先天性静止性夜盲大鼠视觉信号特征及其视觉信号传导通路重塑

发布时间：2018-05-16 21:18

本文选题：先天性静止性夜盲 + 多电极阵列　；参考：《第四军医大学》2013年博士论文

【摘要】：先天性静止性夜盲(congenital stationary night blindness, CSNB)是一种非进展性、以视杆视觉信号系统功能障碍为主的遗传性视网膜病。我们实验室发现的Cacna1f基因自然突变CSNB大鼠,其电生理及行为学证明其克服了基因工程诱导突变动物模式与临床实际差异较大的缺陷,因此可以作为研究CSNB发病机制及视网膜信号通路之间相互关系的一种有效模型。Cav1.4型钙通道为L-型钙通道的亚型之一,其主要的功能单位a1f亚基由Cacnalf基因编码。目前发现Cacna1f基因多种位点突变可损伤视杆和视锥信号传导通路,并与性染色体遗传的不完全型静止性夜盲密切相关。研究已证实Cacna1f基因突变导致的α1f亚基缺陷可使光感受器细胞带状突触数量、形态以及功能发生异常,从而引发光感受器细胞与二级神经元之间视觉信号传导障碍。近期形态学研究发现,自然突变CSNB大鼠视网膜双极细胞所发出的异常突触可伸及外核层外部,外丛状层厚度减少,水平细胞数目减少,而视网膜内层的超微结构则相对正常完整。上述视网膜外层发生的形态学变化很可能影响视网膜内层的视觉信号通路的传导整合功能。然而,CSNB动物视网膜外层所发生的形态学变化及神经递质释放功能障碍对视网膜最内层的神经节细胞会产生何种病理学效应,至今未见诸于报道。多电极阵列系统已广泛应用于视网膜神经元电生理活动记录等实验研究,由于其具备完整性、非侵入性的优点,因而此电生理实验技术弥补了传统的活体外记录与复杂的活体内记录的先天缺陷,目前已经被应用于记录监测光感受器细胞、神经节细胞等视网膜神经元的生理活动。本研究采用多电极阵列记录系统和先天性自然突变大鼠动物模型,根据其视网膜内层信号通路的视觉电生理信号所发生的特征性改变来证明其信号通路发生的重塑,并以此为基础阐明通路重塑的发生机制,为进一步制订治疗方案及采取针对性的视觉干预保护措施提供依据。材料与方法实验采用近交系CSNB大鼠(SPF级,F23,8-10周龄)及正常同龄对照Wt大鼠,将大鼠暗适应12-15小时,深度麻醉后处死大鼠摘取眼球,除去眼前节和视网膜色素上皮层,制备视网膜铺片,并将其神经节细胞面向下贴附于多电极阵列记录芯片,并使其适应生物记录芯片中的灌流环境。药理学实验环节中,实验中所涉及的药品经活性灌注系统进入灌流液。电脑刺激器控制的发光二级管所产生的白光经透镜系统汇聚形成光斑,投射到生物芯片灌流槽之中的视网膜铺片上,给予其同步全野光刺激。多电极阵列系统电极所采集到的神经元电信号经过放大器过滤并放大,存储于电脑系统供后续离线软件分析处理。单电极所采集的电生理信号进行放电分析时,其检测阈值设定为平均幅值的四倍标准差。然后应用Offline Sorter将备选放电波形进行处理,采取其前三个主要组成部分应用NeuroExplore软件进行后续分析：使用K-均值分解类聚算法识别集簇放电；分析单个记录单元的放电序列间距直方图来确定其放电的规则性及峰电位分布,并结合单个记录单元的围刺激时间柱形图来判断放电细胞的种类。电刺激实验环节中,位于阵列中央的28号电极所产生的双阶平衡电流刺激序列诱发视网膜神经元电生理反应,并为阵列中其它电极采集记录,应用相同软件进行分析处理。结果 1.野生型SD大鼠与CSNB大鼠视网膜光刺激诱导场电位施加光刺激程序后,多电极阵列记录系统可从Wt大鼠视网膜铺片采集到典型的光诱发场电位波形,其a波峰值为0.196±0.038mv(n-20)。应用ON-型双极细胞mGluR6受体的特异性拮抗剂APB (100μM/L)不能阻断a-波却可阻断b-波。