不同配伍生物膜对鸡胚绒毛尿囊膜血管生成的影响：寻求最佳组合方式*

背景：如何使生长因子安全的在体内稳定持久的表达,并获得长期的促血管生成效应是亟待解决的问题,这就需要一个良好的药物缓释系统,即可控释的生物模拟系统。 目的：探讨不同配伍的生物膜对鸡胚绒毛尿囊膜血管生成的影响,寻找最佳的组合方式。 设计、时间及地点：生物材料学体外观察,于2008-06/2009-05在江苏省中医院中心实验室完成。 材料：通过交联剂将肝素、中药与Ⅰ型胶原发生共价结合,然后rhVEGF165、碱性成纤维细胞生长因子与肝素发生生理性结合,将血管生成素1制成微球均匀植入生物膜内,即为实验所需生物膜。 方法：将制备的生物膜置于正对观察窗的鸡胚绒毛尿囊膜表面的血管最少处,然后贴在鸡胚绒毛尿囊膜表面上,封口膜封闭假气室,继续孵育。实验组在给予生物膜基础上,依次加入0,10,50,100 ng碱性成纤维细胞生长因子,从中筛选出最显著促进鸡胚绒毛尿囊膜血管新生的组合作为生物膜1;再依次加入100 μL丹参注射液、黄芪注射液、三七总甙注射液、川芎嗪注射液,同法筛选出生物膜2;再依次加入10,50 ng血管生成素Ⅰ,同法筛选出生物膜3作为实验最终结果。同时设立空白对照组,鸡胚绒毛尿囊膜表面未贴生物膜,仅加入PBS。加药7 d后取绒毛尿囊膜,拍摄给药部位,采用Image Pro Plus 5.0.2进行数据收集。 主要观察指标：不同配伍的生物膜对血管新生面积的影响。 结果：①与空白对照组比较,生物膜+0,10,50,100 ng碱性成纤维细胞生长因子各组新生血管面积均明显增加,其中生物膜+100 ng碱性成纤维细胞生长因子组增加幅度最高(P < 0.01),为此将其选为生物膜1。②与生物膜1组比较,生物膜1+三七总甙注射液组、生物膜1+川芎嗪注射液组无明显变化,生物膜1+丹参注射液组、生物膜1+黄芪注射液组新生血管面积分别增加25.48%,63.21%(P均 < 0.01),因此将生物膜1+黄芪注射液组作为生物膜2的最佳组成。③与生物膜2组比较,生物膜2 + 10 ng血管生成素Ⅰ组新生血管面积无明显差异;生物膜2 + 50 ng血管生成素Ⅰ组新生血管面积增加21.49%(P < 0.05),因此将其作为生物膜3。 结论：在实验制备的生物膜基础上,加入100 ng碱性成纤维细胞生长因子、100 μL黄芪注射液组、50 ng血管生成素Ⅰ的组合,具有明显促进鸡胚绒毛尿囊膜血管生成的作用。 关键词：生物膜;鸡胚绒毛尿囊膜;血管生成     

骨髓液促进鸡胚绒毛尿囊膜血管的生成

背景：生长因子能促进侧支血管的发育,且多种因子协同效果更为明显,骨髓液中富含多种生长因子。 目的：观察血管内膜损伤后的骨髓液对鸡胚绒毛尿囊膜血管生成的作用。 方法：受精蛋70枚在(37.5±0.5) ℃条件下孵育,第7天开窗,第8天将存活鸡胚随机分为生理盐水组、正常血清组、正常骨髓液组、损伤血清组、损伤骨髓液组以及血管内皮生成因子组,每组10枚,分别滴加5 μL兔正常血清、5 μL兔骨髓液、5 μL兔血管内膜损害血清、5 μL兔血管内膜损害骨髓液、5 μL生理盐水及0.3 μg 血管内皮生长因子进鸡胚绒毛尿囊膜中,连续3 d。数码相机拍照后平铺于载玻片上,计数鸡胚绒毛尿囊膜新生的血管数目。 结果与结论：与正常血清组相比,正常骨髓液组、血管内膜损害血清组鸡胚绒毛尿囊膜新生的血管总数明显增多,大中血管明显增生;且血管内膜损害血清组大、中血管数更为明显增加。提示正常骨髓液具有明显的促进鸡胚绒毛尿囊膜模型血管生成的作用,其强度优于血管内皮生长因子;血管内膜损伤第7天的血清和骨髓液能够明显的促进鸡胚绒毛尿囊膜上的血管生成,其强度优于血管内皮生长因子组。     

