Chinese Journal of Tissue Engineering Research ›› 2018, Vol. 22 ›› Issue (35): 5700-5705.doi: 10.3969/j.issn.2095-4344.0579
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Zhu Liguo1, Tang Bin1, 2, Wei Xu1, Yin He1
Contact:
Wei Xu, MD, Master's supervisor, Wangjing Hospital of China Academy of Chinese Medical Sciences, Beijing 100102, China
About author:
Zhu Liguo, MD, Chief physician, Doctoral supervisor, Wangjing Hospital of China Academy of Chinese Medical Sciences, Beijing 100102, China
Tang Bin, Master candidate, Wangjing Hospital of China Academy of Chinese Medical Sciences, Beijing 100102, China; Beijing University of Chinese Medicine, Beijing 100029, China
Zhu Liguo and Tang Bin contributed equally to this paper.
Supported by:
the Second Batch of Key Research Projects in the “13th Five-Year Plan” of Chinese Academy of Chinese Medical Sciences, No. ZZ10-022
CLC Number:
Zhu Liguo, Tang Bin, Wei Xu, Yin He. Research progression and evaluation of an animal model of cervical radiculopathy[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(35): 5700-5705.
2.1 直接刺激颈神经根 上肢疼痛、麻木、皮肤感觉障碍和肌力下降是CR患者就诊的主要症状,也是与其他类型颈椎病鉴别的要点。通过手术暴露直接刺激神经根,造成上肢感觉功能和运动功能的异常,是此类模型的主要手段。 2.1.1 瞬时物理压迫神经根 2005年Hubbard等[10]将腰椎神经根压迫的方法运用到大鼠CR模型的研究中,首次通过微血管钳压迫,明确单纯机械刺激对CR中颈 神经根的影响。手术分离大鼠颈后部肌肉及韧带,咬除部分C6/7椎板及关节突,打开硬脊膜,暴露右侧C7神经根,在靠近背根神经节的神经根处分别采用10 gf、60 gf的微血管钳压迫15 min后松开,对照组根据是否打开硬脊膜又分为2组,均未夹钳神经根。造模手术后,通过von Frey细丝刺激前爪掌面记录大鼠在不同压力时的缩足反射次数,检测机械性痛感过敏的程度,以评价大鼠造模成功。该研究发现10 gf和60 gf的压迫产生的缩足次数差异无显著性意义,因此认为短暂压迫颈神经根引起持续性疼痛的力度阈值 < 10 gf。后续Smith等[11]使用该方法也验证了该模型的可靠性。模型术后1 d即可出现明显疼痛过敏现象,并持续7 d以上。其优点是造模周期短,压迫力度和体积均量化,可重复性强。瞬时压迫虽可产生持续的感觉功能异常,但与CR中椎间盘、小关节退变所致的持续物理刺激是否一样还存在争议。 