【作者】 李勇文；

【导师】 黄仁彬；

【作者基本信息】 广西医科大学 ， 药理学， 2010， 博士

【摘要】 目的:弱视是影响儿童视力的主要疾病之一,不及时治疗可以导致终身视力低下,其发病机理尚在探索中。90年代起,国内外学者对药物治疗弱视进行了许多临床性的研究,目前临床上多采用左旋多巴治疗,取得了一定效果。然而,左旋多巴存在副作用多、耐受性差、效果不稳定等缺点。本实验组经过对左旋多巴进行结构改造得到左旋多巴甲酯(LDME)。本研究的目的是观察LDME对斜视性弱视猫模型的治疗作用,评价其治疗弱视的主要药效学作用,探讨LDME的作用机理。方法:正常幼猫30只随机分成6组:左旋多巴甲酯低剂量、中剂量和高剂量组、模型对照组、阳性对照组及正常对照组,每组5只,于4周龄时行眼外直肌切除术造成人工斜视(正常对照组除外),经图形视觉诱发电位(P-VEP)确定形成弱视后,灌胃给与左旋多巴甲酯20mg/kg,40mg/kg, 80mg/kg,阳性对照组给与左旋多巴40mg/kg,正常对照组及模型组均给与等量生理盐水。每天一次,连续30天。①采用图形视觉诱发电位(PVEP)研究左旋多巴甲酯对各组猫视皮层机能的影响;②应用HE尼氏染色、Nissl染色和透射电镜观察左旋多巴甲酯各组猫视皮层17区形态学的改变;③应用原位杂交技术检测视皮质17区c-fos mRNA的表达情况;④应用免疫组化技术检测视皮质17区c-fos蛋白的表达情况;⑤应用免疫组化检测视皮质17区神经生长因子(NGF)的表达情况;⑥用免疫组织化学技术检测视皮质17区NOS阳性细胞的密度。结果:⑴斜视性弱视部分的视觉电生理研究模型对照组(MC)主要以波幅降低及P100潜时延长为特点,MC组潜时延长,潜时较对侧眼及正常对照组双眼的潜时明显延长,差异有显著性(P<0.05);模型对照组每只猫的弱视视觉诱发电位图形P100波幅值与同自身的正常眼及正常组同侧眼比较,P100波幅值变小,差异具有统计学意义(P<0.01)。治疗后各组与模型对照组比较,潜时均有不同程度缩短,以高剂量组最为显著,差异有统计学意义(p<0.01)。经用药物治疗后弱视眼振幅升高,以高剂量组最为显著,与正常对照组比较,无统计学差异(p>0.05)。表明左旋多巴甲酯能明显地改善斜视性弱视猫模型弱视眼的传导和感觉功能。⑵斜视性弱视部分的形态学研究结果模型组(MC)17区视皮层的神经元表现为功能减退,高剂量组、中剂量组、低剂量组及阳性对照组的神经元处于恢复状态,高剂量组的恢复优于阳性对照组、中剂量组、低剂量组。颗粒内层、颗粒外层依次恢复。⑶LDME对视皮质17区c-fos mRNA表达的影响正常对照组猫视皮层c-fos mRNA阳性细胞分布广泛,表达活跃。杂交细胞在各层间的分布情况为:Ⅱ/Ⅲ、Ⅴ/Ⅵ层密度较高,Ⅳ层密度最低。与正常对照组相比较,模型对照组弱视猫视皮层c-fos mRNA杂交细胞在各层均减少,差异有显著性(P<0.01)。与模型对照组相比,治疗组(包括PC、LDMEL、LDMEM、LDMEH)c-fos mRNA杂交细胞在视皮质Ⅱ/Ⅲ、Ⅳ、Ⅴ/Ⅵ层均有增加,除在Ⅳ外,其余各组差异有显著性(P <0.05或P<0.01),而其中以LDMEH组最为明显(P<0.01)。⑷LDME对视皮质17区Fos蛋白表达的影响正常对照组猫视皮质17区全层均见到胞核呈深褐色的阳性细胞,NC猫视皮质17区全层均见高水平的Fos蛋白表达,尤以Ⅱ、Ⅲ、及Ⅵ层密度为高,Ⅳ及Ⅴ层较低。模型对照组猫视皮质全层均可见到阳性细胞,层与层之间的分布模式和正常对照组相似,但各层阳性细胞密度明显降低,差异有统计学意义(P<0.01)。治疗组(包括阳性对照组、左旋多巴甲酯高、中、低剂量组)的阳性细胞的分布基本上都在Ⅱ、Ⅲ、Ⅴ及Ⅵ层,但治疗组的密度高于模型对照组,差异有显著且有统计学意义(P<0.05)。左旋多巴甲酯治疗组与阳性对照组相比较,左旋多巴甲酯治疗组各层阳性细胞分布密度均比阳性对照组高,在一些层上具有统计学意义。⑸LDME对视皮质17区神经生长因子(NGF)表达的影响模型对照组弱视猫VC17区NGF表达特性发生改变,与正常对照组比较,模型对照组内源性NGF免疫阳性细胞数量在各层均减少,两者有显著性差异(p<0.01);通过给与盐酸左旋多巴甲酯治疗后, NGF表达增加,治疗组(包括阳性对照组、左旋多巴甲酯高、中、低剂量组)与模型对照组比较,内源性NGF免疫阳性细胞数量在各层均增加,除LDMEL在Ⅲ、Ⅴ、Ⅵ外,其余差异均有统计学意义(p<0.01或p<0.05);左旋多巴甲酯组与阳性对照组相比较,NGF免疫阳性细胞数量在各层呈上升趋势,且LDMEH在Ⅲ、Ⅵ与阳性对照组相比较差异有统计学意义(p<0.01或p<0.05)。LDMEH与正常对照组比较,NGF免疫阳性细胞数量在各层均少于正常组,但两者比较未见有统计学差异(p>0.05)⑹LDME对视皮质17区NOS表达的影响正常对照组阳性细胞主要分布于灰质的Ⅱ、Ⅲ、Ⅳ、Ⅴ、Ⅵ层和白质中,在Ⅱ/Ⅲ层最多,Ⅴ/Ⅵ层其次,Ⅳ层最少。模型对照组视皮层17区各层NOS阳性细胞的分布模式与正常对照组相似,但各层内及层间NOS阳性纤维明显减少,模型对照组视皮层17区各层NOS阳性细胞密度均显著下降(P<0.05),其中Ⅱ/Ⅲ层及Ⅴ/Ⅵ层NOS阳性细胞密度与正常组对应层次的阳性细胞密度差别具有高度统计意义(P<0.01)。用药后治疗组(包括阳性对照组、左旋多巴甲酯高中低剂量组)各层均有恢复,以LDMEH最为明显。结论:⑴左旋多巴甲酯缩短斜视性弱视猫弱视眼P100波峰潜时及提高P100波幅值,具有改善斜视性弱视猫弱视眼的传导和感觉功能的作用。⑵左旋多巴甲酯对斜视性弱视猫神经元的损伤有较好的治疗效果,高剂量效果最好,能加快复制、转录过程,促进蛋白质的合成,使受损的神经元形态恢复并且能促进胞体突起的形成,促使突触之间的联系增多。⑶左旋多巴甲酯能促进斜视性弱视猫视皮质17区c-fos mRNA的表达;⑷左旋多巴甲酯能促进斜视性弱视猫视皮质17区Fos蛋白的表达。⑸左旋多巴甲酯能增加斜视性弱视猫视皮质17区神经生长因子(NGF)合成与释放。NGF表达增加与PVEP的改变相吻合,证明左旋多巴甲酯能使斜视性弱视猫弱视眼的视功能得到一定的改善。NGF作为一种细胞功能的调控因子,可能在视觉系统发育的可塑性方面发挥重要作用。左旋多巴甲酯通过NGF的作用,参与视觉系统发育的可塑性。⑹左旋多巴甲酯能在一定程度上提高斜视性弱视猫视皮质17区一氧化氮的水平。⑺左旋多巴甲酯斜视性弱视猫有治疗作用,其治疗作用可能与左旋多巴甲酯促进斜视性弱视猫视皮质17区c-fos mRNA、Fos蛋白的表达、增加神经生长因子(NGF)合成与释放及提高斜视性弱视猫视皮质17区一氧化氮的水平有关。更多还原