鸡胚绒毛尿囊膜数字图像血管定量分析

目的：以生物医学工程的角度，研究基于数字图像处理技术的鸡胚绒毛尿囊膜图像血管自动分析方法，设计和研制专用实验检测系统。 方法：实验于2005-06/2007-06分别在上海理工大学医疗器械与食品学院中心实验室和上海中医药大学进行。根据鸡胚绒毛尿囊膜血管图像的特点，研究和开发了包括鸡胚绒毛尿囊膜血管数字图像背景褪色、血管增强、背景和血管定标、标尺校准、血管测量区域划分、血管面积及面积比参数测量等主要分析软件，以及相应的测量分析系统。通过实验检验分析方法和测量参数，并对分析系统进行改进。 结果：建立了有效的鸡胚绒毛尿囊膜数字图像血管自动分析的方法和步骤，研制了专用的实验分析系统。通过药物实验对鸡胚绒毛尿囊膜血管图像进行了参数检测，获取了多组实验数据，并进行了统计分析。统计分析结果显示试验药物有抑制血管生长作用，且作用大小呈剂量依赖关系。 结论：经实验证明基于数字图像处理技术的鸡胚绒毛尿囊膜图像血管自动分析方法能提供可靠的鸡胚绒毛尿囊膜模型定量分析数据，并且在分析速度、测量精度、数据重复性等方面均优于常规人工目测血管计数方法。     

应用鸡胚尿囊绒膜锥虫蓝吸收法评价化学物质的眼刺激性

背景：传统多采用兔眼刺激实验（Draize实验）评价化学物质眼刺激性,即将化学物涂进兔眼睛后观察其变化,这种方法费时,费力,且给动物带来很大痛苦。各国实验室都在寻找动物替代实验的方法。 目的：采用鸡胚尿囊绒膜-锥虫蓝吸收实验作为兔眼刺激实验替代方法评价化学物眼刺激性的可行性。 设计、时间及地点：观察实验,于2008-05/09在中国检验检疫科学研究院毒理学实验室完成。 材料：无特定病原体级受精鸡卵,9日龄,购于北京梅里亚通实验动物技术有限公司。 方法：将1 g/L的锥虫蓝溶液滴加在受化学物质刺激损伤的鸡胚尿囊绒膜上,通过计算鸡胚尿囊绒膜受损伤后吸收锥虫蓝的量,半定量测定化学物质对鸡胚尿囊绒膜的刺激性,并将所得结果与体内兔眼刺激实验评分结果进行比对,作相关性分析。同时预测了5种常见有机溶剂的眼刺激性。 主要观察指标：鸡胚尿囊绒膜受刺激损伤后吸收锥虫蓝的量。 结果：鸡胚尿囊绒膜-锥虫蓝吸收法与体内兔眼刺激实验评分结果相关性很好（R2=0.925 9）。5种常见有机溶剂未稀释时,甘油无眼刺激性,丙酮、乙醇、二甲基亚砜、甲醇均有较高刺激性。当稀释为1%后,丙酮、乙醇、二甲基亚砜无眼刺激性,但甲醇刺激性仍然较高。 结论：鸡胚尿囊绒膜-锥虫蓝吸收法可以代替兔眼刺激试验对化学物质眼刺激性进行评价。     

黄芪注射液对体外血管新生的影响*

背景：血管新生受生长因子的影响,黄芪可与血管内皮生长因子具有协同促血管新生的功效,但机制尚不明确。 目的：通过与单一血管内皮生长因子干预比较,观察中药黄芪对体外血管新生的作用机制。 方法：将黄芪注射液及血管内皮生长因子作用于SD大鼠胸主动脉内皮细胞,应用细胞增殖、细胞迁移、小管形成实验观察黄芪注射液对体外血管新生的促进作用,用Western blot实验检测血管内皮生长因子的表达。 结果与结论：黄芪能明显促进大鼠胸主动脉内皮细胞的增殖(P < 0.01),迁移的细胞数增加(P < 0.01),胸主动脉内皮细胞的小管形成数增加(P < 0.01),并明显促进内皮细胞血管内皮生长因子的表达(P < 0.01)。说明黄芪能明显促进内皮细胞增殖、细胞迁移、小管形成,具有显著的促进体外血管新生作用,其机制可能是通过增加血管内皮生长因子的表达而实现的。     

