Early versus late surgical drainage for obstructive pancreatitis in an experimental model.

BACKGROUND: Chronic pancreatitis (CP) is characterized by intractable abdominal pain, and pancreatic exocrine and endocrine dysfunction. This study investigated whether early surgical drainage of pancreatic duct obstruction leads to improved recovery of pancreatic function compared with late surgical drainage in an experimental model of chronic obstructive pancreatitis. METHODS: Twenty-one piglets underwent pancreatic duct ligation and subsequent longitudinal pancreaticojejunostomy after 3 weeks (early drainage) or 6 weeks (late drainage), and drainage continued for 6 weeks. In controls with CP pancreatic duct ligation was continued for 12 weeks without a drainage procedure. RESULTS: Histological pancreatitis scores decreased with early drainage (P = 0.005), but not with late drainage. Pancreatic secretion of amylase and lipase was restored after early but not late drainage (P = 0.003 and P = 0.048 respectively). Excretion levels of lipase were restored to near-baseline preligation levels after early drainage. Pancreatic endocrine function (glucose tolerance test) showed no insufficiency in either group. CONCLUSION: In this model of early versus late surgical drainage of obstructive pancreatitis, histology grades and pancreatic exocrine function showed improvement in the early drainage group but no recovery in the late drainage group.

Inhibition of classical complement activation attenuates liver ischaemia and reperfusion injury in a rat model.

Activation of the complement system contributes to the pathogenesis of ischaemia/reperfusion (I/R) injury. We evaluated inhibition of the classical pathway of complement using C1-inhibitor (C1-inh) in a model of 70% partial liver I/R injury in male Wistar rats (n = 35). C1-inh was administered at 100, 200 or 400 IU/kg bodyweight, 5 min before 60 min ischaemia (pre-I) or 5 min before 24 h reperfusion (end-I). One hundred IU/kg bodyweight significantly reduced the increase of plasma levels of activated C4 as compared to albumin-treated control rats and attenuated the increase of alanine aminotransferase (ALT). These effects were not better with higher doses of C1-inh. Administration of C1-inh pre-I resulted in lower ALT levels and higher bile secretion after 24 h of reperfusion than administration at end-I. Immunohistochemical assessment indicated that activated C3, the membrane attack complex C5b9 and C-reactive protein (CRP) colocalized in hepatocytes within midzonal areas, suggesting CRP is a mediator of I/R-induced, classical complement activation in rats. Pre-ischaemic administration of C1-inh is an effective pharmacological intervention to protect against liver I/R injury.

Experimental model of obstructive, chronic pancreatitis in pigs.

BACKGROUND: We aimed to develop a reproducible, experimental model of obstructive pancreatitis for future analysis of surgical interventions, and characterized this model using functional, histological and biochemical parameters. ANIMALS AND METHODS: In 10 female pigs the pancreatic duct (PD) was ligated. After 4, 6 or 8 weeks the animals were sacrificed. Before and after ligation, glucose tolerance and intraductal pressure were measured, and pancreatic juice was collected after stimulation with cholecystokinin and secretin. Amylase and lipase activities were analyzed in plasma and juice. Pancreatic tissue was collected for histochemical analysis. RESULTS: Within 4 weeks of ligation, the pancreas appeared atrophic. Intraductal pressure had risen significantly. Acinar-to-ductal metaplasia was accompanied by strong proliferation of stellate cells and increased collagen deposition. Islets of Langerhans appeared smaller and more numerous. Blood amylase and lipase levels were normal and glucose tolerance was unaffected. Pancreatic juice volume and amylase and lipase activities were significantly lower. CONCLUSION: Ligation of the PD in pigs resulted in a marked fibrosing obstructive pancreatitis within 4 weeks, similar to human chronic pancreatitis in regard to clinical, functional, histological and biochemical parameters.

Lesion progression with time and the effect of vascular occlusion following radiofrequency ablation of the liver.

