Chinese Journal of Tissue Engineering Research ›› 2016, Vol. 20 ›› Issue (40): 6067-6073.doi: 10.3969/j.issn.2095-4344.2016.40.021
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Wang Shi-jun1, Li Yu-ting2, Li Chun-de1
Revised:
2016-07-04
Online:
2016-09-30
Published:
2016-09-30
Contact:
Li Chun-de, Chief physician, Doctoral supervisor, Department of Orthopedics, First Hospital, Peking University, Beijing 100034, China
About author:
Wang Shi-jun, M.D., Attending physician, Department of Orthopedics, First Hospital, Peking University, Beijing 100034, China
Li Yu-ting, M.D., Physician, Department of Anesthesiology, First Hospital, Peking University, Beijing 100034, China
Wang Shi-jun and Li Yu-ting contributed equally to this paper.
CLC Number:
Wang Shi-jun, Li Yu-ting, Li Chun-de. Establishment, application and progression of an animal model of cervical spondylosis[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(40): 6067-6073.
2.1 脊髓型颈椎病动物模型 脊髓型颈椎病是颈椎病中最严重的一个类型,是由于颈椎椎体的退变及其周围软组织的退变如椎间盘突出、后纵韧带骨化等引起的。由颈椎间盘突出、骨赘形成、黄韧带肥厚、颈脊髓受压以及颈椎的异常活动等颈椎所呈现的形态学改变所造成的。脊髓型颈椎病的发病率为12%-30%,占颈椎病10%-15%,其发病率可能进一步升高[10]。该病复杂,起病缓慢,脊髓受损后往往出现不可逆的改变,治疗难度大,效果较差[11]。 关于脊髓型颈椎病神经症状的产生过程主要有两种假设。第一种假设认为是由机械性压迫脊髓导致组织损伤,另一种假设认为是由于脊髓内血液循环障碍所导致。目前设计的脊髓型颈椎病动物模型的方法有很多[12-16]。有研究通过植入物法[17],即取大鼠自体第7颈椎棘突于C3-4或C4-5椎间隙植骨建立大鼠脊髓型颈椎病的动物模型,大鼠造模后运动功能有明显改变,体感诱发电位显示神经传导通路异常,病理切片显示脊髓神经细胞有退化改变,说明自体第7颈椎棘突椎间植骨是一种简单、经济、有效的大鼠脊髓型颈椎病动物模型制造方法,为开展脊髓型颈椎病的实验研究提供了重要的动物模型。 拧入螺钉法建立的脊髓型颈椎病动物模型是将螺钉透过椎体钻孔形成椎管占位,拧入合适的螺钉而造成脊髓压迫。应用骨水泥压迫钉建立的脊髓型颈椎病动物模型具有典型脊髓型颈椎病的临床特征[18]。