| Objectives In this paper the increased rabbit intra-ocular pressure method is used to establish an animal model for human retinal ischemia-reperfusion injury(RIRI)simulation. By observation on the morphological change of rabbit retina, protein Bcl-2and Bax expressions of apoptosis gene and change of nerve cells apoptosis, the effect of safflower injection on RIRI protection and cell apoptosis as well as apoptosis related gene Bcl-2 and Bax is discussed in order to provide the clinical treatment of retinal ischemic disease with a theoretical basis.Methods 78 health purebred male Japanese big ear white rabbits weighing(2.4 ± 0.2)kg are selected and then divided randomly into 3 groups: normal group(6), and injury group(36), and treatment group(36).The injury group and treatment group are further divided by reperfusion time into 6h, 12 h, 24 h, 48 h, 72 h, and 5d Group(each group has 6experiment big ear rabbits respectively). The right eyes are selected as experimental eyes.Molding is not required for normal group while retina ischemia-reperfusion injury model is duplicated through anterior chamber intraocular pressure for injury and treatment groups; safflower injection is injected via ear vein to treatment group immediately after the successful modeling(2.0ml/kg), 1/d. Isodose saline solution is injected into Normal and injury groups. Air of 10 ml is injected into each group 6h, 12 h, 24 h, 48 h, 72 h, 5d after the injury via ear vein to kill the experiment rabbits and then immediate removal of experimental eye is performed, followed by fixing embedded sections. HE staining is used to observe the morphological changes of retinal tissue in each time period, while immunohistochemical is employed for detection of apoptosis gene Bcl-2 and Bax expression, and TUNEL method is adopted to assay apoptosis of retinal nerve cells.Image analysis is performed on the experimental results and statistical analysis on data obtained is performed by using SPSS17.0.statistical software.Results 1 HE staining: injury group has a smaller cell population compared with normal groups. After reperfusion for 6h, mild retinal edema occurs and aggravates gradually over time. Each layer presents a loose arrangement of cells, together with vacuolar degeneration.In 24 h group, the retinal edema seems most obvious, while in 48-72 h groups the retinal edema shows a relief, with the number of ganglion cells decreased. In the 5d group, retinal edema disappears while thinning of the retina happens. Treatment group has a smaller number of ganglion cells and its retinal edema is milder than that of injury group. 2detection of retinal cell Bcl-2, and Bax protein expression change by immunohistochemical staining: Bcl-2, and Bax protein positive expression is not seen in normal group, few positive expression can be seen after a perfusion of 6h, mainly in ganglion cell layer; the expression gradually increases with the extending of perfusion time. Positive expression reaches peak at 24 h, few expression can be seen in kernel layer while none can be seen in outer nuclear layer; for 48h-72 h, number of desmin-positive cells gradually declines and it mainly exists in ganglion cell layer; almost no positive cells expression is seen in retina for5 d reperfusion. Bcl-2 and Bax protein expression in the treatment group shares a similar trend with that in injury group. When compared at the same time points, retinal cell Bcl-2expression in the treatment group was significantly higher than that in injury Group(except 5d Group),while the Bax expression was significantly lower than in injury Group(P<0.05). Positive cells enumeration in injury Group and treatment group has a significant difference between different times(P<0.01). 3 detection of apoptosis of retinal nerve cells by TUNEL method: no apoptosis cells are seen in normal group. A few expressions of apoptotic cells can be seen after a reperfusion of 6h. Expression increases after a reperfusion of 12 h while after a reperfusion of 24 h positive expression of apoptosis cells reaches a peak, mainly found in retinal ganglion cell layer and the inner nuclear layer; a reperfusion of 48h-72 h,positive expressions decreases while in case of 5d there are few apoptosis cells. The expression of retinal apoptosis cells in the treatment group shares a basically the same trend with that in injury group, however the expression was significantly less than injury group at each time point. Comparing injury group with treatment group at the same time,the differences in 6h, 12 h, 24 h, 48 h, group are statistically significant(P<0.01); the difference in 72 h group is a statistically significant(P<0.05)while the difference in 5d group is not statistically significant(P>0.05). Apoptosis positive cells enumeration difference in injury Group and treatment group has a statistical significance between different times(P<0.01).Conclusions safflower injection shows a significant protective effect on rabbits with RIRI through a mechanism by which the Bax protein expression is down-regulated while the increased expression Bcl-2 protein expression is up-regulated resulting in the inhibition of nerve cell apoptosis when RIRI occurs. |
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