Ana Maria M. Coelho

Local and systemic effects of hypertonic solution (NaCl 7.5%) in experimental acute pancreatitis.

Objectives: Severe acute pancreatitis (AP) is characterized by hemodynamic alterations and a systemic inflammatory response, leading to a high mortality rate. Treatment of hemorrhagic shock with hypertonic saline solutions significantly reduces mortality through an improvement in the hemodynamic conditions and possibly by an anti-inflammatory effect. Therefore, hypertonic solutions could be effective in AP. Methods: Wistar rats were divided in 4 groups: group C, control, without AP; group NT, AP, without treatment; group NS, treatment with normal saline solution (NaCl 0.9%) 1 hour after AP; group HTS, treatment with hypertonic saline solution (NaCl 7.5%) 1 hour after AP. AP was induced by injection of 2.5% sodium taurocholate into the pancreatic duct. Mean arterial blood pressure (MAP) and heart rate were recorded at 0 and 2, 4, 24, and 48 hours after AP. After induction of AP, animals were killed at 2, 12, 24, and 48 hours for serum amylase, interleukin (IL)-6, and IL-10 analysis, pancreatic tissue culture and histologic analysis, oxidation and phosphorylation of liver mitochondria, pulmonary myeloperoxidase activity (MPO), and mortality study. Results: In animals of groups NS and NT, a significant decrease of MAP was observed 48 hours after AP (NS: 91 ± 3 mm Hg; NT: 89 ± 3 mm Hg) compared with baseline (C: 105 ± 2 mm Hg) and to HTS group (HTS: 102 ± 2 mm Hg; P < 0.05). In animals of group NT, NS, and HTS, serum IL-6 and IL-10 levels were significantly higher at 2 hours after AP compared with the control group. However, IL-6 levels at 12 hours after AP and IL-10 levels at 2 and 12 hours after AP were significant lower in group HTS compared with NS and NT groups (P < 0.05). In group HTS, a decrease of pulmonary MPO activity and of pancreatic infection was observed 24 hours after AP compared with NT and NS groups (P < 0.05). A significant reduction on pancreatic acinar necrosis and mitochondrial dysfunction was observed after 48 hours of AP in animals of group HTS compared with groups NT and NS (P < 0.05). A significant reduction on mortality was observed in HTS (0/14) compared with NS (6/17; 35%) and NT (7/20; 35%). Conclusions: The administration of hypertonic saline solution in experimental AP attenuated hemodynamic alterations, decreased inflammatory cytokines, diminished systemic lesions and pancreatic acinar necrosis, prevented pancreatic infection, and reduced the mortality rate.

MECHANISMS OF THE BENEFICIAL EFFECT OF HYPERTONIC SALINE SOLUTION IN ACUTE PANCREATITIS.

Administration of hypertonic saline (HS) solution to rats with acute pancreatitis (AP) decreases mortality and systemic inflammation. We hypothesized that these effects are related not only to systemic inflammatory reduction, but also to a reduction of the pancreatic lesion. Acute pancreatitis was induced in Wistar rats by injection of 2.5% sodium taurocholate. Animals were divided in groups: without AP, not treated AP, AP treated with NaCl 0.9%, and AP treated with NaCl 7.5%. Trypsinogen activation peptides and amylase activity were increased in ascitic fluid and serum and were not affected by treatment with HS. Pancreatic inflammation was evaluated by increased myeloperoxidase activity, malondialdehyde formation, and histopathology for severity of pancreatic lesions. The HS did not affect these parameters. Expression of cyclooxygenase 2 and inducible nitric oxide synthase was markedly increased in the pancreas of the AP group and was reduced by treatment with HS. This treatment also reduced the levels of TNF-&agr; and IL-6 but not of IL-10 in the pancreatic tissue. These results show that HS modulates cytokine production and expression of enzymes responsible for inflammatory mediator production in the pancreas without affecting the severity of the pancreatic lesions.

