双眼形觉剥夺成年大鼠视皮层可塑性与CSPGs和tPA表达关系的研究

发布时间：2018-03-20 10:34

本文选题：图形视觉诱发电位　切入点：双眼形觉剥夺　出处：《第三军医大学》2011年硕士论文　论文类型：学位论文

【摘要】：人类和哺乳动物出生后,视觉系统能根据周围的视觉环境调整和改变与生俱有的神经联系和突触结构,这一改变发生的最敏感时期,称为视皮层可塑性关键期。以往的研究认为,只有在关键期内弱视才能得到有效治疗,一旦错过则成年期残留的弱视不能得到恢复。但目前的研究与临床观察使这一观点越来越受到质疑,许多基础和临床研究发现,成年动物的视皮层可塑性是被抑制而非完全消失,在一些特定的情况下可被“再激活”。在临床观察中发现,当单眼弱视的成年人,其健眼因疾病或外伤失明后,弱视眼的最佳矫正视力会逐渐提高,甚至可以接近正常,说明人类的成年弱视有治愈的可能。而通过动物研究,更是使用多种方法使成年大鼠视皮层恢复了可塑性。2002年,Pizzorusso等使用chABC酶降解成年期大鼠视皮层内硫酸软骨素蛋白多糖(chondroitin sulphate proteoglycans,CSPGs),首次成功恢复了成年期大鼠视皮层可塑性,并联合健眼反剥夺使成年大鼠弱视眼的视力恢复正常,首次证实了成年期大鼠视皮层可塑性可被“再激活”。随后,其他学者通过成年大鼠10天黑暗饲养、丰富环境法、服用5-羟色胺摄取抑制剂和降低皮层内抑制功能等方法,均成功恢复成年期大鼠视皮层可塑性,这些事实均说明成年期大鼠视皮层可塑性并未完全消失,而是被抑制,通过某些特定的方法,成年期视皮层可塑性可被“再激活”。本实验室以往的研究发现,双眼形觉剥夺可以抑制大鼠γ-氨基丁酸(γ-amino butyric acid, GABA)能抑制性突触传递功能发育,减弱成年大鼠视皮层内神经元抑制性突触传递的强度,调节视皮层抑制性和兴奋性神经递质受体的重新表达和分布。然而,双眼形觉剥夺所致的对GABA抑制环路功能的减弱是通过什么途径进行的,尚未进行进一步研究。以往的研究发现,以CSPGs聚集为主要构成的神经元周围网络(perineuronal net, PNNs)的成熟可以促进GABA抑制性环路功能的成熟,降解PNNs恢复成年大鼠视皮层可塑性后,视皮层内GABA能神经元对其靶细胞的抑制性功能降低,说明PNNs的减少是导致GABA环路抑制功能下降的原因之一。那么双眼形觉剥夺模型所致的GABA抑制性环路的功能降低是否是由于PNNs的减少所导致的呢? 组织型纤溶酶原激活剂(tissue-type plasminogen activator, tPA)是一种丝氨酸蛋白水解酶,是CSPGs天然的降解因子。以往的研究发现,在小鼠视皮层可塑性关键期内,单眼剥夺后,剥夺眼对侧视皮层内tPA的活性增高,说明异常的视觉环境可以影响tPA的活性。那么双眼形觉剥夺模型能不能对视皮层内tPA的活性产生影响呢?结合上述内容我们提出假设:成年大鼠双眼形觉剥夺后,视皮层内tPA活性增高,使CSPGs降解增多,减少PNNs的表达,“再激活”成年大鼠视皮层可塑性。为此,本实验采用以下方法验证此假设: 1、采用图形视觉诱发电位的方法,记录视觉发育可塑性关键期结束前后,视皮层可塑性的变化以及视皮层可塑性结束的时间,然后予以行双眼形觉剥夺,记录不同剥夺时间对视皮层可塑性的影响,确定能稳定激活成年大鼠视皮层可塑性的时间。结果发现:(1)正常出生后5周以内的大鼠,短期3天单眼剥夺即可造成视皮层眼优势的移动,说明大鼠视皮层存在可塑性。(2)正常出生后6周大鼠,短期3天单眼剥夺,不能使眼优势发生移动,说明大鼠视皮层可塑性被抑制。(3)予以7周大鼠双眼形觉剥夺14天,短期3天单眼剥夺可以使视皮层眼优势发生移动,说明双眼形觉剥夺14天可以稳定的再激活成年期大鼠视皮层可塑性。(4)在后面两部分实验,将采用出生后7周大鼠行双眼形觉剥夺14天作为双眼形觉剥夺视皮层可塑性再激活组模型。 2、采用免疫荧光组织化学、免疫蛋白印迹和酶联免疫吸附法,检测在视皮层可塑性关键期前后,及行双眼形觉剥夺14天组大鼠视皮层内tPA的表达及活性变化。结果发现:(1)大鼠出生后1周,视皮层内仅有少量表达及活性,睁眼后其表达及活性明显升高,说明tPA的表达具有视觉经验依赖性。(2)在视皮层可塑性关键期高峰期以内,视皮层内tPA的表达及活性均较高,但到了关键期末和成年期,其表达及活性明显降低,说明其参与了视皮层可塑性,且与视皮层可塑性的终止有关。(3)双眼形觉剥夺组大鼠视皮层内的tPA表达和活性与同周龄及关键期结束前相比均升高,说明双眼形觉剥夺对视皮层内tPA的表达和活性均有影响。 3、采用免疫荧光组织化学双重标记技术,双重标记视皮层可塑性关键期前后及行双眼形觉剥夺14天组大鼠视皮层内tPA和CSPGs的表达变化。结果发现:(1)大鼠出生后1周,视皮层内未见CSPGs表达,睁眼后开始出现,随年龄增长逐渐增加,说明视皮层内CSPGs的表达受到视觉经验和年龄因素影响,与视皮层可塑性的终止有关。(2)双眼形觉剥夺组大鼠视皮层内CSPGs与同周龄大鼠及关键期结束前大鼠相比,其表达明显降低,说明双眼形觉剥夺对视皮层可塑性的影响与CSPGs有关。(3)大鼠视皮层内tPA和CSPGs双标阳性细胞随年龄增加逐渐增多,说明随着年龄增加,tPA对CSPGs水解降低,使CSPGs表达增多,参与了视皮层可塑性关键期的终止。(4)双眼形觉剥夺组大鼠与成年大鼠及关键期结束前大鼠相比,视皮层内tPA和CSPGs双标阳性细胞明显降低,说明双眼形觉剥夺可增加视皮层内tPA的表达和活性,对CSPGs降解增多,减少皮层内PNNs的形成,是“再激活”成年大鼠视皮层可塑性的途径之一。本研究得到以下结论: 1、BFD可成功再激活成年大鼠视皮层可塑性,形觉剥夺和光觉剥夺一样可以终身增强其眼优势可塑性; 2、BFD14天可以使成年大鼠视皮层内tPA的表达和活性增加,加强对CSPGs的降解,减少PNNs的形成,可能是成年大鼠视皮层可塑性被“再激活”的机制之一。
