建立视神经挤压伤的标准方法Yasargil(脑)动脉瘤夹

A standardized method to create optic nerve crush:Yasargil aneurysm clip建立视神经挤压伤的标准方法:Yasargil(脑)动脉瘤夹Levent Sarikcioglu a,*, Necdet Demir b,c, Arife Demirtop ca Department of Anatomy, Akdeniz University, Faculty of Medicine, 07070 Antalya, Turkeya土耳其, 07070 Antalya, Akdeniz大学医学院, 解剖部b Department of Histology and Embryology, Akdeniz University, Faculty of Medicine, 07070 Antalya, Turkeyb 土耳其, 07070 Antalya, Akdeniz大学医学院, 组胚部c Electron Microscopy Unit (TEMGA), Akdeniz University, Faculty of Medicine, 07070 Antalya, Turkeyc 土耳其, 07070 Antalya, Akdeniz 大学医学院, 电镜部Received 4 August 2006; accepted in revised form 18 October 2006Available online 8 December 20062006-8-4收稿；2006-10-18校后发表；2006-12-8在线发表AbstractIt is often difficult to compare results obtained by different investigators on nerve compression injuries, owing to differences in method of pressure application and noncomparable pressure levels. In the present study, we described a new method to crush the optic nerve by using a specially designed and commercially available device. We think that standardization of the compression methods is necessary to compare interlaboratory results._ 2006 Elsevier Ltd. All rights reserved.摘要：由于不同研究者建立神经挤压伤过程中使用方法不同, 压力程度不同, 通常要比较所得出的结果很困难。现在，描述一种新的建立神经挤压伤的方法，此方法采用的设备专门设计且市面有售。我们认为，为了比较各项试验结果，非常有必要将挤压伤的方法标准化。Keywords: Yasargil aneurysm clip; crush injury; optic nerve关键词：Yasargil （脑）动脉瘤夹；挤压伤；视神经 1. Introduction1引言The optic nerve of the rat is a very vulnerable structure (Gellrich et al., 2002). Isolated position of the optic nerve allows the scientist to create lesions conveniently, without damaging other parts of the brain. Another advantage of the optic nerve lesion model is that recovery of the function can be studied in a well-defined anatomical system with specific tests of visual behavior (Sabel and Aschoff, 1993). 鼠视神经结构非常易损伤（Gellrich 等，2002）。视神经位置孤立，这使科学家很方便建立损伤，不会损坏脑的其它部分。视神经损害模型另一优势在于可以在确定的解剖系统中通过针对视觉反应的特定试验研究其功能恢复情况（Sabel and Aschoff, 1993）。 Trauma to the optic nerve caused by either fractures of the midface and/or skull base has been simulated by optic nerve crush injury model (Gellrich et al., 2002). The main drawback of most of the methods described for optic nerve crush is that the injury cannot be quantified at the site of the trauma, but is rather semiquantitatively referred to as pressure in a balloon (Burke et al., 1985, 1986; Cottee et al., 1991), distance between the tips of a forceps (Buys et al., 1995; Duvdevani et al., 1990; Sautter and Sabel, 1993), or pulling force on a micro-sling (Matsuzaki et al., 1982).Gellrich 等人已通过制作视神经挤压伤模型模拟面中部骨折和/或颅底骨折所致视神经损伤（Gellrich 等，2002）。以往所述大部分制作视神经挤压伤模型方法的主要缺点在于创伤位置的损伤不能定量，而是参考气舱中压力（Burke 等，1985，1986；Cottee 等，1991）、镊子尖端之间距离（Buys 等，1995；Duvdevani 等，1990；Sautter和Sabel，1993）、或微悬带上拉力（Matsuzaki等，1982）半定量。In the literature, there are several methods to create optic nerve crush injury. However, the main disadvantages of these methods are standardization and their possible usage by investigators from all over the world. In our previous study we described a simple and reliable device, the Yasargil aneurysmclip, to create sciatic nerve injury. In this current study, our goal was to describe a simple method that simulates trauma to the optic nerve caused by fractures of the midface and/or skull base, in order to investigate the effects of optic nerve crush from a histological point of view.