Cristiano de Jesus Correia

LEUKOCYTE-ENDOTHELIUM INTERACTIONS AFTER HEMORRHAGIC SHOCK/REPERFUSION AND CECAL LIGATION/PUNCTURE: AN INTRAVITAL MICROSCOPIC STUDY IN RAT MESENTERY

ABSTRACT Hemorrhagic shock/reperfusion (HS/R) followed by sepsis triggers systemic microcirculatory disturbances that may induce multiple organ failure. The present study evaluated the effects of HS/R and cecal ligation and puncture, followed by necrotic cecal resection/peritoneal lavage (REL) on leukocyte-endothelium interactions at the mesentery. Eighty-one anesthetized Wistar rats (200-250 g) were randomly assigned to a first injury: (1) control-HS-no hemorrhagic shock/no reperfusion group, (2) HS/blood-HS/R with 25% shed blood, and (3) HS/blood + LR-HS/R with 25% of the shed blood + lactated Ringers solution, 3× shed blood volume. Twenty-four hours post-HS/R, animals were submitted to cecal ligation and puncture and, 24 h thereafter, to REL. Leukocyte-endothelium interactions were assessed by intravital microscopy and intercellular adhesion molecule (ICAM) 1 and P-selectin expression by immunohistochemistry. Lungs were observed for ICAM-1 expression and neutrophil infiltration. Single and double injury induced significant increases in rolling (~2-fold), adherent (~5-fold), and migrated leukocytes (~7-fold); ICAM-1 expression (~1/2-fold), and P-selectin expression (~1/2-fold) at the mesentery compared with control-HS group. REL normalized leukocyte-endothelium interactions at the mesentery in single-injured animals. However, in double-injured rats, adherence and migration of leukocytes decreased but did not normalize. Similar results were observed on ICAM-1 expression and neutrophil infiltration in the lungs from these animals. In conclusion, the current in vivo observation of the mesenteric microcirculation after a double injury followed by REL is a suitable model for the systematic evaluation of the inflammatory reaction at local and distant sites. In addition, data presented herein emphasized the importance of surgical removal of the septic focus in controlling the otherwise lethal sepsis-induced multiple organ dysfunction syndrome.

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.

Pentoxifylline attenuates leukocyte-endothelial interactions in a two-hit model of shock and sepsis.

BACKGROUND This study investigated the effects of pentoxifylline (PTX) combined with resuscitation fluids on microcirculatory dysfunctions in a two-hit model of shock and sepsis. MATERIALS AND METHODS Male Wistar rats (250 g) were submitted to hemorrhagic shock and reperfusion followed by sepsis induced by cecal ligation and puncture. For the initial treatment of shock, rats were randomly divided into: sham, no injury, no treatment; hypertonic saline solution (HS) (7.5%, 4 mL/kg); lactated Ringers solution (LR, 3 × shed blood volume); HS + PTX (4 mL/Kg + 25 mg/kg PTX); and LR + PTX (3 × shed blood volume + 25 mg/kg PTX). After 48 h of being exposed to the double injury, leukocyte-endothelial interactions were assessed by intravital microscopy of the mesentery. Endothelial expression of P-selectin and intercellular adhesion molecule-1 (ICAM-1) was evaluated by immunohistochemistry, as well as lung neutrophil infiltration by histology. RESULTS Lactated Ringers solution induced marked increases (P < 0.001) in the number of rolling leukocytes per 10 min (two-fold), adherent leukocytes per 100 μm venule length (six-fold), migrated leukocytes per 5000 μm(2) (eight-fold), P-selectin and ICAM-1 expression (four-fold), and lung neutrophil infiltration (three-fold) compared with sham. In contrast, PTX attenuated leukocyte-endothelial interactions, P-selectin and ICAM-1 expression at the mesentery when associated with either LR (P < 0.001) or HS (P < 0.05). Neutrophil migration into the lungs was similarly reduced by PTX (P < 0.05). CONCLUSIONS Data presented showed that pentoxifylline attenuates microcirculatory disturbances at the mesenteric bed with significant minimization of lung inflammation after a double-injury model of hemorrhagic shock and reperfusion followed by sepsis.

Influence of brain death and associated trauma on solid organ histological characteristics

PURPOSE To evaluate histopathological alterations triggered by brain death and associated trauma on different solid organs in rats. METHODS Male Wistar rats (n=37) were anesthetized with isoflurane, intubated and mechanically ventilated. A trepanation was performed and a balloon catheter inserted into intracraninal cavity and rapidly inflated with saline to induce brain death. After induction, rats were monitored for 30, 180, and 360 min for hemodynamic parameters and exsanguinated from abdominal aorta. Heart, lung, liver, and kidney were removed and fixed in paraffin to evaluation of histological alterations (H&E). Sham-operated rats were trepanned only and used as control group. RESULTS Brain dead rats showed a hemodynamic instability with hypertensive episode in the first minute after the induction followed by hypotension for approximately 1 h. Histological analyses showed that brain death induces vascular congestion in heart (p<0.05), and lung (p<0.05); lung alveolar edema (p=0.001), kidney tubular edema (p<0.05); and leukocyte infiltration in liver (p<0.05). CONCLUSIONS Brain death induces hemodynamic instability associated with vascular changes in solid organs and compromises most severely the lungs. However, brain death associated trauma triggers important pathophysiological alterations in these organs.

