大鼠皮质脊髓束Luxol Fast Blue染色

目的:探讨显示大鼠皮质脊髓束的特殊染色方法。方法:选用正常成年SD大鼠延髓和脊髓冰冻切片,应用Luxol Fast Blue染色法进行染色。结果:Luxol Fast Blue染色后,在延髓和脊髓切片中可见灰质呈淡红色,白质呈蓝色,延髓锥体染成深蓝色。锥体中Luxol Fast Blue标记的深蓝色阳性纤维,经锥体交叉后至脊髓灰质后连合背侧,沿脊髓后索腹侧深层下行,至荐段后逐渐消失。在延髓锥体和脊髓颈、胸、腰段后索中,深蓝色的Luxol Fast Blue阳性纤维边界清晰,与周围结构区分明显。结论:运用Luxol Fast Blue染色可清楚显示大鼠皮质脊髓束在脊髓内的定位,是一种简便可靠的皮质脊髓束形态学研究方法。

Application of Luxol Fast Blue staining in locating the corticospinal tract in adult rats

BACKGROUND: There are many methods for myelin staining, mordant, or oil-soluble dye or the special reaction of osmic acid with lipoid is used according to different principles. The commonly used methods are classic Well staining, classic lithium carbonate-haematine staining, fast green staining, silver staining, etc. Luxol Fast Blue can brightly stain myelin sheath, and has certain specificity. The background can be very clean if there is proper differentiation, whereas Luxol Fast Blue is cheap and convenient to operate, thus it is an ideal staining reagent for routine myelin sheath. OBJECTIVE: To show the corticospinal tract of normal adult rats with Luxol Fast Blue staining method. DESIGN: A repetitive measurement design. SETTINGS: Institute of Nuerobiology, Nantong University; Department of Rehabilitation Medicine, Affiliated Hospital of Nantong University. MATERIALS: Six healthy adult male SD rats of clean degree, weighing averagely 300 g, were provided by the experimental animal center of Nantong University. 1 g/L Luxol Fast Blue solution was provided by Sigma Company; Leica CM1900 cryostat microtome by Leica Company; Leica DMR microscope by Leica Company. METHODS: The experiment was carried out in the Staff Room of Human Anatomy, Nantong University in May 2005. The rats were given intraperitoneal injection of combined anesthetic (2 mL/kg), then the chest was open for perfusing saline and phosphate buffer containing formamint via heart. Brain and spinal cord were removed after 1 hour then fixed, then changed to phosphate buffer (pH 7.4) containing 300 g/L saccharu at 4 ℃, and stayed overnight, tissue blocks at pyramid, decussation of pyramid and cervical, thoracic, lumbar and sacral segments of spinal cord were removed to prepare continuous horizontal frozen sections (30 μm) after sedimentation, the sections were dried at room temperature. The corticospinal tract of normal adult rats were shown with Luxol Fast Blue staining method, and observed under Leica DMR microscope. MAIN OUTCOME MEASURES: Positive fibers in Luxol Fast Blue staining. RESULTS: After the Luxol Fast Blue staining, the labeled myelinated nerve fibers were bright blue. They located in the pyramid, decussation of pyramid and the ventral part of posterior funiculus in cervical, thoracic, lumbar and sacral segments of spinal cord. CONCLUSION: Luxol Fast Blue staining method may manifest the distribution of corticospinal tract with clear distinct in adult rats.

Early brainstem hemorrhage progression:multi-sequence magnetic resonance imaging and histopathology

According to clinical statistics,the mortality of patients with early brainstem hemorrhage is high.In this study,we established rat models of brainstem hemorrhage by injecting type Ⅶ collagenase into the right basotegmental pontine and investigated the pathological changes of early brainstem hemorrhage using multi-sequence magnetic resonance imaging and histopathological methods.We found that brainstem hematoma gradually formed in the injured rats over the first 3 days and then reduced after 7 days.The edema that occurred was mainly of the vasogenic type.No complete myelin sheath structure was found around the focus of the brainstem hemorrhage.The integrity and continuity of nerve fibers gradually deteriorated over the first 7 days.Neuronal degeneration was mild in the first 3 days and then obviously aggravated on the 7^(th)day.Inflammatory cytokines,interleukin-1β,and tumor necrosis factorαappeared on the 1st day after intracerebral hemorrhage,reached peak levels on the 3^(rd)day,and decreased from the 7^(th)day.Our findings show the characteristics of the progression of early brainstem hemorrhage.