文献中有几种建立视神经损伤的方法。然而这些方法主要的缺点在于标准不统一、来自全世界的研究者是否可能使用。以前我们详细介绍过一种简单而可靠的设备-Yasargil（脑）动脉夹，建立坐骨神经损伤。本次研究目的是阐述一种简单的方法模拟面中部骨折和/或颅底骨折所致视神经损伤，从组织学角度研究视神经挤压伤效果。2. Materials and methods2材料和方法2.1. Animals21动物A total number of 20 female Wistar rats (200e250 g) were randomly divided into two groups (control, experimental). The animals were housed in Makrolon cages (5 per cage) and maintained on a 12-h lightedark cycle; lights on from 7.00e19.00 h. Food and water were provided ad libitum. All procedures were reviewed and approved by animal care and usage committee of Akdeniz University.取20只Wistar母鼠（200250g）,随机分成两组（对照组，试验组）。将动物置于Makrolon笼子中（每笼5只），每12小时循环给予光线，给光时间7：0019：00。随意供应食物和水。所有操作均经过Akdeniz大学动物喂养和使用委员会考查和批准。2.2. Yasargil aneurysm clip22yasargil(脑)动脉瘤夹Yasargil aneurysm clips (Aesculap AG & Co., Tutlingen, Germany) are designed for permanent occlusion of cerebral aneurysms (Yasargil, 2005). These permanent implantable clips are manufactured from a high grade Phynox cobaltechrome alloy, or titanium which conforms to the ISO standards. Closing force of the clips is determined by a computerized electronic gauged scale. Closing force of the Yasargil aneurysm clip (catalog no. FE-752K) used in the present study was 185 gf (grams force) (approximately 1.82 N).yasargil(脑)动脉瘤夹(Aesculap动脉照影公司，Tutlingen,德国)是为脑动脉瘤永久性闭塞而设计的（Yasargil,2005）。这些可植入夹子采用优质、符合ISO标准的Phynox钴铬合金或钛制造。镊子的闭合力由计算机控制的电子刻度表决定。本研究yasargil(脑)动脉瘤夹（目录：NO.FE-752K）采用185g(约1.82N)的闭合力。2.3. Optic nerve crush23视神经挤压伤The rats were anesthetized with a mixture of ketaminee xylazine (ketamine; 80 mg/kg IP, xylazine; 10 mg/kg IP) and their intraorbital optic nerves were exposed after a lateral canthotomy. All surgical procedures were performed under a trinocular operation microscope (Olympus SZ61) with fiber optic illumination. Parts of the lachrymal gland and the eye muscles were dissected 3 mm from the globe. Care was taken to ensure that the crush did not compromise the ophthalmic artery. The clip was placed 2 mm away from the optic nerve head. The dura was left intact and care was taken not to injure the optic nerve except with the subsequent crush. The optic nerve was crushed with a Yasargil aneurysm clip for 30 s. After completion of the crush, the canthotomy was sutured. Then, antibiotic ointment was applied, and the animals were allowed to recover from the surgery.用开他敏-甲苯噻嗪混合剂（开他敏 80mg/kg，甲苯噻嗪 10mg/kg）麻醉小鼠，外眦切开术暴露眶内视神经。所有手术过程在三倍眼内手术显微镜纤维光学照明下完成。距离眼球3mm将泪腺和眼肌分离开，注意不要伤及眼部动脉。动脉瘤夹置于距离视神经头部2mm处。保证硬脑膜完整，注意除随后的挤压伤以外避免损伤视神经。用yasargil(脑)动脉瘤夹挤压视神经30秒，挤压后缝合外眦，然后用抗生素软膏促进动物术后恢复。2.4. Light and electron microscopic evaluation24光学电子显微镜定量One month after the optic nerve crush, the animals were administered an overdose of chloral hydrate intraperitoneally. Each optic nerve was re-exposed and the dural sheath incised longitudinally 1 mm from the globe in both control and experimental groups. The optic nerves posterior to the crush sitewere then collected. Samples were fixed with 4% glutaraldehyde in 0.1 M Sorensens phosphate buffer solution (pH: 7.3), post-fixed with 2% osmium tetraoxide in the same buffered solution, dehydrated through an ascending series of ethanol, and the samples were then embedded in epoxy resin (Araldite CY212, Agar Scientific Ltd., Stansted, UK). Semithin sections (1 mm thickness) stained with toluidine blue were examined with light microscope (Zeiss Axioplan). Then, ultrathin sections (40e60 nm thickness) were contrasted with uranyl acetate and lead citrate, and prepared sections were examined with Zeiss LEO 906E electron microscope.