CSNB大鼠视网膜铺片记录中,光诱发场电位a-波峰值显著减小(0.078±0.026mv, n-20, P0.01), b-波缺如。 2.野生型SD大鼠与CSNB大鼠视网膜电刺激放电反应施加双阶平衡电流刺激后,在野生型大鼠和CSNB大鼠电刺激后原始波形中均可发现两种类型电刺激反应：短潜伏期反应和长潜伏期反应。按照神经节细胞与电流刺激电极的间距将其分为14组：(100μm组,141μm组,200μm组,223μm组,283μm组,300μm组,316μm组,361μm组,387μm组,400μm组,424pm组,447μm组,500μmm组和566μm组)。结果发现神经节细胞的电刺激反应强度随其与位于阵列中央的电流刺激电极间的距离增加而降低；在所分组中,CSNB大鼠视网膜的长潜伏期反应的放电数目均较野生型大鼠减少(P0.01)。 3.野生型SD大鼠与CSNB大鼠视网膜自发性放电反应多电极阵列记录系统从每个视网膜铺片中可同时记录到来自30个-90个神经节细胞的自发放电。分析发现CSNB大鼠视网膜神经节细胞的自发放电频率(9.93±4.14Hz,n=386)显著高于野生型大鼠(2.610.73Hz,n=295, P0.01)。从累积自发放电频率曲线图可以观察到,CSNB大鼠视网膜神经节细胞的自发放电频率曲线较野生型大鼠斜率降低且发生右移。 4.野生型SD大鼠与CSNB大鼠视网膜自发性放电神经节细胞的类型将产生自发性放电反应的神经节细胞归类为四种类型：爆发型放电细胞,常规型放电细胞,非常规型放电细胞,混合型放电细胞。CSNB大鼠视网膜爆发型放电细胞和混合型放电细胞占所有自发放电神经节细胞的比重高于野生型大鼠(Wt：爆发型15.2%,混合型：24.7%,n-20；CSNB:爆发型：24.3%,混合型：31.6%,n-20,P0.01)而非常规型放电细胞占所有自发放电神经节细胞的比重低于野生型大鼠(Wt:51.4%, CSNB:36.8%; P0.01)。 CSNB大鼠视网膜混合型放电神经节细胞的放电总数(246553个,n-20)显著高于Wt大鼠视网膜神经节细胞混合混合型放电细胞的放电总数(112089,n=20,P0.01)。 CSNB大鼠和Wt大鼠视网膜其它三种神经节细胞的放电总数无显著差异(P0.05)。 5.野生型SD大鼠与CSNB大鼠视网膜光诱导放电反应按照对光反应的放电特征可将来自CSNB大鼠和Wt大鼠视网膜的神经节细胞分为六大类：1)给光型(ON-型)；2)撤光型(OFF-型)；3)给-撤光型(ON-OFF型)；4)持续给光型(sustained ON型)；5)持续给-撤光型(sustained ON-OFF型)；6)延迟撤光型(delayed OFF型)。CSNB大鼠视网膜神经节细胞光诱导放电反应的平均频率(16.40±9.97Hz,n-340)较野生型大鼠显著降低(38.6211.78Hz,n=373, P0.01)。与Wt大鼠视网膜相比,CSNB大鼠视网膜的暗适应自发放电(背景噪声)频率较高而光诱导放电反应(视觉信号)频率较低,因此其信号/噪声比(S-N ration)减小。 6.野生型SD大鼠与CSNB大鼠视网膜自发放电的互相关活动在野生型大鼠视网膜中,观察到三种特征性神经节细胞互相关活动类型：短时程互相关活动、中时程互相关活动和长时程互相关活动。而在CSNB大鼠视网膜记录数据中只观察到短时程互相关活动和中时程互相关活动,长时程互相关活动消缺失(n=20)。应用100uM18p-GA可明显降低CSNB大鼠视网膜神经节细胞的自发性放电频率,而其对野生型大鼠视网膜的放电频率无明显影响(n-5)。 