磷脂酰肌醇-3激酶/蛋白激酶B/FoxO1信号通路和白细胞介素17在小鼠实验性自身免疫性脑脊髓炎中的表达变化

目的探讨磷脂酰肌醇-3激酶(PI3K)/蛋白激酶B(Akt)/FoxO1和白细胞介素17(IL-17)与自身免疫性脑脊髓炎(EAE)发病的相关机制。方法将C57BL/6小鼠60只随机分为对照组和模型组(EAE),每组30只。采用髓鞘少突胶质细胞糖蛋白(MOG35~55)联合完全弗氏佐剂诱导建立EAE模型。观察各组小鼠的行为学评分,并于评分4分时取各组小鼠的脊髓、脾脏和外周血。HE染色及Luxol fast blue染色观察脊髓炎性细胞浸润及髓鞘脱失情况;脾细胞在体外用a-CD3和MOG35~55分别刺激7 d,收集其上清液,ELISA检测小鼠脾细胞上清液和血清中细胞因子白细胞介素17(IL-17)和干扰素γ(IFN-γ)的含量;流式细胞术检测小鼠脾脏中CD4+IL-17+细胞的百分比;Western blotting检测IL-17及PI3K/Akt/FoxO1在小鼠脊髓中表达。结果与对照组相比,EAE组小鼠行为学评分明显高于对照组(P<0.05);HE染色及Luxol fast blue染色结果显示,EAE组脊髓病理组织表现出炎性浸润和髓鞘脱失显著增多(P<0.05)。ELISA结果显示,EAE组血清中及体外培养脾细胞的上清中IL-17和IFN-γ的含量明显升高。流式结果表明,EAE组小鼠脾细胞中CD4+IL-17+T细胞的百分比明显增加(P<0.05)。Western blotting结果显示,EAE组小鼠脊髓IL-17、磷酸化Akt(p-Akt)蛋白水平明显升高,但磷酸化FoxO1(p-FoxO1)蛋白水平显著降低(P<0.05)。结论 EAE组小鼠脊髓中促炎因子IL-17分泌增加,可能与PI3K/Akt/FoxO1信号通路活化进而激活T细胞功能有关。

大鼠创伤性脊髓损伤模型的建立

目的:建立一种控制性脊髓挫伤大鼠模型。方法:采用BenchmarkTM立体定位颅脑撞击器制备大鼠创伤性脊髓损伤,并在伤后1、2、3、5、7、142、8 d借助斜板试验及BBB评分评价其运动功能,然后观察损伤部位及其邻近区域的形态学改变。结果:脊髓损伤后大鼠在斜板上维持的角度以及BBB评分显著低于假手术组(P<0.01)。损伤部位可见神经纤维肿胀。灰质部运动神经元肿胀、坏死,尼氏体淡染,甚至溶解。小胶质细胞明显增生。通过铜银染色和Luxol固蓝染色,可见运动神经元溃变以及髓鞘脱失。结论:建立的控制性脊髓挫伤大鼠模型重复性好,适合神经保护药物的药效学筛选。

骨髓间充质干细胞异体移植对小鼠实验性自身免疫性脑脊髓炎的治疗作用

目的探讨大鼠骨髓间充质干细胞(BMSCs)异体移植对小鼠实验性自身免疫性脑脊髓炎(EAE)的治疗作用。方法全骨髓贴壁培养法获得大鼠BMSCs,流式细胞术检测细胞免疫表型,并诱导成骨方向分化。取雌性C57BL/6小鼠,随机分为3组:正常对照组、PBS组和大鼠BMSCs组,用髓鞘少突胶质细胞糖蛋白(MOG)35~55联合完全弗氏佐剂诱导建立EAE模型。小鼠免疫后38d和48d腹腔注射PBS或大鼠BMSCs进行治疗,神经功能评分观察各组小鼠神经功能变化。二次治疗12d后取各组小鼠脊髓、脾脏和外周血。HE染色及Luxol fast blue染色观察脊髓炎性细胞浸润及髓鞘脱失情况;脾脏制成单细胞悬液,细胞CFSE标记后10mg/L刀豆球蛋白(Con A)和MOG_(35~55)刺激培养3d,观察脾细胞增殖情况。ELISA检测大鼠BMSCs移植后外周血细胞因子干扰素γ(IFN-γ)、白细胞介素17(IL-17)含量。结果流式细胞术显示,大鼠BMSCs第3代(P3)细胞表达抗原CD29、CD90、CD106,不表达CD45。体外诱导其能向成骨分化。小鼠发病后,大鼠BMSCs组小鼠神经功能缺损症状较PBS组减轻,评分降低。HE染色和Luxol fast blue染色结果显示,大鼠BMSCs组脊髓炎细胞浸润和脱髓鞘比同时间点PBS组减轻(P<0.05)。Con A和MOG_(35~55)刺激培养后,PBS组和大鼠BMSCs组脾细胞增殖增加,而大鼠BMSCs组又较PBS组降低。与PBS组相比,大鼠BMSCs组血浆细胞因子IFN-γ、IL-17含量降低(P<0.05)。结论全骨髓贴壁培养法能有效分离纯化BMSCs,大鼠BMSCs异体移植对小鼠EAE模型有治疗作用。

