【作者】 李诺；

【导师】 黄丽娜；

【作者基本信息】 湖北中医药大学 ， 中医五官科学， 2014， 博士

【摘要】 研究背景:青光眼是一组威胁和损害视神经视觉功能，以视神经萎缩和视野缺损为共同特征的一种疾病，是主要致盲眼病之一，其病情非常凶险，危害性极大，发病很迅速，世界卫生组织已将其列为第二位致盲眼病[1]，通过激光、药物、手术，降低眼压是目前治疗青光眼的主要方法之一。目标是尽可能减少眼压增高和眼压波动导致的视功能的损害，然而单纯降低眼压并不能阻止青光眼导致的进行的视神经损害，视网膜神经节细胞（retinal ganglion cells,RGCs)和视神经的损伤也很可能由其他病理机制导致。在祖国传统医学中，青光眼属于“五风内障”范围，晚期亦可属于“青盲”、“视瞻昏渺”等范围。其基本病机是由于风火上攻头目，或气郁生火、脾湿生痰、痰火上攻造成肝郁化火、肝胆火炽、痰火上扰、阴偏盛或阳偏盛、气机瘀滞等诸种原因，导致气血失和、经脉失利，目中玄府闭阻，血瘀气滞，瘀滞神水、目系失养发为此病。病位主要在肝，涉及肾、脾。中医药治疗青光眼主要以清火、解郁、活血、利水、祛风、开窍、理气、通络，后期以滋补肝肾、疏肝解郁等法为主[2]。与现代医学认为的由于机械性损伤、缺血性损伤导致的氧化损伤等因素致使视网膜神经节细胞不断死亡及视神经轴突不断减少基本一致[3]。全球青光眼药物治疗的最新研究热点—视神经保护，即通过药物或其他方法使那些还没有受损的或者仅轻度受损的，甚至正处于毒性内环境濒临死亡的RGCs得到存活或继续延长生存时间；然而在临床上，现有的视神经保护药物的治疗效果却非常不理想，究其原因可能是药物在视网膜、视神经局部有效浓度低吸收差，导致疗效不佳。目前视神经保护药物主要通过口服、静脉注射和肌肉注射给药，均是通过全身循环后才能到达眼部组织，不但无法达到有效的治疗浓度，也增加了药物所带来的全身毒副作用。因此寻求一种让神经保护药物更为有效、局部应用于RGCs和视神经的新型给药方式，成为了临床治疗的急迫需求。直接应用于RGCs和视神经的新型给药方式将会被临床所接受。目的当今青光眼治疗还未攻克的核心难点是视网膜神经节细胞的凋亡导致视神经的损害。目前临床治疗的给药方式存在药效不显著及全身毒副作用大的缺点。随着生物技术应用的迅速发展，临床上靶向释放药物的新技术—超声微泡，即通过药物体内定位释放技术，不仅能显著降低药物用量，减轻药物的毒副作用，同时能提高病灶区域内的药物浓度，延缓药物释放、减少给药次数，用药准确从而增强治疗效果。研究已证实白蒺藜皂苷（gross saponins from Tribulus terrestris L，GSTT）与注射用鼠神经生长因子（Mouse Nerve Growth Factor for Injection,mNGF）能明显减少视网膜神经节细胞凋亡增强其存活，对治疗视神经损害起到一定的作用。本课题拟首次开发一种新型超声微泡介导的眼科局部给药系统，同时研究人参皂苷Rg1（Ginsenoside Rg1）对视网膜神经节细胞的影响。本研究通过体外细胞实验，及体内动物实验，应用安全及适宜的超声声强、辐照时间、微泡浓度，研究超声破坏微泡介导药物对视网膜神经节细胞的抗凋亡机制。该给药系统和三种药物单剂的应用，能有效提高视网膜局部的药物浓度，用量准确，减少全身毒副作用，延缓或阻止青光眼视神经损害的发生及发展，给青光眼患者带来更有效的治疗，降低其致盲率，延缓病情恶化，为患者复明带来新的希望。方法1.新生Sprague-Dawley大鼠视网膜神经节细胞（RGCs）体外原代培养与纯度鉴定。2.研究N-甲基-D-天冬氨酸（NMDA）对大鼠原代视网膜神经节细胞（RGCs）细胞凋亡的影响。通过四唑盐（MTT）比色法检测定细胞存活率：①测定不同浓度的NMDA（1000μM、100μM、10μM）对RGCs细胞存活率的影响，选择NMDA造模浓度。②按照步骤①选定的NMDA浓度，通过四唑盐（MTT）比色法测定细胞在30min、24h、48h三个不同时间点的存活率。3.白蒺藜皂苷、注射用鼠神经生长因子、人参皂苷Rg1三种药物对NMDA诱导的大鼠原代视网膜神经节细胞损伤的保护作用。筛选白蒺藜皂苷（10-5μg/ml、10-4μg/ml、10-3μg/ml、10-2μg/ml）、注射用鼠神经生长因子（6×10-2μg/ml、8×10-2μg/ml、10×10-2μg/ml、12×10-2μg/ml）、人参皂苷Rg1（100μg/ml、101μg/ml、102μg/ml、103μg/ml）三种药物对抗RGCs凋亡的最佳有效药物浓度。计算细胞存活率的同时确定这三种药物对RGCs保护的最佳质量浓度（实验中细胞存活率相对最高者所对应的三种药物质量浓度)。4.正常兔眼玻璃体腔注射人参皂苷Rg1的视网膜、视神经安全性研究。健康新西兰大白兔12只，随机分为4组，每组3只（6眼）。实验组分组如下：A组正常对照组玻璃体腔内注射0.9%生理盐水0.1ml；B组玻璃体腔注射低剂量（0.05mg/kg）人参皂苷Rg1，C组玻璃体腔注射中剂量（0.5mg/kg）人参皂苷Rg1，D组玻璃体腔注射高剂量（2.5mg/kg）人参皂苷Rg1。于注药后1周、2周、4周分别行行为学观察，Accupen手持式眼压计每日测量眼压，用裂隙灯显微镜,直接检眼镜,眼部B超、眼前段照相、眼底照相、OCT活体观察兔眼眼前节和眼底变化情况，4周后空气栓塞处死新西兰大白兔，取视网膜、视神经行光学显微镜和透射电镜观察视网膜、视神经组织形态和超微结构。玻璃体腔注药每隔3天用药一次，共28天,第28d处死兔子取材。5.兔眼玻璃体腔注射人参皂苷Rg1后观察经超声辐照微泡干预和未经超声辐照微泡干预，同一时间点不同眼组织内药物浓度的比较及同一眼组织中不同时间点的药物浓度比较。健康新西兰大白兔48只，实验组分组如下：左眼玻璃体腔注射(2.5mg/kg)人参皂苷Rg10.1ml，右眼玻璃体腔注射(2.5mg/kg)人参皂苷Rg10.1ml后注入(45μg/ml)声诺维0.1ml，注射完毕后闭合兔眼涂耦合剂，置超声探头于眼球上方立即用频率1MHZ，声强为0.5W/cm2照射眼球60s，每个时间点6只新西兰大白兔，于注药后30min、1h、2h、3h、4h、6h、12h、24h采用空气栓塞处死兔,分别取房水、玻璃体、视网膜脉络膜、视神经组织进行高效液相色谱法的检测。6.超声靶向击破微泡介导视神经保护药物治疗青光眼性视神经损害的实验研究。采用前房注射0.3%复方卡波姆溶液建立兔慢性高眼压视神经损害动物模型。健康新西兰大白兔24只，随机分为8组：1空白对照组（玻璃体腔内注射0.9%生理盐水0.1ml）、2高眼压模型组（不做处理）、3高眼压模型组+玻璃体腔注射人参皂苷Rg1组（玻璃体腔内注射0.1ml人参皂苷Rg1溶液）、4高眼压模型组+玻璃体腔注射mNGF组（玻璃体腔内注射0.1mlmNGF溶液）、5高眼压模型组+玻璃体腔注射人参皂苷Rg1+超声组（玻璃体腔内注射0.1ml人参皂苷Rg1溶液后给予声强为0.5W/cm2的辐照60s）、6高眼压模型组+玻璃体腔注射mNGF+超声组（玻璃体腔内注射0.1mlmNGF溶液后给予声强为0.5W/cm2的辐照60s）、7高眼压模型组+玻璃体腔注射GSTT+超声+微泡组（玻璃体腔内分别注射0.1ml人参皂苷Rg1溶液和0.1ml微泡混悬液，然后给予声强为0.5W/cm2的辐照60s）、8高眼压模型组+玻璃体腔注射mNGF+超声+微泡组（玻璃体腔内分别注射0.1mlmNGF溶液和0.1ml微泡混悬液，然后给予声强为0.5W/cm2的辐照60s）。各组处理每隔3天一次，共28天,于注药后1周、2周、4周分别行行为学观察，Accupen手持式眼压计测量眼压，用裂隙灯显微镜,眼部B超、眼前段照相，活体观察兔眼眼前节变化情况，4周后空气栓塞处死兔，取兔视网膜和视神经，行兔视网膜病理切片、光镜测量视网膜厚度、视网膜和视神经透射电镜检测、检测视网膜内NO、MDA、SOD的含量。