霍乱毒素对视神经远端切断后视网膜节细胞存活的影响

本研究采用荧光逆行示踪技术观察了玻璃体内注射霍乱毒素对视神经远端切断后的视网膜节细胞存活的影响。结果显示 ,正常视网膜节细胞平均密度为 2 192± 66个 / mm2 ;视神经切断 7d、14 d、2 1d后视网膜节细胞平均密度分别为 15 2 0± 116个 /mm2、73 6± 3 9个 / mm2、466± 5 3个 / mm2 ;玻璃体内注射 Na2 EDTA/ Na Cl对照组在各时间点其视网膜节细胞平均密度与视神经切断组结果相似。玻璃体内注射霍乱毒素后 7d、14 d、2 1d视网膜节细胞平均密度均有提高 ,分别为 164 2± 12 2个 / mm2、10 91±10 7个 / mm2 、64 8± 3 5个 / mm2 ,与视神经切断组及 Na2 EDTA/ Na Cl对照组相比 ,在各时间点上均有显著性差异 (P<0 .0 1)。本研究结果表明霍乱毒素对视神经远端切断后的视网膜节细胞存活有明显的促进作用     

CPT-cAMP对成年金黄地鼠视网膜节细胞存活的影响

目的　探讨眼球玻璃体内注射CPT cAMP对切断视神经后视网膜节细胞存活的影响。方法　用荧光素逆行示踪标记法和定量解剖学技术观察视神经切断 5、7和 14d后成年金黄地鼠视网膜节细胞的密度。结果　 (1)正常视网膜节细胞平均密度为 2 0 0 7± 115 /mm2 ;(2 )视神经切断 5、7、14d后 ,视网膜节细胞平均密度分别下降至 :10 10± 131/mm2 、782± 5 5 /mm2 和2 14± 30 /mm2 ;(3)给予DMSO/生理盐水的对照组在上述各时间段视网膜节细胞平均密度与单纯神经切断组结果相似 ;(4)给予CPT cAMP的实验组在 5、7、14d视网膜节细胞的平均密度分别为 1398± 2 45 /mm2 、12 93± 84/mm2 和 5 0 1± 72 /mm2 ,与视神经切断组和DMSO/生理盐水对照组相比在各时间段上均存在显著性差异 (P <0 0 5 )。结论　CPT cAMP可提高视神经切断后成年金黄地鼠视网膜节细胞的存活。     

Forskolin对成年金黄地鼠视网膜节细胞存活的影响

本研究应用荧光逆行示踪技术观察了Forskolin玻璃体内注射对视神经切断后视网膜节细胞存活的影响。结果表明：（1）正常视网膜节细胞平均密度为2113±120／mm2;（2）视神经切断5、7、14d后,视网膜节细胞平均密度分别下降至：1530±192／mm2、881±160／mm2和181±31／mm2;（3）给予DMSO／生理盐水的对照组在上述各时间组视网膜节细胞平均密度与单纯视神经切断组结果相似;（4）给予Forskolin的实验组在5、7、14d视网膜节细胞的平均密度分别为1863±131／mm2、1639±183／mm2和442±60／mm2,与视神经切断组和DMSO／生理盐水对照组相比,在各时间组均存在显著性差异（P＜0．05）。本研究提示．Forskolin有提高视神经切断后的视网膜节细胞存活的作用。     

Forskolin对成年金黄地鼠视网膜节细胞存活的影响

<正> 本研究应用荧光逆行示踪标记技术,观察Forskolin玻璃体内注射对视神经切断后视网膜节细胞存活的影响.结果:(1)正常视网膜节细胞平均密度为2113±120/mm~2;(2)视神经切断5、7、14d后,视网膜节细胞平均密度分别下降至:1530±192/mm~2、881±160/mm~2和181±31/mm~2;(3)给予DMSO/生理盐水的对照组在上述各时间组视网膜节细胞平均密度与单纯视神经切断组结果相似;(4)给予Forskolin的实验组在5、7、14d视网膜节细胞平均密度分别为1863±131/mm~2、1639±183/mm~2和422±60/mm~2,与视神经切断组和DMSO/生理盐水对照组比在各时间组上均     