两种髓鞘染色方法在Cuprizone诱导脱髓鞘小鼠模型中的比较

目的比较2种神经髓鞘染色方法在Cuprizone小鼠模型脑组织中脱髓鞘的形态学变化,为实验诊断提供病理依据。方法将Cuprizone掺入饲料中喂食小鼠4周,造成神经脱髓鞘模型。取脑后切片,分别用牢固蓝(Luxol Fast Blue)和油红O两种染色方法观察脑纹状体和海马两个切面胼胝体髓鞘脱失变化。结果牢固蓝和油红O两种染色方法均可显示Cuprizone模型小鼠脑内胼胝体部位有脱髓鞘发生,但在纹状体切面油红O染色法能清晰显示髓鞘脱失和炎性细胞浸润,而牢固蓝染色法显示髓鞘脱失与周围组织结构界限不明显。结论与牢固蓝染色法相比,油红O染色法能更清晰显示脱髓鞘,颜色对比明显,是一种简单易行的神经髓鞘染色方法。

冷冻连续切片重建数字人腰髓

目的:探讨数字人脊髓数字化问题,建立一种实用的脊髓三维重建方法。方法:对人腰髓节段的连续冷冻切片直接贴片,进行Luxol Fast Blue染色,采集切片图像并进行图像拼接,Photoshop 7·0软件对图像进行"图像灰度化"、"反相"、"对比度调整"、"图像缩小"等后处理,3D-DOCTOR 4·0软件进行表面重建和体重建。结果:重建出人腰髓节段及其内部的灰质结构具有立体感,并可在计算机上进行自由观察和测量。结论:利用人腰髓连续切片可以进行人体脊髓的三维重建。

Three-dimensional reconstruction of human spinal cord based on histological serial sections

BACKGROUND： It is not possible to reconstruct the inner structure of the spinal cord, such as gray matter and spinal tracts, from the Visual Human Project database or CT and MRI databases, due to low image resolution and contrast in macrosection images. OBJECTIVE： To explore a semi-automatic computerized three-dimensional （3D） reconstruction of human spinal cord based on histological serial sections, in order to solve issues such as low contrast. DESIGN, TIME AND SETTING： An experimental study combining serial section techniques and 3D reconstruction, performed in the laboratory of Human Anatomy and Histoembryology at the Medical School of Nantong University during January to April 2008. SETTING： Department of Anatomy, Institute of Neurobiology, Jiangsu Province Key Laboratory of Neural Regeneration, Laboratory of Image Engineering. MATERIALS： A human lumbar spinal cord segment from fresh autopsy material of an adult male. METHODS： After 4% paraformaldehyde fixation for three days, serial sections of the lumbar spinal cord were cut on a Leica cryostat and mounted on slides in sequence, with eight sections aligned separately on each slide. All sections were stained with Luxol Fast Blue to reveal myelin sheaths. After gradient dehydration and clearing, the stained slides were coverslipped. Sections were observed and images recorded under a light microscope using a digital camera. Six images were acquired at x25 magnification and automatically stitched into a complete section image. After all serial images were obtained, 96 complete serial images of the human lumbar cord segment were automatically processed with ＂Curves＂, ＂Autocontrast＂, ＂Gray scale 8 bit＂, ＂Invert＂, ＂Image resize to 50%＂ steps using Photoshop 7.0 software. All images were added in order into 3D-DOCTOR 4.0 software as a stack, where serial images were automatically realigned with neighboring images and semi-automatically segmented for white matter and gray matter. Finally, simple surface and volume reconstruction were completed on a personal computer. The reconstructed human lumbar spinal cord segment was interactively observed, cut, and measured. MAIN OUTCOME MEASURES： The reconstructed human lumbar spinal cord segment. RESULTS： Compared with serial images obtained from other image modalities, such as CT, MRI, and macrosections from The Visual Human Project database, the Luxol Fast Blue stained histological serial section images exhibited higher resolution and contrast between gray and white matter. Image processing and 3D reconstruction steps were semi-automatically performed with related software. The 3D reconstructed human lumbar cord segment were observed, cut, and measured on a PC. CONCLUSION： A semi-automatically computerized method, based on histological serial sections, is an effective way to 3D-reconstruct the human spinal cord.

冰冻切片的H&E和LFB染色技术的优化及其在自身免疫疾病中的应用

该文旨在探讨基于冰冻切片样品进行苏木素–伊红(hematoxylin-eosin,H&E)染色和快蓝(luxol fast blue,LFB)染色方法的优化及其在实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis,EAE)组织病理分析中的应用。取正常及EAE建模后的C57BL/6小鼠脊髓样品各5份,每份脊髓样品一分为二,分别制成冰冻切片和石蜡切片样品,通过设置不同优化条件对这两组切片进行H&E和LFB染色,比较两组切片染色的结果。石蜡切片经过H&E染色和LFB染色后细胞的形态结构和组织结构清晰,冰冻切片H&E染色和LFB染色后细胞结构清晰度几乎和石蜡切片一致,进一步将该方法应用于分析β-arrestin2基因敲除对于小鼠EAE发生中的病理变化,发现能够很好地重复出基因敲除加重EAE疾病这一现象。冰冻切片染色优化后,基于冰冻切片进行H&E染色和LFB染色可以达到石蜡切片类似的效果,并且实验过程简洁快速,大大缩短了实验时间,提高了效率,可以较好地应用于分析自身免疫疾病病理变化,该优化方法将在自身免疫疾病特别是多发性硬化动物模型的研究中有广泛的应用。