| Objectives:1.To establish corneal endothelial decompensation model on the rhesus monkey and SD rat,and to compare the pros and cons,applications of the two different models.To provide new thoughts in animal models of corneal endothelial decompensation.2.To study the expression of Aqaporin-1(AQP1),Aqaporin-3(AQP3),Aqaporin-5(AQP5),ATPase Na+/K+ and Smac/DIABLO in SD rat corneal endothelial decompensation model induced by PVP-I.To discuss the possible underlying mechanism.Methods:1.To establish the corneal endothelial decompensation model on the rhesus monkey using ultrasound damage and observe the corneal changes clinically and pathologically.2.To establish the corneal endothelial decompensation model on the SD rat using anterior chamber irrigation of 0.5%and 0.25%PVP-I and observe the corneal changes clinically and pathologically.3.To compare the advantages and disadvantages of the two methods to establish the corneal endothelial decompensation model.4.RT-PCR and Western Blot were applied to investigate the mRNA and protein expression of Aqaporin-1(AQP1),Aqaporin-3(AQP3),Aqaporin-5(AQP5),ATPase Na+/K+ and Smac/DIABLO in SD rat corneal endothelial decompensation model induced by PVP-I.Results:1.The success rate of modified ultrasound induced corneal endothelial decompensation model on rhesus monkey was 100.0%.After the endothelium was injured by ultrasound,the changes of cornea occurred gradually in the endothelial layer,stroma,Bowman's membrane and basal epithelial layer.At 4 weeks after surgery,the cornea was severely cloudy with typical bullous changes underneath the epithelium.IVCM results showed that in the early stage,the inter-space of corneal endothelial cells enlarged and mild edema was detected in the endothelial cells and stroma.The cell morphology of stroma altered and activated stromal cells presented with the increase of stromal thickness.Since 2 weeks after surgery,the nerve plexus in Bowman's layer decreased and edema of stroma and endothelial layer increased.At the 3 weeks after surgery,the inter-space of basal epithelial cells increased with a few Langerhans' cells infiltration,while edema of stroma and endothelial layer increased.At the 4 weeks after surgery,large amount of Langerhans' cells presented in basal epithelial layer.Only a few nerve lexus could be seen in Bowman's layer.The stroma and endothelial cells had severe edema.Large amount of activated stromal cells presented.With the prolonged duration after injury,the central corneal thickness in experimental group increased.Compared with the central corneal thickness prior to surgery,the difference was statistically significant.The central corneal thickness in experimental group at different time point was significantly different with it in control group and prior to surgery.At 4w after injury,HE staining indicated that the corneal endothelial layer was invisible.Descemet's membrane was exposed and the corneal stromal thickness increased significantly.Scanning electron microscope showed that large area of Descemet's membrane exposed with very few endothelial cells attached and residual corneal endothelial cells were severely edema.2.The success rate of corneal endothelial decompensation model induced by irrigation of PVP-I into anterior chamber on SD rat was 100.0%both in concentration of 0.5%(group 1)and 0.25%(group 2).Obvious changes of corneal endothelial decompensation were noted at 14 days after surgery in group using concentration of 0.5%PVP-I,while significant changes of corneal endothelial decompensation presented at 30 days after surgery in group using concentration of 0.25%PVP-I.The main complications after surgery included hyphema and eyeball atrophy.The incidence rate of hyphema was 5.0%(5/100 eyes),including 2 eyes in group 1(4.0%)and 3 eyes in group 2(6.0%).At 30 days after surgery,eyeball atrophy was noted in 4.0%of eyes(4/100 eyes),including 3 eyes in group 1(6.0%)and 1 eye in group 2(2.0%).There was no statistically significant difference between the incidence rate of complications in group 1(10.0%)and 2(8.0%)(X2=2.7616,p>0.05).The central corneal thickness(CCT)in group 1 and 2 increased with the duration of follow-up.In group 1,the CCT at 14 days and 30 days was significantly thicker than the CCT prior to surgery(p<0.05).In group 2,the CCT at 30 days was significantly thicker than the CCT prior to surgery and in normal control group(p<0.05).HE staining showed that different degree of the losing corneal endothelial cells in group 1 and 2.The thickening of corneal stromal layer with abundant vessels and vanish of collagen fibers presented.Scanning electron microscope indicated that the exfoliation of corneal endothelial cells with swollen of corneal stroma.Transmit electron microscope indicated that the mitochondrial eminence and edema of axon in corneal tissue.The damages in pathologic examination,Scanning electron microscope and Transmit electron microscope in group 1 was more severe than those in group 2.In SD rat corneal endothelial decompensation model,RT-PCR revealed that the expression of AQP1 mRNA declined first and then increased,reached the summit at 14 days which was 4.7 times of the expression in normal cornea.The expression of AQP3 and AQP5 mRNA declined significantly and reached the valley till 7 days after surgery.The expression of AQP3 and AQP5 mRNA was around 8%and 1%of expression in normal cornea.The expression of Na+K+-ATPase mRNA decreased at early stages and increased at late stages.It reached the valley at 7 days after surgery,which was 1/4 of normal cornea.The summit was reached at 30 days after surgery,which was 1.9 times of expression in normal cornea.Compared with normal corneal tissue,Western-blot showed that the protein expression of AQP1 increased first and then declined.The summit was reached at 7 days after surgery,which was around 7 times of expression in normal cornea.At 30 days,the expression of AQP1 was lower than normal cornea.The protein expression of Na+K+-ATPase increased significantly and reached the summit at 7 days,then declined to similar expression with normal cornea at 14 days.The protein expression of Smac/DIABLO reached the summit at 7 days and remained significantly high at 30 days.Conclusions:1.Modified ultrasound method could induced CED model on rhesus monkey successfully.The pathological physiological procedure of corneal endothelial dysfunction induced by ultrasound in Rhesus monkey can be shown clearly under in vivo confocal microscope.2.Irrigation into anterior chamber using both 0.5%and 0.25%concentration of PVP-I could induce corneal endothelial decompensation model successfully on SD rat.There was possibility of eyeball atrophy in this method.0.5%concentration of PVP-I could induce the corneal endothelial decompensation model more quickly and the clinical manifestation was relatively typical.3.Significant changes were noticed in the expression of protein and mRNA of aquaporin during the development of CED in SD rats.The mRNA expression of AQP1 decreased first then increased significantly while the mRNA expression of AQP3 and AQP5 declined significantly.The protein expression of AQP1 increased significantly then decreased.The mRNA expression of Na+K+-ATPase decreased first then increased,while the protein expression of Na+K+-ATPase increased first and then declined.The mRNA expression of Smac/DIABLO decreased first then increased followed by decrease,while the protein expression of Smac/DIABLO increased constantly.The changes in mRNA and protein expression of AQPs,Na+K+-ATPase and Smac/DIABLOwere noted during the development of CED in SD rats.These findings indicated that AQPs,Na+K+-ATPase and Smac/DIABLO played role in pathophysiological procedure of CED.AQP1 and Na+K+-ATPase might be sensitive indicators of CED formation.Smac/DIABLO might regulate the apoptosis of cells in corneal tissue through mitochondira apoptosis channel and take part in the pathophysiological procedure of CED development.However the exact mechanism of regulation needs further studies. |
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