Lymphatic hyporeactivity and calcium desensitization following hemorrhagic shock

The lymphatic circulation is an important component of the circulatory system. In preliminary studies, we found contractile activity of lymphatic vessels to be decreased during severe shock. In the present study, our aim was to observe changes in lymphatic reactivity to norepinephrine (NE) and to explore the mechanism of calcium sensitivity in rats subjected to hemorrhagic shock (HS). Thirty-two Wistar rats were randomly divided into sham and shock groups, and changes in lymphatic pressure and contractility of mesenteric lymphatics in response to NE were measured at different time points after shock. The results showed that NE-induced changes in lymphatic pressure were decreased at 1 h after shock and that hyporeactivity was maintained for 3 h after shock. The reactivity of mesenteric lymphatics to NE in the shock group at 1 to 2 h after shock was significantly lower than that of the sham group and before shock. The other 49 rats were divided into sham, shock 1 h, and shock 2 h groups for isolation thoracic duct rings. Forty-eight isolated lymphatics per group were used to assay lymphatic reactivity to NE and calcium sensitivity in an isolated vessel perfusion system. The NE concentration-response curves for postshock lymphatic rings (1 or 2 h) and calcium concentration-response curves after shock (2 h) were shifted to the right; isolated lymphatic reactivity to NE and contraction in response to calcium were markedly reduced in shock groups. Lymphatic reactivity to NE and calcium sensitivity were significantly increased in the 2-h shock group following incubation with the calcium sensitizer angiotensin II, and the lymphatic reactivity was reduced after incubation with the calcium sensitivity inhibitor insulin. In conclusion, lymphatic reactivity declines progressively during HS as a result of calcium desensitization. The results suggest that lymphatic hyporeactivity is one of the mechanisms of lymphatic hypocontraction in rats subjected to HS.     

钙失敏在大鼠失血性休克血管低反应性中的作用

目的观察血管平滑肌钙失敏在大鼠失血性休克(HS)血管低反应性中的作用。方法 取失血 性休克大鼠肠系膜上动脉(SMA),利用离体血管环张力测定技术,以血管环对梯度浓度去甲肾上腺素(NE) 的收缩力反映血管反应性,用去极化状态下(120 mmol／L K+)血管环对梯度浓度Ca2+的收缩力反映血管的 钙敏感性,观察失血性休克低反应血管是否存在钙敏感性降低以及钙敏感性调节剂血管紧张素Ⅱ(Ang Ⅱ)、 胰岛素以及Rho-激酶特异性抑制剂Fasudil是否可以通过调节钙敏感性来调节血管反应性。结果 失血性 休克后SMA对NE的反应性和钙敏感性均显著下降,表现为NE的量-效曲线明显右移,NE的最大收缩力 (Emax)和-lg[EC50](pD2)降低(P<0.05或P<0.01);Ca2+的量一效曲线明显右移,Ca2+的Emax和pD2降低 (P<0.05或P<0.01)。具有钙敏感性增强作用的Ang Ⅱ(10-9mol／L)可使NE和Ca2+的量-效曲线左移,使 NE和Ca2+的Emax增高(P<0.05或P<0.01)。而具有钙敏感性抑制作用的胰岛素(100 nmol／L)可使NE 和Ca2+的量-效曲线右移,NE和Ca2+的Emax降低(P<0.05或P<0.01).Fasudil预处理可消除Ang Ⅱ对 NE诱导的血管收缩反应的增强效果,降低钙敏感性。结论 失血性休克血管平滑肌细胞存在钙敏感性降低. 血管平滑肌细胞钙敏感性降低在失血性休克血管低反应性的     

The role of calcium desensitization in vascular hyporeactivity and its regulation after hemorrhagic shock in the rat