结果1.成功建立了简便易行的新生SD大鼠视网膜神经节细胞（RGCs）体外原代培养方法。获得了较为纯化的RGCs，免疫细胞化学法显示RGCs的纯度为90%以上，该方法培养的RGCs体外存活时间可达5d。2. NMDA可诱导大鼠视网膜神经节细胞凋亡。NMDA诱导RGCs损伤后，细胞发生凋亡还是死亡，主要取决于NMDA的浓度，本实验在体外培养RGCs中加入NMDA(100μM)30min，体外模拟视神经损伤后的微环境，四唑盐（MTT）比色试验检测凋亡细胞数，证实100μM的NMDA能成功诱导RGCs发生凋亡。NMDA对于体外培养的大鼠RGCs的兴奋性毒性作用为剂量依赖性，加入10μM、100μM、1000μM NMDA30min后，RGCs的存活率分别为91.46%、64.74％、50.02%。浓度达到100μM及以上时与正常对照组RGCs的存活率比较有明显差异(P<0.05)。加入100μM NMDA分别作用30min、24h、48h的存活率分别为63.3%、55.19%、46.98%，随着时间的延长，RGCs的存活率显著下降；伴随剂量加大，时间延长，RGCs的存活率也随之下降。因此，选择100μM作用30min进行后续实验。3.人参皂苷Rg1可抑制NMDA诱导的视网膜神经节细胞凋亡，促进体外培养的RGCs的存活，保护视网膜神经元。四唑盐（MTT）比色试验显示，经NMDA处理30min的RGCs细胞存活率均有下降，加药组细胞存活率均较NMDA损伤组提高，其中10-4μg/ml GSTT+NMDA组，8×10-2μg/ml mNGF+NMDA组,102μg/ml人参皂苷Rg1+NMDA组，相对于其它剂量组均能提高细胞存活率。三种药物均能促进体外视网膜神经节细胞的存活，具有显著的视神经保护作用。4.正常对照组和不同药物剂量实验组兔眼前后节未见明显异常，玻璃体腔内未见明显炎性混浊，视网膜未见脱离等并发症。正常对照组和实验组光学显微镜观察视网膜组织形态和透射电镜观察视网膜、视神经超微结构均未见明显异常改变。通过行为学、眼前节、眼底、眼部B超及组织学结果均证实，玻璃体腔注射不同浓度的（0.05mg/kg、0.5mg/kg和2.5mg/kg）人参皂苷Rg1，对兔眼视网膜、视神经无明显毒副作用，是安全的。5.单纯玻璃体腔注药组和玻璃体腔注药后经超声辐照微泡干预组，两种不同方法兔眼玻璃体腔注射人参皂苷Rg1后24h内,除各组玻璃体中人参皂苷Rg1含量呈梯度下降,峰值时间为15min,其余眼部组织(房水、视网膜脉络膜、视神经)人参皂苷Rg1含量呈正态曲线变化。其达峰值时间为房水及眼内壁组织视网膜脉络膜先达峰值,均为3h,而眼外壁组织达峰值时间较晚,视神经为4h。将单纯玻璃体腔注药组和玻璃体腔注药后经超声辐照微泡干预组兔眼各组织人参皂苷Rg1达峰值含量进行比较,经超声辐照微泡干预组各组织人参皂苷Rg1峰值含量均明显高于单纯玻璃体腔注药组,具有统计学意义(P<0.05或P<0.01)，说明玻璃体腔注射药物经超声辐照微泡干预后较单纯玻璃体腔注药能够在眼内各组织达到高的药物浓度。将各组各球壁组织人参皂苷Rg1峰值含量进行比较，发现玻璃体腔注射人参皂苷Rg1眼部各组织含量分布为玻璃体＞视网膜脉络膜＞视神经＞房水。除外玻璃体内因直接注射人参皂苷Rg1导致高药物浓度，视网膜、视神经为最能获得理想药物含量的组织，因此玻璃体腔注射人参皂苷Rg1是保证眼后节疾病治疗，特别是视网膜神经保护作用药物浓度的有效方法。超声辐照微泡在此基础上进一步提高了药物利用度。6.兔眼前房注射0.3%复方卡波姆溶液是一种较好的慢性高眼压模型制作方法，成功建立了青光眼性视神经视网膜损害模型。兔视网膜光镜形态学观察：1组兔视网膜神经节细胞呈单层排列，细胞大小不一，轮廓不规则，核深染，细胞边界清晰，细胞排列整齐紧密，分层清楚；2组模型组兔视网膜神经节细胞数目明显减少，各层结构疏松，排列紊乱，层间分界不清晰，视网膜神经节细胞层呈大量空泡样改变，部分细胞严重变性，出现核溶解、核染色浅淡、胞浆染色浅淡，核结构稀疏，核仁固缩及偏位，胞质染色质边集，内质网肿胀，线粒体肿胀，嵴消失，空泡变性。3、4、5、6组兔视网膜神经节细胞数目相对减少，兔视网膜各层结构相对完整，排列轻度紊乱，分层相对清晰，可见散在空泡样变的细胞；7、8组视网膜各层结构较完整，排列较整齐，形态大致正常，内外核层出现少量细胞丢失，局部有轻度空泡样改变。光镜测量兔视网膜厚度，各组兔视网膜厚度的差异均具有统计学意义（P＜0.05）；2组兔视网膜厚度明显薄于1组，差异有统计学意义（P＜0.05）；3、4、5、6组兔视网膜厚度明显厚于2组，差异有统计学意义（P＜0.05）；7、8组兔视网膜厚度明显明显厚于3、4、5、6组，差异有统计学意义（P＜0.05）。兔视网膜超微结构观察：1组正常视网膜神经节细胞核呈类圆形，RGC细胞核膜清晰，染色均匀，核仁明显，内、外核层核染色质颜色清晰，分布均匀，内质网、线粒体等细胞器形态正常，细胞膜完整，感光细胞排列整齐。2组模型组感光细胞线粒体不同程度肿胀、空泡样变；内、外核层结构紊乱；RGC数目减少，肿胀色淡。3、4、5、6组RGC胞核内充满均匀、色浅淡的常染色质，感光细胞排列稍有紊乱，线粒体未见明显肿胀及空泡样变，内核层少数细胞染色质边集，部分核稍有变形，核染色质略显稀疏，颜色稍淡，线粒体轻度空泡变性，可见线粒体嵴，内质网仅见轻度肿胀。7、8组视网膜神经元未见明显坏死及凋亡，RGC染色质分布均匀，核仁明显，粗面内质网基本正常，线粒体改变不明显，光感受器的外节盘膜排列整齐，未见明显溶解。兔视神经超微结构观察：1组正常兔视神经纤维粗细不等,髓鞘完整，排列紧密、规则,呈“发丝”状，轴突外表光滑,包绕轴突的髓鞘板层致密,视神经髓鞘完整,轴浆均匀,轴浆内可见清晰的正常微管、微丝和椭圆形的线粒体；2组模型组神经纤维普遍发生病变,可见轴索消失,髓鞘排列紊乱,节段性松解、溶解、萎缩成团或脱髓变性,胶质增生明显，微管和神经微丝结构消失，线粒体肿胀、空泡变性；3、4、5、6组视神经髓鞘相对不规整，部分髓鞘变薄、疏松呈“洋葱皮”状，轴突内微管和微丝肿胀，但不消失，部分线粒体空泡变性；7、8组视神经髓鞘排列相对致密，髓鞘完整、规则,无脱髓鞘现象，可见微管、微丝，轴突结构接近正常。兔视网膜组织内NO、MDA、SOD含量的检测：2组兔视网膜NO、MDA的含量较1组明显升高，SOD活性较1组明显降低，比较有显著统计学差异(P＜0.05)。3、4、5、6、7、8组NO、MDA的含量较2组明显降低，SOD活性较2组明显升高，比较有显著统计学差异(P＜0.05)。结论SD大鼠RGCs体外培养成功，且RGCs较纯化，是一种较理想的细胞培养实验模型。NMDA对RGCs具有兴奋性毒性作用，可诱导RGCs凋亡；白蒺藜皂苷、注射用鼠神经生长因子及人参皂苷Rg1具有对抗NMDA诱导的RGCs凋亡，保护视网膜神经节细胞，增强其活性的作用；玻璃体腔注射2.5mg/kg的人参皂苷Rg1，对兔眼视网膜、视神经无明显毒副作用，是安全的。兔前房注射复方卡波姆溶液是一种较理想的兔高眼压造模方法。复方卡波姆诱发的兔青光眼模型具有引起眼压中度、稳定升高，持续时间长,方法简单,易于操作和控制等优点，可用于对青光眼性视神经视网膜损害的研究及对抗青光眼药物的研究,是一种较理想的兔青光眼模型。超声靶向破坏微泡能靶向释放药物，增强药物到达眼部的浓度，超声击破微泡介导视神经保护药物人参皂苷Rg1和mNGF治疗高眼压兔视网膜、视神经损害是有效的。超声微泡介导视神经保护药物通过改变视网膜内NO、MDA、SOD的表达量对高眼压兔视神经损害发挥保护作用。更多还原