The Effects of Low and High Tidal Volume and Pentoxifylline on Intestinal Blood Flow and Leukocyte-Endothelial Interactions in Mechanically Ventilated Rats

BACKGROUND: The combination of high PEEP and low tidal volume (VT) decreases some risks of mechanical ventilation, including pulmonary overdistention, damage due to cyclic opening and closing of the alveoli, and inflammatory responses that can lead to multiple-organ dysfunction. We hypothesized that high VT and high PEEP induce mesenteric microcirculatory disturbances and that those disturbances would be attenuated by pentoxifylline, which is anti-inflammatory. METHODS: We anesthetized (isoflurane 1.5%), tracheostomized, and mechanically ventilated 57 male Wistar rats with PEEP of 10 cm H2O and FIO2 of 0.21 for 2 hours. One group received low VT (7 mL/kg), another group received high VT (10 mL/kg), and a third group received high VT plus pentoxifylline (25 mg/kg). We measured mean arterial pressure, respiratory mechanics, mesenteric blood flow, and leukocyte-endothelial interactions. RESULTS: The mean arterial pressure was similar among the groups at baseline (108 mm Hg [IQR 94–118 mm Hg]) and after 2 hours of mechanical ventilation (104 mm Hg [IQR 90–114 mm Hg]). Mesenteric blood flow was also similar between the groups: low VT 15.1 mL/min (IQR 12.4–17.7 mL/min), high VT 11.3 mL/min (IQR 8.6–13.8 mL/min), high-VT/pentoxifylline 12.4 mL/min (10.8–13.7 mL/min). Peak airway pressure after 2 hours was lower (P = .03) in the low-VT group (10.4 cm H2O [IQR 10.2–10.4 cm H2O]) than in the high-VT group (12.6 cm H2O [10.2–14.9 cm H2O]) or the high-VT/pentoxifylline group (12.8 cm H2O [10.7–16.0 cm H2O]). There were fewer adherent leukocytes (P = .005) and fewer migrated leukocytes (P = .002) in the low-VT group (5 cells/100 μm length [IQR 4–7 cells/100 μm length] and 1 cell/5,000 μm2 [IQR 1–2 cells/5,000 μm2], respectively) and the high-VT/pentoxifylline group (5 cells/100 μm length [IQR 3–10 cells/100 μm length] and 1 cell/5,000 μm2 [IQR 1–3 cells/5,000 μm2], respectively) than in the high-VT group (14 cells/100 μm length [IQR 11–16 cells/100 μm length] and 9 cells/5,000 μm2 [IQR 8–12 cells/5,000 μm2], respectively). CONCLUSIONS: Low VT with high PEEP was lung-protective, and early pentoxifylline reduced the inflammatory response to high VT with high PEEP (and presumed lung overdistention) during mechanical ventilation.

Effects of ethyl pyruvate on leukocyte-endothelial interactions in the mesenteric microcirculation during early sepsis treatment.

OBJECTIVES: Experimental studies on sepsis have demonstrated that ethyl pyruvate is endowed with antioxidant and anti-inflammatory properties. This study aimed to investigate the effects of ethyl pyruvate on leukocyte-endothelial interactions in the mesenteric microcirculation in a live Escherichia coli-induced sepsis model in rats. METHODS: Male Wistar rats were administered an intravenous suspension of E. coli bacteria or were subjected to a sham procedure. Three hours after bacterial infusion, the rats were randomized into the following groups: a control group without treatment, a group treated with lactated Ringers solution (4 mL/kg, i.v.), and a group treated with lactated Ringers solution (4 mL/kg, i.v.) plus ethyl pyruvate (50 mg/kg). At 24 h after bacterial infusion, leukocyte-endothelial interactions were investigated using intravital microscopy, and the expression of P-selectin and intercellular adhesion molecule-1 was evaluated via immunohistochemistry. White blood cell and platelet counts were also determined at baseline and 3 h and 24 h after E. coli inoculation. RESULTS: The non-treated and lactated Ringers solution-treated groups exhibited increases in the numbers of rolling leukocytes (∼2.5-fold increase), adherent cells (∼3.0-fold), and migrated cells (∼3.5-fold) compared with the sham group. In contrast, treatment with Ringers ethyl pyruvate solution reduced the numbers of rolling, adherent and migrated leukocytes to the levels observed in the sham group. Additionally, the expression of P-selectin and intercellular adhesion molecule-1 was significantly increased on mesenteric microvessels in the non-treated group compared with the sham group (p<0.001). The expression of both adhesion molecules was reduced in the other groups, with ethyl pyruvate being more effective than lactated Ringers solution. Infusion of bacteria caused significant leukopenia (3 h), followed by leukocytosis with granulocytosis (24 h). There was also an intense and progressive reduction in the number of platelets. However, no differences were observed after treatment with the different solutions. CONCLUSIONS: The presented data suggest that ethyl pyruvate efficiently reduces the inflammatory response in the mesenteric microcirculation in an experimental model of sepsis induced by live E. coli and is associated, at least in part, with down-regulation of P-selectin and intercellular adhesion molecule-1.