The objectives of the present study were to investigate the role of calcium desensitization in vascular hyporeactivity, and the regulatory effects of Rho-kinase, protein kinase C (PKC), and protein kinase G (PKG) on calcium sensitivity. The vascular reactivity and calcium sensitivity with superior mesenteric artery (SMA) from hemorrhagic shock rat were observed by measuring the contraction initiated by norepinephrine (NE) and Ca2+ under depolarizing conditions (120 mmol/L K) in an isolated organ perfusion system. Angiotensin II (Ang-II) and Fasudil, the Rho-kinase agonist and inhibitor, phorbol 12-myristate 13-acetate (PMA) and staurosporine, the PKC agonist and inhibitor, 8Br-cGMP and KT-5823, the PKG agonist and inhibitor, and Calyculin A, myosin light chain phosphatase (MLCP) inhibitor were used as tool agents. The results indicated that vascular reactivity and calcium sensitivity were decreased after hemorrhagic shock. The cumulative dose-response curve of SMA to NE and Ca2+ after shock was shifted to the right. Ang-II (10 mol/L) could improve the decreased vascular reactivity by increasing the calcium sensitivity of SMA, and insulin (100 nmol/L) could further decrease the vascular reactivity by decreasing the calcium sensitivity of SMA. These results suggested that the vasculature after shock was desensitized to calcium, which played an important role in the onset of vascular hyporeactivity after shock. PMA and KT-5823 could increase the sensitivity of SMA to Ca2+ and made the cumulative dose-response curve shift to the left. In contrast, Fasudil, staurosporine, and 8Br-cGMP decreased the sensitivity of SMA to Ca2+ and made the cumulative dose-response curve of Ca2+ shift to the right. Calyculin A (10 mol/L) pretreatment further enhanced Ang-II, and PMA induced increase of calcium sensitivity, yet weakened the 8Br-cGMP-induced decrease of calcium sensitivity. Taken together, the data suggest that Rho-kinase, PKC, and PKG are involved in the regulation of calcium sensitivity of vascular smooth muscle after hemorrhagic shock, and their regulatory effects on calcium sensitivity of vasculature are possibly related to MLCP.     

Changes of Rho kinase activity after hemorrhagic shock and its role in shock-induced biphasic response of vascular reactivity and calcium sensitivity

The purpose of the present study is to investigate the changes of Rho kinase activity and its role in biphasic response of vascular reactivity and calcium sensitivity after hemorrhagic shock. The vascular reactivity and calcium sensitivity of superior mesenteric artery (SMA) from hemorrhagic shock rats were determined via observing the contraction initiated by norepinephrine (NE) and Ca under depolarizing conditions (120 mmol/L K) with isolated organ perfusion system. At same time, Rho kinase activity in mesenteric artery was measured, and the effects of Rho kinase activity-regulating agents, angiotensin II (Ang-II), insulin, and Y-27632, on vascular reactivity and calcium sensitivity were also observed. The results indicated that the vascular reactivity and calcium sensitivity were increased at early shock (immediate and 30 min after shock) and decreased at late shock (1 and 2 h after shock). The maximal contractions of NE and Ca were significantly increased (P < 0.05 or P < 0.01) at early shock. But they were significantly decreased at late shock (P < 0.05 or P < 0.01). Rho kinase activity was significantly increased at early shock (immediate after shock) (P < 0.05) but significantly decreased at 1 and 2 h after shock (P < 0.05 or P < 0.01). It was positively correlated with the changes of vascular reactivity and calcium sensitivity. Insulin decreased the increased contractile response of SMA to NE and Caat early shock (P < 0.05 or P < 0.01). Angiotensin II increased the decreased contractile response of SMA to NE and Ca at 2-h shock (P < 0.05 or P < 0.01); Y-27632, Rho kinase-specific antagonist, decreased the contractile response of SMA to NE and Ca at 2-h shock, and abolished Ang-II induced the increase of vascular reactivity and calcium sensitivity. The results suggest that Rho kinase may be involved in the biphasic change of vascular reactivity and calcium sensitivity after hemorrhagic shock. Rho kinase may regulate vascular reactivity through the regulation of calcium sensitivity. Rho kinase-regulating agents may have some beneficial effects on shock-induced vascular hyporeactivity.     

ICI174,864对失血性休克大鼠全身及局部血管反应性变化的作用

目的 观察大鼠不同程度失血性休克时全身和局部血管反应性变化及δ阿片受体特异性拮抗剂IC1174,864对失血性休克大鼠血管反应性的影响。方法 56只Wistar大鼠,戊巴比妥钠麻醉(30mg／kg)。实验分两部分。第一部分实验用32只大鼠,随机分为手术对照组、1h低血压组,2h低血压组和3h低血压组,每组8只动物;动物经股动脉插管放血至血压40mmHg(1mmHg=0．133kPa)分别维持1、2和3h,然后回输全部残余失血,观察回输血后1、2和3h血压和肠系膜上动脉(SMA)对去甲肾上腺素(NE,3pg,／kg)的反应性变化。第二部分实验用24只大鼠,随机分为失血性休克对照组、IC1174,864 0．5mg／kg干预组和1．0mg／kg干预组,每组8只动物;动物经股动脉插管放血至血压40mmHg,维持2h,回输残余失血,观察给予IC1174,864后1、2和4h血压和SMA对NE(3μg／kg)的升压和收缩反应。结果 失血性休克后全身(血压对NE的升压反应)和局部(SMA对NE的收缩反应)血管反应性显著降低,且呈一定的程度和时间依赖性,即休克程度越重,时间越长,血管反应性降低越多。单纯回输失血不能纠正血管的低反应性,IC1174,864对休克血管的低反应性有不同程度的恢复作用,且呈一定的剂量依赖关系。结论 失血性休克可诱导全身和局部血管反应性降低,并与休克程度和时间密切相关,全身和局部血管反应性的降低呈现一定的平行关系。δ阿片受体特异性拮抗剂IC1174,864对失血性休克大鼠的血管低反应性有一定的恢复作用。     