视神经挤压伤后一个月，向动物腹膜内注射超剂量的水合氯醛。再次暴露所有视神经，对照组和试验组均距离眼球1mm处纵向切开硬脑膜鞘，然后收集损伤位置后面的视神经，用4戊二醛将样本固定在0.1M Sorensens磷酸盐缓冲溶液（PH:7.3）中，再用2的四氧化锇固定在同一缓冲液中,用浓度递增的酒精脱水，然后将样本植入环氧树脂（Araldite CY212,Agar Scientific Ltd.,Stansted,UK）中 。用光学显微镜(Zeiss Axioplan)检测甲苯胺蓝染色的正常厚度一半的切片（1um厚），然后用乙酸双氧铀和枸橼酸铅衬比超薄切片（4060nm）,用Zeiss LEO 906E电子显微镜检测所制备切片。3. Results3结果3.1. Application of surgery31手术方法In our pilot study, the optic nerve was crushed by three approaches, supraorbiral, lateral, and medial. In medial approach, it was difficult to use the Yasargil aneurysm clip to reach the optic nerve, since there was the lacrimal duct system in this region. In supraorbital approach, it was also difficultto reach the optic nerve. The frontal lobe of the brain and the skull bones make the surgery difficult. The easier approach was the lateral approach. By lateral chantotomy, a small incision was performed and the optic nerve was exposed, then it was crushed with a Yasargil aneurysm clip (Fig. 1).在初步研究中，我们通过三个入路制作视神经挤压伤：眶上，外侧，内侧。因为局部有泪管系统，采用内侧入路时很难使用yasargil(脑)动脉瘤夹接触视神经。采用眶上入路时同样很难接触视神经,脑额叶和颅骨使手术难以进行。外侧入路方法比较简单，从侧面做一个小切口，暴露视神经，然后即可用yasargil(脑)动脉瘤夹挤压伤视神经（图1）。3.2. Light and electron microscopic evaluation32光学电子显微镜定量The optic nerve was consisted of both myelinated and unmyelinated nerve fibers. Neither myelin debris nor damaged fibers was detected in control group in light microscopic observations. Myelinated and unmyelinated fibers showed well-shaped appearance (Fig. 2A, B). 视神经由有髓、无髓纤维组成。用光学显微镜在对照组中既观察不到髓鞘碎片也观察不到损伤的纤维。有髓、无髓神经纤维形态表现完好（图2A,B）。Light microscopic evaluations revealed that numerous damaged myelin residues were present in the experimental group at the 30th postoperative day. Additionally, electron microscopic observations revealed that optic nerve crush resulted in severe degradation of the fibers of the optic nerve (Fig. 2C, D).光学显微镜显示，试验组术后30天出现大量损伤的髓鞘残余物。此外，电子显微镜观察到视神经挤压伤导致严重的视神经纤维降解（图2C,D）。4. Discussion4讨论 Compression of nerve fibers can cause various clinical symptoms depending on the cause, magnitude and duration of the compression trauma (Rydevik and Lundborg, 1977). The pathophysiology of these various lesions is not fully understood and it has been debated whether the ischemia, secondary to compression, or the mechanical deformation of nerve fibers per se is the more significant etiological factor. Compression injury was, at first, attributed to ischemia due to following reasons: (1) nerve function was known to be dependent on blood supply and perfusion was impaired during compression; (2) nerve conduction failure did not occur even when in vitro nerve was compressed under high pressures (Powell and Myers, 1986); (3) nerve injury was greater with increased durations of compression; and (4) large myelinated fibers were especially vulnerable to ischemic injury. While the role of the ischemia has been stressed by some authors (Merrington and Nathan, 1949; Powell and Myers, 1986), the significance of the mechanical nerve fiber