体外培养下可持续分泌人血管抑素和内皮抑素基因工程细胞对血管生成的抑制效应

背景：以往实验构建了可持续分泌人血管抑素和内皮抑素的基因工程细胞,即人血管抑素/293细胞和人内皮抑素293细胞,并已证明可持续分泌人血管抑素蛋白和人内皮抑素蛋白。但尚不了解其分泌物是否能发挥血管生成的抑制作用。 目的：观察以基因工程技术构建的人血管抑素/293细胞和人内皮抑素/293细胞对鸡胚尿囊膜血管新生的抑制效应。 设计、时间及地点：随机对照动物实验,于2006-06/2007-01在解放军总医院老年医学研究所细胞生物学研究室完成。 材料：Hek/293细胞为人胚胎肾细胞,由解放军军事医学科学院提供。人血管抑素/293细胞和人内皮抑素/293细胞由课题组前期构建。SPF级鸡胚购自北京维通利华实验动物有限公司。 方法：将鸡胚消毒后,置于37 ℃无菌恒温箱中孵育5 d,在距胚头1 cm两条卵黄静脉之间的卵壳投影部位磨切暴露尿囊膜,制成假气室,用无菌滤纸封闭窗口,制成鸡胚尿囊膜模型,备用。用灭菌蒸馏水配制体积分数为0.5%甲基纤维素溶液,制成甲基纤维素小杯。卵壳开窗后第3天,分别吸取浓缩的293细胞、人血管抑素/293细胞、人内皮抑素/293细胞培养上清液各20 μL,或人血管抑素/293细胞和人内皮抑素/293细胞培养上清液各10 μL,加于甲基纤维素小杯中,将其置于鸡胚尿囊膜的两条大血管之间的无血管区。 主要观察指标：观察尿囊膜血管生成的变化。 结果：共培养9 d时,与单纯293细胞上清液组相比,人血管抑素/293细胞上清液或人内皮抑素/293细胞上清液组,甲基纤维素小杯内及邻近区的鸡胚尿囊膜血管明显发育不良,血管分布稀疏,数量明显减少,其一级血管数量虽未明显减少,但其管径较细,而二级血管和三级以下的血管数量明显减少、管径明显变细,伸入尿囊膜边缘区的血管数量减少,管径明显变细。与人血管抑素/293细胞组或人内皮抑素/293细胞组相比,人血管抑素/293细胞＋人内皮抑素/293细胞组的一级血管数量和管径未见明显变化,而其二级血管和三级以下血管数量明显减少,血管树消失。 结论：人血管抑素/293细胞和人内皮抑素/293细胞的分泌物对鸡胚尿囊膜的血管新生均具有明显的抑制作用,且联合用药抑制作用增强。     

血脂康通过减少CD68、脂质沉积和新生血管生成增加α肌动蛋白和胶原稳定斑块

目的 研究血脂康对动脉硬化斑块稳定性的作用及机制.方法 将小鼠分为对照组和血脂康组.染色斑块、巨噬细胞CD68、脂肪、新生血管CD31、血管内皮生长因子(vascular endothelial growth factor,VEGF)、血管生成素1(recombinant angiopoietin-1,ANGPT-1)、血管生成素2(recombinant angiopoietin-2,ANGPT-2)、平滑肌α肌动蛋白和胶原.结果 血脂康组斑块、CD68、脂肪、CD31和VEGF减少(P＜0.05),而ANGPT-1、平滑肌α肌动蛋白和胶原增加.结论 血脂康通过减少斑块内巨噬细胞、脂质、新生血管生成并增加平滑肌α肌动蛋白和胶原稳定动脉硬化斑块.     