失血性休克大鼠血管平滑肌钙敏感性变化及其在休克双相血管反应性变化中的作用

目的观察失血性休克后血管平滑肌钙敏感性是否存在双相变化,以及钙敏感性的双相变化与血管反应性双相变化的关系。方法取失血性休克大鼠肠系膜上动脉(SMA),采用离体血管环张力测定技术,观察在失血性休克后不同时间点(休克即刻、休克30min、休克1h、休克2h)大鼠SMA血管环对梯度浓度去甲肾上腺素(NE)的收缩反应性,以及在去极化状态下(120mmol/LK+)血管环对梯度浓度钙的收缩反应性变化(钙敏感性),分析血管反应性变化与钙敏感性变化的关系;同时观察钙敏感性增强剂血管紧张素(Ang)和钙敏感性抑制剂胰岛素对血管反应性的影响。结果休克早期(即休克即刻和休克30min时)SMA对NE和钙的反应性明显升高,量效曲线明显左移,最大收缩力(Emax)明显升高(P均<0.05);随着休克时间的延长,血管环对NE和钙的反应性均逐渐下降,到休克2h均已明显降低,其量效曲线明显右移,E-max明显降低(P<0.05或P<0.01);休克后不同时间点血管反应性变化与钙敏感性变化呈显著正相关(r=0.9624,P<0.05)。具有钙敏感性增强作用的Ang(1×10-9mol/L)可明显升高休克2h血管环对NE和钙的反应性(P<0.05或P<0.01),而有钙敏感性抑制作用的胰岛素则可降低休克早期(休克即刻)血管环对NE和钙的反应性(P<0.05或P<0.01)。结论失血性休克血管平滑肌细胞存在钙敏感性的双相变化,血管平滑肌细胞钙敏感性双相变化在失血性休克血管反应性的双相变化中起重要作用。     

Mechanisms of Rho kinase regulation of vascular reactivity following hemorrhagic shock in rats

Our previous research showed that Rho kinase took part in the regulation of vascular hyporeactivity after shock. The objective of the present study was to investigate its mechanism. With isolated superior mesenteric artery (SMA) from hemorrhagic shock rats, we studied the relationship of Rho kinase regulating vascular reactivity to calcium sensitivity and myosin light chain phosphatase (MLCP) and myosin light chain kinase (MLCK). The vascular reactivity and calcium sensitivity of SMA were observed by measuring the contraction initiated by accumulative norepinephrine (NE) and calcium under depolarizing condition (120 mM K(+)) with an isolated organ perfusion system. Hypoxia-treated vascular smooth muscle cells (VSMCs) were used to study the effects of Rho kinase on the activity of MLCP and MLCK and the phosphorylation of 20-kDa myosin light chain (MLC(20)). Myosin light chain (20 kDa) phosphorylation of VSMC in mesenteric artery was detected by immunoprecipitation and Western blotting. The activity of MLCP and MLCK was assayed by enzymatic catalysis. The contractile response of VSMC was measured by the ratio of accumulative infiltration of fluorescent isothiocyanate-conjugated bovine serum albumin through transwell. The results indicated that the vascular reactivity and calcium sensitivity of SMA to NE and calcium following hemorrhagic shock and the contractile response of VSMC to NE following hypoxia were significantly decreased. Angiotensin II (Ang-II), the Rho kinase stimulator, significantly improved hypoxia or hemorrhagic shock-induced decrease of vascular reactivity and calcium sensitivity. These effects of Ang-II on vascular reactivity were abolished by Y-27632, the specific Rho kinase inhibitor. Calyculin A, the MLCP inhibitor, further enhanced Ang-II-induced increase of calcium sensitivity, but ML-9, the MLCK inhibitor, had no effect. Further studies showed Ang-II reversed the hypoxia-induced increase of MLCP activity and increased the hypoxia-induced decrease of MLC(20) phosphorylation in VSMC. It was suggested that Rho kinase played an important role in the regulation of vascular reactivity after hemorrhagic shock. The mechanisms may be related to its calcium sensitivity regulation. Rho kinase up-regulates calcium sensitivity of VSMC possibly through inhibiting the activity of MLCP and increasing the phosphorylation of MLC(20).     