deformationwas emphasized by others (Gelfan and Tarlov, 1956; Ochoa et al., 1972). The optic nerve axons may be severed by either crushing or cutting the optic nerve. In rabbits and rats this operation does not usually result in ischemic death of the retina, provided that the lesion is more than about 1 mm behind the eyeball. The central retinal artery in these animals enters the optic nerve just behind the optic disc. Posteriorly, the optic nerve contains no large internal vessels and appears to receive its blood supply through its meningeal covering (Ruskell, 1964). Postoperative changes in the retina and optic pathwayscan therefore be attributed with some confidence to the degeneration of axon (Kiernan, 1985). In the present study, the clip was placed 2-3 mm away from the optic nerve head and care was taken to ensure that the crush did not compromise the ophthalmic artery. Therefore, we think that the central retinal artery did not get affected from the compression.由于压伤原因、强度、持续时间不同，压迫神经纤维可以引起不同临床症状(Rydevik and Lundborg, 1977). 目前尚无法完全了解各种损伤的病理生理学，以往曾就压迫神经后引起的局部缺血和神经纤维本身机械变形两者那个是更重要的致病因素做过讨论。起初，把挤压伤归因于局部缺血，原因在于：（1）神经功能依靠血液供给，挤压使灌注受损；（2）即使活体外高压力挤压神经，神经传导也未发生故障(Powell and Myers, 1986)；（3）挤压持续时间延长，神经损伤加重；（4）巨大的有髓神经纤维在局部缺血情况下特别容易损伤。一些著作者已经强调过局部缺血的作用(Merrington and Nathan, 1949; Powell and Myers, 1986)，也有人强调过神经纤维机械变形的重要性(Gelfan and Tarlov, 1956; Ochoaet al., 1972)。挤压或切割均可以离断视神经纤维轴索。在对兔子和大鼠所做实验中，只要损伤在眼球下超过1mm，通常不会导致视网膜局部缺血坏死。首先，这些动物的视网膜中央动脉于视神经盘后进入视神经；其次，视神经内部没有大血管，可能是通过脑脊膜接受血液供应(Ruskell, 1964)。视网膜和视路的术后改变一定程度上可归因于轴索退化(Kiernan, 1985)。此次研究动脉瘤夹置于距离视神经乳头23mm处，同时注意确保挤压不会损伤眼动脉，因此我们认为视网膜中央动脉未受到挤压的影响。Although the crush itself is an acute injury, the propagation of damage is a chronic event. This model can therefore simulate the spreading of neurodegenerative diseases, but not their initiation. Existing models for glaucoma are associated with increased intraocular pressure (Schwartz, 2004; Yoles et al.,1997). Each model has its specific advantages and disadvantages, and it is only by accumulating the relevant data from several models that it will be possible to make valid inferences for clinical situations. It is worth noting that whatever information was obtained from the optic nerve crush model turnedout to be valid for other models as well (Schwartz, 2004; Schwartz and Cohen, 2000). It has been reported that chronic degeneration caused by an increase in intraocular pressure would better simulate the characteristics of glaucoma than crush injury (Schwartz, 2004; Schwartz and Cohen, 2000).虽然挤压伤本身是急性损伤，损伤的传播却是慢性过程。因此本模型可以模拟神经变性疾病的扩展，但是不能模拟神经变性疾病的起始反应。而现有的青光眼模型伴随眼内压增加(Schwartz, 2004; Yoles et al., 1997).。各个模型各有利弊，只有通过积累几个模型的相关数据才能对临床表现做有效推断。值得注意的是，从视神经挤压伤模型中所获得的信息经证明对其他模型也有确实根据(Schwartz, 2004; Schwartz and Cohen, 2000)。据报导，眼内压增加引起慢性退化比挤压伤能更好地模拟青光眼的特征(Schwartz, 2004; Schwartz and Cohen, 2000)。