BACKGROUND: The effectiveness of radiofrequency ablation (RFA) under selective vascular occlusion and its effects on architecture and viability of normal liver parenchyma was studied in a porcine model. METHODS: RFA was applied in the liver under general anaesthesia in 18 pigs. Six animals were killed immediately after the procedure and 12 at 24 h. RFA was performed sequentially under four conditions: (1) without vascular occlusion, (2) during occlusion of the hepatic artery, (3) during occlusion of the portal vein and (4) during occlusion of the hepatic artery and portal vein. Liver biopsies from the treated area were stained for conventional histological examination, reduced nicotinamide adenine dinucleotide diaphorase and 5'-nucleotidase activity. RESULTS: Vascular occlusion significantly increased the size of the coagulation centre after RFA. Combined portal venous and arterial occlusion had no additional effect on lesion size compared with venous or arterial occlusion alone. After 24 h, deterioration of viability was observed in the parenchyma up to 3 cm from the coagulated area. CONCLUSION: The efficacy of RFA in liver increases with occlusion of the portal vein or hepatic artery. The extent of secondary heat-induced necrosis in liver parenchyma should be considered for determination of the final size of the ablated area.

Pronounced effect of minor changes in body temperature on ischemia and reperfusion injury in rat liver.

This study examined the effects of 1 degrees C hypo- or hyperthermia on in vivo liver ischemia and reperfusion (I/R) injury in 15 fasted male Wistar rats. Rats were ventilated, and rectal temperature was maintained at 36, 37 (normothermic), or 38 degrees C. In all rats, 70% liver ischemia was induced by clamping the afferent vessels to the median and left lateral lobes for 60 min, and reperfusion was allowed for 90 min. Changes in plasma aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alpha-glutathione S-transferase (alpha-GST) levels were measured, hemodynamics and bile secretion were monitored, and arterial blood-gas analysis was performed. All ventilated rats showed a normal pH, arterial PCO(2), and arterial PO(2). AST, ALT, and alpha-GST levels were significantly higher in the 38 degrees C group when compared with the 36 and 37 degrees C groups after ischemia. No differences in bile secretion were found between all groups. Histopathological alterations were in agreement with AST, ALT, and alpha-GST levels in plasma. We conclude that a decrease of only 1 degrees C in body temperature significantly attenuates liver I/R injury, whereas an increase of 1 degrees C significantly increases liver I/R injury.

Complement activation induced by ischemia-reperfusion in humans: a study in patients undergoing partial hepatectomy.

BACKGROUND/AIM: Activation of the complement system is induced by ischemia-reperfusion (I/R) in animal models. Whether I/R also induces complement activation in humans is not known. Here, we investigated complement activation in patients undergoing major liver resection. METHODS: In 11 of 17 patients, the hepatoduodenal ligament was clamped, making the liver transiently ischemic (HEMI+; mean ischemia time, 42 +/- 18 min); 6 patients were operated without clamping (HEMI-). Activation at plasma level (circulating activation products) was studied in blood samples collected prior to surgery and 5, 24 and 48 h thereafter. Parameters analyzed were C4b/c and C3b/c, C4d and C3d, C3a, as well as complexes between complement and C-reactive protein (CRP), which reflect CRP-induced complement activation. Activation at tissue level (C3 and C4 fixation) was studied in liver biopsies obtained before and after resection. RESULTS: In plasma, post-operative levels of C4b/c and C3b/c were not different from baseline levels in both groups. Mean plasma levels of C4b/c and C3b/c were significantly decreased at 24 h post-surgery in the HEMI+ group (p=0.02 and p=0.07). At the same time, levels of C4d-CRP and C3d-CRP were significantly increased (p<0.01 for both parameters). At tissue level, activated complement fragments were observed intracellularly in some pericentral hepatocytes. In I/R livers, large numbers of hepatocytes were positively stained for all complement activation products. CONCLUSIONS: Our data show that in situ complement activation via the classical route occurred during liver resection and that ischemia and/or reperfusion may have contributed to activation. Levels of complement activation products in the circulation were low, showing that transient ischemia had no severe influence on systemic complement activation, suggesting a locally contained response.

