【作者】 于大志；

【导师】 江华；

【作者基本信息】 第二军医大学 ， 外科学， 2010， 博士

【摘要】 一、研究目的随着临床外科学尤其是显微外科的发展,吻合血管和神经的游离组织移植手术越来越广泛,为了达到重建受区功能和避免供区功能障碍的目的,肌肉的部分移植也变得越来越重要,这就需要我们尽可能详细地了解肌内神经和血管在肌肉内的分布、走行,以及它们之间的相互关系。研究肌内神经分布的方法有解剖、计算机重建和Sihler’s肌内神经染色法。前两种方法存在着许多不足之处,但Sihler’s染色技术能在保持肌肉大体形态的情况下使整个标本变得透明或者半透明,将神经主干及其分支染成蓝紫色,神经在肌肉内的分布及走行清晰可见,被认为是研究肌内神经的理想方法。研究肌内血管分布的方法主要是血管灌注造影剂,在X线下拍片显示。造影剂有很多,应用较多的是硫酸钡和氧化铅,但由于氧化铅的毒性,使得硫酸钡成为主要造影剂,配合钼靶拍照,使得结果清晰明了。以前学者研究肌内神经和血管时都是分开进行的,也就是在一块肌肉上研究肌内神经,而在另外一块肌肉上研究肌内血管,这样就不能准确地反映两者之间的关系。也有学者报道了在同一块肌肉上研究肌内神经和血管的方法,但是没有达到理想的效果。本研究旨在探讨在同一块肌肉上研究肌内神经和血管分布的方法,并在临床常用肌瓣上进行应用。二、研究方法方法一：家兔5只过量麻醉处死后,经腹主动脉向远侧灌注硫酸钡乳胶。乳胶凝固后分离出股薄肌、股二头肌和腓肠肌,继续进行Sihler’s肌内神经染色。具体步骤如下：(1)固定(Fixation):10%的甲醛溶液固定标本3周,液体变混浊时更换液体；(2)浸软和除色素(Maceration and Depigmentation):固定后的组织在流水下冲洗半小时,置入3%氢氧化钾(每100毫升加入3%双氧水0.2毫升),2-3天换液体一次直至肌肉淡黄色褪尽,肌肉发白呈半透明,此过程一般需要2-3周；(3)脱钙(Decalcification):肌肉流水下冲洗半小时入溶液,每5天换液一次,脱钙后的肌肉会丧失透明度或皱缩,此过程需要2-3周；(4)染色(Staining):脱钙后的肌肉蒸馏水浸泡1小时,中间更换蒸馏水2次,浸入Sihler’sⅡ溶液,此过程约需2-3周；(5)脱色(Destaining):染色后,肌肉重新入Sihler’sⅠ溶液中(液体变成黄褐色时及时更换新的Sihler’sⅠ溶液),当肌肉纤维脱色,神经主干及肌肉内分支变成蓝紫色时停止,此过程约需20小时；(6)中和(Neutralization):脱色后的肌肉蒸馏水浸泡1小时,入0.05%碳酸锂溶液(需要加热才能溶解)中浸泡2小时,中间轻轻搅动数次；(7)透明(Clearing):中和后的肌肉逐渐入40%,60%,80%,100%甘油中,每一个浓度浸泡3-4天;(8)保存：最后标本避光保存于100%甘油中(加入少量的麝香草酚)。染色结束后,将标本放置于有机玻璃托盘,标本边缘空白处放置两枚金属标记物,在保持肌肉形态不变的情况下分别进行肌内神经光学背光拍照和钼靶拍照,得到图片A和B,最后在计算机上根据金属标志物将两幅图片完全重叠,画出肌内血管和神经的走行分布模式图,达到在同一块肌肉上同时显示肌内神经和血管的目的。方法二：1、家兔5只过量麻醉处死后,经腹主动脉向远侧灌注半透明红色乳胶。乳胶凝固后分离出股薄肌、股二头肌和腓肠肌。2、新鲜童尸3具,经腹主动脉分别向近侧和远侧灌注半透明红色乳胶。乳胶凝固后分离出背阔肌和股薄肌。将以上分离出的肌肉标本继续进行Sihler’s肌内神经染色。染色步骤同上。染色结束后,在X线观片灯上可以清楚地看到肌内神经和血管,神经显示为蓝紫色,血管显示为红色。这样就能直观地显示神经和血管的准确信息以及它们之间的相互关系,达到了在同一块肌肉的同一张照片上同时显示肌内神经和肌内血管的目的。三、结果方法一中所得到的肌肉标本的图片A和图片B清晰明了,从图片A中能看到肌内神经和肌内血管的信息,图片B中的线条信息全部是肌内血管的分支信息,通过图片重叠,可以将图片A中有关血管的信息辨别出来,剩余的线条信息就是肌内神经的信息。方法二所得到的图片中肌内神经呈现蓝紫色,而肌内血管呈现红色,两者对比明显,使得结果更加准确直观。可以看出研究中的两种方法均能够在同一块肌肉上同时显示肌内神经和肌内血管的信息,清晰直观地显示神经和血管在肌肉内的走行以及两者之间的关系,而且通过这两种方法所得到的结果是一致的。它不仅适用于动物实验,在新鲜尸体上同样具有良好的结果：1、股深动脉分支与闭孔神经前支在进入股薄肌之前有一段相同的走行,两者共同组成血管神经蒂进入肌肉,在肌肉内的走行有明显的伴行关系,据此可以将股薄肌分成数个肌亚部。而股动脉分支则单独走行,与神经分支没有明显的伴行关系。2、胸背动脉和神经在背阔肌外即组成血管神经蒂,入肌点相同,入肌后各自的分支也相互伴行。据此我们可以将背阔肌分成3-4个肌肉亚部,每个肌肉亚部都有相对稳定的血管神经支配。四、结论1、本研究中所发现的两种方法均能达到了预期的目的。1、方法一中通过图片重叠,可以将肌内神经和肌内血管进行区别,绘制出肌内神经和血管的模式图。2、方法二中所得到的图片中,肌内神经呈现蓝紫色,而肌内血管呈现红色,两者颜色差别明显,使得结果更加准确直观。这两种方法均首次清晰直观地显示了同一块肌肉的肌内神经和血管的分布、走行以及两者之间的关系,相比以前的研究方式来说是一个重大创新,对于解剖学和临床医学具有重大而深远的意义。2、这两种方法各有优势。当研究的肌肉比较小,或者肌肉比较扁阔时,建议用比较简单快捷的第二种方法；当研究的肌肉的体积比较大时,建议用第一种方法,以便能更好地区分肌内的神经和血管,使研究结果更加准确。3、通过本研究中的方法对肌肉标本进行研究,具有重要的意义：(1)是对解剖学的重要补充,完善解剖学的内容；(2)指导临床选择理想的手术入路,避免不必要的神经血管损伤；(3)对常用肌肉进行肌亚部划分,指导临床进行部分肌肉移植,既重建受区功能,又避免供区功能障碍；(4)指导临床用同一块肌肉重建几个部位的功能,节约供区资源。4、发现了一些肌内神经血管分布的规律：(1)一块肌腹通常由一条神经主干支配,而血液供应通常有两条以上；(2)当供应肌肉的血管有两条以上时,神经一般伴随肌肉的主要供应血管入肌；(3)当神经血管共同组成神经血管蒂一起入肌时,它们进入肌肉后的分支,分布是紧密伴行的。更多还原

【Abstract】 BackgroundInformation on the distribution of intramuscular nerve and vessel branches within skeletal muscles is very important. This will be very useful to anatomists, physiologists and clinicians.In the study of the intramuscular nerves, anatomical dissection has been the most commonly used method, but it has been limited by the inability to trace the nerve fibers from the extramuscular branches to the intramuscular terminal branches, as the latter are very fine and invisible even under the dissecting microscop. Furthermore, direct dissection would damage muscle fiber and disrupt the normal anatomical relationship between muscle and nerve fibers. Computer reconstruction of serial histological sections, another alternative, is not only time-consuming but also inaccurate due to distortion during tissue cutting, staining, orientation, and reconstruction. Sihler’s stain technique was devised by Sihler in the 1800s. Researchers have modified the technique for various purposes and named it Sihler’s stain in honor of its originator. Sihler’s stain allows a whole specimen to be rendered relatively transparent, even if calcified, while the nerves were counterstained dark purple and were visible to their terminal branches. This is the best method for the intramuscular nerve