Chih-Chin Shih

Therapeutic effects of hypertonic saline on peritonitis-induced septic shock with multiple organ dysfunction syndrome in rats.

Objective:Significant mortality in patients with sepsis results from the development of multiple organ dysfunction syndrome. Small-volume resuscitation with 7.5% NaCl hypertonic saline has been proposed to restore physiologic hemodynamics in hemorrhagic shock. Therefore, we hypothesized that hypertonic saline resuscitation could alleviate the development of multiple organ dysfunction syndrome in sepsis induced by cecal ligation and puncture. Design:Randomized, prospective animal experiment. Setting:Academic research laboratory. Subjects:Male Wistar rats. Interventions:The animals were randomly allocated to one of four groups: 1) sham operation (0.9% NaCl, 4 mL/kg intravenously, at 3 hrs after laparotomy); 2) sham operation plus hypertonic saline (7.5% NaCl, 4 mL/kg intravenously, at 3 hrs after laparotomy); 3) cecal ligation and puncture (0.9% NaCl, 4 mL/kg intravenously, at 3 hrs after cecal ligation and puncture); and 4) cecal ligation and puncture plus hypertonic saline (7.5% NaCl, 4 mL/kg intravenously, at 3 hrs after cecal ligation and puncture). Measurements and Main Results:Cecal ligation and puncture for 18 hrs was associated with circulatory failure (i.e., hypotension and vascular hyporeactivity to norepinephrine), multiple organ dysfunction syndrome (examined by biochemical variables and histologic studies), and 18-hr mortality. Hypertonic saline not only ameliorated the deterioration of hemodynamic changes but also attenuated neutrophil infiltration in the lung and the liver of septic animals. Hypertonic saline increased the survival rate at 9 and 18 hrs compared with the cecal ligation and puncture group. Moreover, hypertonic saline reduced plasma nitric oxide and interleukin-1&bgr; and organ O2−· levels in rats that underwent cecal ligation and puncture. Conclusions:Hypertonic saline prevented circulatory failure, alleviated multiple organ dysfunction syndrome, and decreased the mortality rate in animals receiving cecal ligation and puncture. These beneficial effects of hypertonic saline may be attributed to reducing the plasma concentration of nitric oxide and interleukin-1&bgr; as well as the organ O2−· level and decreasing lung neutrophil infiltration and liver necrosis. Our study suggests that hypertonic saline could be a potential and inexpensive therapeutic agent in the early sepsis of animals or patients.

Abnormal Activation Of Potassium Channels In Aortic Smooth Muscle Of Rats With Peritonitis-induced Septic Shock

This study was conducted to examine the role of membrane hyperpolarization in mediating vascular hyporeactivity induced by cecal ligation and puncture (CLP) in endothelial-denuded strips of rat thoracic aorta ex vivo. The CLP for 18 h elicited a significant fall of blood pressure and a severe vascular hyporeactivity to norepinephrine as seen in severe sepsis. At the end of the in vivo experiments, thoracic aortas were removed from both CLP-treated and control rats. After removal of the endothelium, aortic segments were mounted in myographs for the recording of isometric tension and smooth muscle membrane potential. The membrane potential recording showed that a hyperpolarization was observed in the CLP-treated rats when compared with the control rats. This hyperpolarization was reversed by iberiotoxin (a large-conductance Ca2+-activated K+ channel blocker), 4-aminopyridine (a voltage-dependent K+ channel blocker), barium (an inward rectifier K+ channels blocker), N-(1-adamantyl)-N&vprime;-cyclohexyl-4-morpholinecarboxamidine hydrochloride (a pore-forming blocker of adenosine triphosphate (ATP)-sensitive K+ channels [KATP]), or methylene blue (a nonspecific guanylyl cyclase [GC] inhibitor). However, this hyperpolarization was not significantly affected by apamin (a small-conductance Ca2+-activated K+ channel blocker), glibenclamide (a sulfonylurea blocker of KATP), N&ohgr;-nitro-l-arginine methyl ester (a NOS inhibitor), or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (an NO-sensitive GC inhibitor). In addition, the basal tension of the tissues obtained from CLP rats was increased simultaneously, whereas membrane potential was reversed. In contrast, none of these inhibitors had significant effects on the membrane potential or the basal tension in control tissues. Thus, we provide electrophysiological and functional evidence demonstrating that an abnormal activation of K+ channels in vascular smooth muscle in animals with septic shock induced by CLP. Our observations suggest that the activation of large conductance Ca2+-activated K+ channels, voltage-dependent K+ channels, inward rectifier K+ channels, and KATP channels, but not small conductance Ca2+-activated K+ channels, contributes to CLP-induced vascular hyporeactivity. Furthermore, the hyperpolarization in septic shock induced by CLP is likely via non-NO-sensitive GC pathway.

