The Expression Of AQP4 In The Rat Retina After Chronic Experimental Elevation Of Intraocular Pressure

introductionThe concept of aquaporin was originated from Koefoe's research in 1953, and supported by many physiologists's experiments after that. 13 members of aquaporins having been Identified so far in mammals. AQPs are expressed in many fluid transport tissues, such as kidney tubules and glandular epithelia, as well as in non-fluid -transporting tissues, such as epidermis, adipose tissue and astroglia. Their classical role is facilitating trans-epithelial fluid. AQPs are also involved in swelling of tissue under stress, as in the injured cornea and the brain in stroke, tumor and infection. AQPs might also be involved in neural signal transduction, cell volume regulation and organellar physiology. The expression of AQP4 was found up-regulated in the ischemic and ischemic-reperfusion injuries, and the suppression of their expression could relieve the brain edema and damages. The similar phenomenon was also found in the ischemic-reperfusion model of the globe. In present study, we detected the expression of AQP4 in rat retina after chronic elevation of intraocular pressure and try to find out the potential role of it in this model.Materials1,Experimental animal90 healthy adult male wistar rats without diseases of eyes, weighed 250g-300g were selected in the present study.2,Reagentchloral hydrate nalytical reagent chloroform avantin ALC retroviridase RNase-free water [Oligo (dT) 15 ] (0.5μg/μl) dNTPs Mix heat stability Taq DNA PM 100bp DNA ladder Tris EDTA EB Agarose DEPC TaKaRa RNA PCR kit Pcr primer polylysine hematoxylin SABCkit APES H2O2 Rabbit—AQP43,equipmentTonopen applanometer Underwater electric coagulator MVE XC47/11-6 nitrogen canister deep freeze refrigerator DIAX900 tissue bomogenator FA 1604Selectronic balance XW-80A swirl commingler Multiphor II mini-electrophoresis apparatus UVIprogelatum image analysis system GeneQuant RNA/DNA analysator DUPONT SUPER T-21 high speed refrigerated centrifuge Biometra gradient PCR apparatus 2035 Jung Brocut section cutter OLYMPUS B201 ;microscope and imaging system refrigerator,homeothermiaMethods1,establishment of the modelan increase of IOP was induced by coagulating two episcleral veins unilaterally in rats with electric coagulator.2,RT-PCR (reverse transcriptase polymerase chain reaction)(1) Total RNA isolationTotal RNA was isolated using RNAout isolation system. The concentration and purity of total RNA was quantified by ultraviolet spectrophotometry by measuring OD260/OD280. preparation was discard if they had a ratio of optic density at 260nm/280nm lower than 1.6.(2) RT ( reverse transcription)The RNA preparation was subjected to reverse transcription using the reverse system under the condition of 42℃30 minutes, 99℃5minutes, 5℃5minutes.(3) cDNA amplificationThe first strand of cDNA was amplified with forward and reverse primer designed with primer Express3.0 software. (4) electrophoresisThe PCR product was determined by loading it on 2% agarose gels and subjecting them to electrophoresis.3,Immunohistochemistry and hematoxylin -eosine stainingglobs were enucleated and dissected into calix opticus, then fixed in fixative, dehydrate through an ethanol series, and embedded in paraffin. Serial sections were cut, then incubated with antibodies, followed by color development achieved in DAB, counterstained by hematoxylin eosine, then mounted on glass slides for observation. The method of HE staining was similar with IHC except for the inbubating with antibodies and antibodies' repairing.4,Statistical analysisQuantitative data were expressed as means±SE, and were analyzed with one-way analysis of variance (ANOVA) followed by Bonferroni test for multiple comparisons among experimental groups with control groups. Statistical analysis was performed using the SPSS 13.0statistical software. The results were considered statistically different at p<0.05.Results1,RT-PCRAQP4 mRNA expressed in normal rat retina, and the expression of it increased in IOP elevation model rats with the time going on. The peak expression of it was on the 7th day after IOP elevated and then decreased but still maintained at a higher level. The expression among experimental groups was statisticlly different compared with the control group except for the 3th day group.2,IHC(Immunohistochemistry)AQP4 was located in GCL, IPL , INL, OUL and ONL, both in normal and model rat retina. The expression of it was similar detected by means of immunohistochemistry and RT-PCR. 3,HEBy morphology observation of HE staining slides, we found the thickness of model rat retina changed significantly versus the control group, thickening at the fist stage and thinning at last duing the period of IOP elevation.The expression of AQP4 was intimately correlated with the thickness of rentina.DiscussionAQP4 was first cloned and confirmed as the member of AQPs family. It reside widely in mammals. In eye globe, it distribute in the nonpigmented epithelium of ciliarybody and retina.It's classical role is facilitating trans-epithelial fluid transport . AQP4 is also involved in swelling of tissues under stress, as in the brain in stroke, tumor and infection. Recent analysis has revealed unexpected cellular roles of AQP4: it might also be involved in neural signal transduction, cell volume regulation and organellar physiology.The patheogenesy of glaucoma is mainly mechanical theory and ischemic theory. It tends to consider the two mechanisms contribute to the glaucomatous damage together. The rationale for study of retinal neuroprotection in AQP4 deficiency was protection of brain tissue in AQP4 deficiency after ischemic stroke produced by middle cerebral artery occlusion. AQP4-expressing glial cells in brain have a similar supportive relationship to neurons as do the AQP4-expressing Mu¨ller cells in retina to ganglion cells and bipolar cells. The ECS in retina contains ions, neurotransmitters, and various matrix macromolecules, forming the microenvironment bathing Mu¨ller cells, bipolar cells, and ganglion cells, and facilitating cell- cell communication by diffusible solutes. On theoretical grounds, AQP4 deletion in Mu¨ller cells and reduced Mu¨ller cell water permeability could reduce initial cell swelling after ischemic damage, as well as alter ECS volume and composition after ischemia. As in other electrically excitable tissues, cell swelling and altered ECS homeostasis may be important early determinants of retinal neuronal cell injury and apoptotic cell death. In the present study, we found the correlation between AQP4 and the thickness of retina. It suggests that AQP4 might involve in the retina swelling and apoptotic signal transduction and possibly participate in the retina damage of glaucoma. The suppression of the AQP4 might be a new treatment for glaucoma.Conclusion1,AQP4 located at the GCL, IPL, INL,OUL, and a little at ONL. not only in normal globe of rat but also in the rat model.2,The expression of AQP4 was up-regulated and intimately correlated with thethickness of the retina after the IOP being elevated , this suggest that AQP4 might involved in damage of model retina. The function of AQP4 indicates that it might participate the damage of model by several mechanisms.