Nogo-A及受体NgR对大鼠视皮层可塑性调控作用的研究
发布时间：2018-09-19 18:34
【摘要】：第一章Nogo-A及受体NgR在正常发育及单眼剥夺模型大鼠视皮层中表达的动态变化 目的探讨正常发育过程及单眼剥夺模型大鼠视皮层中Nogo-A及受体NgR的mRNA和蛋白质表达的动态变化及意义。 方法84只新生SD大鼠随机分为6组：0日龄正常组(NorPO,12只)、14日龄正常组(NorP14,12只)、28日龄正常组(NorP28,12只)、60日龄正常组(NorP60,18只)、28日龄模型组(MDP28,12只)以及60日龄模型组(MDP60,18只),模型组大鼠于出生后21天缝合右侧眼睑建立单眼形觉剥夺模型。各组动物均依照实验设计的时间点处死并取左侧视皮层组织,提取总RNA及蛋白质,采用RT-PCR及免疫印迹Western blot方法检测视皮层中Nogo-A及NgR的表达,了解正常发育及单眼剥夺模型大鼠视皮层中Nogo-A及NgR的动态表达趋势,同时通过免疫荧光组织化学方法检测早期单眼剥夺对Nogo-A及NgR蛋白表达影响的层特异性。 结果正常新生大鼠(NorPO)视皮层中即有Nogo-A/NgR mRNA及蛋白质的表达,其表达量随年龄增长而明显上调。其中Nogo-A mRNA及蛋白质于出生后28天(NorP28)到达高峰并持续至成年期(NorP60); NgR mRNA于出生后14天(NorP14)到达高峰并持续至成年期(NorP60),而NgR蛋白质表达上调则较mRNA滞后,于出生后28天(NorP28)到达高峰并持续至成年期(NorP60)。早期单眼剥夺后,模型组(MDP28、MDP60)大鼠剥夺眼对侧视皮层中Nogo-A/NgR mRNA及蛋白质表达较同日龄正常组(NorP28、NorP60)轻度减少,差异均无统计学意义(P0.05)。此外,MDP28组与MDP60组大鼠比较剥夺眼对侧视皮层中Nogo-A/NgR mRNA及蛋白质表达水平差异无统计学意义(P0.05)。免疫荧光组织化学方法检测显示Nogo-A及NgR蛋白在NorP60组及MDP60组大鼠视皮层Ⅱ-Ⅲ层,Ⅳ层,Ⅴ层,Ⅵ层中均有表达,早期单眼剥夺可使MDP60组大鼠剥夺眼对侧视皮层中第Ⅱ-Ⅲ层、Ⅳ层Nogo-A及NgR免疫阳性细胞密度较NorP60组明显下调。 结论大鼠Nogo-A/NgR mRNA及蛋白质自出生到成年期在视皮层中持续表达,且在视觉发育关键期后期达高峰,视觉发育早期单眼剥夺可致其表达下调,呈现出一定的视觉经验依赖性。因此,Nogo-A/NgR在视觉发育早期可能主要参与轴突生长及导向；同时调控视皮层可塑性关键期的终止,并对成年视皮层可塑性发挥一定的抑制作用。 第二章NgR拮抗剂NEP1-40对成年单眼剥夺模型大鼠视皮层可塑性的再激活作用 目的观察NgR拮抗剂NEP1-40对成年单眼剥夺模型大鼠剥夺眼对侧视皮层结构及功能可塑性再激活的影响,进一步阐明Nogo-A/NgR信号系统对成年视皮层可塑性的调控作用。 方法将120只新生SD大鼠随机分为两大组即正常组和单眼剥夺模型组。按给药方式不同,正常组又分为正常对照组(Nor)、正常+PBS治疗组(Nor+PBS)、正常+NEP1-40治疗组(Nor+NEP);单眼剥夺模型组又分为模型对照组(MD)、模型+PBS治疗组(MD+PBS)以及模型+NEP1-40治疗组(MD+NEP),每组各20只。模型组大鼠于出生后21天缝合右侧眼睑建立单眼形觉剥夺模型,于45日龄时打开剥夺眼,对各组大鼠行闪光视觉诱发电位(F-VEP)检测,确定单眼剥夺模型建立成功后,对需给药的各组大鼠按组别给予0.02mg/μl的NEP1-40或0.01M的PBS侧脑室注药治疗,每天1次,每次10μl,持续7天。于52日龄时对各组大鼠再次行F-VEP检测后处死动物,取侧脑室组织行尼氏染色明确给药部位,取左侧视皮层组织进行尼氏染色观察神经元形态及数量、高尔基染色观察树突棘密度、透射电镜观察突触超微结构变化。 结果(1)尼氏染色观察侧脑室及视皮层组织结构：未接受侧脑室注药治疗的MD组大鼠,大脑皮层组织结构完整,未见注射针头的痕迹；接受过侧脑室注药治疗的MD+PBS组及MD+NEP组大鼠,可见注射针头经大脑皮层向侧脑室方向穿过的痕迹。此外,接受过侧脑室注药治疗的MD+PBS组及MD+NEP组大鼠剥夺眼对侧视皮层层次分明,结构清晰,神经细胞数量及形态较MD组无明显差异。 (2)高尔基染色观察视皮层神经元树突棘密度：MD组大鼠剥夺眼对侧视皮层神经元树突棘密度较Nor组显著降低(P0.05)。Nor+PBS组及Nor+NEP组大鼠视皮层神经元树突棘密度与Nor组比较差异均无统计学意义(P0.05); MD+NEP组大鼠剥夺眼对侧视皮层树突棘密度较MD组及MD+PBS组明显增加(P0.05),且与Nor组比较差异无统计学意义(P0.05)；MD+PBS组与MD组大鼠剥夺眼对侧视皮层神经元树突棘密度比较差异无统计学意义(P0.05),而与Nor组比较差异有统计学意义(P0.05)。 (3)透射电镜观察视皮层神经元突触界面结构参数：与Nor组比较,MD组大鼠剥夺眼对侧视皮层神经元突触间隙增大,突触活性区长度缩短,突触界面曲率减小,突触后致密物厚度变薄(P0.05)。Nor+PBS组及Nor+NEP组大鼠视皮层神经元突触界面结构参数的各项指标与Nor组比较差异均无统计学意义(P0.05)；MD+NEP组大鼠剥夺眼对侧视皮层神经元突触界面结构参数的各项指标均较MD组、MD+PBS组明显改善(P0.05),与Nor组相比除突触间隙外(P0.05),其余各项参数差异无统计学意义(P0.05)。MD+PBS组与MD组大鼠比较剥夺眼对侧视皮层神经元突触界面结构参数的各项指标差异均无统计学意义(P0.05),而与Nor组比较差异有统计学意义(P0.05)。 (4)闪光视觉诱发电位评估大鼠客观视功能：45日龄时F-VEP检测示,MD组、MD+PBS组及MD+NEP组与Nor组比较,P波潜伏期延长,波幅降低(P0.05)；而Nor+PBS组及Nor+NEP组大鼠P波的潜伏期及波幅与Nor组比较差异均无统计学意义(P0.05)。52日龄时F-VEP检测示,MD组与Nor组比较大鼠右眼F-VEP的P波潜伏期延长,波幅降低(P0.05)。Nor+PBS组及Nor+NEP组大鼠右眼F-VEP的P波潜伏期及波幅与Nor组比较差异均无统计学意义(P0.05); MD+NEP组与MD组、MD+PBS组大鼠比较右眼F-VEP的P波潜伏期及波幅差异有统计学意义(P0.05),而与Nor组比较差异无统计学意义(P0.05); MD+PBS组与MD组大鼠比较,右眼F-VEP的P波潜伏期及波幅差异无统计学意义(P0.05),而与Nor组比较差异有统计学意义(P0.05)。 结论NgR拮抗剂NEP1-40可使成年单眼剥夺模型大鼠剥夺眼对侧视皮层神经元的树突棘密度、突触界面结构参数得以恢复,F-VEP的P波潜伏期及波幅恢复至正常水平,重新“激活”被抑制的视皮层结构及功能可塑性。这一研究结果为Nogo-A/NgR系统参与视皮层可塑性的调控提供了直接证据,同时为成年弱视患者提供新的治疗途径奠定了理论基础。
[Abstract]:Chapter 1 Dynamic Changes of Nogo-A and NgR Expression in the Visual Cortex of Normal Developmental and Monocular Deprivation Rats
Objective To investigate the dynamic changes and significance of Nogo-A and NgR mRNA and protein expression in visual cortex of normal development and monocular deprivation rats.
Methods 84 neonatal SD rats were randomly divided into 6 groups: normal group (NorPO, 12 rats), normal group (NorP14, 12 rats), normal group (NorP28, 12 rats), normal group (NorP60, 18 rats), model group (MDP28, 12 rats) and model group (MDP60, 18 rats) at the age of 20 days after birth. Form deprivation model.Each group of animals was executed according to the experimental design time point and the left visual cortex was taken out to extract total RNA and protein.The expression of Nogo-A and NgR in visual cortex was detected by RT-PCR and Western blot. Immunofluorescence histochemistry was used to detect the layer specificity of the effect of early monocular deprivation on the expression of Nogo-A and NgR proteins.