【Abstract】 Background Glaucoma is a group of threat and damage to the opticnerve visual function and optic nerve atrophy and visual fielddefect is a common feature of the disease, is one of the mainblinding eye disease, its dangerous, dangerous, quick, the worldhealth organization has put it as a second blinding eye disease[1],the treatment of glaucoma, including medicines, laser and operation,its main goal is to reduce the intraocular pressure, reduce theincreased intraocular pressure and the intraocular pressurefluctuation caused by visual impairment, but simply loweringintraocular pressure is not enough to prevent glaucoma optic nervecaused by progressive damage and other pathological mechanism arealso likely to lead to retinal ganglion cells (retinal ganglioncells, RGCs) and optic nerve injury. Glaucoma in traditionalChinese medicine belongs to the "five winds cataract" range, thelate may also belong to the "green blind","visual deep faint toescape", etc. Attack on its basic pathogenesis is due to wind fireleader, or qi depression, spleen wet fire ShengTan caused liverdepression, phlegm, fire attack, courage lively, phlegm YuHua fire fire, Yin and Yang sheng, the disorder of qi activity, etc. Variouskinds of reasons, led to estrangement of qi and blood, meridians,xuan fu in the eyes closed, and qi stagnation and blood stasis, thegod of water stasis, alongwith the disease. Disease mainly in theliver, spleen, kidney. Treating glaucoma mainly by lowering, XieYu,invigorate the circulation of traditional Chinese medicine, water,wind, begin to understand, and qi, t2dm, late to nourishing liverand kidney, and liver XieYu method such as[2].With modern medicinethinks because of mechanical damage, ischemic injury causeoxidative damage of retinal ganglion cells death and optic nerveaxons declining almost the same[3]. With drugs or other means so thatthose who has not been damaged, only partly damaged, even is in atoxic environment near death in RGCs are alive and continue toextend the survival time, the new research focus in the worldglaucoma medication-optic nerve protection; However the existingoptic nerve protection drugs in clinical treatment effect is notideal, the reason may be the local effective drug in the retina,optic nerve concentration is low, leading to poor curative effect.Current drug dosing mode optic nerve protection, including oral,intravenous injection and muscle injection, etc., are all throughthe systemic circulation before they can reach the eye tissues, notonly unable to therapeutic effective concentrations are achieved,also increase the systemic side effects from drugs. Thereforesought to make a neuroprotective drugs more effectively, localapplication in the new dosing method of RGCs and optic nerve, becomethe urgent needs of clinical therapy.Objective Retinal ganglion cell apoptosis lead to optic nervedamage, is the core of today’s glaucoma treatment has yet to conquer the difficulties. Current dosing way of clinical treatment effectwas not significant and systemic side effects of big faults.Ultrasound microbubble is a new technology, targeted release drugsby drug in the body positioning release technology, not only cansignificantly reduce drug dosage, reduce drug adverse always use,at the same time can increase the drug concentration in lesion areaand delay the drug release, reduce dosing frequency, to enhance theefficacy of treatment. Research has confirmed that the whitethistle