丹参对缺血肢体关节软骨及滑膜损伤的影响

目的　通过动物实验观察丹参关节内注射对关节缺血再灌注损伤后退行性关节炎的防治作用。方法　 6 3只新西兰大白兔随机分成对照、缺血 2h ,缺血 5h用丹参和不用丹参 5个组 ,分别于缺血、再灌注 1d和 1、 4、 8、 12、 16周取股骨外髁软骨及滑膜行大体和光镜观察 ,以及再灌注后 8周和 16周软骨厚度测量。结果　实验组关节软骨、滑膜早期有缺血再灌注损伤 ,并逐渐演变成早期退行性关节炎改变 ,而这些病变用丹参组明显轻于未用丹参组。结论　丹参能有效地减轻关节软骨及滑膜缺血再灌注损伤及退行性改变。     

肢体缺血再灌注后关节软骨的组织学观察

目的: 通过动物实验观察肢体缺血再灌注后关节软骨的病理变化, 证实缺血再灌注导致关节软骨的损伤, 并探讨关节缺血再灌注损伤与骨性关节炎形成的关系。方法: 健康中国本地兔42只, 随机分为正常对照组, 缺血4h组和缺血10h组, 分别于肢体缺血再灌注后0、1d和1、5、10周,取4只兔8条腿做标本, 行光镜观察, 及图像分析仪处理。结果: 早期(24h以内) 关节软骨的组织学改变在光镜下表现不明显。再灌注5周时损伤最为明显, 此后短期内(10周内) 软骨损伤不再加重。软骨组织形态学的定量观察,股骨外髁关节软骨全层厚度明显增加(P<0 .01), 非钙化层增加不明显, 而钙化层厚度则明显增加(P< 0. 01)。结论: (1) 证实了缺血再灌注导致关节软骨的损伤; (2) 关节缺血再灌注损伤也是导致骨性关节炎形成的重要原因。     

肢体缺血-再灌注后关节软骨病理变化

目的 了解肢体缺血-再灌注(IR)后关节软骨的病理变化特点，探讨共可能的损伤机制。方法 35只成年健康新西兰大白兔随机分为7组，行左侧后肢缺血8h(右侧为对照)，于再灌注后1d、3d、1周、2周、4周、8周和12周取膝关节胫骨平台软骨组织，行大体、光学显微镜、透射电镜和扫描电镜观察。结果 对照侧肢体关节软骨表面平整，细胞排列整齐，形态正常?肢体缺血．再灌注后1周、2周关节软骨细胞变件、坏死，排列紊乱，数目减少，软骨表面“垄沟”变浅；4周、8周软骨表面粗糙，表层细胞脱落；12周软骨表面溃疡形成，胶原暴露，同时可见新生的软骨细胞。结论 肢体缺血．再灌注可导致关节软骨细胞损伤，软骨形态发生变化，与术后慢性关节炎的形成有密切关系。     

丹参与缺血肢体关节软骨及滑膜损伤的影响

目的 通过动物实验观察丹参关节内注射对关节缺血再灌注损伤后退行性关节炎的防治作用。方法 ６３只新西兰大白兔随机分成对照，缺血２ｈ，缺血５ｈ用丹参和不同丹参５个组，分别于缺血，再灌注１ｄ和１、４、８、１２、１６周取股骨外髁软骨及滑膜行大体和光镜观察，以及再灌注后８周和１６周软骨厚度测量。结果 实验组关节软骨，滑膜早期有缺血再灌注损伤，并逐渐演变成早期退行性关节炎改变，而这些病变用对丹参组。结论 丹参     

肢体缺血再灌注损伤对关节滑膜的影响

目的探讨肢体缺血再灌注(IR)对关节滑膜的损伤及其机制。方法80只新西兰大白兔随机分成正常组、缺血组和再灌注组(缺血8h后再灌注);分别于缺血后4、6、8、10h,再灌注1、3、6、12、24、72h和1、2、4、8、12周。取滑膜组织测定丙二醛(MDA)、超氧化物歧化酶(SOD)及乳酸的含量,观察形态学变化并作滑膜组织血管标记和多形核细胞(PMN)计数。结果IR早期滑膜组织有不同程度的变性和坏死,在随后的修复过程中毛细血管出现严重的狭窄和闭塞;SOD在缺血阶段即明显降低,持续到IR后8周;IR组MDA显著高于对照侧、缺血组和正常组;自缺血4h～IR2周,乳酸含量持续在高水平;PMN计数:IR组显著高于对照组。免疫组化标记显示IR8～12周血管数较健侧减少(P<0.05)。结论IR损伤早期以组织的变性坏死为主,之后患侧逐渐演变成慢性滑膜炎和创伤性关节炎。修复过程中微循环障碍及代谢产物长期滞留和PMN作用可能是影响其修复的重要因素。     

