| PURPOSEIn recent years, many more researches have been carried out to study theapoptosis of retinal cells resulted from all kinds of optic nerve injuries of the reti-na. The death of nerve cells has been found in the final phase of diseases whichaffect retina and optic nerve function, most of which are apoptosis. Therefore, itis important to suppress and regulate the apoptosis and its relevant gene to treatthese kinds of diseases. Now, the animal model of retinal ischemia-reperfusioninjury is regarded as an important method to study the retina injury and the ap-optosis of the retina cells. In the process of retinal ischemia-reperfusion injury,after the perfusion, vacuoles and edema gradually appear in the inner nine lay-ers, where the number of cells decrease and the cells begin to be apoptosis inthe later stage. The apoptosis cell can be found in the ganglion cell layer and in-ner nuclear layer after transient ischemia of the rat's retina. There are manykinds of apoptosis relevant gene express abnormally after the retinal ischemia-reperfusion. So the ratio of apoptosis cells and the expression change of apopto-sis relevant gene can be one of reliable markers to evaluate nerve protective a-gent.During the process of apoptosis, the expression of gene that promotes andsuppresses apoptosis exists, such as caspase-3 and Bcl-2 that play importantroles in the apoptosis, caspase-3 is an important gene promoting apoptosis,whose activity can be suppressed to prevent apoptosis. Bcl-2 is a classic anti-apoptosis gene.P38 MAPK is a member of MAPK family, which pertains to a kind of pro- tein kinase under stress and is activated in pathological condition to be involvedin the cell apoptosis. STAT is a transcription factor activated by polypeptide a-dapters such as cytokine and growth factor, which is a key composition of theJAK-STAT signal transduction passage. It is generally believed that the over-expression of STAT can suppress the apoptosis.In latest decades, it has been attached more attention how to protect opticnerve after retinal injury and degeneration to refrain the optic function from dete-riorating by researchers who have currently found that many endogenetic and ec-togenetic agent, especially the Calcium channel blocking agent, play positiveroles in this process.Cell calcium influx and inner calcium overloading of cell universally exist inmany kinds of organisms suffering ischemia-reperfusion injury, leading to cellapoptosis inevitably. Most researchers prefer to prevent calcium influx to sup-press the apoptosis. It has been reported that Nimodipine, a L type of electronicgating Calcium channel blocking agent, can block the calcium passage alterna-tively. At present, there has been no report that Nimodipine affect apoptosis andits relevant gene and signal transduction passage. This study focuses on whetherL type of electronic gating Calcium channel blocking agent can prevent retinaand optic nerve from retinal ischemia-reperfusion injury or not and its possiblesignal transduction passage, expected to find out appropriate retina nerve protec-tive agent to serve the clinical treatment.MATERIALS AND METHODS1st PART: animal models and specimens preparationRandomize 142 White Wistar Rat (weight 250-270 gram and gender notbe limited) into 4 groups: Group A is normal control group(7) ; Group B is testcontrol group of retinal ischemia-reperfusion (45) ; Group C is group of Nimo-dipine(45); Group D is prosthetic surgery group of low-pressure perfusion(45). Methods to make animal models: Group B: raising inner pressure of eyeto 110mmHg and watching directly the appearance of retinal ischemia, decrea-sing inner pressure of eye to 20 mmHg 1 hour later or below and retinal artery reopening, ie the reperfusion occurring. Group C: injecting Nimodipine to cau-dal vein, performing methods of group B half an hour later, injecting the samedose of Nimodipine before the moment of reperfusion. Group D: methods beingthe same as that of group B with the difference of inner pressure of eye, 20 mm-Hg.To dispose 5 rats (10 eyes) in each group B, C, D at the moment afterreperfusion 0 hour, 2 hours, 6 hours, 12 hours, 24 hours, 72 hours and 168hours respectively. Four eyes were fixed by 4% formaldehydeum polymerisatumincluding 1‰DEPC and embedded by paraffin wax after splitting into half fromcross section; then making 4μm section for immuneohistochemistry assay andhybridization in situ assay. The peeled retina of the other 6 eyes after extractionwere frozen in -70℃for RT-PCR assay. To dispose 2 rats(4 eyes) at the mo-ment after reperfusion 0 hour, 2 hours, 6 hours, 12 hours, 24 hours, 72 hoursat the moment after reperfusion 0 hour, 6 hours, 12 hours, 24 hours, 72 hoursrespectively; peeling retina to freeze for flow cytometer assay. 