The Relationship Between The Changes Of PAP1 Gene Expression And The Apoptosis In Rat Retinal Cells After Retinal Ischemia-reperfusion Injury

PART I The rat model of retinal ischemia-reperfusion injury can be established and the apoptosis of the retinal cells was detected.[Objective]To establish a rat model of retinal ischemia-reperfusion (RIR) imjury by transiently elevated intraocular pressure (IOP) and to detect the apoptosis of the retinal cells.[Methods]72 sprague-Dawley (SD) rats were divided randomly into control group and RIR group .Each group Was consisted of 6 subgroups (3h,6h,12h,24h,72h and 7d after RIR; n=6).Retinal ischemia was induced of a needle into the anterior chamber connected to a saline column.Elevated IOP at 110 mmHg was maintained for 60 minutes.Morphological changes of the model' s retinas were study using nissel staining. The apoptosis of the retinal cells were evaluated dynamically by in situ terminal deoxynucleotidyl transferase-mediated biotin-deoxyuridine triphosphate nick-end labeling (TUNEL).[Results]1. The sign of retinal ischemia and reperfusion were clearly investigated in the model of RIR.2.The morphological changes of the rats retinas:The edema and vaculoatoin in inner plexiform layer (IPL)presented at 6h and 12h after RIR; karyopycnosis presented at 24h after RIR; The thickness of retinal IPL in RIR group at 7d was significantly thinner than that of control group (P< 0.01).3.The apoptosis of the retinal cells: The TUNEL-postive cells presented mainly in the retinal ganglion cell layers and the inner nuclear layers, and few postive cells in external nuclear layers.Few TUNEL postive cells were observed in the normal retina; Compared with the control group, the mumber of TUNEL postive cells in RIR group was significantly increased at the time points of 6h to 72h after reperfusion, respectively (P<0.01).The number of TUNEL postive cells reached a maximum at 24h and followed by a decreased at 72h after reperfusion.[Conclusions]The model of retinal ischemia-reperfusion with apoptosis of the retinal cells can be successfully established by elevated intraocular pressure at 110 mmHg and maintained for 60 minutes. PART II The expression of PAPl gene in rat retinaAfter RIR[Objective]To observe the expression of PAP1 gene in rat retina and the change of PAP1 gene expression after RIR.[Methods]120 Sprague-Dawley (SD) rats were divided randomly into cotrol group and RIR group. Each group was consisted of 6 subgroups (3h,6h,12h,24h,72h,and 7d after RIR n=10) Retinal ischemia was induced by transiently elevated Intraocucar pressure through the insertion of a needle into the anterior chamber connected to a saline column。Elevated the intraocular perssure at 110 mmHg was maintained for 60 minutes.The transcription levels and the changes of the expression of PAP1 gene in the retina after RIR were dynamically detected by semiquantitative reverse transcriptase-polymerase chain (RT-PCR) and in situ hybridization.[Results]1. There is the expression of PAP1 gene in the retina in the control group.The expression of PAP1 gene was detected in the GCL and INL of the retina sections.2.Compared with the control group,the transcription levels of PAP1-mRNA significantly increased as early as 3h after RIR(P < 0.01)and then significantly decreased at 6h,12h,24h(P<0.01).But, the expression amount of PAP1-mRNA signifcantly increased at 72h> 7d after RIR(P<0.01).3.The expression levels of PAP1 gene was detected in situ hybridization technique .Compared with the control group,the expression anount of PAP1 gene significantly increased at 3h(P<0.01),and then decreased at 6h,12h and 24h(P<0.01).The expression amount of PAP1 gene significantly increased at 72h,7d after RIR(P<0.01),the expression of PAP1 gene was detected in GCL and INL of the retina sections .[Conclusion]The expression of PAP1 gene was detected in the retina.There is the changes of the PAP1 gene expression amount in the retina after RIR. PART III PAP1 gene expression and the apoptosis in ratRetinal cells after RIR[Objective]To investigate the possible mechanism by exploring the relationship between the changes of PAP1 gene expression and the apoptosis in rat retinal cells after RIR.[Metheds]The model of RIR was established by elevated intraocular pressure at 110mmHg and maintained for 60 minutes.48 SD rats were randomly divided into control group and RIR group.Each group was consisted of 6 subgroups (i.e. 3h,6h,12h,24h,72h and 7d after RIR, n=4) , The apoptosis of retinal cells were evaluated dynamically by in situ terminal deoxynucleotidyl transferase mediated biotin -deoxyuridine triphosphate nick-end labeling (TUNEL).[Results]1 .Compared with the control group,the number of TUNEL postive cells in RIR group was significantly increased at the time points of 6h to 72h after reperfusion,respectively(P < 0.01).The number of TUNEL postive cells was not significantly higher than the control group at the time points of 3h and 7d after reperfusion(P>0.05).2.Compared with the control group ,the expression amount of PAP1-mRNA in RIR group was significantly increased at the time points of 3h(P<0.01);and the expression amount of PAP1-mRNA in RIR group was significantly decreased at the time points of 6h 12h and 24h (P< 0.01)and then increased significatly at the time points of 72h and 7d (P< 0.01).3. There was negative correlation between the expression of PAP1 gene and the apoptosis in retinal cells (r= - 0.679, P<0.01) .[Conclusion] PAP1 gene may be involved in the mechanisms of retinal ischemia-reperfusion injury and cell apoptosis. PAP1 gene maybe protects the retina.