the b-wave amplitude of ERG decreased after enhancing perfusion stopped.The morphological changes of rat retina was swelling and edema formation after AOI.The co-expression of AQP4 and Kir4.1 was significanly seen in the inner layer of rat retina in control group.While in the experimental group,the co-expression of AQP4 and Kir4.1 in rat retina was decreased or completely loss,especially in perivascular and vitreous-fasing membrane domains.The changes of AQP4 and Kir4.1 co-expression was correlated with the RWC increase and ERG b wave amplitude decrease in rat retina after AOI.And the expression of laminin was decreased significantly after AOI.While the changes ofα-syntrophin expression was only slightly altered as compared with the control.In vitro the GFAP expression of cells in experinmental group was increased after cultured under different pressure incubation.Compared with the control grooup,the cells in experimental group was swelling and the volume of the cells increased after AOI,and their viability was decreased.The co-expression of AQP 4 and Kir4.1 in the memmbrane of Müller cells in experimental group was decreased significantly,and the decrease of AQP4 and Kir4.1 co-expression was co-relative with the levels of pressure.While,the co-expression of AQP4 and Kir4.1 in different content laminin-1 cultured Müller cells was only slightly decreased,as compared with the control.Conclusions:In vivo,the ERG b wave amplitude decreased significantly after AOI.While the co-expression of AQP4 and Kir4.1 was decreased along with a reduce of the laminin,their intracellular anchoring molecular in rat retina after AOI.In vitro the decreased of AQP4 and Kir4.1 co-expression resulted in the Müller cell injuries and there was a co-relationship between increase of AQP4 and Kir4.1 co-expression and the content of lainin-1 in cultuer medium.These results indicate that the decrease of AQP4 and Kir4.1 co-expression in Müller cells memmbrane may play an important role in retinal edema and visuion dysfunction after AOI,and laminin may play an important role in the anhoring of AQP4 and Kir4.1 in Müller cells special memmbrane.