Effects of Octreotide Pretreatment in Experimental Acute Pancreatitis

Background: Severity of systemic lesions and mortality of experimental acute pancreatitis (AP) are reduced after pancreatic enzyme content reduction induced by cerulein administration. Octreotide has been used both prophylatically and therapeutically in AP. The possible effects of octreotide on pancreatic enzyme content and its influence on pulmonary lesions of experimental AP were assessed in this study. Methods: Wistar male rats were divided in two branches: Branch I – Animals divided into three groups: Group Sa (n = 10) intravenous saline infusion; Group Ce (n = 10) intravenous cerulein infusion, (0.133 µg/kg–1·h–1) and Group Oc (n = 10) SC octreotide (10 µg/kg–1). Trypsin, elastase and amylase pancreatic contents as well as serum amylase were determined thereafter in all three groups; Branch II – Rats treated as in branch I, were submitted to sodium taurocholate AP (Groups Sa+AP, Ce+AP and Oc+AP). Two hours thereafter amylase and TAP assays were performed in serum, ascites and pancreatic tissue in eight animals of each group. Pulmonary histology was studied by morphometry 24 h after AP in the remaining animals. Results: Increased serum amylase and pancreatic enzyme contents were observed in octreotide-treated animals when compared to animals receiving saline or cerulein. After AP increases of serum and ascitic fluid amylase and of pancreatic TAP were observed in octreotide pre-treated animals when compared to saline and cerulein groups. Pulmonary interstitial and alveolar edema after AP was significantly increased in rats receiving octreotide as compared to the cerulein group. Conclusion: Octreotide administration acutely increases the enzymatic content of the pancreas and thus may have a potential deleterious influence in the evolution of AP.

Postconditioning ameliorates lipid peroxidation in liver ischemia-reperfusion injury in rats

PURPOSE Liver ischemia-reperfusion injury is a phenomenon presents in events like liver resections and transplantation. The restoration of blood flow may leads to local and systemic injury. Several techniques have been developed in order to avoid or ameliorate ischemia-reperfusion injury in clinical situations. The application of a stutter reperfusion after the ischemic event (postconditioning) could alters the hydrodynamics and stimulates endogenous mechanisms that attenuate the reperfusion injury. The present study was designed to evaluate the potential protective effect of postconditioning in a model of ischemia-reperfusion in rats. METHODS Hepatic anterior pedicle of median and left anterolateral segments were exposed and clamped for 1 hour. Two hours later, clamp was released in two different ways: Control Group (n=7): clamp was release straightforward; Postconditioning Group (n=7): clamp was released intermittently. Lipid peroxidation (malondialdehyde) and expression of the glutathione-s-transferase-alpha-3 gene were studied. RESULTS Lipid peroxidation was significantly decreased in ischemic and non-ischemic liver by postconditioning. GST- alpha3 gene was overexpressed in post-conditioned group, but not significantly. CONCLUSION Postconditioning induced hepatoprotection by reducing lipid peroxidation in the ischemic and non-ischemic liver.

Inhibition of cyclooxygenase-2 in experimental severe acute pancreatitis

BACKGROUND The standard treatment for acute pancreatitis (AP) is still based on supportive care. The search for a new drug that could change the natural history of the disease is a continuing challenge for many researchers. The aim of this study is to evaluate the effect of a cyclooxygenase-2 (COX-2) inhibitor on experimental AP in rats. METHODS The animals were divided into 2 groups: Group 1 (n = 30)-animals with taurocholate-induced AP treated with parecoxib (40 mg/kg). Group 2 (n = 30)-animals with taurocholate-induced AP that received saline. The COX-2 inhibitor (parecoxib) was injected immediately after AP induction, through the penis dorsal vein. The parameters evaluated were histology, serum levels of amylase, IL-6 and IL-10, and mortality rate. RESULTS The serum levels of IL-6 and IL-10 in the parecoxib-treated group were lower than the control group. The amylase serum levels and the mortality rate remained unchanged in the treated animals. Histologic morphology also was unaltered, except for fat necrosis, which was higher in parecoxib-treated rats. CONCLUSION Inhibition of Cox-2 decreases the systemic release of inflammatory cytokines, but has a poor effect on the direct pancreas injury caused by taurocholate.