Nitric oxide contributes to abnormal vascular calcium regulation and reactivity induced by peritonitis-associated septic shock in rats

Calcium plays an important role in determining vascular smooth muscle tone. Norepinephrine (NE)-induced vascular contraction contains two components: 1) Ca2+ release from the sarcoplasmic reticulum as the fast phase and 2) Ca2+ influx via a voltage-dependent calcium channel as the slow phase. This study used functional isometric tension recording to evaluate mediators contributing to abnormal NE-induced Ca2+ handling and reactivity in isolated thoracic aortas from septic rats. Sepsis was induced by cecal ligation and puncture (CLP), and thoracic aortas were removed at 18 h after CLP. Our results showed that rats that received CLP for 18 h manifested severe hypotension and vascular hyporeactivity to NE in vivo. This vascular hyporecativity to NE was also observed in the aorta obtained from CLP-induced sepsis rat. Both the fast and slow phases of NE-induced contraction were reduced in aortas from sepsis rats. To clarify what possible mediators contribute to the abnormal Ca2+ handling in aortas from sepsis animals, inhibitors of Ca2+ channel and release were used. Inhibition by 2-aminoethoxy-diphenyl borane, ryanodine, and cyclopiazonic acid of the NE-induced contraction in Ca2+-free solution was greater in the aorta from sepsis rats and inhibitions of cyclopiazonic acid and ryanodine, but not of 2-aminoethoxy-diphenyl borane, were attenuated by NOS inhibitor N&ohgr;-nitro-l-arginine methyl ester. In addition, the attenuation of NE-induced contraction by nifedipine in the aorta was also greater in the CLP group. Our results suggest that abnormal NE-induced Ca2+ handling associated with vascular hyporeactivity in the CLP-induced sepsis is caused by a major decrease in sarcoplasmic reticulum function and a minor impairment of voltage-dependent Ca2+ channels on membrane to Ca2+ handling, at least, in the aorta, and this could be attributed to an overproduction of NO in sepsis.

RhoA/Rho-Kinase and Nitric Oxide in Vascular Reactivity in Rats with Endotoxaemia

RhoA/Rho-kinase (RhoA/ROK) pathway promotes vasoconstriction by calcium sensitivity mechanism. LPS causes nitric oxide (NO) overproduction to induce vascular hyporeactivity. Thus, we tried to examine the role of RhoA/ROK and NO in the regulation of vascular reactivity in different time-point of endotoxaemia. Male Wistar rats were intravenously infused for 10 min with saline or E. coli endotoxin (lipopolysaccharide, LPS, 10 mg/kg) and divided to five groups (n = 8 in each group): (i) Control, sacrificed at 6 h after saline infusion; (ii) LPS1h, sacrificed at 1 h after LPS infusion; (iii) LPS2h, sacrificed at 2 h after LPS infusion; (iv) LPS4h, sacrificed at 4 h after LPS infusion; and (v) LPS6h, sacrificed at 6 h after LPS infusion. LPS1h and LPS2h were regarded as early endotoxaemia, whereas LPS4h and LPS6h were regarded as late endotoxaemia. Indeed, our results showed that LPS reproduced a biphasic hypotension and sustained vascular hyporeactivity to noradrenaline (NA) in vivo. Interestingly, this hyporeactivity did not occur in ex vivo during early endotoxaemia. This could be due to increases of aortic RhoA activity (n = 5, P<0.05) and myosin phosphatase targeting subunit 1 phosphorylation (n = 3, P<0.05). In addition, pressor response to NA and vascular reactivity in early endotoxaemia were inhibited by ROK inhibitor, Y27632. Furthermore, plasma bradykinin was increased at 10 min (24.6±13.7 ng/mL, n = 5, P<0.05) and aortic endothelial NO synthase expression was increased at 1 h (+200%. n = 3, P<0.05) after LPS. In late endotoxaemia, the vascular hyporeactivity was associated with aortic inducible NO synthase expression (n = 3, P<0.05) and an increased serum NO level (n = 8, P<0.05). Thus, an increased RhoA activity could compensate vascular hyporeactivity in early endotoxaemia, and the large NO production inhibiting RhoA activity would lead to vascular hyporeactivity eventually.

