急性眼高压后大鼠视网膜节细胞选择性死亡与视网膜局部血供的关系
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摘要
目的:检测急性眼高压后视网膜中央部和周边部血液供应的改变是否有差异;如果这两个部位的血供改变有差异,进一步检测这种差异改变与节细胞选择性死亡的关系。
方法:第一部分:90只成年健康SD大鼠(体重200～220g)分三步进行实验。首先,为了找到最优的灌注条件,我们在60只动物中测试了由四种明胶浓度和五种灌注压力组合而成的二十种灌注条件。基于这60只动物的结果,我们希望找到能充分显示视网膜中央部和周边部血管的最优明胶墨汁灌注方法。其次,进一步比较第一步实验得出的最优灌注方法与常用的vWf免疫荧光法在大鼠视网膜血管标记效果方面的异同。最后,观察了第一步实验得出的最优灌注方法对同一组织中神经元/胶质细胞标记的影响。
第二部分:通过先前改良的明胶墨汁灌注和荧光微球注射方法从形态和功能两方面检测急性眼高压后大鼠视网膜中央、中间和周边部血液供应的时空变化。
第三部分:通过荧光金逆行标记系统检测了急性眼高压后再灌过程中节细胞丢失的时空变化;在此基础上,结合第二部分的资料,进行节细胞的丢失率与局部血供增加率之间的线性相关分析和线性回归分析。
结果:第一部分:先在140%大鼠平均动脉压(mean arterialpressure,MAP)下灌注含3%明胶的37℃墨汁20ml,再在180%MAP下灌注含5%明胶的37℃墨汁20 ml能充分显示大鼠视网膜血管。该明胶墨汁灌注方法对视网膜血管的显示效果在切片和铺片上均优于常用的von Willebrand factor(vWf)免疫组化方法,而且该方法不影响视网膜神经元和胶质细胞的免疫组化标记和视网膜节细胞的荧光金逆行标记。
第二部分:急性眼高压后再灌过程中视网膜中央、中间和周边部血供都是先增加、再降低、然后逐渐恢复;而且整个再灌过程中视网膜中央部和中间部血供增加率都大于周边部。
第三部分:急性眼高压后再灌对视网膜中央和中间部节细胞存活的影响相似;急性眼高压后再灌过程中,尤其是再灌1d内,视网膜周边部节细胞丢失率明显高于中央部和中间部。
急性眼高压后视网膜中央、中间和周边部节细胞的丢失率与其局部血供的增加率显著相关,尤其在再灌1d内;急性眼高压后周边部节细胞的丢失率与局部血供增加率的相关程度明显高于中央部和中间部。
结论:①改良的明胶墨汁灌注方法对大鼠视网膜血管的标记效果优于vWf标记方法,而且能在同一视网膜上与神经元/胶质细胞双标记;②急性眼高压后大鼠视网膜局部血供的改变和节细胞丢失都具有时空特性;③急性眼高压后大鼠视网膜节细胞的选择性死亡与视网膜局部血供的差异改变有关,而且这种相关关系具有时相特异性和区域特异性。
Purpose:To detect whether the changes of local blood supply in rat retinae following acute high intraocular pressure is differential from the central to the peripheral.And if yes,the relationship between the inhomogenous changes of local blood supply and the selective loss of retinal ganglion cells(RGCs) in rat retinae was further investigated.
Methods:Part 1:90 adult Sprague-Dawley rats(200-250 g) were used in this study.Firstly,60 rats were subjected to 20 combinations(n=3 in each combination) of different gelatin concentrations(0%,1%,3%and 5%) and perfusion pressures(100%,120%,140%,160%and 180%MAP) during retinal perfusion.Based on the data from these 60 rats,the optimal perfusion condition of gelatin-ink,under which the central and peripheral retinal vessels were completely filled,was gotten.Secondly,12 rats were used for evaluation of the retinal vessel labeling outcome of the gelatin-ink perfusion method under the optimal condition(n=6) by using the vWf immunostaining(n=6) as control.Finally,we investigated whether this improved labeling technique affected the following labeling of neurons and glial cells in the same retinae
Part 2:The temporal-spatial changes of blood supply in the central, middle and peripheral retinae following the acute introeular high pressure were detected by the above improved gelatin-ink perfusion method and fluorescent microsphere injection method.
