【作者】 赵津；

【导师】 史中立；

【作者基本信息】 河北医科大学 ， 人体解剖与组织胚胎学， 2010， 硕士

【摘要】 肢体缺血再灌注(IR)损伤常发生于肢体挤压伤、肢体高位离断再植、腹主动脉瘤手术等患者的转归过程中。肢体缺血的时间越长,IR损伤表现越重,严重者可引起肢体坏死,不得已而行截肢手术。更严重的是,由缺血区组织细胞产生释放的活性氧物质及炎症介质可引发全身性IR损伤,进而引起肾等多器官功能障碍。因此,及早采取有效措施防治肢体IR损伤是整体救治的重要环节。研究表明,信使分子一氧化碳(CO)具有抗炎、抗氧化损伤的重要功效,在机体遭遇器官IR损伤的病理过程中,CO发挥细胞保护作用。本课题组先前的研究证实,给予适量外源性CO可有效减轻大鼠肢体IR损伤。但前期研究所用动物模型的肢体缺血时间为4 h,缺血程度相对较轻。给予外源性CO对肢体严重IR损伤有何影响,尚需进一步观察研究。目的:本实验拟通过夹闭大鼠股动脉8h再开放的方法复制肢体严重IR损伤模型,使模型动物吸入含CO的医用空气后,观测血清乳酸脱氢酶(LDH)、肌酸激酶(CK)、肢体湿干(W/D)重比值和病理学指标的变化,以探讨吸入CO对肢体严重IR损伤的效应,为最终将吸入CO的办法应用于防治肢体严重IR损伤提供实验依据。方法:(1)动物分组:健康、清洁、雄性SD大鼠56只,随机分为IR组(24只)、IR +CO(RC)组(24只)、假手术(S)组(8只)。在IR、RC组中,8只用于10 d存活率的观测;8只于再灌注6 h时取材,另8只于再灌注12 h时取材,用于LDH、CK、肢体W/D和病理学指标的观测。S组中,4只与再灌注6 h组对比观测,另4只与再灌注12 h组对比观测。(2)模型复制:本实验采用无创血管夹在双侧后肢根部夹闭股动脉,并用橡皮筋绑扎后肢根部,以此造成双侧后肢缺血。根据文献提示,并经本实验证实,缺血达8 h以上者再灌注后血压下降,呈现休克表现。据此,本实验将缺血时间确定为8 h,以此建立肢体严重IR动物模型。S组动物模拟手术过程,但不造成肢体缺血。(3)吸入CO的方法:RC组动物吸入CO,把动物置于特制的器皿中,通入含有CO的医用空气,CO的体积分数为0.075%。于再灌注前2 h时开始持续吸入含有CO的医用空气,直至再灌注后4 h,共计吸入6 h。S、IR组暴露于空气中,自由呼吸。(4)10 d存活率观测:IR、RC组各取8只大鼠,于再灌注过程中,观测尾动脉收缩压的变化和10 d存活率。(5)血清LDH、CK活性测定:IR、RC组,分别于再灌注6 h、12 h时,S组于对应的手术后时间点,在深度麻醉下经右心房采血各5mL,静置,离心,取上清液送全自动生化分析仪检测血清LDH、CK活性。(6)肢体W/D重比值测定:上述动物经心房采血后,把左侧后肢从髋关节处截断,用电子分析天平测量其湿重,在60℃电热恒温培养箱中干燥7天,待水分完全析出后测其干重,算得W/D。(7)病理学观察:从髋关节处截取上述动物右侧后肢,对肢体肿胀程度、皮下渗出情况及坏死粘连程度、肌肉颜色、质地等进行大体观察。其后,剥去皮肤,将小腿部分进行固定,把小腿三头肌切成组织块,石蜡包埋、切片,HE染色,光镜观察肌组织病理学变化。结果:(1)大体观察结果:S组,肢体形态大小正常,皮下无出血及渗出物,肌肉颜色暗红、弹性好。IR组,肢体明显肿胀,皮下组织内、肌间隔内有血色渗出液,并可见大量浅黄色、胶状渗出物,肌肉色泽深暗,成褐色,弹性差。RC组,肢体的肿胀程度较IR组明显减轻,皮下组织、肌间隔内血色渗出液及胶状渗出物明显减少,肌肉颜色较IR组显苍白,弹性中等。(2)肢体W/D重比值:S组,W/D最低,IR组W/D最高,RC组W/D居中。IR组和RC组相比,RC组和S组相比,均有统计学差异(P<0.05)。(3)HE染色观察结果:S组,骨骼肌细胞形态正常,胞浆均匀、红染,胞核无变形,核仁清晰;肌束明显,胞间无炎性细胞浸润。IR组,骨骼肌细胞胞体增大,部分肌细胞变性、坏死,肌浆溶解、破坏,胞核消失,呈空泡状;肌束断裂,胞间有大量的中性粒细胞浸润,并广泛出血。与再灌注6 h相比,再灌注12 h者病理表现更为严重。RC组,可见部分肌细胞变性,肌浆含有空泡,呈灶状溶解,大部分肌细胞肿大程度减轻,肌浆染色接近正常,胞核染色及位置趋近正常,未见核固缩;骨骼肌细胞间有少量单核细胞浸润,出血减轻。(4)血清LDH和CK活性检测结果:6 h取材者:IR组和RC组相比,有统计学差异(P<0.05);RC组和S组相比,无统计学差异(P>0.05);12 h取材者:IR组和RC组相比,RC组和S组相比,均有统计学差异(P<0.05)。(5)10 d存活率:IR组,再灌注后大鼠尾动脉收缩压开始降低,其下降过程可区分为四个时相:第一时相,再灌注0-2 h,为血压快速下降期,至2 h前后,尾动脉平均收缩压为8.6708±0.5992 kpa;第二时相,再灌注2-4 h,下降幅度较小,为血压相对稳定期;第三时相,再灌注4-8 h,为血压缓慢下降期,至再灌注8 h时尾动脉平均收缩压为5.3259±0.3832 kpa;第四时相,再灌注8 h-死亡,为持续低血压期,尾动脉收缩压波动在5 kpa上下,动物处于持续休克状态,再灌注13-14 h间有6只大鼠死亡,另外2只再灌注至20 h前后死亡,10 d存活率为0。RC组,尾动脉收缩压的下降速率较慢、下降幅度较小,与IR组的四个时相比照:第一时相,血压也快速下降,但下降幅度显著变小,再灌注至2 h前后,尾动脉平均收缩压为10.3843±1.3111 kpa;第三时相,血压下降的速率和幅度均明显降低,至再灌注8 h时尾动脉平均收缩压为8.3300±0.4224 kpa;第四时相,血压未出现下降,且稳中有升,至再灌注13 h时尾动脉平均收缩压为9.4157±0.3305 kpa,8只大鼠中1只在再灌注至20 h前后死亡,其余7只再灌注10 d后仍存活,且状态良好,10 d存活率为7/8。结论:(1)大鼠肢体缺血8 h者,再灌注过程中血压明显下降,出现休克表现,10 d存活率为0。据此认为,使大鼠两侧后肢持续缺血8 h行再灌注,以此可复制肢体严重IR模型。(2)及早给予外源性低浓度CO,可稳定动物血压,防止休克发生,显著提高肢体严重IR损伤大鼠的生存率。(3)及早给予外源性低浓度CO,可显著减轻肢体IR损伤,降低肢体的伤残率。更多还原