不同剂量氯化锂对成年大鼠视神经切断后视网膜神经节细胞的保护作用

目的:研究不同剂量氯化锂(LiCl)对成年大鼠视神经切断后视网膜神经节细胞(RGCs)存活的作用。方法:眶内切断72只成年雌性SD大鼠左侧视神经且残端留置荧光金(FG)后,随机分为生理盐水对照组和低剂量(30 mg/kg/d)、中剂量(60 mg/kg/d)、高剂量(85 mg/kg/d)氯化锂实验组。术前1 d及术后每天腹腔注射生理盐水或不同剂量的氯化锂溶液,直至术后2 d、7 d或14 d处死动物。平铺视网膜后取样计数FG逆行标记的存活节细胞,并由此计算出每一视网膜内节细胞的平均密度。结果:术后2 d各剂量氯化锂组节细胞平均密度与对照组相比无显著性差异(P>0.05)。当存活时间增至7 d时,各剂量氯化锂组节细胞密度均明显高于对照组(P<0.01),且中剂量组节细胞密度增高最为显著。术后14 d时,低剂量与中剂量组节细胞密度仍显著高于对照组(P<0.01),但高剂量组节细胞密度与对照组相比无统计学差异(P>0.05)。结论:腹腔内注射氯化锂可显著促进成年大鼠视神经切断后节细胞的存活,这种神经保护作用为剂量依赖性。     

霍乱霉素对成年金黄地鼠视网膜节细胞存活的影响

<正> 近端切断成年金黄地鼠视神经,视网膜节细胞大量死亡,两周后达90%.本研究应用荧光逆行示踪标记技术,观察玻璃体内注射霍乱毒素对视神经切断后视网膜节细胞存活的影响.结果表明:(1)正常视网膜节细胞平均密度为2007±115/mm~2;(2)切断视神经5、7、14d后,视网膜节细胞平均密度分别下降至:1147±209/mm~2、837±123/mm~2和201±73/mm~2;(3)给予Na_2EDTA/NaCL的对照组在上述各时间组视网膜节细胞平均密度与单纯视神经切断组结果相似;(4)给予霍乱毒素的实验组在5、7、14d视网膜节细胞的平均密度分别为1436±150/mm~2、1192±129/mm~2     

不同穴位和频率的电针刺激对成年大鼠视神经切断后视网膜神经节细胞存活的作用

目的:研究不同穴位和频率的电针刺激对成年大鼠视神经切断后视网膜神经节细胞(节细胞)存活的作用。方法:54只成年SD大鼠接受右侧视神经眶内切断术及节细胞荧光金(Fluoro Gold,FG)逆行性标记后,随机分为9组,对照组(含2、7、14 d组)及不同穴位、频率和伤后存活时间组合的电针组(含假穴4/20 Hz电针7 d组、百会穴4/20 Hz电针7 d组、睛明穴2 Hz电针7 d组、睛明穴4/20 Hz电针2、7、14 d组),每组6只动物。结果:(1)睛明穴4/20 Hz电针7 d组存活节细胞平均密度显著高于对照、假穴和百会穴组(P<0.01),而假穴和百会穴组与对照组间无显著性差异;(2)睛明穴2 Hz与睛明穴4/20 Hz电针7 d组节细胞密度虽无显著性差异,但均显著高于对照7 d组(P<0.01);(3)分别以4/20 Hz电针刺激睛明穴2、7、14 d,与对照组相同时间点相比,仅在伤后7 d出现节细胞密度的显著增高(P<0.01)。结论:以4/20 Hz与2 Hz两种不同频率的电针刺激睛明穴,均能在成年大鼠视神经切断后7 d延缓节细胞的死亡。就本研究所观察的不同穴位和频率而言,电针的神经保护作用取决于针刺穴位而非电针频率。     