肢体缺血再灌注后MMP-3在关节软骨中的表达

目的:了解肢体缺血再灌注后关节软骨内基质金属蛋白酶3(matrixmetalloproteinase- 3,MMP- 3)的表达情况。方法:35只成年健康新西兰大白兔,体重为(3. 0±0 . 5 )kg ,随机分为7组,行左侧后肢缺血8h(右侧为对照) ,于再灌注后1、3d及1、2、4、8和12周取膝关节胫骨平台软骨组织,石蜡切片,免疫组化SP法检测关节软骨内MMP -3的表达。结果:肢体缺血再灌注早期,MMP -3在关节软骨出现强阳性表达,肢体缺血再灌注后3d组阳性细胞较对照侧明显增多,至2周组达峰值(P <0 . 0 1) ,之后逐渐回落,12周组仍高于对照侧(P <0 . 0 5 )。结论:肢体缺血再灌注后,关节软骨内合成并分泌了MMP 3,在软骨基质的降解中发挥重要作用。     

透明质酸载碱性成纤维细胞生长因子对兔膝关节缺血再灌注损伤防治作用的实验研究

目的：通过动物实验观察碱性成纤维细胞生长因子(bFGF)与透明质酸(HA)关节腔内注射对关节缺血再灌注损伤及退行性关节炎的防治作用。方法：采用夹闭兔股血管8h的方法模拟缺血再灌注模型。将48只新西兰大白兔随机分为对照组(A组)，HA治疗组(B组)和FGF／HA治疗组(C组)，在关节腔内注射相应的药物。用光镜和电镜观察关节滑膜和软骨的病理改变，观察并半定量计算软骨蛋白多糖(PGs)的变化，检测滑膜丙二醛(MDA)的含量。结果：C组的软骨和滑膜病理改变明显轻于A组和B组，在C组中，滑膜MDA的含量明显低于其它两组，而PGs的含量明显高于其它两组：结论：联合应用bFGF和HA能明显减轻兔膝关节缺血再灌注损伤和退行性改变，其作用要明显优于单用HA。     

MMP-3/TIMP-1在大鼠肢体再灌注后关节软骨损伤的作用

目的　探讨缺血再灌注后关节软骨中MMP - 3/TIMP - 1比例变化与软骨损伤的关系。方法　采用大鼠后肢股动脉夹闭的方法模拟缺血再灌注的动物模型 ,用Wistar大鼠 4 0只 ,随机分成正常对照组 (NG)、肢体单纯缺血组 (IG)和缺血再灌注组 (IR)。运用免疫组化技术 ,分别测定TIMP - 1和MMP - 3在关节软骨中不同时相的表达变化并进行半定量分析 ,观察关节软骨病理改变及蛋白多糖 (PG)的变化。结果　缺血再灌注后 ,关节软骨中的MMP - 3和TIMP - 1表达均有增加 ,但MMP - 3增加的幅度大于TIMP ,导致MMP - 3/TIMP - 1比值增大 ,与再灌注后引起的关节软骨损伤相关。结论　MMP/TIMP的失平衡表达是导致缺血再灌注后关节软骨损伤的重要因素     

MMP-3／TIMP-1在大鼠肢体再灌注后关节软骨损伤的作用

目的 探讨缺血再灌注后关节软骨中MMP-3／TIMP-1比例变化与软骨损伤的关系。方法 采用大鼠后肢股动脉夹闭的方法模拟缺血再灌注的动物模型，用Wistar大鼠40只，随机分成正常对照组(NG)、肢体单纯缺血组(IG)和缺血再灌注组(IR)。运用免疫组化技术，分别测定TIMP-1和MMP-3在关节软骨中不同时相的表达变化并进行半定量分析，观察关节软骨病理改变及蛋白多糖(PG)的变化。结果 缺血再灌注后，关节软骨中的MMP-3和TIMP-1表达均有增加，但MMP-3增加的幅度大于TIMP，导致MMP-3／TIMP-1比值增大，与再灌注后引起的关节软骨损伤相关。结论 MMP／TIMP的失平衡表达是导致缺血再灌注后关节软骨损伤的重要因素。     