重组腺病毒介导TIMP1基因对类风湿关节炎大鼠滑膜新生血管的影响

背景：滑膜血管翳形成后可分泌一系列酶如基质金属蛋白酶等,可促进血管新生和炎症反应,形成软骨和骨的破坏。 目的：研究重组腺病毒介导的基质金属蛋白酶组织抑制因子1(TIMP1)基因对类风湿关节炎模型大鼠关节炎症及滑膜血管新生的影响。 设计、时间及地点：基因治疗动物体内实验,于2005-07/2007-06在扬州大学完成。 材料：HEK-293A细胞系购于中科院上海细胞生物研究所细胞库,ECV-304细胞为自备,带有绿色荧光蛋白(GFP)基因或/和基质金属蛋白酶组织抑制因子1基因的重组腺病毒(rAD-GFP-TIMP1及rAD-GFP)的病毒贮存液由南京师范大学分子医学实验室惠赠。清洁级4~6周龄SD大鼠40只。 方法：将rAD-GFP-TIMP1和rAD-GFP扩增、纯化,用紫外分光光度法检测重组腺病毒的颗粒数。用rAD-GFP-TIMP1感染ECV-304细胞检测病毒活力。30只大鼠注射Ⅱ型胶原建立类风湿关节炎大鼠模型,分为：①模型组注射PBS。②模型+ rAD-GFP-TIMP1组注射rAD-GFP-TIMP1。③模型+RAD-GFP组注射rAD-GFP,均为膝关节腔内注射;10只大鼠注射生理盐水造模做对照组。1次/周,4周。 主要观察指标：①rAD-GFP-TIMP1及rAD-GFP在HEK-293A细胞中扩增、纯化后病毒的颗粒数及GFP蛋白表达。②各组大鼠每周进行1次关节炎指数评分。③于造模第4周处死大鼠取膝关节行滑膜病理免疫组织化学染色及微血管密度检测。④取血清采用Western Blot检测大鼠体内基质金属蛋白酶组织抑制因子1、血管内皮生长因子、基质金属蛋白酶1,2,3,9的表达。 结果：①纯化后的rAD-GFP-TIMP1和rAD-GFP滴度分别为：5.7×1012,4.8×1012 pfu/mL;A260 nm/A280 nm均> 1.3;用rAD-GFP-TIMP1感染ECV-304细胞获得成功提示病毒活力正常。②感染rAD-GFP-TIMP1的关节炎大鼠血清中基质金属蛋 白酶组织抑制因子1获得了高表达,rAD-GFP-TIMP1治疗组大鼠血清中基质金属蛋白酶组织抑制因子1的相对含量明显高于模型组和rAD-GFP治疗组。③rAD-GFP-TIMP1治疗组的关节炎指数评分明显低于模型组。④感染rAD-GFP-TIMP1可使滑膜新生血管数目明显减少,同时能抑制基质金属蛋白酶1,3,9的表达,而对血管内皮生长因子和基质金属蛋白酶2的抑制作用不明显。 结论：①感染rAD-GFP-TIMP1的大鼠体内基质金属蛋白酶组织抑制因子1基因获得了有效的表达。②感染rAD-GFP-TIMP1能明显降低模型大鼠的关节炎指数评分。③血管内皮生长因子,基质金属蛋白酶1,2,3,9共同参与促进关节炎大鼠滑膜血管新生的形成和发展,rAD-GFP-TIMP1可能通过抑制基质金属蛋白酶1,3,9的表达而发挥抗血管新生的作用。     

注射用丹参多酚酸对大面积脑梗死患者血管新生促进作用及对脑灌注改善作用的观察研究

目的 通过观察大面积脑梗死患者治疗前后血管新生相关因子及脑灌注情况的变化,探讨注射用丹参多酚酸对侧支循环形成及对神经功能改善的作用.方法 选取大面积脑梗死患者44例,将所有受试者按1:1比例随机分为丹参多酚酸治疗组和常规治疗组(对照组),每组样本含量为22例.两组均给予脑梗死脱水降颅压、脑保护治疗,丹参多酚酸治疗组在以...     

Interaction of platelets with collagen modified by irradiation or partial proteolysis

Neutral salt-soluble rat skin collagen modified by X-irradiation of its oxygen-free solution, or by treatment with trypsin, was found to have a decreased ability to polymerize and to aggregate platelets. On the other hand, collagen fibrils preformed by thermal polymerization retained their original platelet aggregating activity irrespective of irradiation or treatment with trypsin.It has been concluded that platelet aggregating activity of soluble collagen is closely related to its ability to polymerize. The radiation-induced impairment of this property of soluble collagen does not appear to be correlated with the changes in carbohydrate or free ε-amino group content. With the dose range applied, these changes were negligible.     