saponins (gross saponins from Tribulus terrestris L, GSTT)with injectable Mouse Nerve Growth Factor (Mouse Nerve GrowthFactor for Injection, mNGF) can significantly reduce the apoptosisof retinal ganglion cells enhance their survival, have the effectof treatment of optic Nerve damage. This topic proposed for thefirst time to develop a new type of ultrasound mediated microbubbleeye local drug delivery system, and studies the ginseng saponinsRg1(Ginsenoside Rg1) effects on retinal ganglion cells. This studythrough experiments in vitro safety and is suitable for ultra soundintensity, irradiation time, concentration of microvesicles andultrasound microbubble mediated drug antiapoptotic mechanisms ofretinal ganglion cells. The drug delivery system and three kindsof single dose of drug application, can effectively improve theretinal local drug concentration, reduce the systemic side effects,delay or prevent the occurrence/development of glaucoma optic nervedamage, brings more effective treatments for glaucoma patients toreduce the blindness rate, comes and brings new hope for patients.Methods:1.Newborn SD rat retinal ganglion cells in vitro primaryculture and identification.2.The N-methyl-D-aspartate (NMDA) on the original generation of retinal ganglion cell apoptosis of RGCs cell lines. Bytetrazolium salt colorimetry to detect cell survival rate：①Determination of different concentrations of NMDA (1000μM、100μM、10μM) effects on RGCs cell survival rate，select NMDA buildingconcentration.②According to the step①the selected NMDAconcentration by tetrazolium salt colorimetry to detect cell in30min,24h,48h survival at three different time points.3.Screening of GSTT (10-5μg/ml、10-4μg/ml、10-3μg/ml、10-2μg/ml),injectable mNGF (6×10-2μg/ml、8×10-2μg/ml、10×10-2μg/ml、12×10-2μg/ml), Ginsenoside Rg1(100μg/ml、101μg/ml、102μg/ml、103μg/ml) of three drugs against RGCs apoptosis in the besteffective concentration.Calculation of cell survival rate at thesame time to determine these three drugs to protect RGCs optimalmass concentration (experiment cell survival rate relatively thehighest of the three kinds of drug concentration).④Determinedby MTT colorimetric determination of different concentrations ofmicrovesicles (22.5μg/ml、45μg/ml、90μg/ml), different volumeratio of microvesicles solution (1%,5%,10%) of RGCs cell survivalrate, to observe different concentration and volume ofmicrovesicles on RGCs cell growth activity.4. Normal rabbit eye glass body cavity injection Rg1ginsenosidesin the retina, optic nerve safety studies.12only healthy NewZealand white rabbit, randomly divided into4groups, each group3(6eyes). In experimental group is as follows: A group of normalcontrol group0.1ml glass cavity of0.9%saline injection; GroupB glass body cavity injection of low dose (0.05mg/kg) Rg1ginsenosides, group C glass body cavity injection dose (0.5mg/kg)in Rg1ginsenosides, group D glass body cavity injection of high dose (2.5mg/kg) Rg1ginsenosides. Within1week,2weeks after theinjection medicine,4weeks respectively behavior observation,Accupen handheld tonometer daily measuring intraocular pressure,with slit lamp microscope and direct ophthalmoscope, eye Bultrasonic, anterior segment photography, fundus photography, OCTvivo observation section and fundus changes in rabbit eyes eyes,air embolism executed after4weeks of