【作者】 刘苏；

【导师】 吕广明；

【作者基本信息】 南通大学 ， 人体解剖与组织胚胎学， 2006， 硕士

【摘要】 第一部分皮质脊髓束在大鼠脊髓内的精确定位目的：确定大鼠皮质脊髓束在延髓和脊髓白质内的走行和精确定位。方法：选用成年SD大鼠，采用Luxol fast blue(LFB、)染色法和protein kinase Cγ(PKCγ)特异性抗体免疫组织化学染色法在冠状面、矢状面、横断面三种不同切面上对正常大鼠皮质脊髓束进行解剖定位。结果：在延髓锥体中，LFB染色标记的深蓝色的有髓纤维经锥体交叉至脊髓颈、胸、腰、骶段后索腹侧，与周围淡染的纤维易于区别；PKCγ阳性反应产物呈棕色，分布于延髓腹侧面的锥体中，在延髓锥体下部绝大部分交叉到背侧面，在脊髓白质内沿后索腹侧下行，至脊髓的骶尾段。同时延髓和脊髓背角神经元中也有PKCγ免疫阳性物质存在，在脊髓前索和外侧索未见有PKCγ阳性反应产物。LFB阳性纤维和PKCγ免疫阳性反应产物在延髓锥体和脊髓后索内的定位分布与大鼠皮质脊髓束在延髓和脊髓中的位置和走行相一致。结论：运用LFB染色和PKCγ特异性抗体免疫组织化学染色可以清晰对正常大鼠皮质脊髓束进行精确解剖定位，为进一步研究皮质脊髓束的损伤和功能重建提供形态学基础。第二部分大鼠皮质脊髓束半横段损伤模型的建立目的：成功制作大鼠皮质脊髓束半横断损伤模型。方法：选择性切割大鼠延髓左侧锥体，建立大鼠皮质脊髓束半横断损伤模型，采用斜板实验进行行为学检测，运用LFB染色法、生物素化葡聚糖胺(BDA)神经示踪法和PKCγ免疫组织化学染色法对模型进行形态学评判。结果：皮质脊髓束损伤组大鼠术后右侧前后肢瘫痪，斜板实验显示运动功能受限；LFB染色可以看出损伤部位左侧皮质脊髓束无Luxol Fast Blue浓染神经纤维束；BDA神经示踪法结果显示在锥体交叉平面，仅见一束BDA阳性反应产物经右侧交叉至左侧，在脊髓后索的左侧半下行至骶尾段，而在损伤平面以下，未见有BDA阳性反应产物经左侧锥体交叉至右侧，在整个脊髓后索的右侧半未见有BDA阳性反应产物。PKCγ免疫组织化学染色结果发现延髓锥体右侧半有一束PKCγ阳性反应产物经右侧交叉至左侧，脊髓后索腹侧部左侧半有PKCγ阳性反应产物，而脊髓后索腹侧部右侧半未见有PKCγ阳性反应产物。结论：选择在延髓切断一侧锥体制作皮质脊髓束半横断损伤模型，定位准确，重复性好，结果可靠，是研究皮质脊髓束的可塑性和神经轴突再生的理想动物模型。BDA神经束路示踪法和PKCγ免疫组织化学染色法可作为皮质脊髓束半横断损伤模型的形态学指标。第三部分大鼠皮质脊髓束半横断损伤后皮质脊髓束超微结构的变化目的：观察皮质脊髓束半横断损伤后皮质脊髓束超微结构的变化。方法：选择性切割大鼠延髓左侧锥体，建立皮质脊髓束半横断损伤模型，模型制作后4d、14d、28d，采用透射电镜观察受损皮质脊髓束超微结构的变化。结果：皮质脊髓束半横断损伤后，受损皮质脊髓束髓鞘和轴索发生肿胀，形态不规则。随着时间的延长，溃变进行性加重，受损皮质脊髓束主要表现为髓鞘破坏、溶解及轴索脱髓鞘病变、胞浆浓缩、细胞器增多及空泡样变性。脊髓颈、胸、腰段受损皮质脊髓束的溃变比损伤部位严重。结论：皮质脊髓束半横断损伤后，受损皮质脊髓束中的髓鞘和轴索发生进行性溃变。更多还原

