Fernando Luiz Zanoni

INSULIN REGULATES CYTOKINES AND INTERCELLULAR ADHESION MOLECULE-1 GENE EXPRESSION THROUGH NUCLEAR FACTOR-κB ACTIVATION IN LPS-INDUCED ACUTE LUNG INJURY IN RATS

Diabetic patients have increased susceptibility to infection, which may be related to impaired inflammatory response observed in experimental models of diabetes, and restored by insulin treatment. The goal of this study was to investigate whether insulin regulates transcription of cytokines and intercellular adhesion molecule 1 (ICAM-1) via nuclear factor-&kgr;B (NF-&kgr;B) signaling pathway in Escherichia coli LPS-induced lung inflammation. Diabetic male Wistar rats (alloxan, 42 mg/kg, i.v., 10 days) and controls were instilled intratracheally with saline containing LPS (750 &mgr;g/0.4 mL) or saline only. Some diabetic rats were given neutral protamine Hagedorn insulin (4 IU, s.c.) 2 h before LPS. Analyses performed 6 h after LPS included: (a) lung and mesenteric lymph node IL-1&bgr;, TNF-&agr;, IL-10, and ICAM-1 messenger RNA (mRNA) were quantified by real-time reverse transcriptase-polymerase chain reaction; (b) number of neutrophils in the bronchoalveolar lavage (BAL) fluid, and concentrations of IL-1&bgr;, TNF-&agr;, and IL-10 in the BAL were determined by the enzyme-linked immunosorbent assay; and (c) activation of NF-&kgr;B p65 subunit and phosphorylation of I-&kgr;B&agr; were quantified by Western blot analysis. Relative to controls, diabetic rats exhibited a reduction in lung and mesenteric lymph node IL-1&bgr; (40%), TNF-&agr; (∼30%), and IL-10 (∼40%) mRNA levels and reduced concentrations of IL-1&bgr; (52%), TNF-&agr; (62%), IL-10 (43%), and neutrophil counts (72%) in the BAL. Activation of NF-&kgr;B p65 subunit and phosphorylation of I-&kgr;B&agr; were almost suppressed in diabetic rats. Treatment of diabetic rats with insulin completely restored mRNA and protein levels of these cytokines and potentiated lung ICAM-1 mRNA levels (30%) and number of neutrophils (72%) in the BAL. Activation of NF-&kgr;B p65 subunit and phosphorylation of I-&kgr;B&agr; were partially restored by insulin treatment. In conclusion, data presented suggest that insulin regulates transcription of proinflammatory (IL-1&bgr;, TNF-&agr;) and anti-inflammatory (IL-10) cytokines, and expression of ICAM-1 via the NF-&kgr;B signaling pathway.

Mesenteric microcirculatory dysfunctions and translocation of indigenous bacteria in a rat model of strangulated small bowel obstruction

PRUPOSE Bacterial translocation has been shown to occur in critically ill patients after extensive trauma, shock, sepsis, or thermal injury. The present study investigates mesenteric microcirculatory dysfunctions, the bacterial translocation phenomenon, and hemodynamic/metabolic disturbances in a rat model of intestinal obstruction and ischemia. METHODS Anesthetized (pentobarbital 50 mg/kg, i.p.) male Wistar rats (250–350 g) were submitted to intestinal obstruction or laparotomy without intestinal obstruction (Sham) and were evaluated 24 hours later. Bacterial translocation was assessed by bacterial culture of the mesenteric lymph nodes (MLN), liver, spleen, and blood. Leukocyte-endothelial interactions in the mesenteric microcirculation were assessed by intravital microscopy, and P-selectin and intercellular adhesion molecule (ICAM)-1 expressions were quantified by immunohistochemistry. Hematocrit, blood gases, lactate, glucose, white blood cells, serum urea, creatinine, bilirubin, and hepatic enzymes were measured. RESULTS About 86% of intestinal obstruction rats presented positive cultures for E. coli in samples of the mesenteric lymph nodes, liver, and spleen, and 57% had positive hemocultures. In comparison to the Sham rats, intestinal obstruction induced neutrophilia and increased the number of rolling (~2-fold), adherent (~5-fold), and migrated leukocytes (~11-fold); this increase was accompanied by an increased expression of P-selectin (~2-fold) and intercellular adhesion molecule-1 (~2-fold) in the mesenteric microcirculation. Intestinal obstruction rats exhibited decreased PaCO2, alkalosis, hyperlactatemia, and hyperglycemia, and increased blood potassium, hepatic enzyme activity, serum urea, creatinine, and bilirubin. A high mortality rate was observed after intestinal obstruction (83% at 72 h vs. 0% in Sham rats). CONCLUSION Intestinal obstruction and ischemia in rats is a relevant model for the in vivo study of mesenteric microcirculatory dysfunction and the occurrence of bacterial translocation. This model parallels the events implicated in multiple organ dysfunction (MOD) and death.