IBMX对成年地鼠视神经切断后视网膜节细胞存活的影响

<正> IBMX(3-异丁基-1-甲基黄嘌吟,3-isobutyl-1-methyl-xanthine)是胞内cAMP降解酶-磷酸二酯酶(PDE)的非特异性抑制剂,可通过抑制cAMP的降解而有效提高胞内cAMP的水平,而cAMP是细胞内重要的第二信使,可以转导多种因子的跨膜信号.最近有报道,IBMX通过提高胞内cAMP水平抑制HL60细胞的凋亡,目前对IBMX是否可以促进视网膜节细胞的存活及其促进中枢神经元存活的作用机制不清楚,它是否通过提高胞内cAMP水平,后者再作用于神经营养因子或其它胞内信号系统的某些位点而起作用,目前还不清楚.本研究应用荧光逆行标记技术,观察玻璃体内注射IBMX对视神经切断后视网膜节细胞存活的影响.结果:(1)正常视网膜节细胞平均密度为1975±283/mm~2(2)视神经切断5、7、14 天后,视网膜节细胞平均密度分别下降至:1010±131/mm~2,782±55/mm~2和214±30mm~2;(3)给予DMSO/生理盐水的对照组在上述各时间段视网膜节细胞平均密度与单纯视神经切断组结果相似;(4)给予IBMX的实验组在5、7、14天视网膜节细胞的平均密度分别为1402±69/mm~2;1182±194mm~2和483±17/mm~2,与视神经切断组和DMSO/理盐水对照组相比在各时间段上均存在显著     

黄嘌呤抗视神经切断后视网膜细胞凋亡的作用

目的　探讨眼球玻璃体注射 3 异丁基 1 甲基 (IBMX)对切断视神经后视网膜细胞凋亡的作用。方法　用荧光核染料 332 5 8和琼脂糖凝胶电脉技术观察切断视神经 5、7和 1 4d后成年金黄地鼠视网膜细胞凋亡的变化。结果　 ( 1 )视神经切断 5、7、1 4d后 ,节细胞层 (GCL)细胞凋亡密度 (cell/mm2 )分别为 94 60± 2 9 40、81 2 0± 1 5 1 9和 31 2 0± 1 1 43,给予IBMX组在上述时间点凋亡密度为 67 80± 1 3 83、60 2 0± 9 68和 2 7 0 0± 9 5 7。IBMX 7d组与对照组比较有显著差异 (P <0 0 5 )。 ( 2 )琼脂糖凝胶电脉显示切断 7d的视网膜细胞DNA抽提物显示典型的DNA梯形条带。其它各间间点 ( 5d和 1 4d)以及玻璃体内注射IBMX均未见典型DNA梯形条带。结论　IBMX有抗视神经切断后视网膜细胞凋亡的作用     

黄芪甲苷对成年大鼠视神经切断后视网膜节细胞存活的影响

目的:探讨黄芪甲苷(AST)在成年大鼠视神经切断后对视网膜节细胞(RGCs)存活的影响。方法:动物分为正常组、单纯切断视神经组、AST处理组和生理盐水对照组。用荧光金(FG)逆行示踪标记法及定量解剖学技术观察正常和经AST处理的SD大鼠于视神经切断后5、7、14 d的RGCs的密度。结果:正常组RGCs平均密度为(2 230±156)/mm~2。单纯视神经切断组RGCs平均密度与生理盐水对照组相比较,无显著性差异;AST处理组与单纯切断视神经组和生理盐水对照组相比较,在各个时间点上均存在显著性差异。结论:黄芪甲苷可提高成年大鼠视神经切断后视网膜节细胞短期存活。     

MK-801和L-NA对成年仓鼠视网膜节细胞轴突损伤后存活与再生的影响

目的 研究NMDA受体阻断剂MK-801和一氧化氮合酶抑制剂L-NA对成年仓鼠视网膜节细胞(下称节细胞)轴突切断后存活和再生的影响。方法 切断动物左侧视神经后分两组：存活组存活2、7或14d；再生组视神经眶侧断端同一段坐骨神经吻合后存活28d。所有实验动物自视神经切断前1d开始，每日接受腹腔注射MK-801和／或LNA直至处死。结果存活节细胞均数在MK-801组与对照组间无显著性差异，但L-NA组在术后2和7d节细胞数较对照组显著增加，合用MK-801／LNA较单用MK-801或L-NA使更多节细胞存活。而MK-801或L-NA对节细胞轴突在周围神经移植物内的再生均无明显作用。结论 lmg／kg剂量的MK-801对节细胞的存活无明显作用，但同时阻断NMDA受体和抑制一氧化氮合酶比单纯抑制一氧化氮合酶对节细胞有更强的神经保护作用。1．0mg／kgMK-801或4．5mg／kgL-NA对节细胞轴突的再生无明显促进作用。     