类似的瞬时压迫干预在国内也有所报道,梅荣军 等[12]认为,因CR的根性症状多属于臂丛神经的支配范围,且C5-8前支及T1前支大部分汇聚为臂丛神经,故用眼科显微止血钳夹住大鼠臂丛神经根部的神经分支各1次,止血钳扣紧1扣,持续约30 s,以期模拟出CR的大鼠模型。并认为从颈前锁骨下入路对比颈后入路、颈前锁骨上入路,具有操作简便、创伤小、切口恢复快的优势。但文中未具体叙述采用何种感觉或运动功能检测的评价标准,以验证该CR模型造模方案是否成功;其次止血钳挟持力度无法定量控制。更重要的是,该方案压迫臂丛神经根更类似于臂丛神经卡压综合征,和临床中椎间盘突出或小关节增生压迫颈神经根造成CR的发病机制存在差异,该手术模型是否符合CR的发病机制有待商榷。 2.1.2 瞬时物理压迫结合化学刺激 2007年Rothman等[13]利用铬酸处理后的羊肠线比普通肠线的组织吸收时间长,可持续且缓慢的引起炎症反应的特点,在Hubbard等[10]研究的基础上,同样以大鼠为实验动物,进一步增加铬制羊肠线作为颈神经根的化学免疫刺激物。先采用10 gf微血管钳压迫C7神经根模拟机械刺激,后将4段2 mm长的3-0铬制羊肠线放置于紧挨C7背根神经节的神经根上,模拟化学免疫刺激。与Hubbard等采用相同的动物行为评价方法,分别比较2种方法单独使用以及两者联用的差异。最后发现短暂机械压迫或化学刺激均能引起同侧的神经根型症状,且两者联用时根性疼痛及痛觉过敏的症状要显著高于单一方法。 此方案的优点是微血管钳的力度和时间、羊肠线的长度和规格均有明确定量,物理压迫及化学刺激等干预因素的一致性较强。羊肠线可一定程度弥补之前实验中微血管钳只能短暂压迫的缺点,但其可被组织吸收,仍无法完全模拟慢性持续压迫的病理特点;再者,羊肠线的异体免疫反应与椎间盘中髓核对神经根的炎性刺激是否类似,尚需进一步验证。 2.1.3 持续物理压迫 2006年窦夏睿等[14]首次运用椎管插线法在大鼠上建立CR模型。暴露椎体后部肌肉韧带后,将长约1.5 cm,直径0.5 mm的尼龙渔线纵向放置于左侧C7-T1神经根腋下,从后方对颈神经根造成压迫,产生痛阈和体感诱发电位的改变。因该模型主要压迫的是支配感觉功能的脊神经后根和背根神经节,故大鼠运动功能未产生明显异常,这也符合临床中CR以疼痛麻木等感觉异常发生率最高的特点。随后被Sun等[15]成功复制并验证了可行性。虽然实验中的尼龙线经过乙醇和多聚赖氨酸溶液处理后,可增加组织的黏附性,但仍不能保证尼龙线对颈神经根保持固定持续地压迫,而不产生位移使神经根压迫消失或造成脊髓压迫。因尼龙线卡压的程度较轻,同时大鼠神经的自身修复能力强,在2周左右感觉功能基本恢复,所以此方案只适合CR急性期的研究。 针对椎管插线法模型病程持续短,且存在自愈性等问题,谢炜等[16]在该模型的基础上进行改良,增加为2根尼龙渔线,从头尾分别穿入,压迫对神经根,一定程度上解决了椎管插线法模型中前肢神经根性症状自愈的问题。 杨大志等[17-18]最早于1998年采用自体骨卡压的方式,在猫上建立颈神经根持续受压的CR模型。手术暴露右侧C7,C8神经根及其椎间孔内口,用牙髓钻破坏椎间孔周围骨皮质后,将咬除的棘突剪成 3 mm×10 mm骨条,在中间处对折呈V形骨块,直视下从椎间孔内口向外,嵌插到神经根通道的骨性管道内及侧隐窝后方,左侧做正常对照。通过运动诱发电位和体感诱发电位检测,及X射线片、CT、MRI等评价造模成功。该模型优点是运动及感觉功能均可出现异常,并可产生持续的神经根性症状,比较符合CR的临床特点,且自体骨排异性低,术后恢复快。但该模型需要取自体椎板加工成V型骨块,手术操作精细性较高,只适合猫等体型较大的实验动物,因此研究成本偏高。 孔微微[19]、Liu等[20-21]在大鼠上采用V或L型不锈钢柱模拟椎间盘突出及椎后小关节增生的物理刺激,称为颈神经根疼痛模型。该模型基于经典大鼠疼痛模型中的慢性压迫背根神经节模型,但将其压迫部位由腰椎背根神经节换成了颈椎。其不锈钢柱表面光滑,直径0.63 mm、长4 mm(可由6号钝针头弯折相应角度后,裁剪而成),手术暴露后,对C6/C7和C7/T1椎间孔内的颈神经根及背根神经节进行持续压迫,可致大鼠同侧上肢出现显著的机械性痛敏和热痛敏。