精氨酸血管加压素恢复失血性休克大鼠血管反应性和钙敏感性与蛋白激酶C亚型的关系

目的 探讨精氨酸血管加压素(AVP)对失血性休克大鼠血管反应性和钙敏感性的恢复作用与蛋白激酶C(PKC)亚型的关系.方法 取失血性休克大鼠肠系膜上动脉(SMA)并去除内皮,利用离体血管环张力测定技术,观察了AVP(5×10-11、5×10-10和5×10-9 mol/L)调节对休克SMA对去甲肾上腺素(NE)反应性和钙敏感性的作用及其与PKCα、δ亚型的关系.结果 AVP(5×10-11、5×10-10和5×10-9 mol/L)可明显恢复休克后的血管反应性和钙敏感性,使SMA对NE和Ca2+的量一效曲线明显左移,血管环产生的最大收缩张力(Emax)升高(P均＜0.01),且呈一定的剂量依赖关系,各剂量组间比较差异均有统计学意义(P均＜0.05).而特异性的PKCα、δ亚型抑制剂G66976(5×10-6 mol/L)和Rottlerin(10-5 mol/L)均可拮抗AVP(5×10-10 mol/L)诱导的休克后血管反应性和钙敏感性升高,抵消了AVP诱导的NE和Ca2+的量一效曲线左移,使NE的Emax明显降低(P＜0.05或P＜0.01).结论 AVP能剂量依赖性地升高失血性休克大鼠的血管反应性和血管平滑肌钙敏感性,其机制可能与PKCα、δ亚型激活有关.     

Hemorrhage-induced vascular hyporeactivity to norepinephrine in select vasculatures of rats and the roles of nitric oxide and endothelin

Hemorrhage-induced vascular hyporeactivity to norepinephrine (NE) and the possible effector roles of nitric oxide (NO) and endothelin (ET) were investigated in different vascular beds of rats. Under urethane anesthesia, rats (n = 7 per group) were hemorrhaged to a mean arterial pressure (MAP) of 50 mm Hg for 60 min. A group of rats was pretreated with either NG-nitro-L-arginine methyl ester (10 mg/kg), an NO synthase inhibitor, or PD142893 (0.1 mg/kg), an ET receptor antagonist 15 min before the end of the hypotensive period. Operated, euvolemic rats served as controls. The responses of MAP and the blood flow of the superior mesenteric (SMA), celiac (CA), left renal (LRA), and left femoral arteries (LFA) to NE (3 microg/kg, i.v.) were measured at baseline (prehemorrhage), at the end of the hypotensive period (0 h), and at 1, 2, and 4 h after the end of the hypotensive period. The pressor responses to NE on MAP at 0, 1, 2, and 4 h in the 60-min hemorrhage groups were reduced to 45.9%, 37.8%, 29.2%, 18.4% of baseline pressor response, respectively. At these same times, the fall in blood flow in response to NE in SMA, CA, LRA, and LFA was significantly blunted (P < 0.01). This loss of responsiveness in CA and LFA was more severe than in SMA and LRA (P < 0.05-P < 0.01). Pretreatment with L-NAME or PD142893 significantly improved the pressor response of MAP and the blood flow responses of the four arteries to NE (P < 0.01). Hypotension at 50 mm Hg for 60 min resulted in an apparent loss of vascular reactivity to NE, and the four vasculatures studies were not affected to the same extent. In addition, NO and ET appear to contribute to the loss of vascular reactivity in different vasculatures in hemorrhagic shock.     

