Liangming Liu

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 &mgr;g/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.

Ideal permissive hypotension to resuscitate uncontrolled hemorrhagic shock and the tolerance time in rats.

Background: Studies have shown that permissive hypotension for uncontrolled hemorrhagic shock can result in good resuscitation outcome. The ideal target mean arterial pressure (MAP) and the tolerance time for permissive hypotension have not been determined. Methods: To elucidate the ideal target MAP and tolerance time for permissive hypotension with uncontrolled hemorrhagic shock rats, the effects of different target MAPs (40, 50, 60, 70, 80, and 100 mmHg) and 60-, 90-, and 120-min permissive hypotension (50 mmHg) on uncontrolled hemorrhagic shock were observed. Results: Rats in normotensive groups (80 and 100 mmHg) had increased blood loss (101%, 126% of total blood volume), decreased hematocrit, decreased vital organ (liver and kidney) and mitochondrial function, and decreased animal survival rate (1 of 10). Rats in the 50- and 60-mmHg target MAP groups had decreased blood loss (52% and 69%, respectively), good hematocrit and vital organ and mitochondrial function, stable hemodynamics, and increased animal survival (8 of 10 and 6 of 10, respectively). Rats in the 40-mmHg target MAP group, although having decreased blood loss (39%), appeared to have very inferior organ function and animal survival (2 of 10). Animal survival (1 of 10) and vital organ function in the 120-min permissive hypotension group were significantly inferior to the 60- and 90-min groups. The 60- and 90-min groups had similar animal survival (8 of 10 and 6 of 10) and vital organ function. Conclusion: A target resuscitation pressure of 50–60 mmHg is the ideal blood pressure for uncontrolled hemorrhagic shock. Ninety minutes of permissive hypotension is the tolerance limit; 120 min of hypotensive resuscitation can cause severe organ damage and should be avoided.

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

ABSTRACT 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 Ca2+ 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 Ca2+ 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 Ca2+at early shock (P < 0.05 or P < 0.01). Angiotensin II increased the decreased contractile response of SMA to NE and Ca2+ 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 Ca2+ 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.

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 (MLC20). 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 MLC20 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 MLC20.

Effects of the balance in activity of RhoA and Rac1 on the shock-induced biphasic change of vascular reactivity in rats.

Objective:To investigate the effects of the balance in activity of RhoA and Rac1 on the shock-induced biphasic change in vascular reactivity and the related mechanism. Background:Vascular reactivity after hemorrhagic shock shows a biphasic change. RhoA and Rac1 are the main members of a family of Rho GTPases; whether and how they participate in the regulation of the biphasic change in vascular reactivity after shock is not known. Methods:The relationship of the balance of the activity RhoA and Rac1 with the changes in vascular reactivity after hemorrhagic shock, the effects of artificially changing the balance of RhoA and Rac1 activity on vascular reactivity, and the roles of Rho kinase and p21-activated kinase (PAK) in RhoA/Rac1 regulation of vascular reactivity were observed in isolated superior mesenteric arteries (SMAs) from hemorrhagic shocked rats and hypoxia-treated vascular smooth muscle cells (VSMCs). Results:The reactivity of SMAs and VSMCs to norepinephrine after shock or hypoxia was positively correlated with changes in the RhoA and Rac1 activity ratio. Artificially changing the balance in activity of RhoA and Rac1 significantly changed the shock-induced biphasic response of vascular reactivity. Specific antagonist of Rho kinase and PAK (Y-27632 and PAK-18) respectively abolished the effect of activation of RhoA and Rac1. Activation of RhoA significantly increased the activity of Rho kinase and inhibited the activity of Rac1 in SMAs. Rac1 activation significantly increased the activity of PAK and decreased the activity of RhoA. Conclusions:The balance in the activity of RhoA and Rac1 participated in the biphasic vascular reactivity seen after hemorrhagic shock. RhoA and Rac1 regulation of the vascular reactivity after shock are closely related to Rho kinase and the PAK pathway. RhoA regulates vascular reactivity mainly through activation of Rho kinase and inhibition of Rac1. Rac1 regulates vascular reactivity mainly through inhibition of RhoA and activation of PAK. These findings have potential significance for the treatment of vascular hyporesponsiveness.