胡志俊等[19]用平头不锈钢螺钉从大鼠C4椎体前侧拧入压迫颈脊髓腹侧,根据螺钉侵占脊髓深度不同划分为轻、中、重压迫,造模后持续保留螺钉30 d,造成脊髓持续受压,建立了不同程度压迫的大鼠颈部脊髓慢性压迫性损伤模型,通过脊髓损伤程度联合行为学评分,脊髓神经传导功能障碍诱发电位动态检测以及大鼠脊髓形态、病理组织学检查结果证实造模成功,并系统观察了大鼠脊髓慢性损伤后的一系列病理机制改变,深化了对脊髓慢性损伤的认识。拧入螺钉法建立脊髓型颈椎病动物模型制作简便,可同时用于病理学检查、SEP、CT及MRI等检查,可重复性强,是综合研究脊髓型颈椎病相关数据的可靠模型。 王军等[20]将不同大小吸水性压迫材料聚乙烯醇丙烯酰胺互穿网络水凝胶植入C5-C7椎板下,制作慢性颈脊髓压迫动物模型,MRI检查显示压迫组大鼠出现不同程度的椎管狭窄和脊髓压迫,压迫组大鼠脊髓压迫节段发现较多凋亡细胞,说明构建的大鼠慢性颈脊髓压迫模型符合脊髓型颈椎病的病理改变。采用压力球囊法致慢性颈脊髓压迫具有操作简单易行,不易伤及脊髓,不易感染,死亡率低,安全性及成功率较高,对脊髓压迫实验模型的观察指标比较客观可靠,可重复性强等特点,用它皆可制作不同程度的慢性压迫脊髓损伤实验动物模型,脊髓压迫程度可根据实验目的不同进行调节,为脊髓压迫性损伤机制的研究提供了一种理想的动物实验模型。 注射法建立的脊髓型颈椎病动物模型是用注射器直接将髓核溶解剂如透明质酸酶或木瓜凝乳蛋白酶等化学物质注射髓核内,髓核溶解剂可消化髓核中的多肽蛋白原分子基质,导致髓核脱水、皱缩,最终溶解髓核而制成。该法具有可在非开放手术下进行、创伤小、操作简单等优点,缺点是髓核内注射的药物可影响以后生物化学检测的准确性。张红利等[21]观察颈椎横突旁注射硬化剂制作兔颈椎病模型的效果。用硬化剂注射法造模后取兔血清,酶联免疫吸附实验检测动物血清肿瘤坏死因子、P物质、神经肽、降钙素基因相关肽含量。结果显示模型组血清肿瘤坏死因子、P物质、神经肽含量升高,且3次检测呈逐渐升高趋势,降钙素基因相关肽含量降低,3次检测呈逐渐降低趋势。实验证实颈椎横突旁注射硬化剂可能通过炎症因子刺激引起神经递质释放,导致颈椎周围疼痛,它是制作颈椎病动物模型的较实用方法。 颈型颈椎病是颈椎病最常见类型之一,有数据表明青少年颈型颈椎病的发病率目前呈上升趋势。根据流行病学调查,颈型颈椎病的发生与长时间低头屈曲位造成的异常应力负荷有关。对颈型颈椎病动物模型的研究是揭示颈椎病病因学和发病机制的基础。张欣等[22]设计一种无创颈型颈椎病动物模型的制备方法并验证其可行性,将新西兰雄性大白兔置于改良后的兔盒中,剔去颈部毛发,敷以自制保湿冰袋,调节保湿冰袋松紧并将其下端扣住兔盒,使兔的颈部成低头屈曲位45°,将吹风机出风口对准兔颈部,风力5级,每次造模2 h,每天造模2次,共4 h。造模后进行X射线影像学观察,光镜下组织形态学观察,结果显示颈椎生理曲度变直,椎间隙变窄,椎体边缘有轻微骨质增生,椎间盘髓核脱水皱缩,质量减轻。纤维环产生放射状裂隙,胶原纤维变性、紊乱甚至断裂;退变髓核中肿瘤坏死因子浓度显著升高,说明此造模法符合人颈椎病发病规律,避免了致病因素单一,操作简便易行,造模方法对所观察组织无损伤等干扰,有利于颈型颈椎病防治的进一步研究。 2.2 神经根型颈椎病动物模型 神经根型颈椎病是指颈椎间盘退行性改变及在外力作用下引发的颈椎间盘内外动力平衡失调,刺激或受压周围组织结构神经根引发的综合征。神经根型颈椎病的主要原因是椎间盘退行性改变、继发性病理改变,导致单侧或双侧脊神经根受刺激或受压所致,发病率占颈椎病的60%[23-24]。神经根型颈椎病具有较典型的根性症状,其疼痛范围与颈脊神经所支配的区域相一致。头颈部长期处于单一姿势位置,颈部活动受限比较明显,表现出疼痛症状更为显著。建立公认的重复性强的实验动物模型,从多方面进行深层次探索,找出神经根型颈椎病最本质的发病机制是目前众多数学者研究的热点[25-26]。 采用尼龙渔线对颈神经根及背根节的卡压可以建立稳定的神经根型颈椎病根性痛的动物模型,正中神经短潜伏期诱发电位是神经根损伤及好转的可靠检测手段[27]。 