Paradoxical effects of brain death and associated trauma on rat mesenteric microcirculation: an intravital microscopic study

OBJECTIVE: Experimental findings support clinical evidence that brain death impairs the viability of organs for transplantation, triggering hemodynamic, hormonal, and inflammatory responses. However, several of these events could be consequences of brain death–associated trauma. This study investigated microcirculatory alterations and systemic inflammatory markers in brain-dead rats and the influence of the associated trauma. METHOD: Brain death was induced using intracranial balloon inflation; sham-operated rats were trepanned only. After 30 or 180 min, the mesenteric microcirculation was observed using intravital microscopy. The expression of P-selectin and ICAM-1 on the endothelium was evaluated using immunohistochemistry. The serum cytokine, chemokine, and corticosterone levels were quantified using enzyme-linked immunosorbent assays. White blood cell counts were also determined. RESULTS: Brain death resulted in a decrease in the mesenteric perfusion to 30%, a 2.6-fold increase in the expression of ICAM-1 and leukocyte migration at the mesentery, a 70% reduction in the serum corticosterone level and pronounced leukopenia. Similar increases in the cytokine and chemokine levels were seen in the both the experimental and control animals. CONCLUSION: The data presented in this study suggest that brain death itself induces hypoperfusion in the mesenteric microcirculation that is associated with a pronounced reduction in the endogenous corticosterone level, thereby leading to increased local inflammation and organ dysfunction. These events are paradoxically associated with induced leukopenia after brain damage.

HYPERTONIC SALINE SOLUTION REDUCES MESENTERIC MICROCIRCULATORY DYSFUNCTIONS AND BACTERIAL TRANSLOCATION IN A RAT MODEL OF STRANGULATED SMALL BOWEL OBSTRUCTION

ABSTRACT We examined the effects of hypertonic saline (HS) on inflammatory, metabolic variables, and bacterial translocation (BT) in rats submitted to intestinal obstruction and ischemia (IO). Male Wistar rats were submitted to IO and treated, 2 h thereafter, with lactated Ringer’s (LR) (4 mL/kg per 5 min, i.v.) or HS (7.5% NaCl, 4 mL/kg per 5 min, i.v.). Twenty-four hours after IO, rats were also submitted to enterectomy/enteroanastomosis to resection of necrotized small bowel. Leukocyte-endothelial interactions were investigated by intravital microscopy and the expression of P-selectin and intercellular adhesion molecule 1 by immunohistochemistry. Bacterial cultures of mesenteric lymph nodes, liver, spleen, and blood were used to evaluate BT. Levels of chemokines (cytokine-induced neutrophil chemoattractants 1 and 2), insulin, and corticosterone were determined by enzyme-linked immunosorbent assay. Intestinal histology, serum urea and creatinine levels, and hepatic enzymes activities were performed to evaluate local and remote damage. Relative to IO and LR-treated rats, which exhibited increases in the number of rolling (1.5-fold), adhered (3.5-fold) and migrated (9.0-fold) leukocytes, and increased expression of P-selectin (3-fold) and intercellular adhesion molecule 1 (3-fold) on mesenteric microcirculation, treatment with HS followed by enterectomy reduced leukocyte-endothelial interactions and expression of both adhesion molecules to values attained in sham rats. Serum chemokines were normalized after treatment with both solutions followed by enterectomy. Hypertonic saline–treated rats demonstrated a significant reduction in BT to 50% in liver and spleen samples and bacteremia (14%), compared with 82% of BT in liver and spleen samples of IO and LR-treated rats and bacteremia (57%). Local intestinal damage was attenuated, and renal and hepatic function preserved by treatment with HS followed by enterectomy. Survival rate increased to 86% up to 15 days. Data presented suggest that HS solution followed by enterectomy reduces mesenteric microcirculatory dysfunctions and BT, attenuating local and remote damage in a model of strangulated small bowel obstruction.

Microcirculatory effects of local and remote ischemic preconditioning in supraceliac aortic clamping.