其感觉异常术后1 d即可出现,4 d达到最大值,可持续2周。不锈钢柱简免了自体骨块的加工过程,便于在老鼠等小型动物的运用。此方案中不仅可简化操作,还降低了实验费用,同时也能避免自体骨吸收等潜在问题,比较适合研究推广。 2.1.4 持续物理压迫结合化学刺激 1998年张军 等[22-24]首次在大鼠上采用甲醛定量滤纸片法模拟持续物理压迫和化学刺激。手术分离肌肉打开椎板,暴露脊髓及双侧C6,C7神经根后,将浸泡有甲醛的定量试纸压迫于双侧C6,C7神经根腋下。通过行为学评分、体感电刺激测定等方法证实模型成立。 此模型的优点是定量滤纸作不易吸收,可持续压迫神经根,甲醛对组织具有刺激性,能引起神经根的炎性反应,从物理和化学双重方面引起神经根症状较符合目前CR病理机制的主流学说。但方案中试纸的规格没有叙述,且试纸浸泡后所含甲醛溶液的含量难以定量和统一。再者,甲醛对神经根造成的化学刺激是否与椎间盘及髓核的炎性刺激影响一样有待研究。虽然该模型存在一定问题,但经过后期实验推广,不断被其他人的相关研究采用并验证了可靠性。说明此模型在手术操作、实验费用方面有很大的优势,在国内的CR基础研究中有一定的地位。 2.2 模拟颈椎退变间接刺激颈神经根 CR发病多因工作学习等原因而长期采用的不良姿势和体位产生肌肉劳损,颈部肌肉力量减弱或力量分配不均,导致椎间盘及椎后小关节受力局部受力过大而突出或增生,进而压迫颈神经根[25]。多项研究利用此原理,不直接刺激神经根,而通过相关手段调整肌肉力量,改变颈椎正常的生物力学特征,使椎间盘和小关节退变,为CR中神经根压迫提供外在基础,以此建立CR模型。 2.2.1 非手术干预肌肉韧带 2012年杨开洋等[26]以“颈部力学平衡失调”为原则,将应航等[27]的兔椎间盘退变造模方案应用于CR的研究中。将家兔固定于特制固定架,使兔颈椎低头屈曲45°,5 h/d,连续2个月。通过肌电图测量尺神经和正中神经的F波传导速度评价造模效果。研究中未进行动物行为学评定而仅进行肌电图检测,不能充分评价模型是否均出现感觉或运动功能异常等神经根性症状。且造模需要连续2个月每天对家兔进行操作,工作量较大。 2.2.2 手术干预肌肉韧带 2015年金哲峰等[28]以“动静力失衡”为原则,在王拥军等[29]椎间盘退变大鼠模型的基础上,增加使颈椎小关节突退变的干预手段。通过切断大鼠的颈背部深群颈夹肌和头、颈、寰最长肌,切除颈骼肋肌与头半棘肌,切断C2-C7棘上和棘间韧带,造成颈部后伸无力,模拟CR患者因长期低头产生的颈椎生理曲线改变和椎间盘退变突出。再采用Ⅱ型胶原蛋白酶注射至椎后小关节,使关节突退变增生。2种手段模拟颈椎间盘及椎后关节突关节的双重退变,建立慢性CR的大鼠模型。手术后2个月观察大鼠烦躁、食欲、用嘴反复舔前爪、倦怠、易激惹、活动频繁等行为,并检测模型右侧正中神经的感觉、运动神经传导速度,评价该CR大鼠模型是否造模成功。 该CR模型从颈椎病发病过程的起始阶段开始,模拟了颈椎病中的颈椎曲度改变及椎后小关节退变,为神经根受压提供可能的条件。文章中没有列举出现反复舔前爪等前肢症状的大鼠只数或比例,不能确保所有模型大鼠均出现上肢的神经根性症状。其次因没有针对特定颈神经根进行干预,故不能明确受压的颈神经节段及根性症状的神经分布区域,而文中未解释电生理只测量大鼠右侧正中神经的原因。 此类间接刺激神经根的造模方案通过手术或非手术模拟CR患者肌肉韧带的劳损,引起颈椎增生退变而压迫颈神经根。优点是符合颈椎病正常的发展过程,较真实地还原患者颈后部肌肉劳损、疼痛僵硬,椎间盘突出、小关节增生,颈椎生理曲度改变等临床表现。缺点首先是干预措施仅为神经根刺激提供了外在条件,而不能保证模型均发展成颈椎病中的神经根型,故还需采用更为完善地动物根性症状评价措施,进一步验证模型地可靠性;其次该方法造模周期较长,动物饲养成本较高,从时间和经济方面也限制了其应用。"
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