| Hypoxia ioning protects against retinal ischemia reperfusion injury in rats AbstractpreconditObjectivePart1: To investigate the expression of HIF-1αand P53 after hypoxia and find the correlation between them.Part2: To investigate the expression of hif-1α, apoptosis of retinal cells and the role ofhif-1αin apoptosis in rats' retinal ischemia-reperfusion injury.Part3: To examine whether hypoxia preconditioning(HPC)can decrease ischemia reperfusion(I/R)injury in retina and investigate the role of hif-1α. Investigate apoptosis in rats' retinal injury after hypoxia preconditioning.Part4: To investigate the expression of hif-1α, in rats' retinal ischemia-reperfusion injury, after intravitreal injection idio-inhibitor Wortmannin and hypoxia preconditioning.MethodsPart1: To investigate the expression of HIF-1αand P53 after hypoxia and find the correlation between them. Normal pressure hypoxia animal model: hypoxia gmary was made of organic glass, the vlume is 100cm×60cm×60cm, the thickness of the organic glass is 4cm, the flanks were enveloped by chloroform, so as to open the grnary to take foods, water and rats. Open a diameter 1cm outing gas hole in the surface of the gmary. Make a inlet in the flank to receive Nitrogen gas and wind through T-tube. To control the flow rate glass float-type flowmeter was used. The animals were divided into two groups, each group 28. One group was breed in normoxia room and the other was breed in hypoxia room. four animals were executed after breeding 0hour,2 hours,6hours,8hours,12hours,24hours,48hours. HE staining was done to observe the common change of the rat retina. Immunohistrochemical staining detected the expression of HIF-1αand P53 protein. Through electron microscope the ultra-structure of the retinal can be observed. Buffy-yellow drop located in cell nucleus and cytoplasm is the positive expression. Using Image Analysis System analyze the pictures. Statistical analysis was performed using SPSS 13.0 software. Variance analysis and t-test were used to deal with the date. All values were expressed as means and SE. Statistical significance was accepted when the P-value was less than 0.05.Part2: To investigate the expression of HIF-1α, apoptosis of retinal cells and the role of HIF-1αin apoptosis in rats' retinal ischemia-reperfusion injury. 54 healthy adult Wistar rats (sex is not limited, the weight from 170g-220g, offered by experimental animal department of China medical university) were divided randomly into 3 groups. Group A (normal control group, n=6); Group B (ischemia-reperfusion group, n=42), for each rat anterior chamber was puncture in random eye, these rats were further divided into 6 sub-groups according to different time point of reperfusion (0,2,6,8,12,24,48 hours), each sub-group had 6 rats; Group C (experimental control group, n=6), 6 rats were randomly selected to do puncture of anterior chamber in random eye of two as controls.The rat model of experimental retinal ischemia-reperfusion injury was established by increasing the intraocular pressure to 110mmHg (1kPa=7.5mmHg) in rat eyes. At different time points of post ischemia, the expression of HIF-1 a of the retina was detected by immunohistochemical staining, and apoptosis of the retinal cell was detected by terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphatebiotin nick end labeling.To make the model of ischemia-reperfusion injury in retinal in rats the animals were anesthetized by peritoneal injection 20%chloral hydrate. Decicaine was used to do superficial anesthesia. Rats' heads were fixed to do puncture of anterior chamber at temple limbus of cornea and the distal end of pinhead was connected with Saline bottle which was elevated to 150cms above the animal.Overdose narcotic was used to execute the mice after anterior chamber were filled at different time, removed the eyes and fixed them in routine fluids. Tissue was then anhydrated and paraffinized. Section was made 4μm in thickness. The distribution of apoptosis was detected by using TUNEL with in situ cell death staining kit. The procedures were according to instructions. Immunohistochemical staining and TUNEL staining image were evaluated by analysis assay ofHIF-1αIOD and apoptosis IOD.Buffy-yellow drop located in cell nucleus and cytoplasm is the positive expression. Using Image Analysis System analyze the pictures. Statistical analysis was performed using SPSS 13.0 software. Variance analysis and t-test were used to deal with the date. All values were expressed as means and SE. Statistical significance was accepted when the P-value was less than 0.05.Part3: To examine whether hypoxia preconditioning(HPC)can decrease ischemia reperfusion(I/R)injury in retina and investigate the role of hif-1α. Investigate apoptosis in rats' retinal injury after hypoxia preconditioning. retinal ischemia was induced in Wistar rats by increasing the intraocular pressure to 110mmHg for 60 minutes via cannulation into the anterior chamber. retinal ischemia hypoxia preconditioning for 4hours constituted the preconditioning stimulus. Rats first underwent HPC 4hours. After 4hours later they underwent a 60 minutes of ischemia 