studies.In 1895, Roentgen discovered x-rays, and the first angiogram was produced by Haschek in 1896 after injecting chalk into the arteries of a cadaveric hand. From then on, all kinds of contrast media were tried, and the most useful of these methods have been barium sulfate and lead oxide injection techniques. Barium sulfate is a well-known radiographic contrast medium.In 1920, Gough injected barium sulfate mixed with gelatin or latex into vessels, and obtained success. In 1936, Salmon perfected the lead oxide technique to study muscle and skin vascular anatomy. So barium was soon replaced by lead oxide as the standard contrast agent for the study of very fine vascular networks. However, the barium sulfate technique has been reappraised lately. In 1999, some authors have improved the technique and obtained angiograms of very high quality by using mammography techniques. Nowadays, barium sulfate technique becomes the simple and effective method。The intramuscular nerve and blood vessels were observed independently before 1994, when Taylor injected radiopaque lead oxide mixture into blood vessels, followed by the anatomical dissection of nerves. In each case the tissue was radiographed with and without the wire markers that was labeled for nerve branches. In this way, a subtraction picture could be obtained by superimposing the radiographs for those cases where it proved difficult to distinguish the wire marker from the vessels. Although the intramuscular nerve branches and blood vessels were displayed in same skeletal muscle, the nerve branches within the skeletal muscle was still shown by anatomical dissection,which has inability to trace the nerve fibers from extramuscular branches to the intramuscular terminator. Indeed, direct dissection would damage muscle fiber and disrupt the normal anatomical relationship between muscle and nerve fibers.In this experiment, we will invent a method which can display the intramuscular nerve and vessel branches simultaneously.MethodsMethod one:Five rabbits were used in this study. After sacrifice with an overdose of chloral hydrate, the abdominal aorta and inferior vena cava were exposed through median abdominal incision. Subsequently, an abdominal aortic catheterization was performed downward to infuse physiological saline, whilst the inferior vena cava was opened. In the process, the lower limb was massaged to promote the removal of congestion. The infusion of physiological saline was stopped when the venous effluent was clear. The perfusion fluid which composed of liquid silicon rubber and superfine barium sulfate was perfused into the abdominal aorta using a 10ml syringe. The injection was maintained for additional one minute until the syringe underwent sentient elastic resistance. In this study, about 6.8ml mixture was used to each rabbit. After successful perfusion, the abdominal aorta was ligated to avoid outflow of mixture. Afterward, the specimens were immersed in 10% formaldehyde solution for two days, and then the gracilis, biceps femoris and gastrocnemius were incised and subjected to modified Sihler’s stain. During the incision, the complete neurovascular pedicle must be reserved. After the procedur mentioned above, the specimens were then stained by Sihler’s technique. In brief, muscle specimens were fixed in 10% unneutralized formalin for about 3 weeks. For further processing, muscles were washed with running water for about half an hour and then placed in a solution of 3% potassium hydroxide (KOH). For depigmentation,0.2 ml of 3% hydrogen peroxide was added per 100 ml of aqueous KOH. The total duration for immersion of the muscles was determined by the size of muscle and varied from 2 to 3 weeks. The specimens were then transferred into Sihler’s Solution I which contained 1 part glacial acetic acid,2 parts glycerin, and 12 parts of 1% aqueous chloral hydrate for about 2 to 3 weeks. This solution was changed every 5 days. The specimens were then stained by immersing