Results The expression of Nogo-A/NgR mRNA and protein in visual cortex of normal neonatal rats (NorPO) was up-regulated with age. Nogo-A mRNA and protein peaked at 28 days after birth (NorP28) and lasted until adulthood (NorP60); NgR mRNA peaked at 14 days after birth (NorP14) and lasted until adulthood (NorP60). However, the up-regulation of NgR protein expression was lagged behind that of mRNA, reaching its peak at 28 days after birth (NorP28) and continuing to adulthood (NorP60). After early monocular deprivation, the expression of Nogo-A/NgR mRNA and protein in the contralateral visual cortex of the deprived rats in the model group (MDP28, MDP60) was slightly lower than that in the normal control group (NorP28, NorP60). In addition, there was no significant difference in the expression of Nogo-A/NgR mRNA and protein between MDP 28 and MDP 60 deprived rats (P 0.05). Immunofluorescence histochemistry showed that Nogo-A and NgR proteins were expressed in layers II-III, IV, V and VI of the visual cortex of NorP 60 and MDP 60 rats. The densities of Nogo-A and NgR immunoreactive cells in the contralateral visual cortex of MDP60 rats were significantly lower than those in NorP60 rats.
Conclusion Nogo-A/NgR mRNA and protein are continuously expressed in the visual cortex from birth to adulthood, and reach a peak at the late critical stage of visual development. Monocular deprivation in early visual development can lead to down-regulation of Nogo-A/NgR expression, showing a certain degree of visual experience dependence. At the same time, it regulates the termination of the critical period of visual cortex plasticity and inhibits the adult visual cortex plasticity.
The second chapter is about the reactivation of NgR antagonist NEP1-40 on the plasticity of visual cortex in adult monocular deprivation rats.
Objective To observe the effects of NgR antagonist NEP1-40 on the structural and functional plasticity reactivation of the contralateral visual cortex in adult monocular deprivation rats, and further elucidate the regulatory role of the Nogo-A/NgR signaling system on the plasticity of the adult visual cortex.
Methods 120 neonatal SD rats were randomly divided into two groups: normal group and monocular deprivation model group. According to different administration methods, the normal group was divided into normal control group (Nor), normal + PBS treatment group (Nor + PBS), normal + NEP1-40 treatment group (Nor + NEP), monocular deprivation model group was divided into model control group (MD), model + PBS treatment group (MD + PBS) and model. The model group was established by suturing the right eyelid 21 days after birth. The deprived eyes were opened at 45 days of age. The flash visual evoked potential (F-VEP) was detected in each group. After the successful establishment of the deprived model, the rats in each group were given 0.02mg/mu according to the group. L NEP 1-40 or 0.01M PBS were injected into the lateral ventricle once a day, 10 ml each time for 7 days. At the age of 52, the rats in each group were sacrificed after F-VEP test again. The lateral ventricle tissues were taken for Nissl staining to determine the site of administration. The left visual cortex tissues were taken for Nissl staining to observe the morphology and number of neurons. The dendrites were observed by Golgi staining. The ultrastructure of synapses was observed by transmission electron microscope.
Results (1) Nissl staining was used to observe the structure of the lateral ventricle and visual cortex: The cortex of MD rats without the treatment of lateral ventricle injection was intact, and there was no evidence of needle penetration; MD+PBS rats and MD+NEP rats with the treatment of lateral ventricle injection showed that the needle penetrated the lateral ventricle through the cerebral cortex. In addition, MD+PBS group and MD+NEP group, which had been treated by intraventricular injection, had distinct layers and clear structures of contralateral visual cortex. There was no significant difference in the number and morphology of nerve cells between MD+PBS group and MD+NEP group.
(2) Golgi staining to observe dendritic spine density of optic cortex neurons: The dendritic spine density of deprived contralateral optic cortex neurons in MD group was significantly lower than that in Nor group (P 0.05). The dendritic spine density of optic cortex neurons in Nor+PBS group and Nor+NEP group was not significantly different from that in Nor group (P 0.05). Compared with MD group and MD+PBS group, the density of dendritic spines in MD+PBS group increased significantly (P 0.05), and there was no significant difference between MD+PBS group and Nor group (P 0.05); there was no significant difference between MD+PBS group and MD group in dendritic spines density of deprived contralateral visual cortex neurons (P 0.05), but there was significant difference between MD+PBS group and Nor group (P 0.05).
