本文選題：圖形視覺誘發(fā)電位　切入點(diǎn)：雙眼形覺剝奪　出處：《第三軍醫(yī)大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：人類和哺乳動(dòng)物出生后,視覺系統(tǒng)能根據(jù)周圍的視覺環(huán)境調(diào)整和改變與生俱有的神經(jīng)聯(lián)系和突觸結(jié)構(gòu),這一改變發(fā)生的最敏感時(shí)期,稱為視皮層可塑性關(guān)鍵期。以往的研究認(rèn)為,只有在關(guān)鍵期內(nèi)弱視才能得到有效治療,一旦錯(cuò)過則成年期殘留的弱視不能得到恢復(fù)。但目前的研究與臨床觀察使這一觀點(diǎn)越來越受到質(zhì)疑,許多基礎(chǔ)和臨床研究發(fā)現(xiàn),成年動(dòng)物的視皮層可塑性是被抑制而非完全消失,在一些特定的情況下可被“再激活”。 在臨床觀察中發(fā)現(xiàn),當(dāng)單眼弱視的成年人,其健眼因疾病或外傷失明后,弱視眼的最佳矯正視力會(huì)逐漸提高,甚至可以接近正常,說明人類的成年弱視有治愈的可能。而通過動(dòng)物研究,更是使用多種方法使成年大鼠視皮層恢復(fù)了可塑性。2002年,Pizzorusso等使用chABC酶降解成年期大鼠視皮層內(nèi)硫酸軟骨素蛋白多糖(chondroitin sulphate proteoglycans,CSPGs),首次成功恢復(fù)了成年期大鼠視皮層可塑性,并聯(lián)合健眼反剝奪使成年大鼠弱視眼的視力恢復(fù)正常,首次證實(shí)了成年期大鼠視皮層可塑性可被“再激活”。隨后,其他學(xué)者通過成年大鼠10天黑暗飼養(yǎng)、豐富環(huán)境法、服用5-羥色胺攝取抑制劑和降低皮層內(nèi)抑制功能等方法,均成功恢復(fù)成年期大鼠視皮層可塑性,這些事實(shí)均說明成年期大鼠視皮層可塑性并未完全消失,而是被抑制,通過某些特定的方法,成年期視皮層可塑性可被“再激活”。本實(shí)驗(yàn)室以往的研究發(fā)現(xiàn),雙眼形覺剝奪可以抑制大鼠γ-氨基丁酸(γ-amino butyric acid, GABA)能抑制性突觸傳遞功能發(fā)育,減弱成年大鼠視皮層內(nèi)神經(jīng)元抑制性突觸傳遞的強(qiáng)度,調(diào)節(jié)視皮層抑制性和興奮性神經(jīng)遞質(zhì)受體的重新表達(dá)和分布。然而,雙眼形覺剝奪所致的對(duì)GABA抑制環(huán)路功能的減弱是通過什么途徑進(jìn)行的,尚未進(jìn)行進(jìn)一步研究。以往的研究發(fā)現(xiàn),以CSPGs聚集為主要構(gòu)成的神經(jīng)元周圍網(wǎng)絡(luò)(perineuronal net, PNNs)的成熟可以促進(jìn)GABA抑制性環(huán)路功能的成熟,降解PNNs恢復(fù)成年大鼠視皮層可塑性后,視皮層內(nèi)GABA能神經(jīng)元對(duì)其靶細(xì)胞的抑制性功能降低,說明PNNs的減少是導(dǎo)致GABA環(huán)路抑制功能下降的原因之一。那么雙眼形覺剝奪模型所致的GABA抑制性環(huán)路的功能降低是否是由于PNNs的減少所導(dǎo)致的呢? 組織型纖溶酶原激活劑(tissue-type plasminogen activator, tPA)是一種絲氨酸蛋白水解酶,是CSPGs天然的降解因子。以往的研究發(fā)現(xiàn),在小鼠視皮層可塑性關(guān)鍵期內(nèi),單眼剝奪后,剝奪眼對(duì)側(cè)視皮層內(nèi)tPA的活性增高,說明異常的視覺環(huán)境可以影響tPA的活性。那么雙眼形覺剝奪模型能不能對(duì)視皮層內(nèi)tPA的活性產(chǎn)生影響呢?結(jié)合上述內(nèi)容我們提出假設(shè):成年大鼠雙眼形覺剝奪后,視皮層內(nèi)tPA活性增高,使CSPGs降解增多,減少PNNs的表達(dá),“再激活”成年大鼠視皮層可塑性。 