7. CSNB大鼠视网膜视觉信号传导通路的重塑按照单个神经节细胞ON-型放电反应频率占其光诱导放电反应总频率的比例,将其归为三类：1)ON反应主导型神经节细胞(比率0.5)；2)OFF反应主导型神经节细胞(比率0.5)；3)均衡反应型神经节细胞(比率=0.5)。较野生型大鼠,CSNB大鼠视网膜中的OFF反应主导型神经节细胞数占光诱导放电神经节细胞总数的比例增加(Wt:60.6%; CSNB:78.1%),而ON反应主导型神经节细胞数所占的比例减小(Wt:31.5%; CSNB:18.2%)。对照两种大鼠视网膜神经节细胞光诱导ON-型和OFF-型反应的潜伏期发现：CSNB大鼠的ON-型和OFF-型反应潜伏期均较野生型大鼠显著延长(P0.05)。应用用CNQX+PDA可明显阻断CSNB大鼠和野生型大鼠视网膜光诱导OFF-型反应：在两种视网膜中,撤光型神经节细胞(OFF RGCs)和给光-撤光型神经节细胞(ON-OFF RGCs)的OFF反应均可被阻断剂消除,而延迟撤光型神经节细胞(delayed OFF RGCs)和持续给光-撤光型神经节细胞的OFF反应无显著变化。施加药物鸡尾酒后,在CSNB大鼠视网膜中,光诱导神经节细胞放电反应的平均频率降低而ON-型反应平均频率增加(P0.01),且ON-型反应的潜伏期显著减小(P0.05)；Wt大鼠视网膜中的光诱导神经节细胞放电反应的平均频率降低,但ON-型反应的平均频率和潜伏期无显著变化(P0.05)。结论 1.在视网膜光诱发反应场电位记录中,CSNB大鼠视网膜光诱发场电位仍可记录到a-波,尽管其峰值显著减小,说明CSNB大鼠光感受器细胞至少保存了部分活性和功能；其光诱发场电位波形中b-波缺失表明CSNB大鼠视网膜视觉信号传导在第一级神经元与第二级神经元之间发生严重障碍。 2.在电刺激放电反应记录中,CSNB大鼠视网膜的长潜伏期反应的放电频率较野生型大鼠降低,表明Cacnalf基因突变后来源于视网膜外层的间接突触刺激驱动减少。 3. CSNB大鼠视网膜神经节细胞自发放电的频率升高表明：虽然CSNB大鼠视网膜外层视觉信号传导发生障碍且光感受器细胞功能存在缺陷,但其视网膜内层的自发性电生理活动亢进。 4. CSNB大鼠视网膜混合型放电细胞占所有自发放电神经节细胞的比重升高,且混合型放电细胞的放电总数显著增加,因此可以判定活跃的混合型放电细胞是CSNB大鼠视网膜自发性电生理活动亢进的主要原因。 5.信号/噪声比下降表明CSNB大鼠视网膜分离有效视觉信号与背景噪声的反应能力降低,其神经节细胞处理编码视觉信号的有效度和可信度发生缺陷,且直接影响到视网膜视觉信号传通路的效率。 6. CSNB大鼠视网膜记录数据中长时程互相关活动消缺失,可能是其视网膜L-型钙通道失功能导致化学递质释放障碍导致视网膜外层的多突触环路信息输入减少或消失所造成。隙连接在CSNB大鼠视网膜中神经元协同放电活动中发挥重要作用,且其表达水平或活性功能发生上调。 7. CSNB大鼠视网膜光感受器细胞和ON-型双极细胞发生视觉信号传导障碍后,其下游的视觉信号传导通路确实发生了重塑：光诱导放电反应频率降低主要归因于其ON-型放电反应减弱,OFF通路在内层视网膜视觉信号传导中占据主导地位,而ON通路与神经节细胞之间发生了某种程度的分离和障碍。综上所述,CSNB大鼠视网膜外层神经递质释放障碍与内层视觉信号传导通路重塑之间联系紧密,明确其视觉信号传导通路重塑的机制和时机可为下一步制订视觉保护干预措施、改进完善治疗方案提供依据。
[Abstract]:The congenital stationary night blindness ( CSNB ) is a non - progressive , inherited retinal disease based on visual signal system dysfunction .