Effect of sodium arsenite on iNOS expression and vascular hyporeactivity associated with cecal ligation and puncture in the rat

Induction of the heat shock response protects animals from either endotoxemia or peritonitis. In endotoxemia, heat shock protein (HSP) induction is associated with reversal of vascular hyporeactivity and inhibition of iNOS expression. Recent studies suggest differences in the inflammatory mechanisms during endotoxemia and peritonitis animal models and their response to therapeutic interventions. We therefore studied the effect of the HSP inducer sodium arsenite (SA) on vascular reactivity and iNOS expression in rats undergoing cecal ligation and puncture (CLP). CLP resulted in suppression of the pressor effect of norepinephrine (NE) in vivo (measured by changes in blood pressure in response to NE boluses) and ex vivo (changes in contraction force in isolated mesenteric arteries in response to NE concentrations), and in the expression of iNOS protein. Pretreatment of the rats with SA resulted in reversal of CLP-induced vascular hyporeactivity in vivo and ex vivo, and inhibition of iNOS expression after 22 h. SA pretreatment improved 7-day survival after CLP from 18.2% to 70% (P < 0.005). Glucocorticoid receptor inhibition did not affect the effect of HSP induction on iNOS expression. The similarity of the effect of HSP on vascular reactivity and iNOS expression in two distinct sepsis models suggests that this effect may be clinically important and that a causative relationship between HSP induction, iNOS inhibition, and reversal of vascular reactivity is likely.     

蛋白激酶C和蛋白激酶G对失血性休克大鼠血管平滑肌细胞钙敏感性的调节作用

目的探讨蛋白激酶C(PKC)、蛋白激酶G(PKG)对失血性休克大鼠血管平滑肌细胞钙敏感性的调节作用。方法经股动脉放血使平均动脉压维持在40mmHg(1mmHg=0.133kPa)2h制备失血性休克大鼠模型。采用离体血管环张力测定技术,测定在失血性休克大鼠肠系膜上动脉(SMA)血管环去极化状态下(120mmol/LK+)对梯度浓度Ca2+的收缩反应性(钙敏感性)变化;同时观察PKC、PKG活性调节剂对钙敏感性的影响。测定大鼠SMA的PKC、PKG活性变化,分析PKC、PKG活性变化与血管钙敏感性变化间的关系。结果休克2hSMA血管环对钙敏感性明显降低,其量-效曲线明显右移,最大收缩力(Emax)明显降低(P均<0.01)。PKC激动剂PMA1×10-7mol/L可明显提高休克血管环对钙敏感性,PKC拮抗剂staurosporine1×10-7mol/L可降低休克血管环对钙敏感性(P<0.05或P<0.01);PKG激动剂8Br-cGMP1×10-4mol/L可使休克血管钙敏感性降低,其拮抗剂KT-58231×10-6mol/L可提高休克血管钙敏感性(P均<0.05)。休克2hSMA的PKC活性明显降低,PKG活性明显升高(P<0.05和P<0.01),分别与休克血管钙敏感性变化呈正相关和负相关(P均<0.01)。结论PKC和PKG参与了失血性休克血管平滑肌细胞钙敏感性的调控,PKC可上调血管平滑肌细胞的钙敏感性,PKG可下调其钙敏感性。     

Trauma-hemorrhage-induced neutrophil priming is prevented by mesenteric lymph duct ligation

Our objective in this study was to test the hypothesis that priming of neutrophils (PMN) in vivo by trauma-hemorrhagic shock (T/HS) is mediated by factors carried in intestinal lymph that prime PMNs by enhancing their responses to inflammatory mediators. Previous studies have shown that T/HS-induced lung injury is mediated by factors contained in mesenteric lymph and that ligation of the main mesenteric lymph duct (LDL) can prevent T/HS-induced lung injury. Since T/HS-induced lung injury is associated with PMN infiltration, one mechanism underlying this protective effect may be the prevention of PMN priming and activation. Therefore, we assessed the ability of T/HS to prime PMN responses to inflammatory agonists, and the ability of mesenteric lymph duct division to protect against such T/HS-induced PMN priming in an all-rat system. PMN were collected from male rats 6 h after laparotomy (trauma) plus hemorrhagic shock (30 mmHg for 90 min; T/HS) or trauma plus sham shock (T/SS). Uninstrumented rats were used as controls (UC). In a second set of experiments, rats were subjected to T/HS with or without mesenteric lymph duct division. PMN were then stimulated with chemokine (GRO, MIP-2) and lipid (PAF) chemoattractants, and cell calcium flux was used to quantify responses to those agonists. T/SS primed PMN responses to GRO, MIP-2. and PAF in comparison to UC rats, but the addition of shock (T/HS) amplified PMN priming in a significant manner, especially in response to GRO. Mesenteric lymph duct division prior to T/HS diminished PMN priming to the levels seen in T/SS. This reversal of priming was significant for GRO and GRO/MIP-2 given sequentially, with the other agonist regimens showing similar trends. The results support the concept that trauma and hemorrhagic shock play important additive roles in inflammatory PMN priming. Entry of gut-derived inflammatory products into the circulation via mesenteric lymph seems to play a dominant role in mediating the conversion of physiologic shock insults into immunoinflammatory PMN priming. Shock-induced gut lymph priming enhances PMN responses to many important chemoattractants, most notably the chemokines, and mesenteric lymph duct division effectively reverses such priming to priming levels seen in trauma without shock.     