INTRODUCTION Supraceliac aortic clamping in major vascular procedures promotes splanchnic ischemia and reperfusion (I/R) injury that may induce endothelial dysfunction, widespread inflammation, multiorgan dysfunction, and death. We tested the hypothesis that local or remote ischemic preconditioning (IPC) may be protective against injury after supraceliac aortic clamping through the modulation of mesenteric leukocyte-endothelial interactions, as evaluated with intravital microscopy and expression of adhesion molecules. METHODS Fifty-six male Wistar rats (weight, 190 to 250 g), were divided into four groups of 14 rats each: control-sham surgery without aortic occlusion; I/R through supraceliac aortic occlusion for 20 minutes, followed by 120 minutes of reperfusion; local IPC through supraceliac aortic occlusion for two cycles of 5 minutes of ischemia and 5 minutes of reperfusion, followed by the same protocol of the IR group; remote IPC through infrarenal aortic occlusion for two cycles of 10 minutes of ischemia and 10 minutes of reperfusion, followed by the same protocol of the IR group. Seven animals per group were used to evaluate in vivo leukocyte-endothelial interactions in postcapillary venules with intravital microscopy and another seven animals per group were used to collect mesentery samples for immunohistochemistry demonstration of adhesion molecules expression. RESULTS Supraceliac aortic occlusion increased the number of rolling leukocytes with slower velocities and increased the number of adherent leukocytes to the venular surface and leukocyte migration to the interstitium. The expression of P-selectin, E-selectin, and intercellular adhesion molecule-1 was also increased significantly after I/R. Local or remote IPC reduced the leukocyte recruitment in vivo and normalized the expression of adhesion molecules. CONCLUSIONS Local or remote IPC reduces endothelial dysfunction on mesenteric microcirculation caused by I/R injury after supraceliac aortic clamping.

Inhibition of Autonomic Storm by Epidural Anesthesia Does Not Influence Cardiac Inflammatory Response After Brain Death in Rats

BACKGROUND After brain death (BD) donors usually experience cardiac dysfunction, which is responsible for a considerable number of unused organs. Causes of this cardiac dysfunction are not fully understood. Some authors argue that autonomic storm with severe hemodynamic instability leads to inflammatory activation and myocardial dysfunction. OBJECTIVES To investigate the hypothesis that thoracic epidural anesthesia blocks autonomic storm and improves graft condition by reducing the inflammatory response. METHODS Twenty-eight male Wistar rats (250-350 g) allocated to four groups received saline or bupivacaine via an epidural catheter at various times in relation to brain-death induction. Brain death was induced by a sudden increase in intracranial pressure by rapid inflation of a ballon catheter in the extradural space. Blood gases, electrolytes, and lactate analyses were performed at time zero, and 3 and 6 hours. Blood leukocytes were counted at 0 and 6 hours. After 6 hours of BD, we performed euthanasia to measure vascular adhesion molecule (VCAM)-1, intracellular adhesion molecule (ICAM)-1, interleukin (IL)-1β, tumor necrosis factor (TNF)-α, Bcl-2 and caspase-3 on cardiac tissue. RESULTS Thoracic epidural anesthesia was effective to block the autonomic storm with a significant difference in mean arterial pressure between the untreated (saline) and the bupivacaine group before BD (P < .05). However, no significant difference was observed for the expressions of VCAM-1, ICAM-1, TNF-α, IL-1β, Bcl-2, and caspase-3 (P > .05). CONCLUSION Autonomic storm did not seem to be responsible for the inflammatory changes associated with BD; thoracic epidural anesthesia did not modify the expression of inflammatory mediators although it effectively blocked the autonomic storm.

Mesenteric hypoperfusion and inflammation induced by brain death are not affected by inhibition of the autonomic storm in rats

OBJECTIVES: Brain death is typically followed by autonomic changes that lead to hemodynamic instability, which is likely associated with microcirculatory dysfunction and inflammation. We evaluated the role of the microcirculation in the hemodynamic and inflammatory events that occur after brain death and the effects of autonomic storm inhibition via thoracic epidural blockade on mesenteric microcirculatory changes and inflammatory responses. METHODS: Male Wistar rats were anesthetized and mechanically ventilated. Brain death was induced via intracranial balloon inflation. Bupivacaine (brain death-thoracic epidural blockade group) or saline (brain death group) infusion via an epidural catheter was initiated immediately before brain death induction. Sham-operated animals were used as controls (SH group). The mesenteric microcirculation was analyzed via intravital microscopy, and the expression of adhesion molecules was evaluated via immunohistochemistry 180 min after brain death induction. RESULTS: A significant difference in mean arterial pressure behavior was observed between the brain death-thoracic epidural blockade group and the other groups, indicating that the former group experienced autonomic storm inhibition. However, the proportion of perfused small vessels in the brain death-thoracic epidural blockade group was similar to or lower than that in the brain death and SH groups, respectively. The expression of intercellular adhesion molecule 1 was similar between the brain death-thoracic epidural blockade and brain death groups but was significantly lower in the SH group than in the other two groups. The number of migrating leukocytes in the perivascular tissue followed the same trend for all groups. CONCLUSIONS: Although thoracic epidural blockade effectively inhibited the autonomic storm, it did not affect mesenteric hypoperfusion or inflammation induced by brain death.