New Zealand white rabbit,retina, optic nerve line of optical microscope and transmissionelectron microscope morphology and ultrastructure of the retina,optic nerve. Glass body cavity injection medicine treat once every3days, a total of28days,28d executed rabbits out.5. Glass body cavity injection Rg1ginsenosides in rabbit eyes afterobserved by ultrasound irradiation micro bubble intervention andwithout ultrasonic irradiation micro bubble intervention, at thesame time point comparison of different eye tissues drugconcentration and different time points in the same eye tissueconcentration of drug.48only healthy New Zealand white rabbit,the experimental group are as follows: left eye glass body cavityinjection (2.5mg/kg) ginseng saponin Rg10.1ml, right eye glassbody cavity injection (2.5mg/kg) ginseng saponin Rg10.1ml afterinjection (45mu g/ml) sound novy0.1ml, closed after the injectionof rabbit eyes with coupling agent, the ultrasonic probe in the eyewith a frequency of1MHZ, immediately above the sound intensityis0.5W/cm2illuminate eye60s, each time point6New Zealand whiterabbit, at30min after drug infusion,1h,2h,3h,4h,6h,12h,24h by air embolism executed rabbit, respectively in aqueoushumor and vitreous body, retina detached, optic nerve tissue forhigh-performance liquid chromatography (HPLC) testing. 6. Targeted ultrasound break the cell mediated optic protectionexperimental study drug in treatment of glaucoma optic nerve damage.Anterior chamber injection of0.3%compound carbomer solution isadopted to establish the rabbit animal model of chronic highintraocular pressure optic nerve damage. Healthy New Zealand whiterabbit24, were randomly divided into8groups: blank control group1(0.1ml glass cavity of0.9%saline injection),2high intraocularpressure model group (do not do processing),3high intraocularpressure model group+glass body cavity injection of ginsengsaponin Rg1groups (0.1ml glass cavity injection of ginseng saponinRg1solution),4high intraocular pressure model mNGF+glass bodycavity injection group (0.1mlmNGF solution glass cavity injection),5high intraocular pressure model group+glass body cavityinjection of ginseng saponin Rg1+ultrasound group (0.1ml glasscavity injection of ginseng saponin Rg1solution after giving soundintensity is0.5W/cm2irradiation60s),6high intraocularpressure model+glass body cavity injection mNGF+ultrasound group(0.1mlmNGF glass cavity injection solution after giving soundintensity is0.5W/cm2irradiation60s),7high intraocularpressure model group+glass body cavity injection GSTT+microbubble ultrasound group (glass cavity injection respectively0.1and0.1ml Rg1ginsenosides solution ml cell suspension liquid,then give the sound intensity is0.5W/cm2irradiation60s),8highintraocular pressure model group+glass body cavity injection mNGF+microbubble ultrasound group (glass cavity injection respectively0.1and0.1ml mlmNGF solution cell suspension liquid, then givethe sound intensity is0.5W/cm2irradiation60s). Each processingonce every three days, a total of28days,1week,2weeks after the injection drug,4weeks respectively behavior observation,Accupen handheld tonometer intraocular pressure measurement, withslit lamp microscope, eye B ultrasonic, anterior segment, liveobservation of rabbit eyes at present, the change of the section,air embolism executed after4weeks of