(3) Observation of synaptic interface parameters by transmission electron microscopy: Compared with Nor group, the synaptic gap of deprived contralateral visual cortex neurons in MD group increased, the length of synaptic active area shortened, the curvature of synaptic interface decreased, and the thickness of postsynaptic dense substance thinned (P 0.05). There was no significant difference in the parameters of synaptic interface between MD + NEP group and Nor group (P 0.05). The parameters of synaptic interface of deprived contralateral visual cortex neurons in MD + NEP group were significantly improved compared with MD group and MD + PBS group (P 0.05). There was no significant difference in the parameters of synaptic interface between BS group and MD group (P 0.05), but there was significant difference between BS group and Nor group (P 0.05).
(4) Flash Visual Evoked Potentials Assessment of Objective Visual Function in Rats: At 45 days of age, F-VEP detection showed that compared with Nor group, MD+PBS group, MD+NEP group and MD+PBS group, P wave latency was prolonged and amplitude was decreased (P 0.05), but there was no significant difference between Nor+PBS group and Nor+NEP group in P wave latency and amplitude (P 0.05). The results showed that the P wave latency and amplitude of F-VE P in the right eye of MD group and Nor group were prolonged and decreased (P 0.05). There was no significant difference between Nor+PBS group and Nor+NEP group in the P wave latency and amplitude of F-VE P in the right eye (P 0.05). There was no significant difference in P wave latency and amplitude between MD+PBS group and MD group (P 0.05), but there was significant difference between MD+PBS group and Nor group (P 0.05).
Conclusion NgR antagonist NEP1-40 can restore the dendritic spine density, synaptic interface structure parameters, F-VE P latency and amplitude to normal level, and re-activate the inhibited visual cortex structure and functional plasticity in adult monocular deprivation rats. Systematic involvement in the regulation of visual cortex plasticity provides direct evidence, and provides a theoretical basis for the treatment of adult amblyopia.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R77
本文编号：2250974
[Abstract]:Chapter 1 Dynamic Changes of Nogo-A and NgR Expression in the Visual Cortex of Normal Developmental and Monocular Deprivation Rats
Objective To investigate the dynamic changes and significance of Nogo-A and NgR mRNA and protein expression in visual cortex of normal development and monocular deprivation rats.
Methods 84 neonatal SD rats were randomly divided into 6 groups: normal group (NorPO, 12 rats), normal group (NorP14, 12 rats), normal group (NorP28, 12 rats), normal group (NorP60, 18 rats), model group (MDP28, 12 rats) and model group (MDP60, 18 rats) at the age of 20 days after birth. Form deprivation model.Each group of animals was executed according to the experimental design time point and the left visual cortex was taken out to extract total RNA and protein.The expression of Nogo-A and NgR in visual cortex was detected by RT-PCR and Western blot. Immunofluorescence histochemistry was used to detect the layer specificity of the effect of early monocular deprivation on the expression of Nogo-A and NgR proteins.
Results The expression of Nogo-A/NgR mRNA and protein in visual cortex of normal neonatal rats (NorPO) was up-regulated with age. Nogo-A mRNA and protein peaked at 28 days after birth (NorP28) and lasted until adulthood (NorP60); NgR mRNA peaked at 14 days after birth (NorP14) and lasted until adulthood (NorP60). However, the up-regulation of NgR protein expression was lagged behind that of mRNA, reaching its peak at 28 days after birth (NorP28) and continuing to adulthood (NorP60). After early monocular deprivation, the expression of Nogo-A/NgR mRNA and protein in the contralateral visual cortex of the deprived rats in the model group (MDP28, MDP60) was slightly lower than that in the normal control group (NorP28, NorP60). In addition, there was no significant difference in the expression of Nogo-A/NgR mRNA and protein between MDP 28 and MDP 60 deprived rats (P 0.05). Immunofluorescence histochemistry showed that Nogo-A and NgR proteins were expressed in layers II-III, IV, V and VI of the visual cortex of NorP 60 and MDP 60 rats. The densities of Nogo-A and NgR immunoreactive cells in the contralateral visual cortex of MDP60 rats were significantly lower than those in NorP60 rats.
Conclusion Nogo-A/NgR mRNA and protein are continuously expressed in the visual cortex from birth to adulthood, and reach a peak at the late critical stage of visual development. Monocular deprivation in early visual development can lead to down-regulation of Nogo-A/NgR expression, showing a certain degree of visual experience dependence. At the same time, it regulates the termination of the critical period of visual cortex plasticity and inhibits the adult visual cortex plasticity.
The second chapter is about the reactivation of NgR antagonist NEP1-40 on the plasticity of visual cortex in adult monocular deprivation rats.