Effects of small-volume hypertonic saline on acid-base and electrolytes balance in rats with peritonitis-induced sepsis.

ABSTRACT Our previous study has demonstrated that hypertonic saline (HS) given at 3 h after cecal ligation and puncture (CLP) surgery alleviates circulatory failure, multiple organ dysfunction syndrome, and mortality rate in rats. However, only few data exist on the application of HS in acid-base and electrolyte imbalance of sepsis. In addition, early one-dose HS administration seems to have only modest improvement on mortality rate. Thus, we evaluated the effects of HS on acid-base equilibrium and electrolyte balance in CLP-induced sepsis model and further compared with the effects of two- and one-dose HS administration. Male Wistar rats received CLP or sham operation followed by the administration of saline or HS (7.5% NaCl, 4 mL/kg, intravenously at 3 and 9 h after laparotomy or CLP). The changes in hemodynamics, biochemical variables, blood gas, electrolytes, organ histology, and plasma levels of nitric oxide (NO) and interleukin 1&bgr; (IL-1&bgr;) were examined during the 18-h observation. Hypertonic saline given either at 3 h (one-dose administration) or at 3 and 9 h (two-dose administration) after CLP attenuated circulatory failure, multiple organ dysfunction syndrome, metabolic acidosis, hyperkalemia, neutrophil infiltration, and 18-h mortality. Moreover, both one- and two-dose HS administrations significantly diminished plasma NO and IL-1&bgr; levels in CLP rats. However, only the two-dose HS administration significantly improved hyponatremia and hypocalcemia in septic rats. Beneficial effects of HS in septic rats may be attributed to not only reducing plasma levels of NO and IL-1&bgr;, but also improving metabolic acidosis and electrolyte imbalance. In addition, two-dose HS administration could reverse electrolyte imbalance caused by CLP.

Possible biomarkers of early mortality in peritonitis-induced sepsis rats

BACKGROUND Sepsis induced by cecal ligation and puncture (CLP) is accompanied by circulatory failure, multiple organ dysfunction syndrome, metabolic acidosis, and electrolyte imbalance in rats. However, it remains uncertain which parameters can be used to predict the mortality of septic rats. Thus, the aim of this study was to examine which possible biomarkers were associated with mortality in the CLP-induced sepsis model. MATERIALS AND METHODS After the carotid artery and vein were cannulated, rats were subsequently subjected to CLP or sham operation. The changes of hemodynamics, biochemical variables, blood gas, and electrolytes were monitored during the 18-h observation. RESULTS The CLP surgery caused circulatory failure, multiple organ dysfunction syndrome, metabolic acidosis, electrolyte imbalance, and death. Compared with survivors, nonsurvivors showed significant difference in (1) blood glucose; (2) lactate dehydrogenase, alanine aminotransferase, aspartate aminotransferase, creatinine, and blood urea nitrogen in serum; and (3) base excess, HCO3(-), PaCO2, potassium, and calcium in whole blood at 9 h after CLP. No significant difference in blood pressure, heart rate, pressor response to noradrenaline, rectal temperature, total protein, albumin, PaO2, and sodium was observed between nonsurvivors and survivors. However, after multifactor dimensionality reduction analysis, the union of HCO3(-) and blood glucose had the biggest testing balanced accuracy. CONCLUSIONS These results indicate that HCO3(-) plus blood glucose serves as the best biomarker of early death in rats with CLP-induced sepsis. Thus, these parameters could guide experimental procedures for making the right interventions when utilizing CLP as a sepsis model in rats.