It is often difficult to compare the results obtained by different investigators on nerve compression injuries, owing to differences in method of pressure application and noncomparable pressure levels. In the present study, we tried to overcome some of these problems by using a specially designed and commercially available device. In our previous study (Sarikcioglu and Ozkan, 2003), this device was used to produce quantitative crush injury to the rat sciatic nerve. One of the most important advantages of this device is that, due to its relatively small size, it can reach the narrow approached areas, such as spinal nerve roots, facial nerve, and optic nerve as studied in the present study. The most important disadvantage of this device is that it cannot be used for graded compression injury. The investigator could only change the duration of the compression. We think that standardization of the compression is necessary to compare interlaboratory results.由于挤压方法和压力水平不同，通常很难比较不同研究者对神经挤压性损伤得出的结果。本研究中，我们使用专门设计且市面有售的设备克服这些问题。在我们以往的研究中(Sarikcioglu and Ozkan, 2003)，曾使用这一设备制造小鼠坐骨神经定量损伤。此设备最大的优点之一在于由于其尺寸相对较小，可以到达入路狭窄的区域，如脊神经根，面神经和本次研究中的视神经。其最大的缺点在于不能用来制作不同等级的挤压伤。研究者仅仅能够改变挤压的持续时间。我们认为为了比较不同试验得出的结果，有必要将压力标准化。Although crush of the intraorbital part of the optic nerve has been extensively performed (Becker et al., 2000; Buys et al., 1995; Campbell et al., 1999; Chen and Weber, 2001; Freeman and Grosskreutz, 2000; Meyer and Miotke, 1990; Okada et al., 2005), crush of the intracranial part of the optic nerve has also been reported. Chierzi et al. (1999) performed intracranial optic nerve crush injuries in wild-type mice and in bcl-2 transgenic mice, which overexpress bcl-2 in neurons, including retinal ganglion cells. These investigators found that 100% of retinal ganglion cells were still viable 1 month after optic nerve crush in bcl-2 transgenic mice, compared with 45% retinal ganglion cell survival after crush in wild-type mice.虽然视神经眶内挤压伤模型已广泛完成(Becker et al., 2000; Buys et al., 1995; Campbell et al., 1999; Chen and Weber, 2001; Freeman and Grosskreutz, 2000; Meyer and Miotke, 1990; Okada et al., 2005),然而颅内部视神经挤压伤模型也有报导。Chierzi等（1999）曾用野生鼠和神经元中过度表达bcl-2的转基因小鼠制作视神经眶内挤压伤模型，其中包括视网膜神经节细胞。研究者发现bcl-2转基因小鼠在视神经挤压伤后一个月视网膜神经节细胞仍然100存活，而野生鼠经挤压伤后视网膜神经节细胞仅有45存活。In the literature, there are several types of indirect or direct injuring models, neither of which allows quantitative or standard application of compression. The balloon method was used on the optic nerve of the cat (Burke et al., 1985, 1986; Cottee et al., 1991) and micro-sling was used on the optic nerve of Japanese monkeys (Matsuzaki et al., 1982). Forceps was also used to cause a defined trauma to the optic nerve of rats (Buys et al., 1995; Duvdevani et al., 1990; Kiernan, 1985; Sautter and Sabel, 1993), mice (Li et al., 1999; Tezel et al., 2004), and opossum (Araujo Couto et al., 2004).文献中，直接或间接损伤模型有好几种类型，但是都没有对压力定量或标准化。过去曾使用气球法制作猫视神经损伤模型 (Burke et al., 1985, 1986; Cottee et al., 1991)、采用微悬带制作日本猴视神经损伤模型 (Matsuzaki et al., 1982)、用镊子制作大鼠 (Buys et al., 1995; Duvdevani et al., 1990; Kiernan, 1985; Sautter and Sabel, 1993) 、小鼠(Li et al., 1999; Tezelet al., 2004)及负鼠(Araujo Couto et al., 2004)视神经损伤模型。Chen and Weber (2001) used a smooth-faced bulldog clamp that exerts approximately 1024 gf (grams force) to crush the optic nerve for 15 s. They used this clamp in the cat. Additionally, ligature is also a popular method to crush the optic nerve as Okada et al. (2005) performed a pulleyeweight system to crush the optic nerve of the cat. They used a 6-0 polyfilament silk suture placed around the optic nerve and tightened the suture with a 20 g weight (0.2 N) for 60 s. They were able to accomplishthe optic nerve approach by removal of the orbital roof. We think that these clamps and the pulleyeweight system cannot be used for optic nerve crush of the small laboratory animalsChen和Weber(2001) 曾选猫作试验动物使用表面光滑可以施加压力约1024g的动脉瘤夹损伤视神经15秒。