Organ blood flow after partial hepatectomy in rats: modification by endotoxin-neutralizing bactericidal/permeability-increasing protein (rBPI23).

BACKGROUND/AIM: Both maintenance of adequate perfusion and regeneration of the remnant liver are important in the recovery of liver function after partial hepatectomy. In previous experiments, we have shown that profound hypotension and liver injury can be attenuated by neutralizing endotoxins. The relative contribution of endotoxemia to changes in liver blood flow and blood flow to other major organs after partial hepatectomy is not known. The aim of this study was to examine the effect of endotoxin neutralization on individual organ blood flows including hepatic artery and splanchnic blood flow after experimental partial hepatectomy and its relation to liver cell proliferation. METHODS: Male Wistar rats underwent either two-thirds partial hepatectomy or sham operation. Treatment consisted of continuous infusion of recombinant N-terminal bactericidal/permeability-increasing protein (rBPI23) or control protein. At 4 h after surgery, organ blood flows were measured using the radiolabeled microsphere technique, and at 24 h, proliferation index in liver tissue was calculated. RESULTS: After partial hepatectomy, blood flows to virtually all organs were significantly lower as compared to values obtained in sham-operated rats. rBPI23 greatly improved hepatic artery flow (p<0.001) but not portal venous flow. These effects of rBPI23 on liver flow preceded an equally enhanced liver cell proliferation (p<0.01). Endotoxin neutralization led to significantly higher flows to some but not all splanchnic organs. Lung perfusion was significantly improved by rBPI23. CONCLUSIONS: Neutralization of endogenous endotoxins improves liver blood flow after partial hepatectomy and also periportal and pericentral liver cell proliferation. This proliferation effect may result from an increased hepatic artery flow. Lung, colon, spleen and pancreas flow but not kidney flow was greatly enhanced by rBPI23.

Quantitative analysis of the functionality and efficiency of three surgical dissection techniques: a time-motion analysis.

The increasing technological complexity of surgery demands objective evaluation of surgical techniques. In particular, alternatives for laparoscopic ligation, such as monopolar coagulation and the relatively new bipolar scissors combining dissection with coagulation, should be analyzed and compared. This study tests the efficacy of quantitative time-motion analysis in evaluating and comparing the functionality and efficiency of dissection and ligation techniques in a clinical setting. Standard dissection with ligation of vessels, bipolar scissors, and monopolar coagulation were consecutively applied to dissect 4 of the small bowel mesentery of pigs, in random order. All actions performed were recorded and analyzed, using a standard action list. The efficiency of each technique was expressed in mean dissection time and number of actions, and the safety in occurrence of complications and severity of microscopic damage. Time-motion analysis evaluated the efficiency objectively and reproducibly (ICC 0.98). Bipolar scissors were significantly more efficient (time 7 +/- 2 min, actions 129 +/- 33) than the standard technique (28 +/- 6, 771 +/- 185) and monopolar coagulation (14 +/- 5, 368 +/- 32) (p < 0.01). Furthermore, bipolar coagulation needed significantly less recoagulation of an oozing vessel (0.5% of the total dissected vessels) than did monopolar coagulation (10.4%), and the damaged zone was significantly smaller (p < 0.05). Significantly less time was spent waiting or exchanging instruments with bipolar scissors than with the standard technique (p < 0.05). This time-motion analysis objectively compared the efficiency and functionality of three surgical dissection techniques during clinical use. Bipolar scissors were more efficient than were both other techniques, and they coagulated vessels more safely than did monopolar coagulation.

In situ analysis of ischaemia/reperfusion injury in rat liver studied in three different models.