14 eyes in GroupA, four eyes for wax specimen and 10 eyes to be frozen for RT-PCR assay andflow cytometer assay.2nd PART: Nimodipine's influence on the pathological change of rat'sretinal ischemia-reperfusion injuryTo observe the change of retina in light microscope and to measure the dis-tance(depth) from inner limiting membrane to outer limiting membrane byMetamorph image analysis software. The location of measurement was 200μmoutside of the nose side of the optic papilla and every section was measured 3times and the average value was obtained. 2 sections were measured in eachspecimen. The pixel figure was converted into micrometer one. The obtained da-ta were performed ANOVA and SNK test by SPSS statistical software.3rd PART: Nimodipine's influence on the cell apoptosis of rat's retinal is-chemia-reperfusion injuryCell suspension obtained by mechanical method and stained by PropidiumIodide one step method and examined by the flow cytometer was analyzed for dis-tribution of cell cycle and situation of cell apoptosis of different kinds of samplesby Cellquest multifunction software to obtain the percentage of apoptosis cell for SPSS statistical analysis.4th PART Nimodipine's influence on the cell apoptosis relevant gene ex-pression after retinal ischemia-reperfusion injury of ratsThe expression of caspase-3 and Bcl-2 were detected in 2 sections of everyspecimen at different time by immunohistochemistry assay. The average opticaldensity of positive cells in unit area was figured by Metamorph software and ana-lyzed by SPSS software.5th PART Nimodipine's influence on the signal transduction system of reti-nal ischemia-reperfusion cells of ratsThe expression of P38MARK and STAT3 mRNA was detected at differenttime by RT-PCR assay and hybridization in situ assay. The results obtained fromhybridization in situ assay was analyzed by Metamorph software and the resultantdata of average absorption optical density was performed ANOVA and SNK testby SPSS statistical software. The image of RT-PCR was analyzed by Kodak 1Dimage analysis system to detect the relative content of the targeted gene mRNAfor statistical test (ANOVA).RESULT1. the pathological change of rat's retinal ischemia-reperfusion injuryIn Group A, there is the normal rat's retina that is divided into 10 layerssimilar to that of human being. In Group B, such occurs 2 hours later after is-chemia-reperfusion as cells vacuoles in ganglion cell layer and inner nuclearlayer, stroma rarefaction, rarefaction in nerve fiber layer and inner plexiformlayer and cell disarray in inner nuclear layer. The above changes are even moreapparent 6 hours later and the retinal edema becomes even more serious 12 hourslater. Nuclear concentration appears in ganglion cell layer 12 hours later, whichis no much difference 24 hours later. 72 hours later, retinal edema relieves andnuclear concentration becomes more in ganglion cell layer and inner nuclear lay-er. 168 hours later, retinal edema disappears, the number of cells in ganglioncell layer and inner nuclear layer drops dramatically, inner plexiform layer at-tenuates and retina is atrophy. In Group C, the change trend of histology is similar to that of Group B, ex-cept that it is more gentle in Group C than in Group B that edema, rarefaction ininner plexiform layer, cells of nuclear concentration and the number of cells andthe retina depth 168 hours later.In Group D, retinal histology seems to be no difference with the normal ret-ina.2. the measurement of retinal depthThe average depth of normal rat's retina is 127μm, from inner limiting lay-er to outer limiting layer of the retina in the wax sections. After retinal ischemia-reperfusion injury, the depth of retina increases 2 hours later and hits thepeak 12 hours later with the average figure 181.62μm, which decreases 72hours later and becomes narrow obviously. In Group C, the depth of retina grad-ually increases 2 hours later but the degree is less than that in Group B. And theaverage figure is 98.56μm 168 hours later after ischemia-reperfusion, which ismuch more different from that in Group B. No change of the depth of retinatakes place in Group D.All obtained data are analyzed by ANOVA, which there is no statistical sig-nificance at the very beginning among and statistical significant difference at dif-ferent time between Group A, D and B, C, Group B and C respectively.3. the result of the flow cytometer assayObvious double-peak appears in Group A and D. Substage (apoptosispeak) G0/G1＜4%.In Group B, substage G0/G1 begins to increase 6 hours later, increasesgradually 12 hours later, hits the peak at the point of 24th hour and decreases 72hours later.In Group C, the trend is similar with that in Group B, which differs in thelower apoptosis peak.The ratio of the apoptosis cell analyzed by Cellquest software is dealt withstatistical test (x~2 test), which shows statistical significance (P＜0.01) at dif-ferent time between Group B, C and D.4. the expression of the apoptosis relevant gene①caspase-3 : nucleus appears to be brown in positive expression of retinal caspase-3 by immunohistochemistry assay. The expression of retinal caspase-3 inGroup A and Group D is only restricted in the ganglion cell layer and 0-1/HF.In Group B, scattered positive expression can be seen in the ganglion cell layer2 hours later, which increases in the ganglion cell layer and the inner nuclearhyer 6 hours later, hits the peak 24 hours later, appears to be abundant 72hours later and still exists 168 hours later. In Group C, the expression ofcaspase-3 is similar with that in Group B but the number of positive cells and thedegree of staining are inferior to that in Group B. The statistical analysis of theaverage absorption optical density by the Metamorph image analysis softwareshows there are statistical significance (P＜0.05) at each time point except 0hour and 168th hour between Group A,D and B, Group A,D and C, Group Band C except Group A and D.