【Abstract】 Limb ischemia reperfusion (IR) injury frequently encountered in limb crush injury, limb replantation of high mutilation, abdominal aortic aneurysm surgery with patients in the transfer process. The longer limb ischemia, the more sever IR injury in the performance, can cause severe limb necrosis and amputation at last. More seriously, the ischemic tissue cells release reactive oxygen substances and inflammatory mediators can lead to systemic IR injury, causing kidney and multiple organ dysfunction. Therefore, take effective measures to prevent limb IR injury earlier is an important part of the overall treatment. Studies have shown that Carbon monoxide (CO) has important functions under physiological and pathophysiological conditions. Recent tudies suggested anti-inflammatory and anti-apoptot. CO plays a cell protection effects in the pathological process of organ IR injury of the body. Our laboratories and others have demonstrated that the administration of exogenous carbon monoxide (CO) at appropriate concentrations can effectively reduce the IR injury of rat limbs. However, the animal limb ischemia time of pre-institute model was4h, ischemia was a lighter degree.How exogenous CO on the severe limb IR injury requires further observation and study.Objective: This study was intended to block the femoral artery 8 h in rats and re-open method of copying a serious limb IR injury model, after making the model animal inhalation medical air containing CO, observe the serum lactate dehydrogenase (LDH), creatine kinase (CK), limb wet-dry (W/D) weight ratio and pathological changes of indicators to provide final experimental basis for preventing and treatmenting serious limb IR injury through the approach of inhalating CO.Methods: (1) Animal groups: healthy, clean and 56 male SD rats were randomly divided into IR group (24 rats), IR + CO (RC) group (24 rats), Sham operation (S) group (8 rats). In the IR, RC group, 8 rats for the observation of 10 d survival rate. 8 rats were taken after reperfusion for 6 hours, and the other 8 rats were taken after reperfusion for 12 hours. LDH, CK, limb W/D and pathological indicators were tested to detect the skeletal muscle function. S group, 4 rats were observed compared with 6h reperfusion group, the other 4 rats were observed compared with 12h reperfusion group. (2) Model reproduction: this study used a non-invasive blood vessel occlusion the femoral artery at the root of the two hind limbs, and used rubber bands lashing the root of limbs, thus causing bilateral hindlimbs ischemia. According to the literature prompted and this experiment confirmed, the blood pressure decreased after more than 8h ischemia and reperfusion, showing the performance of shock. Accordingly, this experiment made the ischemic time as 8h, in order to establish animal models of sever limb IR injury. S group was simulated surgical procedure, but does not result in limb ischemia. (3) CO inhalation method: animals were placed in a special container in RC group, then passed into the medical air containing CO, CO volume fraction is 0.075%. Before reperfusion of 2h, the animais started to breathe CO continuously, until after reperfusion of 4h, a total of 6h. S, IR group exposed to air, breathed freely. (4) 10d survival rate observed: IR, RC group depicted 8 rats, observed the changes of tail artery systolic pressure and 10d survival in the course of reperfusion. (5) Serum LDH, CK