Animal models of liver ischaemia and reperfusion are frequently used to study the consequences on liver cells of transient oxygen deprivation. In 3 different rat models we studied ischaemia/reperfusion effects on liver cell membrane integrity, cytoplasmic enzyme proteins and enzyme activities by in situ histochemical techniques. In vivo ischaemia, as well as no-flow hypoxia, or N2-induced hypoxia in isolated perfused livers, reduced the activity of 5'-nucleotidase, a sensitive marker for plasma membrane damage in hepatocytes. As little as 2 minutes of reoxygenation in each model resulted in leakage of soluble enzymes from parenchymal and non-parenchymal liver cells, as shown by decreased protein level and activity of cytoplasmic enzymes. Whereas a multifocal decrease was observed after in vivo reperfusion, a decrease was found in all periportal and midzonal cells after blood-free reoxygenation. As judged by alkaline phosphatase activity and immunohistochemistry, an influx of inflammatory cells was not found in the in vivo model. Our findings indicate that reoxygenation itself, rather than restoration of flow, accounts for the loss of soluble enzymes from liver cells after a period of hypoxia. In situ detection of enzyme protein and activity proved useful for the examination of very early ischaemia/reperfusion effects on rat liver cells.

Endotoxin- and cytokine-mediated effects on liver cell proliferation and lipid metabolism after partial hepatectomy: a study with recombinant N-terminal bactericidal/permeability-increasing protein and interleukin-1 receptor antagonist.

This study was performed to clarify the mechanisms underlying post-resection changes in liver cell proliferation and metabolism. To assess the role of gut-derived endotoxaemia and endogenous cytokines in these changes, the effects of peri-operative treatment with either the lipopolysaccharide-neutralizing bactericidal/permeability-increasing protein or interleukin-1 receptor antagonist were investigated at 24 h after two-thirds hepatectomy in rats. Peri-operative treatment with either agent caused enhanced expression of proliferating cell nuclear antigen (PCNA) and reduced lipid accumulation. Activity of the hexose monophosphate shunt was significantly decreased after partial hepatectomy and restored by interleukin-1 receptor antagonist only. After partial hepatectomy, bile canalicular alkaline phosphatase activity was significantly increased in pericentral zones and redistributed to both bile canalicular and sinusoidal membranes of hepatocytes. These effects were not significantly influenced by either treatment. It is concluded that endotoxin restricts liver cell proliferation and leads to lipid accumulation following partial hepatectomy, and that interleukin-1 is a principal mediator in these processes. Furthermore, interleukin-1 mediates a repression of the pentose phosphate pathway. These changes may be of significance with respect to liver function, at least in the early phase after partial hepatectomy.

Endotoxin and interleukin-1 related hepatic inflammatory response promotes liver failure after partial hepatectomy.

Impairment of various functions of the liver and concomitantly increased levels of parameters of liver damage, a clinical entity termed liver failure, is commonly seen after partial hepatectomy. We investigated in a rat model whether damage of the remnant liver was due to local inflammatory responses, and related to endotoxin or interleukin-1 (IL-1). To address this question, the effects of partial hepatectomy on infiltration of immunocompetent cells and expression of major histocompatibility complex (MHC) class II antigen of macrophages in the remnant liver was studied using immunohistochemical techniques. Specific intervention with recombinant N-terminal bactericidal/permeability-increasing protein (rBPI23) to neutralize endotoxin and with IL-1 receptor antagonist (IL-1ra) to block IL-1 activity was used to examine the respective roles of endotoxin and IL-1. After partial hepatectomy, we found an influx of neutrophils, an increased expression of MHC class II antigens, and morphologic changes of Kupffer cells consistent with activation. These inflammatory events coincided with increased serum levels of markers of liver damage (aspartate aminotransferase, alanine aminotransferase, ammonia). Both neutralization of endotoxin and blocking of IL-1 activity reduced hepatic inflammation and reduced serum levels of aminotransferases and ammonia. In addition, liver cell proliferation as assessed by staining for proliferating cell nuclear antigen (PCNA) expression was significantly enhanced when either endotoxin or IL-1 effects were blocked. Thus, our results suggest that local hepatic inflammatory responses inhibit liver cell proliferation and promote liver failure, presumably by affecting the functional capacity of the remnant liver.