Serine proteases are involved in the pathogenesis of trauma-hemorrhagic shock-induced gut and lung injury

The objective of this work was to test the hypothesis that Intraluminal serine proteases are involved in trauma-hemorrhagic shock (T/HS)-induced intestinal and lung injury. Male Sprague-Dawley rats were administrated the serine protease inhibitor (6-amidino-2-naphthyl p-guanidinobenzoate dimethanesulfate, Nafamostat) either intraluminally into the gut or intravenously after a laparotomy (trauma) and then subjected to 90 min of hemorrhagic shock (T/HS) or sham shock (T/SS). Intestinal and lung injury was assessed at 3 h after resuscitation with Ringer's lactate solution. In a second set of experiments, mesenteric lymph was collected from the groups of rats subjected to T/HS or T/SS and its ability to activate normal neutrophils was tested. Lung permeability, pulmonary myeloperoxidase levels, and the bronchoalveolar lavage fluid protein to plasma protein ratio were increased after T/HS but were significantly decreased in the T/HS rats receiving intraluminal (P < 0.05), but not intravenous, nafamostat. Likewise, T/HS-induced intestinal villus injury was less in the nafamostat-treated shock rats (P < 0.05). Last, the ability of T/HS mesenteric lymph to increase PMN CD11b expression or prime neutrophils for an augmented respiratory burst was significantly reduced by the intraluminal administration of nafamostat. Because intraluminal nafamostat reduced T/HS-induced gut and lung injury as well as the neutrophil activating ability of intestinal T/HS lymph, the presence of serine proteases in the ischemic gut may play an important role in T/HS-induced gut and hence lung injury.     

肌内皮缝隙连接对失血性休克大鼠血管收缩反应的影响

目的 研究肌内皮缝隙连接（MEGJ）对失血性休克大鼠内皮依赖性和非依赖性血管收缩反应的影响。方法 56只SD大鼠按随机数字表法分为去甲肾上腺素（NE）组和杨梅黄酮组；NE组分为正常对照以及休克0、0．5、1和2h5个亚组；杨梅黄酮组分为正常对照以及休克0、0．5、1、2、3和4h7个亚组。各组取其肠系膜上动脉（SMA）制成血管环，分别测量在18α-甘草次酸（18α-GA）作用前后血管环对NE和杨梅黄酮反应性的变化。结果 SMA血管环对NE的反应性表现为：休克0h和0．5h组明显高于正常对照组和其他休克时间点组，休克1h和2h组明显低于正常对照组和休克0h和0．5h组；18α-GA对NE诱导的SMA血管环收缩反应性无明显影响。SMA血管环对杨梅黄酮的反应主要表现为：休克0、0．5、1和2h组明显高于正常对照组，于休克2h达最高点；休克3h和4h组明显低于正常对照组，运用18α-GA阻断MEGJ后，各组血管环的收缩反应性较18α-GA作用前明显下降，其中以休克1、2和3h组下降幅度明显大于其他组。结论 失血性休克后早期，内皮依赖和非依赖性的血管收缩反应均有不同程度代偿性增加，休克1h和2h表现为非内皮依赖性血管收缩反应下降，休克3h和4h表现为内皮依赖性血管收缩反应明显降低；MEGJ在休克后内皮依赖性血管收缩反应的变化中起重要调节作用。     

Hypertonic saline resuscitation limits neutrophil activation after trauma-hemorrhagic shock

