Morphological Studies On Muscles That Were Commonly Used In Clinical Settings Using Simultaneous Displaying Techniques

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.