24 hours later. The light microscopy(LM ), transmission electron microscopy(EM )and the expression of HIF-1αwas observed and TUNUL in rat retinas were measured 0,2,6,8,12,24,48 hours later after 60 minutes ischemia. Buffy-yellow drop located in cell nucleus and cytoplasm is the positive expression. Using Image Analysis System analyze the pictures. Statistical analysis was performed using SPSS 13.0 software. Variance analysis and t-test were used to deal with the date. All values were expressed as means and SE. Statistical significance was accepted when the P-value was less than 0.05.Part4:experimental group (A): PI-3K idio-inhibitor Wortmannin0.01ml was injected intravitreal; experimental control group (B): Ringer's so lation 0.01ml was injected intravitreal; The rat model of experimental retinal ischemia-reperfusion injury was created by increasing the intraocular pressure to 110mmHg (1kPa=7.5mmHg) in rat eyes. At different times post ischemia reperfusion injury, the immunohistochemical staining and RT-PCR were done to observe the expression of HIF-1α. Buffy-yellow drop located in cell nucleus and cytoplasm is the positive expression. Using Image Analysis System analyze the pictures. Statistical analysis was performed using SPSS 13.0 software. Variance analysis and t-test were used to deal with the date. All values were expressed as means and SE. Statistical significance was accepted when the P-value was less than 0.05.ResultPart1: HIF-1αand P53 been expressed in rat's retinal under hypoxia condition. The expression of P53 arriva to the peak after HIF-1α. there is obviously positive correlation between HIF-1αand P53, the correlation is 0.903. The retinal apoptosis arrival to the peak in electron microscope, just at that time the expression of P53 achieve to the peak.Part2: There was no HIF-1αexpression in normal control group and experimental control group. Hif-1αwas expressed in ischemia-reperfusion group at 2,6,8,24,48hours. The expression reached to the peak at the 12 hours after retinal ischemia-reperfusion, the expression of HIF-1αdeclining was detected in 12,24,48hours group in gangling cell layer of retina and inner nuclear layer.The TUNEL positive cells become more after ischemia 60min and 12,24hours reperfusion in GCL (ganglion cell layer) and INL(inner nuclear layer) layers. But we can see little TUNEL positive cells after 48 hours. The expression of TUNEL arrive to the peak after HIF-1α. There is obviously positive correlation between HIF-1αand TUNEL.Part3: Ischemia induced histologic damage while HPC can prevent performed 24 preventable disease ischemia. In cantrast to the sham PC group, the histologic damage was little after HPC. and the expression of HIF-1αwas significantly increased(P＜0.01). The result showed that the peak of the expression of HIF-1αduring the ischemia-reperfusion of retina is 12h group. The IOD is 7.48±1.35. The peak of the expression of HIF-1αduring the ischemia-reperfusion of retina after hypoxia preconditioning is 8h group. The IOD is 9.83±1.57. Compared the peak of two groups T=2.985, p=0.031＜0.05 statistical significance was accepted. The peak of the expression of TUNEL during the ischemia-reperfusion of retina is 24h group. The IOD is 8.3±1.0. The peak of the expression of TUNEL during the ischemia-reperfusion of retina after hypoxia preconditioning is 8h group. The IOD is 4.627±1.92. Compared the peak of the two groups T=3.443, p=0.018＜0.05 statistical significance was accepted.Part 4: In experimental control group: HIF-1αwas expressed in the cells of retinal ganglion layer and inner nuclear layer at 6 hours post ischemia. The expression attained the highest level at the 12 hour after retinal ischemia-reperfusion then the expression was declined. In experimental group: the expression of HIF-1αwere obviously decreased and attained the highest level at the 12 hour then declined. In control group: the expression was low, and the level is almost unchanged. The IOD of HIF-1αduring the ischemia-reperfusion of retina after hypoxia preconditioning was significant different from that of Wortmannin T=13.46 P=0.000ConclusionPatt1: HIF-1αand P53 been expressed in rat's retinal under hypoxia condition. The expression of P53 arrival to the peak after HIF-1α. There is obviously positive correlation between HIF-1αand P53. The retinal apoptosis arrival to the peak in electron microscope , then the expression of P53 achieve to the peak.Part2: Expression of HIF-1αin the rats' retina is greatly enhanced after ischemia-reperfusion, which may be involved in the retinal injury;The injury of retinal neurons occurs partly in the form of apoptosis. The expression of HIF-1αmay play an important role in cell apoptosis.Part3. HPC provides protection against retinal I / R injury during the period of time.Part4: The expression of HIF-1αafter ischemia-reperfusion injury and HPC in rat retina come true through PI-3K pathway. If we block PI-3K pathway the expression of HIF-1αwill be decreased. But from the result we can see Wortmannin can not block the expression completely, we may guess expression of HIF-1αbe completed through many way.
|
PDF Full Text Request