Parameters of optic nerve electrical stimulation affecting neuroprotection of axotomized retinal ganglion cells in adult rats

We previously showed the enhancement of survival of retinal ganglion cells (RGCs) by electrical stimulation (ES) of the optic nerve (ON) stump in adult rats. To elucidate the mechanisms underlying the survival enhancement, we determined whether the neuroprotective effect of ES is affected by the following parameters: stimulation time, frequency of current pulses and starting of ES. ES for 10 min immediately after ON transection was not effective in increasing the number of surviving RGCs 7 days after the transection, but that for 30 min was effective. ES at 20 Hz was the most effective, when applied just after axotomy. When the starting of ES to the ON was shifted either 3 h after or 4 h before the axotomy, the neuroprotective effect of ES was not observed. These results suggest that the electrical activation of RGCs and/or the transected ON interfere with early events after axotomy that leads to RGC death.     

Differential effects of intravitreal optic nerve and sciatic nerve grafts on the survival of retinal ganglion cells and the regeneration of their axons

We have investigated the effects of intravitreal sciatic nerve (SN) and/or optic nerve (ON) grafts on the survival and the axonal regeneration of retinal ganglion cells (RGCs). Following transection of the ON, approximately 40% RGCs survived at 7 days post-axotomy (dpa). Results showed that the intravitreal ON graft significantly promoted the survival of RGCs at 7 dpa (39,063 vs 28,246). Intravitreal SN graft, however, did not rescue axotomized RGCs at 5, 7 or 14 dpa. Axotomized RGCs could be induced to regenerate axons along a segment of SN graft attached to the proximal stump of ON. On average, 608 axotomized RGCs were induced to regenerate axons along the attached SN graft. The presence of intravitreal SN graft promoted about 100% increase in the number of regenerating RGCs (1,227) relative to the control groups. The intravitreal ON graft, surprisingly, also induced about 100% more regenerating RGCs (1220) than in the control group. When SN and ON grafts were co-transplanted into the vitreous, about 200% more regenerating RGCs (1916) were observed than in the control group. These findings illustrated that the intravitreal ON graft rescued axotomized RGCs and enhanced the regeneration of retinal axons. This is the first report to show that ON promotes RGC axonal regeneration. The intravitreal SN graft did not rescue RGCs but promoted axonal regeneration. The differential effects of intravitreal ON and SN grafts on the survival and the RGC regeneration suggest that these might be two independently operating events.     

Cilostazol promotes survival of axotomized retinal ganglion cells in adult rats

Cilostazol (CLZ), a selective inhibitor of cyclic nucleotide phosphodiesterase 3, has been shown to reduce neuronal cell death after a transient cerebral infarction. The mechanism for this reduction was suggested to be an elevation of intracellular cAMP or an inhibition of tumor necrosis factor alpha. Optic nerve injury leads to retinal ganglion cell (RGC) death possibly from a deprivation of neurotrophic factors and/or the down-regulation of intracellular cAMP. The purpose of this study was to determine if CLZ can rescue RGCs after optic nerve transection by inhibiting cyclic nucleotide phosphodiesterase 3. To examine this, the mean densities of surviving RGCs after optic nerve transection were determined in retinas that received an intravitreal injection of CLZ and in retinas that received vehicle. Our results showed that the density of surviving RGCs in the retina with intravitreal CLZ were significantly higher than that with vehicle injection on day 7. The CLZ was effective in promoting the survival at more than 0.05% concentration. The neuroprotective effects induced by 0.05% CLZ could be observed even 14 days after optic nerve transection. Furthermore, combined application of protein kinase A (PKA) inhibitor, KT5720 (10 microM) and 0.05% CLZ significantly decreased the density of surviving RGCs compared to that with only 0.05% CLZ. Based on these data, we concluded that CLZ enhances the survival of axotomized RGC in vivo, possibly depending on the activation of PKA pathway.     