②Bcl-2: the positive expression of retinal Bcl-2 shows brown granules innerve fiber layer, inner plexiform layer, outer plexiform layer, the rod and conecells layer and among the nuclear between the inner nuclear layer and the outernuclear layer (cytoplasm and cell process). There is almost no expression inGroup A and D. In Croup B, positive expression can be seen in the nerve fiberlayer, ganglion cell layer and inner plexiform layer 2 hours later after reperfu-sion, which gradually deepens in stain as time goes on, extends to outer layer ofretina to outer plexiform ultimately, hits the peak 24 hours later and graduallydecreases 72 hours later. In Group C, the change trend with time is similar withthat in Group B but the stain is deeper than that in Group B. Obvious stain canbe seen in the nerve fiber layer and the inner plexiform layer 2 hours after reper-fusion and a large amount of Bcl-2 deposit can be seen in the outer limitingmembrane and the rod and cone cell layer 24 hours later, which stir can notreach the basic line a week later. Statistical analysis is performed after the im-age analysis by Metamorph software to show statistical significance betweenGroup B and C, Group B and A, Group C and A, D.5. the expression change of P38 MAPK and STAT3 mRNA(1) the result of P38 MAPK RT-PCRThere are only a little expression in retina of Group A and D. In Croup B,the expression of P38 MAPK begins to increase 2 hours after reperfusion, which hits the peak 12 hours later, lasts to 24th hours, decreases gradually 72 hourslater and still exists 168 hours later. In Group C, the trend is similar to that inGroup B but the emergence time and peak value are inferior to that in Group B,which shows the expression has been suppressed.(2) the result of STAT-3 RT-PCRThe time range of the result of STAT-3 RT-PCR is similar to that of theresult of P38 MAPK but the expression in Group C is superior to that in GroupC.Statistical analysis of the results of RT-PCR shows that there are statisticalsignificance (P＜0.05) between Group A, D and B, C, Group B and C.(3) P38 MAPK mRNA hybridization in situSeveral cytoplasm stain can be seen in the ganglion cell layer of normal ret-ina but the number of positive cells begins to increase 2 hours later after reperfu-sion in Group B. And all stains has been transferred into cell nuclear, hittingthe peak 6-12 hours later and many positive nuclear can still be seen 168 hourslater. The trend in Group C is similar to that in Group B but the expression isinferior to that in Group B. The statistical significance of each group is similar tothe result of RT-PCR after Metamorph software analysis and SPSS statistics.(4) The expression pattern of STAT3 mRNA hybridization in situ is similarto that of P38 MAPK but the expression is higher in Group C than Group B.CONCLUSION1. Retinal ischemia-reperfusion injury can lead to retinal edema and celllose, hits to the peak 24 hours later and lasts for a week or above, resulting inthe obvious atrophy of retina in later stage. L type of electronic gating Calciumchannel blocking agent, Nimodipine, can obviously protect retinal ischemia-reperfusion injury, prevent the retinal cells after ischemia-reperfusion from dyingand relieve the retinal atrophy.2. Retinal nerve cells after retinal ischemia-reperfusion injury appear to beapoptosis and the number of the apoptosis is most 24 hours later. L type of elec-tronic gating Calcium channel blocking agent, Nimodipine, can decrease the number of apoptosis cells.3. Promoting apoptosis gene, caopase-3, and suppressing apaptosis gene,Bcl-2 are expressing in retina after retinal ischemia-reperfusion injury. Nimo-dipine can down-regulate the expression of caspase-3 and up-regulate the expres-sion of Bcl-2, which indicates the Nimodipine' s protection for retinal ischemia-reperfusion injury is realized by influencing the relevant gene expression.4. F38 MAPK participates in the apoptosis signal transduction of retinalnerve cells in retinal ischemia-reperfusion injury and Nimodipine safeguardsthe retina by suppressing the expression of P38 MAPK.5. Similar to Bcl-2, STAT3 is a key factor in retina anti-apaptosis signaltransduction. Nimodipine safeguard the retina by up-ragulating the expression ofSTAT3.
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