Hepatic damage during acute pancreatitis in the rat

We studied the alterations in the metabolism of liver mitochondria in rats with acute pancreatitis. Male Wistar rats were allocated to a control group (group I) and to five other groups corresponding to 2, 4, 12, 24 and 48 h after the induction of acute pancreatitis by the injection of 5% sodium taurocholate into the pancreatic duct. Sham-operated animals were submitted to the same surgical steps except for the induction of acute pancreatitis. Mitochondrial oxidation and phosphorylation were measured polarographically by determining oxygen consumption without ADP (basal respiration, state 4) and in the presence of ADP (activated respiration, state 3). Serum amylase, transaminases (ALT and AST) and protein were also determined. Ascitic fluid, contents of amylase, trypsin and total protein were also determined and arterial blood pressure was measured in all groups. In ascitic fluid, trypsin and amylase increased reaching a maximum at 2 and 4 h, respectively. Serum amylase increased at 2 h reaching a maximum at 4 h. Serum transaminase levels increased at 12 and 24 h. After 2 h (and also 4 h) there was an increase in state 4 respiration (45.65 +/- 1.79 vs 28.96 +/- 1.50) and a decrease in respiration control rate (3.53 +/- 0.09 vs 4.45 +/- 0.08) and in the ADP/O ratio (1.77 +/- 0.02 vs 1.91 +/- 0.01) compared to controls (P < 0.05). These results indicate a disruption of mitochondrial function, which recovered after 12 h. In the 48-h groups there was mitochondrial damage similar to that occurring in ischemic lesion. Beat-to-beat analysis (30 min) showed that arterial blood pressure remained normal up to 24 h (111 +/- 3 mmHg) while a significant decrease occurred in the 48-h group (91 +/- 4 mmHg). These data suggest biphasic damage in mitochondrial function in acute pancreatitis: an initial uncoupled phase, possibly secondary to enzyme activity, followed by a temporary recovery and then a late and final dysfunction, associated with arterial hypotension, possibly related to ischemic damage.

Anti-Inflammatory Effects of Peritoneal Lavage in Acute Pancreatitis

Objectives: Intraperitoneal administration of trypsin stimulates the production of cytokines from peritoneal macrophages. Removing the pancreatitis-associated ascitic fluid from the peritoneal cavity may decrease the systemic inflammatory response in acute pancreatitis (AP). We investigated the effect of peritoneal lavage on the systemic inflammatory response in severe AP. Methods: Acute pancreatitis was induced in Wistar rats by 5% taurocholate intraductal injection. Peritoneal lavage was performed for 4 hours after onset of AP. At 4 hours after induction of AP, serum samples were assayed for amylase and inflammatory cytokines (tumor necrosis factor &agr;, interleukin-6 [IL-6], and IL-10). Expression of pancreatic cyclooxygenase-2 and inducible nitric oxide synthase, liver mitochondrial function, and pulmonary myeloperoxidase activities were determined. Results: Peritoneal lavage after AP led to a decrease in serum levels of tumor necrosis factor &agr; and IL-6 and an increase in IL-10. In the pancreas, this treatment reduced cyclooxygenase-2 and inducible nitric oxide synthase expression. Liver mitochondrial dysfunction was also reduced. There were no differences on serum amylase levels and pulmonary myeloperoxidase between groups with AP. Conclusions: Peritoneal lavage has a systemic anti-inflammatory effect in severe AP and may be able to decrease the severity of severe AP.