Objective To observe the effects of NgR antagonist NEP1-40 on the structural and functional plasticity reactivation of the contralateral visual cortex in adult monocular deprivation rats, and further elucidate the regulatory role of the Nogo-A/NgR signaling system on the plasticity of the adult visual cortex.
Methods 120 neonatal SD rats were randomly divided into two groups: normal group and monocular deprivation model group. According to different administration methods, the normal group was divided into normal control group (Nor), normal + PBS treatment group (Nor + PBS), normal + NEP1-40 treatment group (Nor + NEP), monocular deprivation model group was divided into model control group (MD), model + PBS treatment group (MD + PBS) and model. The model group was established by suturing the right eyelid 21 days after birth. The deprived eyes were opened at 45 days of age. The flash visual evoked potential (F-VEP) was detected in each group. After the successful establishment of the deprived model, the rats in each group were given 0.02mg/mu according to the group. L NEP 1-40 or 0.01M PBS were injected into the lateral ventricle once a day, 10 ml each time for 7 days. At the age of 52, the rats in each group were sacrificed after F-VEP test again. The lateral ventricle tissues were taken for Nissl staining to determine the site of administration. The left visual cortex tissues were taken for Nissl staining to observe the morphology and number of neurons. The dendrites were observed by Golgi staining. The ultrastructure of synapses was observed by transmission electron microscope.
Results (1) Nissl staining was used to observe the structure of the lateral ventricle and visual cortex: The cortex of MD rats without the treatment of lateral ventricle injection was intact, and there was no evidence of needle penetration; MD+PBS rats and MD+NEP rats with the treatment of lateral ventricle injection showed that the needle penetrated the lateral ventricle through the cerebral cortex. In addition, MD+PBS group and MD+NEP group, which had been treated by intraventricular injection, had distinct layers and clear structures of contralateral visual cortex. There was no significant difference in the number and morphology of nerve cells between MD+PBS group and MD+NEP group.
(2) Golgi staining to observe dendritic spine density of optic cortex neurons: The dendritic spine density of deprived contralateral optic cortex neurons in MD group was significantly lower than that in Nor group (P 0.05). The dendritic spine density of optic cortex neurons in Nor+PBS group and Nor+NEP group was not significantly different from that in Nor group (P 0.05). Compared with MD group and MD+PBS group, the density of dendritic spines in MD+PBS group increased significantly (P 0.05), and there was no significant difference between MD+PBS group and Nor group (P 0.05); there was no significant difference between MD+PBS group and MD group in dendritic spines density of deprived contralateral visual cortex neurons (P 0.05), but there was significant difference between MD+PBS group and Nor group (P 0.05).
(3) Observation of synaptic interface parameters by transmission electron microscopy: Compared with Nor group, the synaptic gap of deprived contralateral visual cortex neurons in MD group increased, the length of synaptic active area shortened, the curvature of synaptic interface decreased, and the thickness of postsynaptic dense substance thinned (P 0.05). There was no significant difference in the parameters of synaptic interface between MD + NEP group and Nor group (P 0.05). The parameters of synaptic interface of deprived contralateral visual cortex neurons in MD + NEP group were significantly improved compared with MD group and MD + PBS group (P 0.05). There was no significant difference in the parameters of synaptic interface between BS group and MD group (P 0.05), but there was significant difference between BS group and Nor group (P 0.05).
(4) Flash Visual Evoked Potentials Assessment of Objective Visual Function in Rats: At 45 days of age, F-VEP detection showed that compared with Nor group, MD+PBS group, MD+NEP group and MD+PBS group, P wave latency was prolonged and amplitude was decreased (P 0.05), but there was no significant difference between Nor+PBS group and Nor+NEP group in P wave latency and amplitude (P 0.05). The results showed that the P wave latency and amplitude of F-VE P in the right eye of MD group and Nor group were prolonged and decreased (P 0.05). There was no significant difference between Nor+PBS group and Nor+NEP group in the P wave latency and amplitude of F-VE P in the right eye (P 0.05). There was no significant difference in P wave latency and amplitude between MD+PBS group and MD group (P 0.05), but there was significant difference between MD+PBS group and Nor group (P 0.05).
Conclusion NgR antagonist NEP1-40 can restore the dendritic spine density, synaptic interface structure parameters, F-VE P latency and amplitude to normal level, and re-activate the inhibited visual cortex structure and functional plasticity in adult monocular deprivation rats. Systematic involvement in the regulation of visual cortex plasticity provides direct evidence, and provides a theoretical basis for the treatment of adult amblyopia.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R77
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