activity assay: IR, RC groups, respectively getted 5mL blood from the right atrial in the deep anesthesia after reperfused 6 h and 12 h, while S group at the corresponding time points after surgery. The blood was standed, centrifuged, and the supernatant was sended automatic biochemical to analyze serum LDH, CK activity. (6) Limb W/D weight ratio measurement: after getted the blood from the atrium, cutted off the left hind legs from the hip joints of the rats, and used an electronic analytical balance to measure the wet weight of severed limbs. After then, at 60℃, the limbs were dried for 7 days in electrical thermostat. After the limbs were heated to dryness, the dry weight of the severed limbs was measured. The wet/dry weight ratio was thus calculated. (7) Pathological observation: cutted off the right side of hind legs from the hip joints of these animals, Gross observesed the limb swelling degree, subcutaneous exudation, necrosis adhesion conditions and muscle color, texture, etc.Subsequently, shucked the skin, fixed parts of the leg, cutted the triceps muscle of calf into tissue pieces, embedded in paraffin, sliced, HE staining, and observesed pathological changes in muscle tissue with light microscopic.Results: (1) The observation on the whole: The skeletal muscle of S group showed normal form. There were no hemorrhagic effusion and the color was dark red, good elasticity.While the skeletal muscle of IR group showed the color brown and edema, elasticity appeared dysfunction obviously with plenty of hemorrhagic effusion in the spatium intermusculare. And the skeletal muscle showed adhesion, exposure of necrotic yellow tissue and almost lost all its functions. However, the color and edema of muscle in RC Group became improved. Hemorrhagic effusion of RC group turned much less and the color turned pale than IR group, moderate elasticity. (2)Limb W/D weight ratio: S group, W/D were the lowest, IR Group W/D maximum, RC Group W/D centre. IR group Compared with RC group, RC group Compared with S group, both had statistically significant difference (P<0.05). (3) Observed by HE staining: The result of the S group showed that muscle fiber arranged orderly. The shape of skeletal muscle remained normal. Chromatosis of the endochylema was symmetrical. The shape of nuclei was normal. The nucleolus was clear. The muscle bundle was obvious. And there were no inflammatory cells infiltration. However, in IR group, swelling in cytoplasm and karyopyknosis in some cells were found. The degeneration and necrosis were significantly in the muscle fibers. The sarcoplasm dissolved and the nuclei disappeared. Muscle fiber arranged derangement and there was more cytoplasmic vacuolization in IR group. The muscle bundle fractured and HE staining of the untreated muscles revealed massive neutrophils infiltration and wide hemorrhage.Compared with reperfusion 6h,the pathology of reperfusion 12h performed more sever.In RC group, there were pieces of cells showing degeneration and vacuolus. Most muscle cells showed slight swelling and focal