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.

Avaliação inicial dos gradientes sistêmicos e regionais da pCO2 como marcadores de hipoperfusão mesentérica

RACIONAL: Apesar dos recentes avancos nos metodos de imagem e no cuidado dos doentes criticos, a taxa de mortalidade do abdome agudo vascular nas ultimas duas decadas continua praticamente inalterada. OBJETIVOS: Avaliar as alteracoes imediatas dos gradientes regionais da pCO2 induzidas pela isquemia e reperfusao mesenterica. Determinar se outros marcadores sistemicos de hipoperfusao esplâncnica sao capazes de detectar precocemente as alteracoes circulatorias ocorridas na mucosa intestinal apos oclusao da arteria mesenterica superior. METODOS: Foram utilizados sete caes machos sem raca definida (20,6 ± 1,1 kg), submetidos a oclusao da arteria mesenterica superior por 45 minutos, sendo os animais observados por periodo adicional de 2 horas apos a reperfusao. Variaveis hemodinâmicas sistemicas foram avaliadas por meio de cateter arterial e Swan-Ganz. A perfusao do sistema digestorio foi avaliada pela medida do fluxo sanguineo da veia mesenterica superior e da serosa jejunal (fluxometro ultra-sonico). Oferta, taxa de extracao e consumo intestinal de oxigenio (DO2intest, TEO2intest e VO2intest, respectivamente), pH intramucoso (tonometria a gas) e os gradientes veia mesenterica-arterial e mucosa-arterial da pCO2 (Dvm-apCO2 e Dt-apCO2, respectivamente), foram calculados. RESULTADOS: A oclusao da arteria mesenterica superior nao esteve associada a alteracoes hemodinâmicas sistemicas, mas pode-se observar aumento significativo do Dvm-apCO2 (1,7 ± 0,5 para 5,7 ± 1,8 mm Hg) e do Dt-apCO2 (8,2 ± 4,8 para 48,7 ± 4,6 mm Hg). Na fase de reperfusao observou-se reducao da DO2intest (67,7 ± 9,9 para 38,8 ± 5,3 mL/min) e consequente aumento da TEO2intest de 5,0 ± 1,1% para 12,4 ± 2,7%. Nao houve correlacao entre os gradientes da pCO2 analisados. CONCLUSAO: A tonometria permite detectar de maneira precoce a reducao de fluxo intestinal. Alem disso, pudemos demonstrar que as variacoes dos gradientes regionais e/ou sistemicos da pCO2 nao sao capazes de avaliar a magnitude da reducao de fluxo da mucosa intestinal durante o fenomeno de isquemia e reperfusao mesenterica.

17β-Estradiol protects against lung injuries after brain death in male rats

BACKGROUND Brain death elicits microvascular dysfunction and inflammation, and thereby compromises lung viability for transplantation. As 17β-estradiol was shown to be anti-inflammatory and vascular protective, we investigated its effects on lung injury after brain death in male rats. METHODS Wistar rats were assigned to: sham-operation by trepanation only (SH, n = 7); brain death (BD, n = 7); administration of 17β-estradiol (280 μg/kg, iv) at 60 minutes after brain death (BD-E2, n = 7). Experiments were performed 180 minutes thereafter. Histopathological changes in the lung were evaluated by histomorphometry. Gene expression of inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS), and endothelin-1 was measured by real-time polymerase chain reaction. Protein expression of NO synthases, endothelin-1, platelet endothelial cell adhesion molecule-1 (PECAM-1), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), BCL-2, and caspase 3 was assessed by immunohistochemistry. Cytokines were quantified by enzyme-linked immunosorbent assay. RESULTS Treatment with 17β-estradiol after brain death decreased lung edema and hemorrhage (p < 0.0001), and serum levels of cytokine-induced neutrophil chemoattractant-1 (CINC-1; p = 0.0020). iNOS (p < 0.0001) and VCAM-1 (p < 0.0001) also diminished at protein levels, while eNOS accumulated (p = 0.0002). However, gene expression of iNOS, eNOS, and endothelin-1 was comparable among groups, as was protein expression of endothelin-1, ICAM-1, BCL-2, and caspase 3. CONCLUSIONS 17β-Estradiol effectively reduces lung injury in brain-dead rats mainly due to its ability to regulate NO synthases. Thus, the drug may improve lung viability for transplantation.