Effects of hypertonic saline and lactated Ringer's solutions on bacterial translocation in a rat model of intestinal obstruction and ischemia

Clinical evidence suggests that bacterial translocation (BT) may not be the primary cause in the development of sepsis and multiple organ dysfunction. However, BT has an important role in the activation of the immune system. Therapies have been extensively investigated to improve tissue perfusion and reduce intestinal ischemia. The aim of this study is to evaluate the effects of hypertonic saline (HSS) 7.5% and lactated Ringers (LR) solutions on intestinal BT in rats that underwent intestinal obstruction and ischaemia (IO).

Bilateral sympathectomy improves postinfarction left ventricular remodeling and function

Objectives: To evaluate the influence of bilateral or left sympathectomy on left ventricular remodeling and function after myocardial infarction in rats. Methods: Myocardial infarction was induced in rats by ligation of the left anterior descending coronary. Seven days later, rats were divided into 4 groups: the myocardial infarction, myocardial infarction with left sympathectomy, myocardial infarction with bilateral sympathectomy, and sham groups. After 8 weeks, left ventricular function was evaluated with the use of a pressure‐volume conductance catheter under steady‐state conditions and pharmacological stress. Infarct size and extracellular matrix fibrosis were evaluated, and cardiac matrix metalloproteinases and myocardial inflammatory markers were analyzed. Results: The myocardial infarction and left sympathectomy group had an increased end diastolic volume, whereas the bilateral sympathectomy group had a mean end‐diastolic volume similar to that of the sham group (P < .002). Significant reduction in ejection fraction was observed in the myocardial infarction and left sympathectomy group, whereas it was preserved after bilateral sympathectomy (P < .001). In response to dobutamine, left ventricular contractility increased in sham rats, rising stroke work, cardiac output, systolic volume, end‐diastolic volume, ejection fraction, and dP/dt max. Only bilateral sympathectomy rats had significant increases in ejection fraction (P < .001) with dobutamine. Fibrotic tissue and matrix metalloproteinase expression decreased in the bilateral sympathectomy group compared to that in the myocardial infarction group (P < .001) and was associated with left ventricular wall thickness maintenance and better apoptotic markers in noninfarcted myocardium. Conclusions: Bilateral sympathectomy effectively attenuated left ventricular remodeling and preserved systolic function after myocardial infarction induction in rats.

Evidence of bone marrow downregulation in brain‐dead rats

Experimental findings support the evidence of a persistent leucopenia triggered by brain death (BD). This study aimed to investigate leucocyte behaviour in bone marrow and blood after BD in rats. BD was induced using intracranial balloon catheter inflation. Sham‐operated (SH) rats were trepanned only. Thereafter bone marrow cells were harvested every six hours from the femoral cavity and used for total and differential counts. They were analysed further by flow cytometry to characterize lymphocyte subsets, granulocyte adhesion molecules expression and apoptosis/necrosis [annexin V/propidium iodide (PI) protocol]. BD rats exhibited a reduction in bone marrow cells due to a reduction in lymphocytes (40%) and segmented cells (45%). Bone marrow lymphocyte subsets were similar in BD and SH rats (CD3, P = 0.1; CD4, P = 0.4; CD3/CD4, P = 0.4; CD5, P = 0.4, CD3/CD5, P = 0.2; CD8, P = 0.8). Expression of L‐selectin and beta2‐integrins on granulocytes did not differ (CD11a, P = 0.9; CD11b/c, P = 0.7; CD62L, P = 0.1). There were no differences in the percentage of apoptosis and necrosis (Annexin V, P = 0.73; PI, P = 0.21; Annexin V/PI, P = 0.29). In conclusion, data presented suggest that the downregulation of the bone marrow is triggered by brain death itself, and it is not related to changes in lymphocyte subsets, granulocyte adhesion molecules expression or apoptosis and necrosis.