rabbit, rabbit retina andoptic nerve, line of rabbit retina pathological section, usingmeasuring retinal thickness, retina and optic nerve transmissionelectron microscopy (sem) test, test the content of NO, MDA, SODretina.Result:1. Got more purified rat retinal ganglion cells, and by cellimmunochemical examination, the purity of more than90%.2. NMDA cells for in vitro culture of rat RGCs excitatory toxiceffect is dose dependent, add10μM、100μM、1000μM NMDA after30min, RGCs survival rates were91.46%,64.74%and50.02%respectively. Concentration with the normal control group of100microns or more the RGCs survival rate have obvious difference (P<0.05). Join100mu M NMDA role respectively for30min,24h,48h survival rates are63.3%,55.19%and63.3%respectively, as theextension of time, the RGCs survival rate decreased significantly;With increasing doses, prolonged, RGCs of survival. Therefore,select100microns effect30min to follow-up experiments.3.Determined by MTT assay showed that30min after NMDA processingof RGCs cell survival rate were decreased, cell survival rate plusdrug group were increased the NMDA group, including10-4μg/ml GSTT+NMDA group,8×10-2μg/ml mNGF+NMDA group,102μg/ml of GinsenosideRg1groups, compared to other dose group could increase the cellsurvival rate, selected the corresponding concentration forsubsequent experiments. 4.Rg1ginsenosides can suppress the apoptosis of retinal ganglioncells induced by NMDA, promote the survival of the in vitro culturedRGCs, protect retinal neurons. Tetrazolium salt (determined by MTTcolorimetric test showed that30min after NMDA processing of RGCscell survival rate were decreased, and cell survival medicine groupwere improved the NMDA injury group, including10-4mu g/ml GSTT+NMDA group,8x10-2mu g/ml mNGF+NMDA group,102mu g/ml Rg1ginsenosides+NMDA group, relative to other dose group couldimprove the cell survival rate. Three kinds of drugs can promotethe survival of retinal ganglion cells in vitro, has significantoptic nerve protective effect.5. Pure vision medicine group and the glass body cavity medicineinjection by ultrasound irradiation after micro bubbleintervention group, two different methods of rabbit eyes visionwithin24h after injection of Rg1ginsenosides, in addition to thegroups in the vitreous Rg1ginsenosides content gradient descent,the peak time for15min, the rest of the eye tissues (aqueous humor,retina, choroid, optic nerve) ginseng saponin content of Rg1correlation curve changes. Its up to the peak time for aqueous humorand internal organization with retinal choroid at first peak, bothfor3h, and the outer wall of eye tissue of peak time late, theoptic nerve is4h. Will simply body cavity medicine injection groupand the glass body cavity medicine injection by ultrasoundirradiation after micro bubble intervention group organizationsRg1ginsenosides in rabbit eyes of peak levels, by ultrasoundirradiation microbubble intervention group organizations Rg1ginsenosides peak levels were significantly higher than that ofpure glass body cavity medicine injection group, with statistical significance (P <0.05or P <0.01), suggesting the glass body cavityinject drugs by ultrasound irradiation micro bubble after theintervention is relatively simple glass body cavity medicineinjection can reach high drug concentration in each organizationin the eye. Move