Comparative enzyme histochemistry of the early and term rat decidua with special attention to decidual regression.

As the early rat decidua is believed to fulfil functions other than the late or basal decidua, the question as to whether this difference is reflected in decidual cell metabolism was investigated. Using cryosections of pregnant rat uteri of the 10th, 15th and 21st gestational day, activities of oxyradical-forming enzymes and hydrolases were analysed histochemically. The enzyme activities of decidual stromal cells and fibroblasts of the metrial gland exhibited three main fluctuations. One group of enzyme activities did not change during gestation, a second group decreased or disappeared, and a third group increased or was expressed in the late decidua only. Enzymes of the purine and polyamine pathway, including oxyradical-forming oxidases, were absent from early mesometrial decidual cells, but were highly active in the late regressing decidua and metrial gland. Some acid hydrolases and neutral proteases became active in the mature decidua. The possibility that purine-degrading and oxyradical-forming enzymes support decidual as well as metrial gland regression, and thus placental separation, by direct tissue damage and/or by indirect rupture of lysosomal membranes, inducing the release of acid hydrolases, is considered.

Enzyme histochemistry of the regressing rat decidua and metrial gland.

In order to understand more about participation of the basal placental zones in processes of regression and degradation as well as separation on the cellular level, the cell metabolism of the rat decidua and metrial gland was investigated enzyme histochemically in cryosections for activities of oxyradical-forming enzymes and hydrolyzing enzymes. Additionally, plastic sections were studied to facilitate the recognition of cell types. Decidual stromal cells and fibroblasts formed the vast majority amongst many cell types in the decidua and metrial gland. High activities of enzymes involved in purine degradation and oxyradical generation were demonstrated in decidual stromal cells and fibroblasts. Microsomal alanyl aminopeptidase and various acid hydrolases were shown to be extremely active in decidual stromal cells. The abundance of these enzyme activities in the decidua and metrial gland in contrast to other placental areas suggests, that these enzymes may have specialized functions in connection with regression and degradation processes finally contributing to placental separation.

Enzyme reaction rate studies in electromotor neurons of the weakly electric fish Apteronotus leptorhynchus.

A histochemical analysis of reaction rates of a series of enzymes was performed in electromotor neurons of the weakly electric fish Apteronotus leptorhynchus. These neurons were selected because of their functional homogeneity. The high metabolic activity of these cells as well as their large size facilitate cytophotometric analysis in cryostat sections. Sections were incubated for the activity of hexokinase, glucose-6-phosphate dehydrogenase, succinate dehydrogenase, NADPH dehydrogenase, NADPH ferrihaemoprotein reductase and beta-hydroxybutyrate dehydrogenase. All media contained polyvinyl alcohol as tissue stabilizer and Nitro BT as final electron acceptor. Measurements were performed with a Vickers M85a cytophotometer. Linear relationships between the specific formation of formazan (test minus control reaction) and incubation time were obtained for all enzymes although some reactions showed an initial lag phase or an intercept with the ordinate. The relatively high activities of hexokinase, succinate dehydrogenase and the extremely low activity of hydroxybutyrate dehydrogenase indicate that energy is mainly supplied by glycolysis. Glucose-6-phosphate dehydrogenase showed a high activity whereas NADPH reductase and dehydrogenase activity were low in electromotor neurons, indicating that the NADPH generated is largely used for biosynthesis. Despite their synchronous firing pattern activity, electromotor neurons showed a considerable heterogeneity with respect to their metabolic activity.