【Abstract】 PartⅠPrecise Localization of Corticospinal Tract in Spinal Cord in Adult RatsObjective: To detect the precise localization of corticospinal tract in medulla oblongata and white matter of spinal cord in adult rats. Methods: Anatomical localization of the corticospinal tract were revealed by using LFB staining method and protein kinase Cγimmunohistochemistry at coronal, sagittal and transverse sections of medulla oblongata and cervical, thoracic, lumbar and sacral segments of spinal cords in normal adult Sprague-Dawley rats. Results: The labeled myelinated nerve fibers were dark blue and clearly identified with surrounding hypochromatic nerve fibers in the pyramid, decussated to the ventral part of posterior funiculus in cervical, thoracic, lumbar and sacral segments of spinal cord. PKCγimmunoreactivities were brown and expressed in pyramid. Most of PKCγimmunoreactivities decussated posteriorly and projected to the dorsal part of contralateral side of medulla oblongata. In the spinal cord, the PKCγimmunoreactivities were located at the ventral part of dorsal funiculus in cervical, thoracic, lumbar segments of spinal cord and at the middle of dorsal funiculus in sacral segments. In addition, PKCγimmunoreactivities were also expressed in the neurons at dorsal horn of medulla oblongata and different segments of spinal cord. There were no PKCγimmunoreactivities in the ventral funiculus and the lateral funiculus of spinal cord. The distribution of LFB staining fibers and PKCγimmunoreactivities at medulla oblongata and white matter of spinal cord in adult rats were correspondence with the position and courser of corticospinal tract in rodents. Conclusions: Using LFB staining method and PKCγimmunohistochemical staining method can precisely reveal the anatomical localization of corticospinal tract in rats and provide the morphological foundation for studying the lesion and functional recovery of corticospinal tract.PartⅡEstablishment of Corticospinal Tract Semitransection Model in Adult RatsObjective: To establish corticospinal tract semitransection model in adult rats successfully. Methods: Tiltboard test, LFB staining, biotin dextran amine (BDA) tracing technique and PKCγimmunohistochemistry were utilized to assess the function and morphology of corticospinal tract semitransection model. Results: Forelimbs and hindlimbs at right sides were paralyzed after unilateral corticospinal tract semitransection. Tiltboard test showed the motor function of the opposite side of lesion side were limited. There was no LFB dense dye nerve fiber bundle in left pyramid at the level of injury. BDA tracing method and PKCγimmunohistochemistry showed only one bundle of BDA and PKCγpositive fibers decussated from right to left at the level of decussation of pyramid, then descended to sacral segments at the left part of dorsal funiculus of spinal cord. Under the level of injury, there were no BDA and PKCγlabeling nerve fibers decussated from left to right at the level of decussation of pyramid and descended at the right part of dorsal funiculus of spinal cord. Conclusions: Selectively semitransect the unilateral pyramidal tract at pyramid of medulla oblongata is easy to locate the position of corticospinal tract and the models are effective and reproducible. It is an ideal animal model for studying the plasticity and regeneration of corticospinal tract. BDA tracing technique and PKCγimmunohistochemistry are effective morphological assessments of corticospinal tract semitransection model. PartⅢThe Changes of Ultrastructure of Corticospinal Tract after Corticospinal Tract Semitransection in RatsObjective: To investigate the changes of ultrastructure of corticospinal tract after corticospinal tract semitransection in rats. Methods: Electron microscopy were utilized to determine the degeneration of the corticospinal tract at 4, 14, 28 days after corticospinal tract semitransection. Results: The early ultrastructural morphology revealed that the myelin sheath and axons of lesioned corticospinal tract were swelling and irregular in shape. Subsequently, during the extended periods, the ultrastructural changes were myelinoclasis, dissolvation in myelin sheath, and demyelination, shrink of cytoplasm, vacuolar degeneration, increase of organelles in axons progressively. These changes of lesioned corticospinal tract at cervical, thoracic, lumbar segments of spinal cord were more serious than those closed to lesion site. Conclusions: These results indicate that the myelin sheath and axons undergo progressive and extensive degeneration in lesioned corticospinal tract.更多还原