Synergistic potentiation by epinephrine of collagen or thrombin-induced calcium mobilization in human platelets

Changes in cytoplasmic free calcium in response to epinephrine in combination with other agonists were studied in human platelets loaded with the fluorescent calcium indicator, quin 2. In the presence of 1mM external calcium, epinephrine augmented the increase in cytoplasmic free calcium in response to collagen or thrombin, and this was inhibited by yohimbine. In the absence of extracellular calcium, thrombin and collagen only caused a small increase in cytoplasmic free calcium, and the response was not enhanced by epinephrine. High concentrations of epinephrine alone caused a small increase in cytoplasmic free calcium in platelets when calcium was present externally. Inhibition of cyclooxygenase by aspirin markedly reduced the calcium response to collagen and almost completely inhibited potentiation by epinephrine. The calcium response to thrombin was inhibited to a lesser extent by aspirin, and aspirin did not prevent potentiation by epinephrine. These findings suggest that augmentation of the cytoplasmic free calcium level may be involved in the potentiation of platelet responses to agonists by epinephrine, and that metabolites of arachidonic acid make an important contribution to this action of epinephrine.     

On the role of the major platelet membrane glycoproteins in platelet adhesion to collagen

The effect of modification of intact rabbit platelets with chymotrypsin, trypsin or thrombin on platelet adhesion to collagen fibers was determined. Experiments were performed $\text{in vitro}$ with washed platelets suspended in a medium containing EGTA to prevent platelet aggregation. Platelet adhesion was unaffected by chymotrypsin or thrombin treatment but was reduced upon trypsin treatment. The latter effect could be attributed to the ADP released upon trypsin treatment. In the presence of high concentrations of AMP, adhesion was restored to control values. Release also occurred on thrombin treatment but this did not influence adhesion.The effect of the enzymes on platelet membrane glycoproteins was monitored by gel electrophoresis. Chymotrypsin caused the loss, at least, of the regions susceptible to lactoperoxidase-catalyzed iodination and to periodic acid-Schiff (PAS) staining from the major membrane glycoproteins I, II and III of rabbit platelets. Such degradation also occurred to glycoproteins I and II with trypsin, but thrombin was without detectable effect on the major membrane glycoproteins.The results indicate that degradation of the major membrane glycoproteins does not affect adhesion to collagen. Adhesion appears to be mediated either by protease-resistant parts of these molecules or by other membrane entities not readily attacked by proteases.     

Developmental variations of choline acetyl-transferase, dopamine-beta-hydroxylase and monoamineoxidase in chicken embryo and chicken sympathetic ganglia

Medial septal lesions (MSL) abolished hippocampal theta activity and attenuated the overall hippocampal potential level, while frontal cortical lesions (FCL) had no effect on theta rhythm. A decrease in the overall potential level was observed only in some FCL rats. Although spontaneous behavioral patterns, including drinking, face-washing, searching, body movements, awake-and-resting, and slow-wave sleep, were not altered either by MSL or FCL during the 15 min observation, orienting responses to tones, especially searching-type, were reduced during drinking in MSL rats, while no such reductions were observed in FCL and no-lesion control rats. Auditory evoked potentials were not changed either by MSL or FCL. Spontaneous behavioral patterns were not different in FCL rats whether or not the overall hippocampal potential level was decreased.     

N- and R-cadherin expression in the optic nerve of the chicken embryo

Cadherins are a family of molecules mediating Ca²⁺-dependent cell-cell adhesion in various tissues. N - and R-cadherin are expressed in the chick embryonic CNS and differ in their expression pattern during development. Here we focus on the differential expression of N - and R-cadherin in the early optic nerve. N-cadherin is expressed by the retinal neurites growing through the optic nerve. R-cadherin is expressed by the early optic nerve glia, which derives from the optic stalk neuroepithelium and corresponds to an immature form of the type-1 astrocyte described in rat optic nerve. The close contact between the plasma membranes of the retinal neurites and the optic nerve glia is believed to be important in guiding retinal axons through the optic nerve. Using neuroblastoma cell lines transfected with R-cadherin, we demonstrate that the N-cadherin-positive retinal axons can use R-cadherin as a substrate for axon elongation. These results suggest that the R-cadherin expressed by the early optic nerve glia might provide a molecular substrate for the growth of N-cadherin-positive retinal axons through the optic nerve. © 1993 Wiley-Liss, Inc.     

