Visualization Of Blood Vessels By Evans Blue And Its Application In The Study Of Spinal Cord Injury

Spinal cord injury (SCI) often leads to lifelong disability of the patient, resulting in suffering of the patient and burden of the family and society. At present there is still no effective therapy. Therefore it is difficult and important to study the pathology and therapy of SCI. The pathological process includes the primary injury and the secondary injury. The primary injury causes mechanical trauma of cells and tissue, and also diversified noxious agents which are cytotoxic damage adjacent spinal cord segments and lead to the secondary injury. The necrotic wave spreads in the direction of rostra and cauda, and leads to a greater injury several times more than the original injury. Therefore the mechanism, pathological changes and therapy of the secondary injury are of great importance. Ischemia is the important pathological mechanism for the reduction of spinal cord blood will exacerbate SCI. To study ischemia in the secondary spinal cord injury, the angiography that displays the blood vessels and associates with the immunohistochemical staining is the essential study method.At present, methods used to display blood vessels mainly contain ink perfusion, immunohistochemical method, tannic acid-ferric chloride staining, enzyme histochemical method and so on. However, they all have disadvantages, for example, unable to do immunohistochemical staining, troublesome or fluorescence quenching and so on. We use Evans Blue to dispaly blood vessels. Evans Blue is a fluorescent dye, which is blue at the bright field and red under the excitation of green light with fluorescent microscope. It is used to study the breakdown of the blood brain barrier. Evans Blue can bind to plasma albumin forming Evans Blue-albumin complex, and then combine with the receptor for albumin on the membrane of vascular endothelial cell. Therefore Evans Blue can be perfused to display blood vessels theoretically.There are two sections in the experiment. First we established the method. The experiment animals are C57BL/6 mouse and SD(Sprague-Dawley)rat. 12 C57BL/6 mice are divided into 2 groups: control group, uninjury perfusion group. After injection of EB through the femoral vein (the control group are given normal saline instead), the mice are perfused with normal saline and paraformaldehyde, sliced. Then one slice is dehydrated and transparentized with 100% ethanol and fixed with neutral balata, another slice is done HE staining, and the third slice is done immunostaining for GFAP. 15 SD rats are divided into 2 groups: Post-fixation group and Prefixation group. The method for post-fixation group likes C57BL/6 mouse, including control group, 5% Evans Blue group and 8% Evans Blue group. Prefixation group is divided to two groups: 5% gelatin-Evans Blue group and gelatin-Potassium chromium sulfate-Evans Blue-albumin group. After perfusion with normal saline and paraformaldehyde, the rats are respectively injected solutions to display blood vessels. After gelatin becomes hard, the tissue is taken out, sliced, dehydrated and transparentized with 100% ethanol, fixed with neutral balata or done immunohistochemical study. The results can be summarized as follows: When C57BL/6 mice are given 0.5ml 5% Evans Blue, the blood vessels in the spinal cord are clearly displayed. For Post-fixation group of SD rats, 5ml 8% Evans Blue is enough to show the blood vessels clearly. For prefixation group, when given 5% gelatin-Evans Blue, the blood vessels are fully displayed. Immunohistochemical study can be done in gelatin-Potassium chromium sulfate-Evans Blue-albumin group.In the second section, we use the method in spinal cord injury. The experiment animal are SD rats. Two weeks after injury, they are operated respectively by post-fixation and prefixation method. The results indicate the configuration of blood vessels between injury area and normal area, also the breakdown of BBB can be observed. Immunohistochemical study can be done on neighboring slide. In prefixation group, when given 5% gelatin-Evans Blue, the blood vessels are clear and there is little Evans Blue diffusion next to the center of injury. When given GCEA solution, not only the blood vessels are displayed, but also immunohistochemical study can be done. However, the effect of blood vessels is not so good as 5% gelatin-Evans Blue group.In conclusion, we study method to visualize blood vessels with Evans Blue systemically and the results reveal that Evans Blue is a sensitive fluorescent dye to display blood vessels in spinal cord. Different fixation method and additive can bring different effects. Some method can do immunohistochemical study and this is very important for spinal cord research. You can use one method or combined method as you need in research.