each each peak QiuBi organization Rg1ginsenosidescontent comparison, found that the glass body cavity injection Rg1eye organizations ginsenosides content distribution for vitreous> retina detached> optic> aqueous humor. Except vitreous internalcause direct injection Rg1ginsenosides result in high drugconcentration, the retina and optic nerve is the most can obtainideal drug content organization, so the glass body cavity injectionof ginseng saponin Rg1section is to ensure that eye after treatment,especially the retina nerve protective effect of effective drugconcentration method. Ultrasonic irradiation micro bubble on thebasis of further improve the drug use.6. Rabbit anterior chamber injection of0.3%compound carbomerchronic high intraocular pressure model is a better solution method,successful glaucoma optic nerve retinal damage model is established.Rabbit retina using morphological observation:1set of rabbitretinal ganglion cells in monolayer, cell sizes and irregularcontour, nuclear hyperchromatism, cell borders is clear, neat cellsclosely, stratified clear; Number2group model of rabbit retinalganglion cells decreased significantly, the layers of loosestructure, disordered arrangement, is no clear boundary betweenlayers and retinal ganglion cell layer in a large number of vacuoleschange, some cells serious degeneration and karyolysis, shallowlight nuclei and cytoplasm staining shallow light, nuclearstructure sparse, nucleolus pyknosis and offset, cytoplasmic chromatin edge set, endoplasmic reticulum swelling, mitochondriaswelling and cristae disappear, vacuoles degeneration.3,4,5,6groups of rabbit retinal ganglion cells number less, the rabbitretina to be relatively complete, mild disorder, layered relativelyclear, visible in cavity of cell;7,8groups of retinal layersstructure is complete, the arrangement is neat, roughly normalmorphology, inner and outer nuclear layer appear a small amount ofcell loss, partial sample had mild vacuoles change. Measuring thethickness of the rabbit retina, each group of rabbit retinalthickness differences were statistically significant (P <0.05);2rabbit retinal thickness significantly thinner than1group, thedifference was statistically significant (P <0.05);3,4,5,6groups of rabbit retinal thickness thickness obviously in twogroups, the difference was statistically significant (P <0.05);7,8groups of rabbit retinal thickness is very apparent thicknessfrom3,4,5,6groups, the difference was statistically significant(P <0.05). Rabbit retina ultrastructure observation: group1normal retinal ganglion cell nuclei were round, clear RGC cellnuclear membrane, dyeing uniformity, nucleolus, inner and outernuclear layer of nuclear chromatin color clear, uniformdistribution, endoplasmic reticulum, mitochondria and otherorganelles normal form, the cell membrane integrity, neatphotoreceptor cells.2model group photoreceptor cellsmitochondria swelling, bubble and different level; Inner and outernuclear layer structure disorder; Reduced Numbers of RGC, swellingcolor light.3,4,5,6groups of RGC within the nucleus ofuniformity, color pale euchromatin, photoreceptor cells arrangeddisorder, slightly not seen obvious swelling of mitochondria and vacuoles of samples, a few cell chromatin edge set, the kernel layerpart slightly deformed, nuclear chromatin is shown slightly