Pathway specificity of reticulospinal and vestibulospinal projections in the 11-day chicken embryo

The organization of the axonal pathways of reticulospinal and vestibulospinal projections in the 11-day chicken embryo was ascertained through retrograde tracing experiments. An in vitro preparation of the brainstem and cervical spinal cord facilitated precisely localized tracer applications. Single- and double-labelling experiments involving high cervical injections of tracers in combination with selective lesions defined the specific pathways by which different brainstem neurons project to the spinal cord. Coherent, and in many cases distinct, groups of reticulospinal and vestibulospinal neurons could thus be identified on the basis of their position and projection pathway. The organization of these groups and their projections in the 11-day chicken embryo is similar to that in avian and other vertebrate adults and therefore serves as a reference point for studies of pathfinding by bulbospinal axons during early development.     

Identification of D1-dopamine receptor in chicken embryo retina with [125I]SCH 23982

The D1-dopamine receptor in chicken embryo retina was identified with the D1-dopamine receptor specific ligand, [125I]SCH 23982. Binding of [125I]SCH 23982 to both pre-hatched and post-hatched chicken retina was rapid, saturable and of high affinity. The dissociation constant and maximal binding capacity were 795 +/- 25 pM (mean +/- S.E.M., n = 3) and 32.2 +/- 3.8 fmol/mg protein (mean +/- S.E.M., n = 3), respectively for 13-day-old chicken embryo retina, and 785 +/- 58 pM (mean +/- S.E.M., n = 3) and 96.9 +/- 4.1 fmol/mg protein (mean +/- S.E.M., n = 3), respectively for 1-day-old post-hatched chicken retina. The binding properties of the D1-dopamine receptor in chicken retina were similar to those in rat striatum. The maximal binding capacity of the D1-dopamine receptor for [125I]SCH 23982 was increased concomitant with embryonic development, but without any changes in either affinity or pharmacological properties. Dopamine-stimulated adenylate cyclase activity in the retinal homogenates increased concomitant with embryonic development, diminished in the presence of 1 microM SCH 23390 (a D1-dopaminergic antagonist) but remained unaffected by 1 microM YM-09151-2 (a D2-dopaminergic antagonist).     

Met-enkephalin alteration in the rat during chronic injection of morphine and/or midazolam

We have recently reported that the short-acting anesthetic and analgesic drug midazolam can produce analgesia and decrease morphine tolerance and dependence in the rat by interacting with the opioid system. This study was designed to investigate the effect of midazolam, morphine, and both together on met-enkephalin levels in the rat. Male Sprague–Dawley rats were divided into four groups: (1) saline-saline; (2) saline-morphine; (3) midazolam-saline, and (4) midazolam-morphine groups. First, a saline or midazolam injection was given intraperitoneally and after 30 min a second injection of saline or morphine was given subcutaneously once daily for 11 days. Animals were sacrificed on the 11th day 60 min after the last injection to measure met-enkephalin by radioimmunoassay. Morphine tolerant animals showed a significant increase in met-enkephalin levels in the cortex (137%) and midbrain (89%), and a significant decrease in met-enkephalin levels in the pituitary (74%), cerebellum (34%) and medulla (72%). Midazolam treated animals showed a significant decrease in met-enkephalin levels in the pituitary (63%), cortex (39%), medulla (58%), kidneys (36%), heart (36%) and adrenals (43%), and a significant increase in met-enkephalin levels in the striatum (54%) and pons (51%). When morphine and midazolam were injected together, midazolam antagonized the increase in met-enkephalin levels in cortex and midbrain region and the decrease in met-enkephalin level in the medulla region observed in morphine tolerant animals. These results indicate that morphine tolerance and dependence is associated with changes in the concentration of met-enkephalin in the brain. Midazolam may inhibit morphine tolerance and dependence by reversing some of the changes induced in met-enkephalin levels in brain by morphine in morphine tolerant and dependent animals.     

Possible role of collagen in transverse myelitis and chymopapain-induced paraplegia

We studied the effect of nucleus pulposus (NP) on platelet aggregation. Our in vitro experiments showed that NP extract produced platelet aggregation and the addition of collagenase to the NP extract abolished this response. It was further shown that chymopapain did not affect the activity of the extract. We assume that collagen is the active platelet aggregant in the NP extract. Intravascular release of collagen may cause platelet aggregation, vascular obstruction, ischemia, and cord necrosis in a patient with acute transverse myelitis. Intradiskal chymopapain is known to cause transverse myelitis and it is possible that collagen released during the action of the enzyme initiates a similar chain of events.