骨水泥阻塞髓腔后骨内压升高对远侧关节影响的实验研究

[目的]探讨骨水泥阻塞股骨近中段骨干髓腔后骨内压升高对远侧关节组织结构的影响。[方法]将26只实验兔分成4组,随机取出8只不做模型而作为正常对照组,其余18只采取左侧股骨髓腔内灌注聚甲基丙烯酸甲酯(PMMA)骨水泥而右侧不灌注,制作骨水泥阻塞股骨近中段骨干髓腔的动物模型,根据不同观察时间段随机分成术后4(T4)、8(T8)和16周(T16)3个观察组。对正常对照和模型动物实验侧股骨远端软骨、软骨下骨和滑膜组织标本,采用HE染色、甲苯胺蓝染色、免疫组织化学检测及透射电镜方法进行组织结构观察。[结果](1)HE染色:关节软骨、软骨下骨和关节滑膜组织损害随时间呈进行性加重,造模16周关节软骨破坏,骨组织结构损害,滑膜组织增生、肿胀。(2)甲苯胺蓝染色:造模16周关节软骨全层失染。(3)免疫组化:①Ⅱ型胶原:造模16周软骨细胞染色阳性。②TGF-β1:造模16周软骨细胞染色阳性,关节滑膜细胞染色阳性。(4)透射电镜:关节软骨和骨细胞损害随时间呈进行性加重,造模16周部分软骨细胞和骨细胞坏死、崩解。[结论]骨水泥阻塞股骨近中段骨干髓腔后导致股骨远端血循环障碍,骨内压升高,持续骨内高压加重了股骨远端的缺血缺氧状态,使股骨远端骨代谢发生紊乱,最终造成股骨远端骨、软骨和滑膜组织发生了退变或坏死。     

CXC chemokine expression and synthesis in skeletal muscle during ischemia/reperfusion

BACKGROUND: The chemokines keratinocyte-Derived Cytokine (KC) and macrophage inflammatory protein (MIP)-2, murine equivalents of human interleukin 8, have been implicated in remote injury after acute hind limb ischemia/reperfusion (I/R). These studies were designed to determine whether the cytokines responsible for remote tissue injury are also synthesized and accumulate in the ischemic or reperfused hind limb. METHODS: B6, 129SF2/J mice were subjected to either 3 hours of unilateral hind limb ischemia alone (IA) or 3 hours of ischemia followed by 4 or 24 hours of reperfusion (I/R). After IA or I/R, experimental and control (nonischemic) contralateral hind limbs were harvested for analysis of protein content, messenger RNA (mRNA), tissue edema, and viability. RESULTS: IA did not increase KC or MIP-2 mRNA or protein levels. In contrast, I/R resulted in a 15- and 10-fold increase in KC mRNA after 4 and 24 hours of reperfusion, respectively. KC protein levels were increased 10-fold after 4 hours of reperfusion and 30-fold after 24 hours (vs IA or sham; P < .001). MIP-2 mRNA transiently increased 42-fold after 4 hours of reperfusion but decreased to basal levels after 24 hours of reperfusion. Despite the relative increase in MIP-2 mRNA by 4 hours of reperfusion, significantly increased (8- to 10 fold) MIP-2 protein levels were not detected until 24 hours of reperfusion only in the reperfused limbs. Tissue edema was increased significantly (P < .01) compared with sham after just 4 hours of reperfusion and remained increased at 24 hours. Tissue viability decreased 52% after 4 hours of reperfusion and did not change significantly by 24 hours. CONCLUSIONS: Skeletal muscle is a site of significant ongoing chemokine synthesis during reperfusion. The persistent increase in muscle chemokine levels at 24 hours of reperfusion was not associated with increased edema or injury. The role of these chemokines during reperfusion may be further investigated by local or oral administration of chemokines or chemokine receptor antagonists. CLINICAL RELEVANCE: I/R injury remains an important clinical problem across a variety of surgical specialties. In the critical care arena, serum levels of proinflammatory cytokines have been useful in predicting the mortality associated with acute respiratory distress syndrome and sepsis. In this article, the data presented indicate that murine skeletal muscle produces potent proinflammatory neutrophil and macrophage chemokines during reperfusion, but not during ischemia. These findings suggest that measurement of tissue and/or serum levels of chemokines during reperfusion may be an important adjunct to predicting tissue injury along with ongoing inflammation during the clinical course of reperfusion injury. Within the vascular system, severe inflammatory responses are usually associated with thrombotic events. New techniques to noninvasively image thrombin activation (by using magnetic resonance imaging) in reperfused limbs may coincide with the pattern of murine skeletal muscle chemokine expression in humans. The data suggest that reperfusion is when chemokine mRNA and protein synthesis increase. Within the time periods studied in these experiments, the chemokine component of the inflammatory response remained in the reperfused, rather than the systemic nonreperfused, tissue. This observation may underestimate the degree of the systemic response to ischemia because the single mouse hind limb represents only 7% of the mouse total body area, whereas the human limb represents nearly 18% of the adult body area. Despite this shortcoming, these data provide potential temporal and quantitative information regarding the location and magnitude of chemokine synthesis in skeletal muscle during reperfusion.     