為此,本實(shí)驗(yàn)采用以下方法驗(yàn)證此假設(shè): 1、采用圖形視覺誘發(fā)電位的方法,記錄視覺發(fā)育可塑性關(guān)鍵期結(jié)束前后,視皮層可塑性的變化以及視皮層可塑性結(jié)束的時(shí)間,然后予以行雙眼形覺剝奪,記錄不同剝奪時(shí)間對(duì)視皮層可塑性的影響,確定能穩(wěn)定激活成年大鼠視皮層可塑性的時(shí)間。結(jié)果發(fā)現(xiàn):(1)正常出生后5周以內(nèi)的大鼠,短期3天單眼剝奪即可造成視皮層眼優(yōu)勢(shì)的移動(dòng),說明大鼠視皮層存在可塑性。(2)正常出生后6周大鼠,短期3天單眼剝奪,不能使眼優(yōu)勢(shì)發(fā)生移動(dòng),說明大鼠視皮層可塑性被抑制。(3)予以7周大鼠雙眼形覺剝奪14天,短期3天單眼剝奪可以使視皮層眼優(yōu)勢(shì)發(fā)生移動(dòng),說明雙眼形覺剝奪14天可以穩(wěn)定的再激活成年期大鼠視皮層可塑性。(4)在后面兩部分實(shí)驗(yàn),將采用出生后7周大鼠行雙眼形覺剝奪14天作為雙眼形覺剝奪視皮層可塑性再激活組模型。 2、采用免疫熒光組織化學(xué)、免疫蛋白印跡和酶聯(lián)免疫吸附法,檢測(cè)在視皮層可塑性關(guān)鍵期前后,及行雙眼形覺剝奪14天組大鼠視皮層內(nèi)tPA的表達(dá)及活性變化。結(jié)果發(fā)現(xiàn):(1)大鼠出生后1周,視皮層內(nèi)僅有少量表達(dá)及活性,睜眼后其表達(dá)及活性明顯升高,說明tPA的表達(dá)具有視覺經(jīng)驗(yàn)依賴性。(2)在視皮層可塑性關(guān)鍵期高峰期以內(nèi),視皮層內(nèi)tPA的表達(dá)及活性均較高,但到了關(guān)鍵期末和成年期,其表達(dá)及活性明顯降低,說明其參與了視皮層可塑性,且與視皮層可塑性的終止有關(guān)。(3)雙眼形覺剝奪組大鼠視皮層內(nèi)的tPA表達(dá)和活性與同周齡及關(guān)鍵期結(jié)束前相比均升高,說明雙眼形覺剝奪對(duì)視皮層內(nèi)tPA的表達(dá)和活性均有影響。 3、采用免疫熒光組織化學(xué)雙重標(biāo)記技術(shù),雙重標(biāo)記視皮層可塑性關(guān)鍵期前后及行雙眼形覺剝奪14天組大鼠視皮層內(nèi)tPA和CSPGs的表達(dá)變化。結(jié)果發(fā)現(xiàn):(1)大鼠出生后1周,視皮層內(nèi)未見CSPGs表達(dá),睜眼后開始出現(xiàn),隨年齡增長逐漸增加,說明視皮層內(nèi)CSPGs的表達(dá)受到視覺經(jīng)驗(yàn)和年齡因素影響,與視皮層可塑性的終止有關(guān)。(2)雙眼形覺剝奪組大鼠視皮層內(nèi)CSPGs與同周齡大鼠及關(guān)鍵期結(jié)束前大鼠相比,其表達(dá)明顯降低,說明雙眼形覺剝奪對(duì)視皮層可塑性的影響與CSPGs有關(guān)。(3)大鼠視皮層內(nèi)tPA和CSPGs雙標(biāo)陽性細(xì)胞隨年齡增加逐漸增多,說明隨著年齡增加,tPA對(duì)CSPGs水解降低,使CSPGs表達(dá)增多,參與了視皮層可塑性關(guān)鍵期的終止。(4)雙眼形覺剝奪組大鼠與成年大鼠及關(guān)鍵期結(jié)束前大鼠相比,視皮層內(nèi)tPA和CSPGs雙標(biāo)陽性細(xì)胞明顯降低,說明雙眼形覺剝奪可增加視皮層內(nèi)tPA的表達(dá)和活性,對(duì)CSPGs降解增多,減少皮層內(nèi)PNNs的形成,是“再激活”成年大鼠視皮層可塑性的途徑之一。 本研究得到以下結(jié)論: 1、BFD可成功再激活成年大鼠視皮層可塑性,形覺剝奪和光覺剝奪一樣可以終身增強(qiáng)其眼優(yōu)勢(shì)可塑性; 2、BFD14天可以使成年大鼠視皮層內(nèi)tPA的表達(dá)和活性增加,加強(qiáng)對(duì)CSPGs的降解,減少PNNs的形成,可能是成年大鼠視皮層可塑性被“再激活”的機(jī)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.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R77

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