The morphological changes in the outer layer of the retina are likely to affect the conduction integration function of the retinal inner layer . However , the morphological changes in the outer layer of the retina can affect the conduction integration function of the retinal inner layer . However , the morphological changes in the outer layer of the retina and the pathological effects on the ganglion cells in the innermost layer of the retina can be influenced by the morphological changes of the outer layer of the retina .

The multi - electrode array system has been widely used in the electrophysiological activity recording of retinal neurons . Because of its integrity and non - invasive advantage , the electrophysiological experiment has been applied to the physiological activities of retinal neurons such as photoreceptor cells and ganglion cells .

Materials and Methods

In the experiment of pharmacology , the electric signals collected by the electrodes of the multi - electrode array system are filtered and amplified by the lens system , and then applied to the perfusion environment in the bio - recording chip . The electric signals collected by the electrodes of the computer stimulator are filtered and amplified by the lens system .
In the electrical stimulation experiment , the electrophysiologic response of the retinal neurons was induced by the double - order equilibrium current stimulation sequence generated by the electrode 28 at the center of the array , and the recordings were collected for the other electrodes in the array and analyzed by the same software .

Results

1 . Retina induced field potential of wild - type SD rats and CSNB rats

The peak of a - wave was 0.196 卤 0.038mv ( n - 20 ) . The peak of a - wave was significantly decreased ( 0.078 卤 0.026mv , n - 20 , P0.01 ) , b - wave was absent .

2 . Electrical stimulation of electroretinogram in rats with wild - type SD rats and CSNB rats

Two types of electrical stimulation responses were found in the original waveform following the electrical stimulation of wild - type rats and CSNB rats following the application of the double - order equilibrium current stimulation : short latency response and long latency response . They were divided into 14 groups according to the distance between ganglion cells and current stimulation electrodes : ( 100 渭m , 14渭m , 200渭m , 22渭m , 36渭m , 38渭m , 400渭m , 424pm , 447 渭m , 500 渭m and 566 渭m ) . The results showed that the electrical stimulation response intensity of ganglion cells decreased with increasing distance from the current stimulating electrodes located in the center of the array ;
In the group , the number of discharges in the long incubation period of the retina of the CSNB rats was reduced compared with that of the wild type rats ( P0.01 ) .

3 . Spontaneous discharge of retinal spontaneous discharges of wild - type SD rats and CSNB rats

It was found that the spontaneous discharge frequency ( 9.93 卤 4.14Hz , n = 386 ) of retinal ganglion cells in CSNB rats was significantly higher than that of wild type rats ( 2.610 . 73Hz , n = 295 , P0.01 ) . From the cumulative spontaneous discharge frequency graph , the spontaneous discharge frequency curve of the retinal ganglion cells of the CSNB rats was observed to be lower and shifted to the right compared with the wild - type rats .

4 . Type of spontaneous discharges of retinal ganglion cells from wild - type SD rats and CSNB rats

There were four types of ganglion cells producing spontaneous discharge reaction : burst type discharge cells , conventional type discharge cells , non - conventional type discharge cells and mixed type discharge cells . The proportion of the retinal burst type discharge cells and mixed type discharge cells in the CSNB rat was higher than that of the wild type rats ( Wt : explosion type 15.2 % , mixed type : 24.7 % , n - 20 ;
Compared with wild type rats ( Wt : 51.4 % , CSNB : 36.8 % , P0.01 ) . The total number of discharge cells ( 246,53 , n - 20 ) in the retinal ganglion cells of CSNB rats was significantly higher than that of the hybrid discharge cells ( 112089 , n = 20 , P0.01 ) . There was no significant difference in the total number of discharges in the retinal ganglion cells of CSNB rats and Wt rat retina ( P0.05 ) .

5 . Photoinduced discharges of retinal light induced by wild - type SD rats and CSNB rats

The retinal ganglion cells from CSNB rats and Wt rat retina can be divided into six categories : 1 ) to light type ( ON - type ) according to the discharge characteristics of the light reaction ;
2 ) Withdrawal type ( OFF - type ) ;
3 ) A - OFF type ( ON - OFF type ) ;
4 ) sustained - to - light type ( sustained ON type ) ;
5 ) sustained on - off type ( sustained ON - OFF type ) ;
6 ) Delayed OFF type . The average frequency ( 16.40 卤 9.97Hz , n - 340 ) of retinal ganglion cells in CSNB rats was significantly lower than that of wild - type rats ( 38.6211 . 78Hz , n = 373 , P0.01 ) . Compared with the retina of Wt rat , the frequency of spontaneous discharge ( background noise ) in the retina of CSNB rats is high and the frequency of light - induced discharge reaction ( visual signal ) is low , so its signal / noise ratio ( S - N ration ) is reduced .