necrosis. Chromatosis of the endochylema and nucleus were close to normal. CO treatment reduced the number of infiltrating macrophages and neutrophil. Only a few monocytes were seen in the spatium intermusculare. Karyopyknosis were not observed and hemorrhages decreased after the treatment. (4) The test results of serum LDH and CK activity: following 6h referfusion: IR group compared with RC group had a statistically significant difference (P<0.05), RC group compared with S group had no significant difference (P> 0.05); following 12h referfusion: IR group compared with RC group, RC group compared with S group both had statistically significant difference (P<0.05). (5) 10 d survival rate: IR group, The rat tail artery systolic blood pressure began to decrease after reperfusion, the decrease process could be divided into four phases: first phase, reperfusion 0-2 h, as the period of rapid decline in blood pressure, the tail artery average systolic pressure was 8.6708±0.5992 kpa before and after 2 h reperfusion; second phase, The blood pressure reperfusion, decreased to a lesser extent after 2-4 h reperfusion, as the period of relatively stable blood pressure; third phase, reperfusion 4-8 h,as the period of slow decline phase. the tail artery average systolic pressure was 5.3259±0.3832 kpa after 8 h reperfusion; fourth phase, reperfusion 8 h-death, as the sustained hypotension period, tail systolic blood pressure fluctuationed up and down the 5 kpa, animals were in the continuous state of shock, there were 6 rats died between reperfusion 13-14 h and another 2 rats died 20 h reperfusion approximately, 10 d survival rate was 0. RC group, the tail systolic blood pressure had a slower decline rate and a lesser extent, compared with the IR group of the four phase: first phase, the blood pressure also declined rapidly, but had a lesser decline rate, the tail artery average systolic pressure was 10.3843±1.3111 kpa after 2 h reperfusion; third phase, the decline rate and magnitude of the blood pressure were significantly reduced, the tail artery average systolic pressure was 8.3300±0.4224 kpa after 8 h reperfusion; fourth phase, blood pressure does not appear down, and up steadily, the average systolic pressure was 9.4157±0.3305 kpa after 13 h reperfusion , 1 rats of the 8 rats died after 20 h reperfusion approximately, the remaining 7 rats were still alive after 10 d reperfusion, and in a good condition, 10 d survival rate was 7 / 8.Conclusion: (1) Rats limb ischemia 8h, the blood pressure decreased significantly after reperfusion, to show shock, 10d survival rate is 0. These results suggested that the rats continued ischemia 8h on both sides of hind limbs and reperfusion can replicate severe limb IR model.(2) Early inhalation low concentrations of exogenous CO, can stable blood pressure of animals, prevent shock occurring, significantly improve survival rate of severe limb IR injury in rats. (3) Early inhalation low concentrations of exogenous CO, can significantly reduce the limb IR injury, degrade limb disability rate.更多还原