Survival and axonal regeneration of off-center retinal ganglion cells of adult cats are promoted with an anti-glaucoma drug, nipradilol, but not BDNF and CNTF

OFF-center retinal ganglion cells (RGCs) occupy a smaller proportion than ON RGCs when RGCs regenerate axons into a transplanted peripheral nerve. We examined whether the regeneration ability of OFF RGCs in adult cats was promoted when the numbers of regenerating RGCs were increased with brain-derived neurotrophic factor (BDNF)+ciliary neurotrophic factor (CNTF)+forskolin (BCF) or 3,4-dihydro-8-(2-hydroxy-3-isopropylamino)-propoxy-3-nitroxy-2H-1-benzopyran (nipradilol), an anti-glaucoma drug. ON or OFF RGCs were morphologically determined on the basis of their dendritic ramification in the inner plexiform layer using computational analysis. In the normal intact retina the ratio of ON and OFF RGCs (ON/OFF ratio) was 1.25 (55%/44%); whereas, it was 2.61 in regenerating RGCs with saline injection (control) 6 weeks after peripheral nerve transplantation. Estimated numbers of regenerating ON and OFF RGCs were 2149 and 895, respectively. An injection of BCF increased only numbers of ON RGCs into 5766 (2.7-fold to control) but not that of OFF RGCs, n=858. Nipradilol increased both estimated numbers of ON (11,518, 5.4-fold to control) and OFF RGCs (7330, 8.2-fold to control). In the retinas with optic nerve (OpN) transection and intravitreal saline-, BCF- or nipradilol-injection, numbers of ON and OFF RGCs surviving axotomy showed similar trend to that in regenerating RGCs. Thus, nipradilol promoted the survival and regeneration abilities of both of ON and OFF RGCs whereas BCF only did the abilities of ON RGCs. The distribution of tropo-myosin-related kinase B, BDNF receptor, was sparser in the outer two thirds of inner plexiform layer. The lower surviving ability of OFF-RGCs may be attributed in part to the distribution.     

霍乱毒素对成年金黄地鼠视网膜节细胞存活的影响

近端切断成年金黄地鼠视神经,视网膜节细胞大量死亡,两周后达９０％。本研究应用荧光逆行示踪标记技术,观察玻璃体内注射霍乱毒素对视神经切断后视网膜节细胞存活的影响。结果表明：①正常视网膜节细胞平均密度为２００７±１１５／ｍｍ２;②切断视神经５、７、１４ｄ后,视网膜节细胞平均密度分别下降至：１１４７±２０９／ｍｍ２、８７３±１２３／ｍｍ２和２０１±７３／ｍｍ２;③给予Ｎａ２ＥＤＴＡ／ＮａＣＬ的对照组在上述各时间组视网膜节细胞平均密度与单纯视神经切断组结果相似;④给予霍乱毒素的实验组在５、７、１４ｄ视网膜节细胞的平均密度分别为１４３６±１５０／ｍｍ２、１１９２±１２９／ｍｍ２和４１３±９０／ｍｍ２,与视神经切断组和Ｎａ２ＥＤＴＡ／ＮａＣＬ对照组相比在各时间组上均存在显著性差异（Ｐ＜０．０５）,结果表明霍乱毒素有提高视神经切断后节细胞存活的作用     

骨髓间充质干细胞对成年大鼠视网膜节细胞再生的影响

目的：探讨骨髓间充质干细胞(MSCs)对受损成年大鼠视网膜节细胞(RGCs)轴突再生的影响。方法：切断大鼠视神经,缝接自体坐骨神经(AG)。动物分对照组、AG MSCs组、AG MSCs tPNS(三七总皂苷)组。各组动物存活3、4周,用粒蓝逆行标记再生的RGCs,荧光镜下观察视网膜平铺片中再生RGCs的数量变化。免疫组化检测MSCs在玻璃体内的分化。结果：动物存活3、4周,再生的RGCs数目实验组与对照组有显著性差异。AG MSC组和AG MSCs tPNS组,存活的细胞表达Vimentin、NF和GFAP,Laminin无表达。结论：玻璃体植入MSCs可促进受损伤的视网膜节细胞轴突再生。