6 . Cross - correlation of spontaneous discharges between wild - type SD rats and CSNB rats

In the retina of wild type rats , three kinds of characteristic ganglia cell cross - correlation activities were observed : short - term cross - correlation activities , medium - duration cross - correlation activities and long - range cross - correlation activities . In the CSNB rat retinal record data only short - term cross - correlation and medium - time cross - correlation were observed , and the long - range cross - correlation activities were eliminated ( n = 20 ) .

7 . Reconstruction of retinal visual signal transduction pathways in CSNB rats

According to the ratio of the frequency of ON - type discharge reaction of individual ganglion cells to the total frequency of light - induced discharge reaction , they were classified into three groups : 1 ) ON reaction - dominant ganglion cells ( ratio 0.5 ) ;
2 ) OFF reaction leading ganglion cells ( ratio 0.5 ) ;
3 ) Compared with wild type rats , the proportion of the dominant ganglion cells in the retina of the CSNB rat was increased ( Wt : 60.6 % ; CSNB : 78.1 % ) , while the proportion of the dominant ganglion cells in the ON reaction was decreased ( Wt : 31.5 % ; CSNB : 18.2 % ) . The latency of the light - induced ON - type and OFF - type responses in both rat retinal ganglion cells was significantly prolonged compared with that of wild - type rats ( P0.05 ) .
The average frequency of light - induced ganglion cell discharge in the retina of Wt rat was decreased , but the average frequency and latency of the ON - type reaction did not change significantly ( P0.05 ) .

Conclusion

1 . In the field potential recording of retinal light - induced reaction , the retinal light - induced field potential of CSNB rats could still be recorded to a - wave , although its peak decreased significantly , indicating that CSNB rat photoreceptor cells had at least partial activity and function ;
The absence of b - wave in the light - induced field potential waveform indicates that the retinal visual signal transduction of CSNB rats is a serious obstacle between the first - level neurons and the second - level neurons .

2 . In the recording of electrical stimulation , the response to long latency of the retina of CSNB rats was lower than that of wild type rats , suggesting that the indirect synaptic stimulation of the outer retina of CSNB gene was decreased after gene mutation .

3 . The frequency of spontaneous discharge of retinal ganglion cells in CSNB rats showed that although the visual signal transduction of the retinal outer layer of CSNB rats was impaired and the function of photoreceptor cells was defective , the spontaneous electrophysiological activity of the retina inner layer was too high .

4 . CSNB rat retinal hybrid discharge cells accounted for a significant increase in the specific gravity of all spontaneous discharge ganglion cells , and the total number of discharge cells in the hybrid discharge cells was significantly increased , so it was possible to determine that the active hybrid discharge cells were the main reason for the spontaneous electrophysiologic activity of the retina of CSNB rats .

5 . The reduction of signal / noise ratio indicates that the response ability of retinal detachment effective visual signal and background noise in CSNB rats is reduced , and the effective degree and credibility of retinal ganglion cell processing coded visual signal are defective , and the efficiency of retinal visual signal transmission path is directly affected .

6 . The deletion of long - range cross - correlation activities in the retinal record data of CSNB rats may be caused by the loss of the retinal L - type calcium channel , which leads to the reduction or disappearance of the multi - synaptic ring information input in the outer layer of the retina .

7 . After the visual signal transduction of the retinal photoreceptor cells and the ON - type bipolar cells in the CSNB rats , the visual signal transduction pathways downstream of the cells were remolded : the decrease in the frequency of the photoinduced discharge was mainly attributed to the decrease of the ON - type discharge reaction , the OFF pathway dominated the visual signal transduction of the inner retina , while the ON pathway and the ganglion cells had some degree of separation and disorder .

In conclusion , the mechanism and timing of visual signal transduction pathway remodeling in the retinal outer layer of CSNB rats are closely related to the reconstruction of the inner layer visual signal transduction pathway , and the mechanism and timing of the reconstruction of the visual signal transduction pathway can be defined as the basis for the next step of developing visual protection interventions and improving the treatment scheme .
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R85

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