The role of glutamine in skeletal muscle ischemia/reperfusion injury in the rat hind limb model.

OBJECTIVES: Ischemia/reperfusion injury is a commonly occurring event with severe pathologic consequences. Reperfusion initiates both the local and systematic damage in part through rapid oxygen generation. The glutathione system is a major mechanism of reducing this oxidative stress. If this system can be maintained or augmented during this stress then less damage may occur. Glutamine provides the source of glutamate to this system and has been shown to preserve total glutathione levels after injury/ischemia to both hepatic and gut models. To test this effect, we looked at glutamine and its role in ischemia/reperfusion injury in a rat hind limb model. METHODS: Fifty male HSD/Holtzman rats weighing 350-400 g were randomized to receive glutamine (3% sol) or normal saline via intraperitoneal injections. The groups were then subjected to 2 hours of ischemia to their hind limbs using the Tourni-Cot method. Animals were then randomized to reperfusion groups of 30 minutes, 2 hours, and 4 hours. Muscle tissue assays were performed for lipid peroxidation (LPO), total glutathione (GSH), and myeloperoxidase (MPO). Peripheral blood was analyzed for creatinephosphokinase levels (CPK). RESULTS: Animals that received glutamine showed a general trend of less lipid peroxidation products than the normal saline groups. In animals that received glutamine and underwent 2 hours of ischemia and reperfusion times of 0 minutes, 30 minutes, and 2 hours, there were significantly less percent changes in lipid peroxidation products from controls (4.6% vs 48.2%, P <0.05), (18.9% vs 123%, P <0.05), (12.6% vs 115%, P <0.05). A general trend upward was noted in CPK levels in both groups. In animals receiving 2 hours of ischemia and 30 minutes of reperfusion, there was a significantly greater level of creatinephosphokinase (CPK) calculated as percent change from control in the normal saline group as compared with the glutamine group (209.2% vs 92.7%). Myeloperoxidase assay of muscle tissue revealed a progressive increase as the reperfusion times grew. In animals receiving 2 hours of ischemia and 30 minutes of reperfusion, the normal saline group had a significantly larger percent increase from controls than the group that received glutamine (1126.4% vs 108%, P <0.05). Also, in those animals receiving 4 hours of reperfusion, the normal saline group had a significantly higher percent increase in MPO content than the glutamine group (6245% vs 108%, P <0.05). Total glutathione levels decreased rapidly as reperfusion occurred in both the normal saline and glutamine groups. No significant difference between the groups was noted. CONCLUSIONS: Total glutathione levels during reperfusion were not significantly different in the groups receiving glutamine versus normal saline. Glutamine may provide an initial protective effect on reperfusion injury after moderate reperfusion times in the hind limb model as defined by CPK and LPO levels. Glutamine may blunt neutrophil recruitment after longer reperfusion times (4 hours) in the ischemic hind limb. Total glutathione levels decreased significantly after moderate levels of ischemia (2 hours) and reperfusion (30 minutes, 2 hours).     