Determination of the Optimal Mean Arterial Pressure for Postbleeding Resuscitation after Hemorrhagic Shock in Rats

Background: The authors previously found that 50–60 mmHg mean arterial blood pressure (MAP) was an optimal target resuscitation pressure for hemorrhagic shock before bleeding was controlled in rats. However, the optimal target resuscitation pressure for hemorrhagic shock after bleeding has been controlled has not been determined. Methods: A model of uncontrolled hemorrhagic shock was initiated in anesthetized Wistar rats. After 1-h hypotensive resuscitation and bleeding was stopped, rats received fluid resuscitation to different target MAPs (50, 70, or 90 mmHg) with lactated Ringers solution (LR), 6% hydroxyethyl starch (HES), LR+HES (2:1) or LR+whole blood (2:1) for 2 h. Animal survival, hemodynamic parameters, and vital organ functions were observed. Results: After bleeding had been controlled, mildly hypotensive resuscitation at a target MAP of 70 mmHg increased the survival time and survival rate compared with a target MAP of 50 mmHg and 90 mmHg (P < 0.05 or 0.01). Hemodynamic parameters, cardiac output, oxygen delivery, and vital organ function (including mitochondrial function) in 70 mmHg target MAP groups were better than in other two-target pressure groups (P < 0.05 or 0.01). Among the fluids tested, LR+whole blood (2:1) or LR+HES130 (2:1) had better effects than LR or HES alone at each level of target blood pressure. Conclusion: Mildly hypotensive resuscitation is also needed for hemorrhagic shock after bleeding has been controlled, irrespective of whether crystalloids or colloids are used. The optimal target pressure was 70 mmHg in our rat model. A resuscitation pressure that is too low or too high cannot produce a good resuscitative effect.

Short-term, Mild Hypothermia Can Increase the Beneficial Effect of Permissive Hypotension on Uncontrolled Hemorrhagic Shock in Rats

Background: Our previous and other studies have shown that hypotensive or hypothermic resuscitation have beneficial effects on uncontrolled hemorrhagic shock. Whether hypothermia can increase the beneficial effect of hypotensive resuscitation on hemorrhagic shock is not known. Methods: Two-hundred and twenty Sprague-Dawley rats were used to make uncontrolled hemorrhagic shock. Before bleeding was controlled, rats received normotensive or hypotensive resuscitation (target mean arterial pressure at 80 or 50 mmHg) in combination with normal (37°C) or mild hypothermia (34°C) (phase II). After bleeding was controlled, rats received whole blood and lactated Ringers solution resuscitation for 2 h (phase III). The animal survival, blood loss, fluid requirement, cardiac output, and coagulation functions, as well as vital organ function, mitochondrial function, and energy metabolism of liver, kidney and intestines, were noted. Results: Short-term, mild hypothermia before bleeding was controlled increased the beneficial effect of hypotensive resuscitation. Hypothermia further decreased blood loss, oxygen consumption, and functional damage to the liver, kidney, and intestines during hypotensive resuscitation, protected mitochondrial function and energy metabolism (activity of Na+-K+-ATPase), and further improved survival time and survival rate (hypothermic/hypotensive combined group: survival rate, 9/10; survival time, 616 min; normothermic/normotensive group: 1/10, 256 min; hypothermic/normotensive group: 4/10, 293 min). Hypothermia slightly inhibited coagulation function. Conclusion: Mild hypothermia before bleeding is controlled can increase the beneficial effect of hypotensive resuscitation on uncontrolled hemorrhagic shock. The mechanism underlying the benefits of short-term hypothermia may be related to the decrease in oxygen consumption and metabolism, and protection of mitochondrial and organ functions.

Angiopoietins regulate vascular reactivity after haemorrhagic shock in rats through the Tie2-nitric oxide pathway