【Abstract】 Objective: Amblyopia is one of the major diseases affecting children vission, it can lead to lifelong poor vision if not treated in time. The formation of amblyopia involves a variety of factors, and its pathogenesis is still under exploration. From the 90s onwards, domestic and foreign scholars have done many clinical studies on the use of drug treatment of amblyopia. At present, clinically L-dopa is often used for the treatment of amblyopia and achieved some success. However, the L-dopa has some disadvantages such as side effects, more poor tolerance, and effects of instability. We obtained L-dopa methyl ester (LDME) through the structural transformation of L-dopa. The main purpose of this study was to observe the effects of LDME on the treatment of strabismic amblyopic cats. Effects of the LDME were evaluated and the possible mechanisms were discussed.Methods: 30 normal kittens were randomly divided into 6 groups: low dose of L-dopa methyl ester (20mg/kg), medium dose of L-dopa methyl ester (40mg/kg), and high dose of L-dopa methyl ester (80mg/kg,), positive control (L-dopa 40mg/kg), normal control, and model control groups. The surgery for producing iatrogenic convergent strabismus was performed on 4 week old kittens (normal control group excluded). After the confirmation of the development of amblyopia by pattern visual evoked potential, L-dopa methyl ester, L-dopa and normal saline were given to the corresponding animals respectively, once daily continuously for 30 days.In order to study the impact of levodopa methyl ester on the of function of cat visual cortex, we observed the pattern visual evoked potential (PVEP) change with visual electrophysiology instrument. The morphological change of the visual cortex was investigated by the HE stain, nissl stain and transmission electron microscope. The c-fos mRNA expression of visual cortex area 17 was detected with the technology of in situ hybridization. C-fos protein expression in the visual cortex area 17 was detected by immunohistochemistry. We had observed nerve growth factor (NGF) expression in the visual cortex area 17 by immunohistochemical technique. NOS immunocytochemstry staining method was applied accordingly to observe the changes of distribution and cells density across the visual cortex under the light microscope.Results:(1) The visual electrophysiology of strabismic amblyopia cats.The change in model control group (MC) was characterized by P100 amplitude reduction and P100 latency extension. P100 latency delayed in MC, latency was more longer than the contralateral eyes and the eyes in NC, the difference was significant (P<0.05). Compared with the same self normal eyes and the ipsilateral eye in NC, P100 amplitude in MC decreased significantly (P<0.01). After treatment, compared with the MC, P100 latency was reduced to varying degrees, and P100 latency of high-dose group was the most significant, which was statistically