Distinct Patterns of Wnt3a and Wnt5a Signaling Pathway in the Lung from Rats with Endotoxic Shock

Septic shock is a syndrome with severe hypotension and multiple organ dysfunction caused by an imbalance between pro-inflammatory and anti-inflammatory response. The most common risk factor of acute lung injury is severe sepsis. Patients with sepsis-related acute respiratory distress syndrome have higher mortality. Recent studies reveal regulatory roles of Wnt3a and Wnt5a signaling in inflammatory processes. Wnt3a signaling has been implicated in anti-inflammatory effects, whereas Wnt5a signaling has been postulated to have pro-inflammatory properties. However, the balance between Wnt3a and Wnt5a signaling pathway in the lung of rats with endotoxic shock has not been determined. Thus, we investigated the major components of Wnt3a and Wnt5a signaling pathway in the lung of endotoxemic rats. Male Wistar rats were intravenously infused with saline or lipopolysaccharide (LPS, 10 mg/kg). The changes of hemodynamics, biochemical variables, and arterial blood gas were examined during the experimental period. At 6 h after saline or LPS, animals were sacrificed, and lungs were obtained for analyzing superoxide production, water accumulation, histologic assessment, and protein expressions of Wnt3a and Wnt5a signaling pathway. Animals that received LPS showed circulatory failure, multiple organ dysfunction, metabolic acidosis, hyperventilation, lung edema, and high mortality. The lung from rats with endotoxic shock exhibited significant decreases in the levels of Wnt3a, Fzd1, Dsh1, phosphorylated GSK-3β at Ser9, and β-catenin. In contrast, the expressions of Wnt5a, Fzd5, and CaMKII were up-regulated in the lung of endotoxemic rats. These findings indicate the major components of Wnt3a and Wnt5a signaling in the lung are disturbed under endotoxic insult.

Procainamide Inhibits DNA Methylation and Alleviates Multiple Organ Dysfunction in Rats with Endotoxic Shock

Excessive inflammatory and oxidative stress lead to circulatory failure, multiple organ dysfunction, and high mortality in patients with sepsis. Microbial infection-induced DNA hypermethylation is associated with the augmentation of inflammation and oxidative stress. In our previous study, the antiarrhythmic drug procainamide inhibits the expression of DNA methyltransferase 1 (DNMT1) and diminishes IL-6 levels in rats with rhabdomyolysis. Thus, we further evaluated the effects of procainamide on the development of circulatory failure and multiple organ dysfunction in rats with endotoxic shock. Male Wistar rats were intravenously infused with saline or lipopolysaccharide (LPS) followed by procainamide administration. The changes of hemodynamics, blood glucose, biochemical variables, and plasma nitric oxide (NO) levels were analyzed during the experimental period. At the end of experiments, animal organs were also obtained for examining superoxide production, neutrophil infiltration, and DNA methylation status. Our results showed that LPS induced circulatory failure, multiple organ dysfunction, and high mortality rate in endotoxemic rats. Overt neutrophil infiltration and superoxide production, accompanied by the elevations of DNMT1 and 5-methylcytosine levels in the lung of endotoxemic rats were also observed. Treatment of endotoxemic animals with procainamide not only inhibited the increased levels of DNMT1 and 5-methylcytosine but also ameliorated neutrophil infiltration and superoxide production in the lung. In addition, the anti-inflammatory gene, IL27RA, was down-regulated in the LPS group and up-regulated in the LPS + Procainamide group. Procainamide also diminished IL27RA methylation in the lung of endotoxemic rat. Moreover, both DNMT inhibitors procainamide and hydralazine improved hypotension, hypoglycemia, and multiple organ dysfunction of LPS-treated rats. Thus, we suggest that the beneficial effects of procainamide could be attributed to the suppression of DNA methylation, neutrophil infiltration, superoxide production, and NO formation. It seems that this old drug may have new potential uses in infectious diseases, in particular, associated with endotoxemia.