另外，随着Okada等人在2005年完成滑轮砝码系统损伤猫视神经，结扎线也成为损伤视神经的普遍方法。他们通过切除眶顶进入视神经入路，把6-0的多丝缝合丝线置于视神经周围，用20g的压力（0.2N）勒紧缝线60秒。但是我们认为，这些动脉瘤夹和滑轮砝码系统不能用于制作小型试验动物视神经损伤模型。Several reports on optic nerve injury based on crushing of the optic nerve have been published in which the force applied was not stated (Becker et al., 2000; Buys et al., 1995; Campbell et al., 1999; Freeman and Grosskreutz, 2000; Meyer and Miotke, 1990). Taking into account that the optic nerve ofthe rat is a very vulnerable structure, a standardized crush model is mandatory to guarantee defined and reliable conditions for later microscopic or electrophysiological evaluationof the post-traumatic changes in the retinal ganglion cell layer (Gellrich et al., 2002). To solve the standardization problem few attempts have been reported. Duvdevani et al. (1990) developed a Castroviejos cross-action (self-closing) capsule forceps modified by attaching a screw to calibrate the force applied to the optic nerve. They changed the screw nut position to achieve various crush forces. They studied the consequences of the lesion with different severities both electrophysiologically and behaviorally. On the electrophysiological examination, they observed a lesion-dependent loss ofconduction of the compound action potential across the crush site; and more severe the crush, the more severe the initial deficit and the smaller the recovery of the compound action potential. On behavioral examination they observed that the loss of visual function, as defined by rats ability to orienttoward a visual stimulus, and the subsequent recovery also depended on the severity of the lesion. To improve their method, Sautter et al. (1991) attached a micrometer screw to the handle cross-action forceps. These cross-action forceps have also been used by numerous studies (Hanke, 2002; Kipnis et al., 2000; Mawrin et al., 2003; Sautter and Sabel, 1993; Schmitt and Sabel, 1996).以前出版过一些关于视神经挤压性损伤的报导，其中应用的压力没有定量(Becker et al., 2000; Buys et al., 1995; Campbell et al., 1999; Freeman and Grosskreutz, 2000; Meyer and Miotke, 1990)。要考虑到大鼠视神经结构非常脆弱，必须采用标准模型，以确保视网膜神经节细胞层损伤后改变的镜下或电生理学评定条件明确可靠(Gellrich et al., 2002)。报导解决标准化问题的方法很少。Duvdevani等人（1990）发明Castroviejos反作用（闭路）密封舱镊子，安装螺杆以校准加在视神经上的作用力。他们改变螺母位置以获得不同挤压力，研究不同电生理学和行为学严重程度带来的损伤后果。电生理学试验中，他们观察到复合动作电位传导的一种损伤依赖性丧失，压伤越严重，最初的丧失越严重，复合动作电位恢复的程度越少。行为学试验中，观察到视觉功能的丧失似乎取决于大鼠对视觉刺激物的适应能力，随后的恢复也依赖于损伤的严重程度。为改良方法，Sautter等人（1991）将测微螺旋安装到手柄反作用镊上。这种反作用镊也已经被应用到众多研究中(Hanke, 2002; Kipnis et al., 2000; Mawrin et al., 2003; Sautter and Sabel, 1993; Schmitt and Sabel, 1996)。Another attempt to calibrate the optic nerve crush was performed by Klocker et al. (2001). They described a model of crush lesion of the rat optic nerve inducing retrograde retinal ganglion cell degeneration that can be carefully controlled in its extent by a newtonmeter device. They studied the histological consequences of the injury, and also monitored the functional integrity of the retinal ganglion cell projection. They observed that the extent of secondary retinal ganglion cell death increased linearly with the applied crush force. Moreover, visually evoked potentials were used to characterize the con