[Abstract]:Humans and mammals after birth, visual system based on neural connections and synaptic structure of the visual environment to adjust and change the most sensitive period of this change, known as the critical period of visual cortex plasticity. Previous research suggested that only in the crucial period of amblyopia can get effective treatment, once missed the adult amblyopia can not be restored. But the residual period of study and clinical observation of the present so that this view is increasingly being questioned, many basic and clinical studies have found that the visual cortex plasticity in the adult animal was inhibited but not completely disappeared, in some particular situations can be "reactivated".
Found in the clinical observation, when the adult amblyopia, ocular trauma due to disease or blindness, amblyopia best corrected visual acuity will gradually improve, even close to normal, indicating that human adult amblyopia may be cured. And through animal studies, is to use a variety of methods to restore the visual cortex of adult rats the plasticity of.2002, Pizzorusso and chABC using the enzymatic degradation of adult rat visual cortex of chondroitin sulfate proteoglycans (chondroitin sulphate, proteoglycans, CSPGs), the first successful restoration of visual cortex plasticity in adult rats, and combined ocular anti to adult rats deprived of visual acuity of the amblyopic eye returned to normal, confirmed for the first time the adult rat visual cortex plasticity can be "reactivated". Subsequently, other scholars through 10 days of dark reared adult rats, rich in environmental law, taking the 5- serotonin uptake inhibition agent and lower cortex In the suppression methods are successful recovery of adult rat visual cortex plasticity, these facts are that the visual cortex plasticity in adult rats has not completely disappeared, but was inhibited by certain methods. Adult visual cortex plasticity can be "reactivated". Previous studies have found that the laboratory. Binocular form deprivation can inhibit the rat GABA (gamma -amino butyric acid, GABA) can inhibit synaptic transmission function of neurons in visual cortex development, weaken the intensity of inhibitory synaptic transmission in adult rats, regulate the expression and distribution of cortical inhibitory and excitatory neurotransmitter receptors. However, binocular form deprivation induced suppression loop function of GABA is weakened by what way, has not been further studied. Previous studies have found that the accumulation of CSPGs as the main form of neuron network (around perine Uronal net, PNNs) of the mature GABA inhibitory loop can promote functional maturation and degradation of PNNs recovery of visual cortex plasticity in adult rats, GABA can function in the visual cortex of inhibitory neurons to its target cells decreased, indicating that PNNs decrease of GABA loop suppression one reason drop. Then under the function of binocular form deprivation induced inhibition of GABA loop function decrease is due to the decrease of PNNs caused?