Part 3:The temporal-spatial changes of ganglion cell loss in the central,middle and peripheral retinae following the acute introcular high pressure were detected by fluorogold retrograde labelling.Based on these and the data of part two,linear correlation and linear regression between loss percentages of ganglion cells and increased ratios of local blood supply were made.
Results:Part 1:Infusing rats first with 20 ml of 37℃ink plus 3% gelatin at 140%MAP,and subsequently with 20 ml of 37℃ink plus 5% gelatin at 180%rat MAP allowed the gelatin-ink to completely fill the rat retinal vessels.Rat retinal vessels labeled by the perfusion method were more in number than that by vWf immunostaining.Moreover,the improved gelatin-ink perfusion had no effect on and caused no contamination to the following fluorogold labeling or immunostaining of retinal neurons or glial cells in the same tissue.
Part 2:During the reperfusion following acute high intraocular pressure,the blood supply in the central,middle and peripheral retina all first increased,and then decreased,and at last recovered.The increased percentages of local blood supply in the central and middle retina all were higher than that of the peripheral at each survival time points during the whole reperfusion.
Part 3:The reperfusion of retina following acute high intraocular pressure has similar effects on the survival of ganglion cells in the central and middle retina.The loss percentages of ganglion cells in the peripheral retinae was significantly higher than that of the central and middle during the reperfusion,especially on the first day of reperfusion.
There were significant correlation between the loss ratio of ganglion cells and the increased percentages of corresponding local blood supply during the reperfusion following the acute high intraocular pressure, especially on the first day of reperfusion.Moreover,the correlativity in the peripheral retina was significantly higher than that of the central and middle retina during the whole reperfusion.
Conclusion:①Improved gelatin-ink perfusion is a method for accurate morphological characterization of rat retinal vessels,which is superior to vWf immunostaining.The gelatin-ink perfusion technique can double label with neurons/glial cells in the same retinae.②There were temporal-spatial changes in the rat retinal ganglion cell loss and local blood supply after the acute high intraocular pressure.③There is significant correlation between the seletive loss of retinal ganglion cells and inhomogenous changes of local blood supply during the reperfusion following the acute high intraocular pressure.And the correlation has special temporal-spatial features.
方法:第一部分:90只成年健康SD大鼠(体重200～220g)分三步进行实验。首先,为了找到最优的灌注条件,我们在60只动物中测试了由四种明胶浓度和五种灌注压力组合而成的二十种灌注条件。基于这60只动物的结果,我们希望找到能充分显示视网膜中央部和周边部血管的最优明胶墨汁灌注方法。其次,进一步比较第一步实验得出的最优灌注方法与常用的vWf免疫荧光法在大鼠视网膜血管标记效果方面的异同。最后,观察了第一步实验得出的最优灌注方法对同一组织中神经元/胶质细胞标记的影响。
第二部分:通过先前改良的明胶墨汁灌注和荧光微球注射方法从形态和功能两方面检测急性眼高压后大鼠视网膜中央、中间和周边部血液供应的时空变化。
第三部分:通过荧光金逆行标记系统检测了急性眼高压后再灌过程中节细胞丢失的时空变化;在此基础上,结合第二部分的资料,进行节细胞的丢失率与局部血供增加率之间的线性相关分析和线性回归分析。
结果:第一部分:先在140%大鼠平均动脉压(mean arterialpressure,MAP)下灌注含3%明胶的37℃墨汁20ml,再在180%MAP下灌注含5%明胶的37℃墨汁20 ml能充分显示大鼠视网膜血管。该明胶墨汁灌注方法对视网膜血管的显示效果在切片和铺片上均优于常用的von Willebrand factor(vWf)免疫组化方法,而且该方法不影响视网膜神经元和胶质细胞的免疫组化标记和视网膜节细胞的荧光金逆行标记。
第二部分:急性眼高压后再灌过程中视网膜中央、中间和周边部血供都是先增加、再降低、然后逐渐恢复;而且整个再灌过程中视网膜中央部和中间部血供增加率都大于周边部。
第三部分:急性眼高压后再灌对视网膜中央和中间部节细胞存活的影响相似;急性眼高压后再灌过程中,尤其是再灌1d内,视网膜周边部节细胞丢失率明显高于中央部和中间部。
急性眼高压后视网膜中央、中间和周边部节细胞的丢失率与其局部血供的增加率显著相关,尤其在再灌1d内;急性眼高压后周边部节细胞的丢失率与局部血供增加率的相关程度明显高于中央部和中间部。
结论:①改良的明胶墨汁灌注方法对大鼠视网膜血管的标记效果优于vWf标记方法,而且能在同一视网膜上与神经元/胶质细胞双标记;②急性眼高压后大鼠视网膜局部血供的改变和节细胞丢失都具有时空特性;③急性眼高压后大鼠视网膜节细胞的选择性死亡与视网膜局部血供的差异改变有关,而且这种相关关系具有时相特异性和区域特异性。
Purpose:To detect whether the changes of local blood supply in rat retinae following acute high intraocular pressure is differential from the central to the peripheral.And if yes,the relationship between the inhomogenous changes of local blood supply and the selective loss of retinal ganglion cells(RGCs) in rat retinae was further investigated.