thin,the color is a bit light, mild mitochondria vacuoles degeneration,cristae mitochondria, endoplasmic reticulum only mild swelling.7,8group not seen obvious necrosis and apoptosis of retinal neurons,RGC chromatin distributed equably, obvious nucleolus, basic normalrough endoplasmic reticulum, mitochondria, change is not obvious,the photoreceptor outer segment order plate membrane, no obviousdissolved. Rabbit optic nerve ultrastructure observation:1set ofnormal rabbit optic fiber thicknesses, myelin integrity, closelypacked, the rules,"hair" shaped, axons surface smooth, plate ofthe axon myelin layer density, optic nerve myelin integrity,axoplasmic evenly, can be seen in the axoplasm clear normalmicrotubule, microfilament and oval mitochondria;2model groupcommon lesions, nerve fibers visible axon disappear, the disorderedarrangement of myelin sheath, segmental release, dissolved,shrinking clumps or pulp degeneration, gliosis, microtubules andneural structure of microfilament disappeared, swollenmitochondria and vacuoles degeneration;3,4,5,6groups of opticnerve myelin is relatively uniform, part of the myelin sheath thin,loose "onion skin" shaped, axons within microtubules and actinfilaments swollen, but not disappeared, some mitochondria vacuolesdegeneration;7,8groups of optic nerve myelin is arrangedrelatively dense, myelin integrity, rules, no demyelinatingphenomenon, visible microtubule, microfilament, axon structureclose to normal. Rabbit retina tissues of NO, MDA, SOD contentdetection: two groups of rabbit retina of NO, MDA content increasedsignificantly in the1group, SOD activity significantly reduced in the1group, are statistically significant differences (P <0.05).3,4,5,6,7,8groups of NO, MDA content in two groups decreasedobviously, SOD activity is significantly increased in the2groups,with significant statistical difference (P <0.05).Conclusion RGCs conclusion SD rats in vitro, and RGCs is purified,it is a kind of ideal model of the cell culture experiments. NMDAof RGCs excitatory toxic effect, can be induced RGCs apoptosis;White thistle saponins, injectable mouse nerve growth factor andginseng saponin Rg1against the RGCs apoptosis induced by NMDA,protect retinal ganglion cells, enhance the role of the active;Glass body cavity injection of2.5mg/kg of Rg1ginsenosides, noobvious side effects on rabbit retina, optic nerve, is safe. Rabbitanterior chamber injection of compound carbomer solution is a kindof ideal rabbit high intraocular pressure model method. Compoundcarbomer induced rabbit model of glaucoma has cause elevatedintraocular pressure medium, stability, long duration and methodof Jane Sheet, the advantages of easy operation and control, canbe used for the study of glaucoma optic nerve sex retinal damageand glaucoma drugs research, is a kind of ideal of the rabbit modelof glaucoma. Targeted ultrasound can damage the cell targetedrelease drugs, strengthen drug to the concentration of the eye,ultrasonic break the cell mediated optic protection drug Rg1ginsenosides and mNGF therapy high intraocular pressure of rabbitretina, optic nerve damage is effective. Ultrasound microbubblemediated optic protection drugs by changing the retina in the amountof the expression of NO, MDA and SOD in play a role of protectionof high intraocular pressure rabbit optic nerve injury.更多还原