孔微微[28]通过在大鼠C6和C7椎间孔内插入V形钢柱造成颈部背根节慢性压迫,建立颈部背根节慢性压迫模型来模拟颈椎神经根病变的临床症状,应用动物行为学检测方法观察颈部背根节压迫后大鼠前肢对机械、热和冷刺激的反应,采用在体单纤维记录技术观察背根神经纤维的自发放电情况。动物行为学检测结果显示压迫C7和C8背根神经节后,大鼠双侧前肢的机械阈值明显降低,热潜伏期缩短,冷刺激异常性痛增强,提示压迫后动物出现了痛觉过敏和异常痛觉。该模型能模拟颈椎神经根病变引起的疼痛,有利于颈椎神经病变疼痛机制的研究。 按照颈后入路、颈前锁骨上入路、颈前锁骨下入路3种不同方式探讨大鼠神经根型颈椎病实验动物模型的制备方法。结果显示颈前锁骨下入路操作过程更为简单,容易掌握,造模后21 d观察到大鼠的大体形态较好,颈前锁骨下入路操作简单,更加科学、合理,符合大鼠神经根型颈椎病实验研究的需要[29]。采用颈椎神经根压迫法制备神经根型颈椎病模型,血液流变学指标上升,颈椎间盘组织形态变化加重,说明不同时期的神经根型颈椎病大鼠的气虚血瘀证型表现出一定的变化,为临床辨证施治及对该病的研究提供了实验依据[30]。 杨大志[31]通过显露右侧C7、C8和L5、L6神经根及其椎间孔内口,采用椎间孔内自体松质骨植入建立神经根慢性嵌压性损伤模型。造模后早期实验侧肢体出现行为异常,实验侧椎间孔骨痂增多,狭窄加重,神经根受压变形,椎间孔骨性截面积8周后明显减小,造模成功率高,操作简单。 张军等[32]通过机械性压迫和化学性刺激法建立了神经根型颈椎病大鼠模型。该模型可以出现明显的炎症、瘀血、水肿及神经功能传导损伤变化,说明模拟颈神经根炎动物模型,可以对神经根型颈椎病急性期加以反映。模拟颈神经根炎方法是一个新的实验性神经根型颈椎病动物模型的制备方法。另外通过模拟慢性颈椎间盘退变及关节突关节双重退变的动物模型可以对神经根型颈椎病加以反映[33]。 2.3 椎动脉型颈椎病动物模型 椎动脉型颈椎病多是由钩椎关节增生的骨赘从侧方压迫、刺激椎动脉第二段,除固有颈椎病症状外,因椎动脉痉挛或折曲狭窄,引起椎-基动脉供血不足,影响大脑后动脉、小脑动脉、内囊动脉缺血而发生。椎动脉型颈椎病是颈椎病中常见的一种类型,其发病仅次于神经根型颈椎病,临床症状比较复杂,在椎动脉影像学检查前常难以确诊[34-36]。虽然颈椎退行性变和骨赘形成是导致椎-基底动脉缺血的主要原因,但椎动脉型颈椎病的发病机制一直未确定,椎动脉型颈椎病动物模型的建立有助于探讨其发病机制[37]。 李俊华[38]用外科方法造成兔右侧椎动脉受压,观察椎动脉受压动物模型血浆内皮素的变化,结果显示造模兔的血浆内皮素值明显高于正常兔,说明血浆内皮素可作为衡量椎动脉型颈椎病的测量指标之一。该模型的致压因素确切稳定,对动物损伤小,有利于进一步研究,成功率80.2%[39]。 张文芽等[40]通过破坏大鼠颈部动静力平衡系统可引起颈椎间盘退变,退变机制可能与细胞因子核因子κB和白细胞介素6引起的炎症反应有关。颈椎运动是多节段之间相互力学及位移关系的变化,颈椎这样一个稳定的生物力学系统在各种因素的作用下,平衡失调,稳定性遭到破坏,而且继发的病理改变,会引起动静力学平衡失调。力学平衡失调法制备椎动脉型颈椎病动物模型是研究此类颈椎伤病的基础,在其病因与发病机制研究中占据重要地位。郭建阔等[41]采用切除兔颈背部浅、深两层肌肉的方法建立一种椎动脉型颈椎病动物模型,探讨颈椎生物力学失衡与颈椎病发生的关系。病理切片检测结果显示椎动脉管腔变窄,内膜增生,管壁明显增厚,结果出现了椎动脉结构异常。说明颈椎生物力学失衡是颈椎退变的主要机制,采用破坏兔颈部动力平衡的方法可以成功制作椎动脉型颈椎病动物模型。"
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