Influence of Pancreatic Enzyme Content on Experimental Acute Pancreatitis

Introduction Some studies demonstrate the crucial role of proteases in the pathogenesis of acute pancreatitis (AP). Systemic release of proinflammatory cytokines such as tumor necrosis factor–&agr; (TNF-&agr;) and interleukin-6 (IL-6) has been demonstrated in AP, yet the mechanism of activation remains unclear. Furthermore, it is not known if the amount of pancreatic enzyme in the pancreas determines the production of proinflammatory cytokines. Aim To determine whether there is a link between the pancreatic enzyme content and the production of cytokines and consequently the systemic lesions observed in AP. Methodology Forty-seven animals were divided into three groups: group I had a high pancreatic enzyme level (with and without AP), group II had a low pancreatic enzyme level (cerulein infusion: 0.133 &mgr;g·kg−1·h−1)(with and without AP), and group III were the controls. AP was induced by injection of 5% sodium taurocholate into the pancreatic duct. To evaluate the pancreatic enzyme contents before AP, trypsinogen and amylase analysis was carried out on pancreatic tissue collected after the animals were killed. Two hours after induction of AP, concentrations of pancreatic enzymes and trypsinogen activation peptide (TAP) in serum, ascitic fluid, and pancreatic tissue were determined. The ascitic fluid was assayed for TNF-&agr; and the serum was assayed for IL-6 with ELISA kits. Systemic lesions were sought on the basis of hepatic mitochondrial respiratory function measured polarographically. Results and Conclusion The administration of physiological doses of cerulein diminishes the pancreatic enzyme and TAP levels, the production of proinflammatory cytokines, and the liver mitochondrial dysfunction observed in AP, suggesting that the pancreatic enzyme content is an important factor in the severity of AP.

Effect of PAF antagonists on cerulein-induced pancreatitis

The present study was undertaken to investigate the involvement of PAF in acute pancreatitis induced by cerulein in rats. Cerulein (two doses of 20 micrograms/rat, the first s.c. and the second i.v., 1 h apart) induced a significant increase in vascular permeability in the pancreas, evaluated by the Evans blue (EB) extravasation method. Plasma amylase levels were also significantly increased in this group. The PAF antagonists, BN-52021 (5 mg/kg) and WEB-2170 (1 and 10 mg/kg), both significantly reduced the extravasation of EB in the pancrease induced by i.v. injection of PAF (1 microgram/kg). At these concentrations, BN-52021 was effective at inhibiting cerulein-induced pancreatitis (60-70% of inhibition) whereas WEB-2170 had no significant effect. Although the inhibition induced by BN-52021 suggests the involvement of PAF in cerulein-pancreatitis, the lack of effect of WEB-2170 reported here does not allow a definite conclusion. Further studies are needed to elucidate the differential effect of the PAF antagonists.

CO2 abdominal insufflation decreases local and systemic inflammatory response in experimental acute pancreatitis.

Objectives: Acute pancreatitis (AP) is a serious disease that is amplified by an associated systemic inflammatory response. We investigated the effect of CO2 pneumoperitoneum on the local and systemic inflammatory response in AP. Methods: Acute pancreatitis was induced in Wistar rats by 5% taurocholate intraductal injection. Carbon dioxide pneumoperitoneum was applied for 30 minutes before the induction of AP. Inflammatory parameters were evaluated in the peritoneum (ascites, cell number, and tumor necrosis factor &agr; [TNF-&agr;]), serum (amylase, TNF-&agr;, interleukin-6 [IL-6], and IL-10), pancreas (myeloperoxidase [MPO] activity, cyclo-oxygenase 2 and inducible nitric oxide synthase expression, and histological diagnosis), liver, and lung (mitochondria dysfunction and MPO activity). Results: Abdominal insufflation with CO2 before induction of AP caused a significant decrease in ascites volume, cells, and TNF-&agr; in the peritoneal cavity and in serum TNF-&agr; and IL-6 but not IL-10 levels. In the pancreas, this treatment reduced MPO activity, acinar and fat necrosis, and the expression of inducible nitric oxide synthase and cyclo-oxygenase 2. There were no significant differences on serum amylase levels, liver mitochondrial function, and pulmonary MPO between groups. Conclusions: Our data demonstrated that CO2 pneumoperitoneum reduced pancreatic inflammation and attenuated systemic inflammatory response in AP. This article suggests that CO2 pneumoperitoneum plays a critical role on the better outcome in patients undergoing laparoscopic pancreatic surgery.