The optic nerve of the rat is a very vulnerable structure (Gellrich et al., 2000). 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，2000）。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., 2000). 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，2000）。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 aneurysm
clip, 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.
2. Materials and methods
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.
2.2. Yasargil aneurysm clip
Yasargil aneurysm clips (Aesculap AG & Co., Tutlingen, Germany) are designed for permanent occlusion of cerebral aneurysms (Yasargil, 2000). 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).
2.3. Optic nerve crush
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 evaluation
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 site
were then collected. Samples were fixed with 4% glutaraldehyde in 0.1 M Sorensen’s 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 900E 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 Sorensen’s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（40－60nm）,用Zeiss LEO 900E电子显微镜检测所制备切片。
3.1. Application of surgery
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 difficult
to 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).
3.2. Light and electron microscopic evaluation
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).
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).