Therapeutic Effects of Procainamide on Endotoxin-Induced Rhabdomyolysis in Rats

Overt systemic inflammatory response is a predisposing mechanism for infection-induced skeletal muscle damage and rhabdomyolysis. Aberrant DNA methylation plays a crucial role in the pathophysiology of excessive inflammatory response. The antiarrhythmic drug procainamide is a non-nucleoside inhibitor of DNA methyltransferase 1 (DNMT1) used to alleviate DNA hypermethylation. Therefore, we evaluated the effects of procainamide on the syndromes and complications of rhabdomyolysis rats induced by lipopolysaccharide (LPS). Rhabdomyolysis animal model was established by intravenous infusion of LPS (5 mg/kg) accompanied by procainamide therapy (50 mg/kg). During the experimental period, the changes of hemodynamics, muscle injury index, kidney function, blood gas, blood electrolytes, blood glucose, and plasma interleukin-6 (IL-6) levels were examined. Kidneys and lungs were exercised to analyze superoxide production, neutrophil infiltration, and DNMTs expression. The rats in this model showed similar clinical syndromes and complications of rhabdomyolysis including high levels of plasma creatine kinase, acute kidney injury, hyperkalemia, hypocalcemia, metabolic acidosis, hypotension, tachycardia, and hypoglycemia. The increases of lung DNMT1 expression and plasma IL-6 concentration were also observed in rhabdomyolysis animals induced by LPS. Treatment with procainamide not only inhibited the overexpression of DNMT1 but also diminished the overproduction of IL-6 in rhabdomyolysis rats. In addition, procainamide improved muscle damage, renal dysfunction, electrolytes disturbance, metabolic acidosis, hypotension, and hypoglycemia in the rats with rhabdomyolysis. Moreover, another DNMT inhibitor hydralazine mitigated hypoglycemia, muscle damage, and renal dysfunction in rhabdomyolysis rats. These findings reveal that therapeutic effects of procainamide could be based on the suppression of DNMT1 and pro-inflammatory cytokine in endotoxin-induced rhabdomyolysis.

Effects of SPAK on vascular reactivity and nitric oxide production in endotoxemic mice

ABSTRACT Vasoplegia impedes therapeutic interventions to restore vascular tone, leading to severe hypotension, poor tissue perfusion, and multiple organ failure in septic shock. High levels of circulating nitric oxide (NO) play a crucial role in endotoxin‐induced vascular hyporeactivity. Proinflammatory cytokines have been implicated in the induction of inducible NO synthase and overproduction of NO. Anti‐inflammatory therapy can diminish NO formation and improve vascular hyporeactivity in septic shock. STE20/SPS1‐realted proline/alanine‐rich kinase (SPAK) has been reported to activate mitogen‐activated protein kinase and contribute to intestinal inflammation. Thus, we evaluated the roles of SPAK in NO production and vascular hyporeactivity in endotoxemic animals. Male wild‐type and SPAK deficiency mice were intraperitoneally administered vehicle or Escherichia coli lipopolysaccharide (LPS, 50 mg/kg). The changes of systolic blood pressure and plasma nitrate and nitrite levels were measured during the experimental period. Thoracic aortas were exercised to assess vascular reactivity and SPAK expression. In the present study, mice in endotoxin model showed severe hypotension and hyporeactivity to serotonin, phenylephrine (PE), and acetylcholine in the aortic rings. Phosphorylated SPAK expression in the aorta and NO levels in the plasma were also increased in animals with endotoxic shock. However, deletion of SPAK not only reduced the elevation of NO levels but also improved vascular hyporeactivity to serotonin and PE in endotoxemic mice. Taken together, SPAK could be involved in the NO overproduction and vascular hyporesponsiveness to vasoconstrictors in endotoxic shock. Thus, inhibition of SPAK could be useful in the prevention of endotoxin‐induced vascular hyporeactivity. Graphical abstract Figure. No Caption available.