There is evidence suggesting that the ischemic gut is a major source of factors that lead to neutrophil activation, and that neutrophil activation can be reduced by hypertonic saline resuscitation. Thus, we tested whether trauma-hemorrhagic shock-induced neutrophil activation can be reduced by hypertonic saline resuscitation, as well as whether hypertonic saline reduces the ability of mesenteric lymph from shocked animals to activate neutrophils. Male Sprague-Dawley rats subjected to trauma (laparotomy), plus 90 min of shock [mean arterial pressure (MAP) MAP = 30 mmHg] or sham shock were resuscitated with Ringer's lactate or 7.5% hypertonic saline at an equivalent sodium load. Whole blood samples were collected before shock as well as at 1 and 2 h after the end of the shock period for neutrophil CD11b and CD18 expression measurements. In a second set of experiments, mesenteric lymph samples collected from rats subjected to trauma plus hemorrhagic shock (T/HS) or trauma plus sham-shock (T/SS) and resuscitated with Ringer's lactate or hypertonic saline were tested for their ability to modulate PMN CD11b, CD18, or L-selectin expression, as well as prime PMN for an augmented respiratory burst. To avoid confounding results due to interspecies differences, while at the same time looking at potential human responses, both naive rat and human PMN were tested. Both CD11b and CD18 expression were increased in PMN harvested from rats subjected to T/HS and resuscitated with Ringer's lactate solution, but not in T/HS rats resuscitated with hypertonic saline. These results indicate that PMN activation is increased to a greater extent in Ringer's lactate-resuscitated than hypertonic saline-resuscitated animals. Likewise, mesenteric lymph from the T/HS rats resuscitated with Ringer's lactate increased naive rat and human PMN CD11b and CD18 expression to a greater extent than did T/HS lymph from the hypertonic saline-treated rats. Additionally, T/HS lymph from the Ringer's lactate- but not the hypertonic saline-treated rats induced PMN L-selectin shedding. Lastly, T/HS lymph from the Ringer's lactate-treated rats induced the greatest PMN respiratory burst. These results indicate that resuscitation from T/HS with hypertonic saline is associated with less PMN activation than resuscitation with Ringer's lactate, and that factors produced or released by the postischemic intestine and carried in the mesenteric lymph contribute to neutrophil activation after an episode of T/HS.     

Anticoagulants influence the in vitro activity and composition of shock lymph but not its in vivo activity

Many models of trauma-hemorrhagic shock (T/HS) involve the reinfusion of anticoagulated shed blood. Our recent observation that the anticoagulant heparin induces increased mesenteric lymph lipase activity and consequent in vitro endothelial cell cytotoxicity prompted us to investigate the effect of heparin-induced lipase activity on organ injury in vivo as well as the effects of other anticoagulants on mesenteric lymph bioactivity in vitro and in vivo. To investigate this issue, rats subjected to trauma-hemorrhage had their shed blood anticoagulated with heparin, the synthetic anticoagulant arixtra (fondaparinux sodium), or citrate. Arixtra, in contrast to heparin, did not increase lymph lipase activity or result in high levels of endothelial cytotoxicity. Yet, the arixtra-treated rats subjected to T/HS still manifested lung injury, neutrophil priming, and red blood cell dysfunction, which was totally abrogated by lymph duct ligation. Furthermore, the injection of T/HS mesenteric lymph, but not sham-shock lymph, collected from the arixtra rats into control mice recreated the pattern of lung injury, polymorphonucleocyte (PMN) priming, and red blood cell dysfunction observed after actual shock. Consistent with these observations, citrate-anticoagulated rats subjected to T/HS developed lung injury, and the injection of mesenteric lymph from the citrate-anticoagulated T/HS rats into control mice also resulted in lung injury. Based on these results, several conclusions can be drawn. First, heparin-induced increased mesenteric lymph lipase activity is not responsible for the in vivo effects of T/HS mesenteric lymph. Second, heparin should be avoided as an anticoagulant when studying the biology or composition of mesenteric lymph because of its ability to cause increases in lymph lipase activity that increase the in vitro cytotoxicity of these lymph samples.     

Bone marrow failure in male rats following trauma/hemorrhagic shock (T/HS) is mediated by mesenteric lymph and modulated by castration

Bone marrow (BM) suppression occurs following trauma/hemorrhagic shock (T/HS) in experimental animals as well as following severe injury in humans. Although the pathophysiology of BM suppression remains poorly understood, mesenteric lymph is thought to play an important role in T/HS-induced BM suppression; however, the direct effect of mesenteric lymph on BM in vitro has never been studied. In addition, recent studies in rats have also shown that female and castrated male rats are protected against T/HS-induced BM failure. We therefore hypothesized that mesenteric lymph is a source of factor(s) causing direct BM suppression and that the effects of mesenteric lymph are gender dependent. To test this hypothesis, we subjected noncastrated (NC) and castrated (C) male and proestrus female rats to T/HS or trauma sham shock (T/SS). Mesenteric lymph collected 3 h postshock was plated (4% v/v) with BM cells collected from unmanipulated male or female rats for granulocyte-macrophage colony-forming units (CFU-GM) and erythroid burst-forming units (BFU-E) colony growth. The T/HS lymph collected from NC-male rats but not from female rats caused a 50% inhibition of CFU-GM and BFU-E colony growth compared with cells cultured without lymph (P < 0.05 versus all other groups (ANOVA + Tukey). T/HS lymph collected from C-male rats also caused no significant inhibition of CFU-GM and BFU-E colony growth compared with cells cultured without lymph. Female and male BM progenitor cells had a similar response to mesenteric lymph from all groups tested. These results show that mesenteric lymph from NC-male rats suppresses CFU-GM and BFU-E progenitor growth in vitro, whereas the lymph from C-male and female rats did not. The effects of mesenteric lymph were the same regardless of whether the target BM was from male or female rats. The results therefore indicate that BM failure in male rats is directly mediated by factors present within the mesenteric lymph that appear to be modulated by castration, and protection against BM failure in female rats occurs at a systemic rather than a local level. Further studies are needed to elucidate potential therapeutic effects of lymph manipulation in hematopoiesis after injury.     