significant (p<0.01). After treatment with LDME, the amplitude increased, and there was no significant difference (p>0.05) when LDMEH was compared with the normal control group, suggesting that L-dopa methyl ester can significantly improve conduction and sensory function in model of strabismic amblyopia cats. (2) The morphological findings of strabismic amblyopia cats.In the MC, the neurons in the visual cortex area 17 were showed dysfunctionly, but the neurons of LDMEH, LDMEM, LDMEL and PC showed recovering signs, the restoration of LDMEH was more well than PC, LDMEM, and LDMEL. The inner particles and particle outer were recovered.(3) The influence of LDME on c-fos mRNA expression in the visual cortex area 17 of strabismic amblyopia cats.In the 17 area of the visual cortex of NC, the c-fos mRNA positive cells were widely distributed, and expressed actively. the distribution of hybrid cells: the densities ofⅡ/ⅢandⅤ/Ⅵlayer were higher, the density ofⅣlayer lowest. Compared with the normal control group, in the 17 area of the visual cortex of MC, the c-fos mRNA positive neurons were decreased in all layers, and the difference was significant (P<0.01). Compared with the model group, in the treatment group (including the PC, LDMEL, LDMEM, LDMEH) c-fosmRNA positive cells in the visual cortexⅡ/Ⅲ,Ⅳ, andⅤ/Ⅵlayers were increased. Except inⅣ, there was a significant (P<0.05 or P<0.01) in the other differences between the groups.(4) The influence of LDME on Fos expression in the visual cortex area 17 of strabismic amblyopia cats.The Fos positive cells, which nucleus showed dark brown, were seen in the whole floor of visual cortex area 17 of the NC cat. The density of the positive cells in layerⅡ,ⅢandⅥwas higher than that in layerⅣandⅤ. The distribution patterns of Fos positive cells between layers were similar to the NC, but the density of positive cell in each layer was significantly lower than that in NC, and the difference was statistically significant (P<0.01). In the treatment group (including the positive control group, L-dopa methyl ester of high, medium and low-dose group) the distribution of positive cells was basically in layer II, III, V and VI, but the density in each layer of the treatment group was more higher than that in model control group, and the difference was significant (P<0.05). Compared with PC, the density of Fos positive cells in each layers of LDME treatment group was more hiher than that in PC respectively, and some layers had statistical significance.