Tissue type plasminogen activator (tissue-type plasminogen, activator, tPA) is a serine protease, is the natural degradation factor of CSPGs. Previous studies have found that in mouse visual cortex plasticity during the critical period after monocular deprivation in the contralateral cortex deprived eye activity of tPA increased, indicating abnormal visual environment can influence the activity of tPA. Then the binocular form deprivation can affect the activity of tPA in the visual cortex is not? According to the content above we hypothesized that binocular form deprivation in adult rats, tPA activity increases in visual cortex, which increased the degradation of CSPGs, reduce the expression of PNNs, "activate" the visual cortex of adult rats plasticity.
To this end, this experiment uses the following methods to verify the hypothesis:
1, using the method of pattern visual evoked potentials, were recorded before and after the end of the critical period of visual plasticity, as the changes in cortical plasticity and visual cortex plasticity over time, and then treated by binocular deprivation, recording different deprivation in visual cortex plasticity influence, determine the stable activation of visual cortex plasticity in adult the time of rat. The results showed that: (1) normal rats within 5 weeks after birth, 3 days short of monocular deprivation can cause movement of the visual cortex ocular dominance, indicating existence of visual cortex plasticity in rats. (2) normal rats 6 weeks after birth, 3 days short of monocular deprivation, can make eye the advantages of mobile description of rat visual cortex plasticity was inhibited. (3) for 7 weeks in rats of binocular deprivation for 14 days, 3 days short of monocular deprivation can make the visual cortex ocular dominance shift, 14 days that binocular form deprivation can stabilize the reactivation of adulthood The plasticity of visual cortex in rats. (4) in the latter two parts, 7 days after birth, the rats underwent binocular form deprivation for 14 days.
2, using immunohistochemistry, Western blot and enzyme-linked immunosorbent assay testing before and after the critical period of visual cortex plasticity, and binocular form sense changes of expression and activity of 14 days of Deprivation Rats in the visual cortex of tPA. Results showed that: (1) 1 weeks of postnatal rats, treated skin in only a small amount of expression and activity, its expression and activity increased significantly after opening, that depended on the visual experience. The expression of tPA (2) in the critical period of visual cortex plasticity peak within the visual cortex in the expression and activity of tPA were higher, but the key to the final and adult stage, its expression and activity the decreased in visual cortex plasticity, and termination of visual cortex plasticity. (3 eyes) the perception of visual expression and activity of tPA in cortex compared with the same week age and the end of the critical period of increase of deprivation rats, indicating binocular deprivation in visual cortex in tPA The expression and activity are affected.
3, using immunofluorescence double labeling technique, double labeling of critical period of visual cortex plasticity and the perception of eyes before and after 14 days the expression change of Deprivation Rats in the visual cortex of tPA and CSPGs. The results showed that: (1) 1 weeks after birth, there was no CSPGs expression in the visual cortex, began to open eyes after gradually increased with age, indicating that expression of CSPGs in the visual cortex is affected by visual experience and age, and termination of visual cortex plasticity. (2) binocular deprivated rats in the visual cortex of CSPGs with the same week old rats and rats compared to before the end of the critical period, and its expression significantly reduced, that binocular deprivated visual cortex plasticity related to CSPGs. (3) in the visual cortex of tPA and CSPGs positive cells increased gradually with the increase of age rats shows that with the increase of age, tPA on the hydrolysis of CSPGs decreased, the expression of CSPGs increased in visual The termination of the critical period of cortical plasticity. (4) binocular deprivated rats and adult rats and rats compared to before the end of the critical period, tPA and CSPGs positive cells in the cortex decreased significantly, indicating binocular form deprivation can increase the expression and activity of tPA in the visual cortex, the CSPGs degradation increased and reduce the formation of cortex PNNs, is one of the ways to activate the visual cortex plasticity in adult rats.
This study obtains the following conclusions:
1, BFD can reactivate the plasticity of the visual cortex of adult rats, and form deprivation and light deprivation can enhance the plasticity of ocular dominance for a lifetime.
2, BFD14 days can increase the expression and activity of tPA in the visual cortex of adult rats, enhance the degradation of CSPGs and reduce the formation of PNNs, which may be one of the mechanisms of reactivation of plasticity in adult rat visual cortex.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R77

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