聚腺苷二磷酸核糖基聚合酶在大鼠失血性休克后血管低反应性发生中的作用

目的 研究聚腺苷二磷酸核糖基聚合酶(PARP)在大鼠失血性休克后血管低反应性发生中的作用.方法 将SD大鼠随机分为休克组、PARP抑制剂3-氨基苯甲酰胺(3-AB)预处理+休克组和假手术对照组,采用股动脉放血复制失血性休克模型.在体观察给予3 μg/kg去甲肾上腺素(NE)升高血压的幅度;离体测定肠系膜上动脉血管环对NE的反应性;硝酸还原酶法测定血浆和肠系膜上动脉血管环组织匀浆中一氧化氮(NO)的含量.结果 休克模型完成后即刻静脉给NE,休克组血压升高幅度显著低于假手术对照组(P＜0.01);回输血1 h后再次静脉给NE,休克组血压显著低于3-AB预处理+休克组和假手术对照组(P＜0.05和P＜0.01).假手术对照组最大收缩张力[(0.367 1±0.221 3)g/mm]＞3-AB预处理+休克组C(0.286 4±0.153 2)g/mm]＞休克组C(0.185 6±0.11 3)g/mm,P＜0.05或P＜0.01];与休克组比较,3-AB预处理+休克组量一效曲线左移,在NE终浓度为10-7、10-6和10-5 mol/L时其收缩力显著增加(P均＜0.05).3组间血浆NO含量差异均无统计学意义,3-AB预处理+休克组血浆和肠系膜上动脉组织匀浆中NO含量虽较休克组稍有降低,但两组问比较差异无统计学意义.结论 PARP参与了大鼠失血性休克后血管低反应性的发生.     

The beneficial effects of protein tyrosine kinase inhibition on the circulatory failure induced by endotoxin in the rat

Implication of enhanced activity of tyrosine kinases has been established in the pathophysiology of many diseases associated with local (e.g., atherosclerosis) or systemic (e.g., septic shock) inflammation. The main objective of this study was to elucidate whether tyrosine kinase and nitric oxide were involved in endotoxin-induced impairment of vascular responses to sympathetic nerve stimulation (SNS) in rat isolated mesenteric bed. Therefore, the effects of genistein, an inhibitor of protein tyrosine kinase, and L-NAME (N-nitro-L-arginine methyl ester), an inhibitor of nitric oxide synthase, on endotoxin-induced shock were investigated in the thiopental-anesthetized rats. We also studied the effects of endotoxin on the vasoconstrictor responses to SNS in the rat isolated perfused mesenteric bed. Endotoxin injection (10 mg kg(-1), i.p.) produced a marked hypotension and a reduction of the pressor responses elicited by phenylephrine (0.1, 0.3, and 3 microg kg(-1), i.v.). Pretreatment of the rats with either genistein (10 mg kg(-1) i.p., 2 h before endotoxin injection), L-NAME (0.1 mg kg(-1), i.p., 30 min before endotoxin injection), or a combination of both attenuated the hypotension caused by endotoxin. SNS in the rat isolated perfused mesenteric bed caused a frequency-dependent vasoconstrictor response, which was abolished by tetrodotoxin (10(-7) M), prazoscin (10(-7) M), and guanethidine (10(-7)M). In mesenteric vascular beds removed from rats injected with endotoxin, the vasoconstrictor responses to SNS were markedly impaired. Although genistein and L-NAME pretreatment attenuated the vascular hyporeactivity to phenylephrine, they did not improve the impaired SNS response of the isolated vascular bed of endotoxin-treated animals. These results indicate that genistein and L-NAME pretreatment prevent the hypotension and the delayed hyporeactivity to phenylephrine induced by endotoxin, but they failed to restore the vascular hyporeactivity to SNS.