Methods:Part 1:90 adult Sprague-Dawley rats(200-250 g) were used in this study.Firstly,60 rats were subjected to 20 combinations(n=3 in each combination) of different gelatin concentrations(0%,1%,3%and 5%) and perfusion pressures(100%,120%,140%,160%and 180%MAP) during retinal perfusion.Based on the data from these 60 rats,the optimal perfusion condition of gelatin-ink,under which the central and peripheral retinal vessels were completely filled,was gotten.Secondly,12 rats were used for evaluation of the retinal vessel labeling outcome of the gelatin-ink perfusion method under the optimal condition(n=6) by using the vWf immunostaining(n=6) as control.Finally,we investigated whether this improved labeling technique affected the following labeling of neurons and glial cells in the same retinae
Part 2:The temporal-spatial changes of blood supply in the central, middle and peripheral retinae following the acute introeular high pressure were detected by the above improved gelatin-ink perfusion method and fluorescent microsphere injection method.
Part 3:The temporal-spatial changes of ganglion cell loss in the central,middle and peripheral retinae following the acute introcular high pressure were detected by fluorogold retrograde labelling.Based on these and the data of part two,linear correlation and linear regression between loss percentages of ganglion cells and increased ratios of local blood supply were made.
Results:Part 1:Infusing rats first with 20 ml of 37℃ink plus 3% gelatin at 140%MAP,and subsequently with 20 ml of 37℃ink plus 5% gelatin at 180%rat MAP allowed the gelatin-ink to completely fill the rat retinal vessels.Rat retinal vessels labeled by the perfusion method were more in number than that by vWf immunostaining.Moreover,the improved gelatin-ink perfusion had no effect on and caused no contamination to the following fluorogold labeling or immunostaining of retinal neurons or glial cells in the same tissue.
Part 2:During the reperfusion following acute high intraocular pressure,the blood supply in the central,middle and peripheral retina all first increased,and then decreased,and at last recovered.The increased percentages of local blood supply in the central and middle retina all were higher than that of the peripheral at each survival time points during the whole reperfusion.
Part 3:The reperfusion of retina following acute high intraocular pressure has similar effects on the survival of ganglion cells in the central and middle retina.The loss percentages of ganglion cells in the peripheral retinae was significantly higher than that of the central and middle during the reperfusion,especially on the first day of reperfusion.
There were significant correlation between the loss ratio of ganglion cells and the increased percentages of corresponding local blood supply during the reperfusion following the acute high intraocular pressure, especially on the first day of reperfusion.Moreover,the correlativity in the peripheral retina was significantly higher than that of the central and middle retina during the whole reperfusion.
Conclusion:①Improved gelatin-ink perfusion is a method for accurate morphological characterization of rat retinal vessels,which is superior to vWf immunostaining.The gelatin-ink perfusion technique can double label with neurons/glial cells in the same retinae.②There were temporal-spatial changes in the rat retinal ganglion cell loss and local blood supply after the acute high intraocular pressure.③There is significant correlation between the seletive loss of retinal ganglion cells and inhomogenous changes of local blood supply during the reperfusion following the acute high intraocular pressure.And the correlation has special temporal-spatial features.
引文
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