the whole muscle in Sihler’s SolutionⅡ(which is composed of 1 part of Ehrlich’s hematoxylin,2 parts of glycerin, and 12 parts of 1% aqueous chloral hydrate). Usually, the immersion time lasted 2 to 3 weeks. The specimens were then destained in Sihler’s SolutionⅠand the solutions were changed when it became purple. When the muscle fiber becomes semitransparent and the intramuscular nerve branches become purple, they were washed in a solution containing 0.05% lithium carbonate for about 2 hours, so that the colors of the nerves changed from purple to deep blue. The specimens were then transferred to glycerin solutions of increasing concentration (40%,60% and 80% respectively, each transfer lasting for 3-4 days). Finally, specimens were stored in pure glycerin. After stained with modified Sihler’s technique, the skeletal muscles were transferred onto a transparent organic glass tray. And then, two mental markers were placed above and below the specimens. Using a back-light, the muscle specimens were photographed with a digital camera. Subsequently, the specimens were radiographed with Molybdenum Target Photograph Machine to display distribution of the intramuscular arteries. In this process, the position of muscles and metal markers should be maintained. With the help of metal markers, the two photographs can be overlaped completely in a computer. So we can draw the schematic of intramuscular nerve and artery。Method two:In this study, we substitute the fluid which composed of liquid silicon rubber and superfine barium sulfate with red semitransparent liquid silicon rubber. After stained with modified Sihler’s technique, the muscle specimens were placed on a back-light viewbox, and were photographed with a digital camera. From the photograph, we can see clearly that the intramuscular nerve branches were stained purple blue and the artery branches red. Even we can place the muscle specimens into transparent airtight cans for clinical teaching.ResultsMethod one:The results showed that the skeletal muscle were integrated and semitransparent in digital photos. And, the muscle fibers were shown as light blue. The intramuscular nerve branches were exhibited as purple blue or black, while the arterial branches were displayed in black. The distribution of arterial branches was revealed perspicuously in radiograph. Based on the superimposing of digital and radiographic pictures, the orientation and distribution of the intramuscular nerves and arterial branches were shown in one picture, which can enhance the understanding of accurate relationship between the muscle fibers, nerve branches and blood vessels.Method two:Clear photographs were obtained. Intramuscular nerve branches were stained purple blue and artery branches were red.Conclusions1. The two methods were able to achieve the desired purpose. In methods one, with the help of radiophotograph and computer, intramuscular nerve branches can be distinguished from the artery branches, and so we can draw out the schematic of intramuscular nerve and artery. In the method two, the intramuscular nerve branches were stained purple blue and the intramuscular artery branches were red. This is a more direct-viewing method.2. Both methods have their own advantages. When the muscles are relatively small or flat, it is recommended to use the second method which is relatively simple and fast. When the muscles are relatively large, it is recommended to use the first method in order to distinguish the intramuscular nerves and arteries clearly which could make the results more accurate.3. There are important significances to study muscle specimens with our methods.(1). This is an important complement to gross anatomy;(2). Guide surgeons select the ideal surgical approach, avoiding unnecessary neurovascular injury;(3). We can divide muscles into several departments. So we can transplants one of the departments, both repairing functions of the operation area and avoiding dysfunctions of donor site.更多还原