(5) The influence of LDME on NGF expression in he visual cortex area 17 of strabismic amblyopia cats.Compared with the NC, the number density of NGF-immunoreactive cells in MC was reduced, and there was a significant difference between the two groups (p<0.01). Compared with MC, the number density of NGF-immunoreactive cells in each layers of the treatment group (including the PC, L-dopa methyl ester of high, medium and low-dose groups) was increased correspondingly, and the differences were statistically significant (p<0.01 or p<0.05) in addition to LDMEL inⅢ,ⅤandⅥ. Compared with the positive control group, the number density of NGF-immunoreactive cells in each layer of L-dopa methyl ester group was in the upward trend, and the differences in layerⅢandⅥof the LDMEH were statistically significant (p<0.01 or p< 0.05). Compared with NC, the number density of NGF-immunoreactive cells in LDMEH was less than that in the normal group, but there was no statistical difference between the two groups (p>0.05).(6) The influence of LDME on NOS expression in the visual cortex area 17 of strabismic amblyopia cats.The NOS positive cells in the NC were mainly distributed in the gray matter of theⅡ,Ⅲ,Ⅳ,ⅤandⅥlayers and white matter, the number of the NOS positive cells inⅡ/Ⅲwas highest,Ⅴ/Ⅵnext, andⅣat least. The distribution pattern of NOS-positive cells in visual cortex area 17 of MC cats was similar to that of the normal control group, however, within each layer and interlayer the NOS-positive fibers were significantly reduced, and the NOS-positive cell density was also significantly decreased (P<0.05). In the treatment group (including the positive control group, high low-dose L-dopa methyl ester groups) the NOS-positive cell density in each layers was increased, and the LDMEH was most obvious.Conclusions:(1) LDME can shorten the peak latency of P100 and increase P100 amplitude in strabismic amblyopia cats, and make them to recover to normal level, which can improve the function of conduction and perception in strabismic amblyopia cats eyes.(2) LDME has good therapeutic effect on the injured neuron in strabismic amblyopic cats, and can restore the neuron morphology. It also can promote the replication, transcription process, protein synthesis, protuberance of the neurons, and the synaptic transmission.(3). LDME can promote c-fos mRNA expression in area 17 of visual cortex of strabismic amblyopia cat.(4) LDME can promote Fos protein expression in area 17 of visual cortex in strabismic amblyopia cat.(5) LDME ester can increase NGF synthesis and release in area 17 of visual cortex in strabismic amblyopia cat. NGP expression coincides with of the change the PVEP. It is proved that LDME can improve the visual function of strabismic amblyopia cats eyes in certain degree. As a regulatory factor in cell function NGP may be play an important role on developmental plasticity in the visual system. (6) LDME can increase the nitric oxide levels in area 17 of visual cortex of strabismic amblyopia cats to some extent.(7) LDME has therapeutic effects on the strabismic amblyopic cats, and its mechanism may be related to the increases in NGF synthesis and release and the nitric oxide level in visual cortex area 17 of strabismic amblyopic cats.更多还原