Experimental evaluation of oxygen free radical scavengers in the prevention of reperfusion injury to skeletal muscle

A prolonged preoperative ischemic interval decreases the chances for successful replantation of an amputated limb. Skeletal muscle is especially sensitive to periods of prolonged ischemia. It is now hypothesized that significant tissue injury occurs during reperfusion, when oxygen-rich blood contacts anaerobic metabolites forming toxic oxygen free radicals. A replantation model, using the rabbit hind limb tibialis anterior muscle, was developed to assess muscle function and histological appearance following ischemic intervals of five and eight hours. Muscle strength five weeks after injury was used as a functional measurement of tissue damage. The effects of the superoxide free radical scavenger superoxide dismutase (SOD) and the hydroxyl radical scavenger dimethylsulfoxide (DMSO), administered systemically just before reperfusion, were studied. Muscle treated with SOD following five hours of ischemia had essentially normal strength and histological appearance; however, there was no protective effect after eight hours. DMSO treatment had no beneficial effects after five hours of ischemia, but after eight hours DMSO-treated muscle had significantly better function than untreated muscle. Histological examination confirmed the functional results. Clinical treatment of ischemic limbs with free radical scavengers before revascularization may aid in avoiding reperfusion injury and may improve survival and later muscle function.     

脊髓缺血再灌注损伤中运动诱发电位的监测作用

目的　探讨运动诱发电位（ＭＥＰ）对脊髓缺血再灌注损伤中神经功能的监测作用。　方法　对２６ 只大鼠腰骶段脊髓缺血前、缺血１５、２５ 、４０ 分钟及再灌注后５、１５、３０ 分钟、１、２ 和２４ 小时ＭＥＰ变化进行监测。　结果　在缺血１５ 分钟时ＭＥＰ潜伏期明显延长（Ｐ＜ ０．０１） ,波幅在缺血２５ 分钟时明显减小（ Ｐ＜ ０－０１） ,缺血４０ 分钟时波形消失;再灌注后５ 分钟时波形恢复,但潜伏期大于正常（Ｐ＜０－０１） ,波幅小于正常（Ｐ＜０－０１）;再灌注后１５ 分钟至２ 小时波幅恢复正常（Ｐ＞ ０－０５）,潜伏期无恢复;再灌注后２４ 小时潜伏期虽然呈恢复趋势,但与再灌注早期相比,差异无显著性意义,此时波幅又明显下降低于正常（Ｐ＜０－０１） ,再灌注后２４ 小时双下肢运动功能比再灌注早明显降低（ Ｐ＜ ０－０５） 。　结论ＭＥＰ能够准确监测脊髓神经功能在缺血再灌注损伤中的变化     

肢体缺血再灌注损伤的新型动物模型制作

[目的]用充气止血带制作肢体缺血再灌注损伤的新型动物模型,研究其对周围神经和骨骼肌损伤的影响.[方法]选择健康新西兰大白兔6个月龄,30只,体重(3.5 ±0.3) kg,雌雄不限,在家兔左侧后肢环扎充气止血带,于不同时间点松开,造成左侧后肢缺血再灌注损伤的模型.随机分为3组,每组10只.A组:对照组,B组:缺血2h,C组:缺血4h.对照组不扎充气止血带,第1、2、3、4、5、6h检测肢体的神经电生理学指标,B组、C组于再灌注(松开止血带,血供恢复后)的1、2、3、4、5、6h检测肢体的神经电生理学指标,A组于第6h观察骨骼肌的形态,B、C组于再灌注(松开止血带,血供恢复后)的第6h观察骨骼肌的形态,每组于术后第5d评估左侧后肢的行走功能.[结果]随着缺血后再灌注时间的延长,B、C和A组相比较,周围神经的潜伏期延长、波幅降低,传导速度降低,三组之间的潜伏期、波幅、传导速度差异均有统计学意义(P＜0.05),光镜观察骨骼肌可见(B、C组):横纹紊乱、肌纤维断裂、间质血管扩张充血、大量中性粒细胞浸润.[结论]经过缺血期和再灌注损伤的交互作用后,肢体的功能性损伤进一步加重,出现了不可逆的病损.该模型制作对动物的损伤较小,更贴近临床.