AIMS Vascular reactivity shows biphasic changes after severe trauma or shock. Our aim was to elucidate the mechanisms of biphasic-changed vascular reactivity after haemorrhagic shock by observing the regulation of angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) on it. METHODS AND RESULTS Haemorrhagic-shock Sprague-Dawley rats, hypoxia-treated superior mesenteric arteries (SMAs) with intact endothelia, and a cell mixture of vascular smooth muscle cells (VSMCs) and vascular endothelial cells (VECs) were adopted to evaluate the regulatory effects of Ang-1 and Ang-2 on vascular reactivity and their relationship to Tie2 (receptor tyrosine kinase)-Akt-endothelial nitric oxide synthase (eNOS) and Tie2-extracellular signal-regulated kinase (Erk)-inducible nitric oxide synthase (iNOS) signal pathways. Ang-1 expression, Tie2 phosphorylation, and nitric oxide (NO) release were increased at early shock. Exogenous Ang-1 maintained the vascular reactivity of SMAs after early hypoxia. Tie2-blocking antibody and the antagonists of Akt and eNOS antagonized Ang-1-induced maintenance in vascular reactivity and a slight release in NO at the early stage of shock. Ang-2 expression, Tie2 phosphorylation, and NO release were greatly increased at late shock, but exogenous Ang-2 further decreased the vascular reactivity of SMAs after late hypoxia. Tie2-blocking antibody and the antagonists of Erk and iNOS andtagonized the Ang-2-induced decrease in vascular reactivity and a large release of NO at the late stage of shock. CONCLUSION Ang-1 and Ang-2 participated in the regulation of vascular reactivity after haemorrhagic shock. Ang-1 was mainly responsible for the hyperreactivity at early shock through the Tie2-Akt-eNOS pathway and an appropriate amount of NO release. Ang-2 was mainly responsible for the hyporeactivity at late shock through the Tie2-Erk-iNOS pathway and the release of a large amount of NO.

Small doses of arginine vasopressin in combination with norepinephrine "buy" time for definitive treatment for uncontrolled hemorrhagic shock in rats.

ABSTRACT Implementation of fluid resuscitation and blood transfusion are greatly limited in prehospital or evacuation settings after severe trauma or war wounds. With uncontrolled hemorrhagic shock rats, we investigated if arginine vasopressin (AVP) in combination with norepinephrine (NE) is independent (or slightly dependent) of fluid resuscitation and can “buy” time for the subsequently definitive treatment of traumatic hemorrhagic shock in the present study. The results showed that AVP (0.4 U/kg) alone or with NE (3 &mgr;g/kg) with one-eighth and one-fourth volumes of total blood volume of lactated Ringer’s infusion significantly increased and maintained the mean arterial pressure. Among all groups, 0.4 U/kg of AVP + NE (3 &mgr;g/kg) with one-eighth volume of lactated Ringer’s infusion had the best effect: it significantly increased and maintained hemodynamics and prolonged the survival time. This early treatment strategy significantly improved the effects of subsequently definitive treatments (after bleeding controlled): it increased the subsequent survival, improved the hemodynamic parameters, improved the cardiac function, and increased the tissue blood flow and oxygen delivery. These results suggested that early application of small doses of AVP (0.4 U/kg) + NE before bleeding control can “buy” time for the definitive treatment of uncontrolled hemorrhagic shock, which may be an effective measure for the early treatment of traumatic hemorrhagic shock.

Protein kinase C isoforms responsible for the regulation of vascular calcium sensitivity and their relationship to integrin-linked kinase pathway after hemorrhagic shock.

BACKGROUND The aim of this study was to investigate the protein kinase C (PKC) isoforms involved in the regulation of vascular calcium sensitivity after hemorrhagic shock, the related mechanism, and the role of integrin-linked kinase (ILK). METHODS Using superior mesenteric artery from hemorrhagic shock rats and hypoxia-treated vascular smooth muscle cells, the effects of PKC isoforms agonists and antagonists on vascular calcium sensitivity, their relationship with myosin light chain phosphatase (MLCP), myosin light chain (MLC20) phosphorylation, and ILK were observed. RESULTS The results indicated that PKCα and ε agonists, thymelea toxin and carbachol, restored shock-induced decrease of vascular calcium sensitivity; PKCα antagonist, Gö-6976 and PKCε pseudosubstrate inhibition peptide, aggravated shock-induced calcium desensitization, whereas the agonists and antagonists of PKCδ and ζ had no effects on shock-induced calcium desensitization. PKCα and ε agonists reversed the increased MLCP activity and the decreased MLC20 phosphorylation induced by shock or hypoxia. ILK inhibitor abolished the effects of PKCα and ε agonists on vascular calcium sensitivity, MLCP activity, and MLC20 phosphorylation. CONCLUSION These findings suggested that PKCα and ε may be the main isoforms responsible for the regulation of vascular calcium sensitivity after hemorrhagic shock, which enforce their regulation through MLCP-MLC20 pathway, and ILK may be a downstream molecule of PKCα and ε.