Reperfusion injury is greater with delayed restoration of venous outflow in concurrent arterial and venous limb injury.

BACKGROUND: Complex limb trauma often involves combined arterial and venous injury, and the resultant ischaemia-reperfusion injury (IRI) causes both local and remote organ injury. This study assessed the influence of the timing of restoration of venous drainage on IRI. METHODS: Male New Zealand white rabbits (n = 36) were randomized into six groups: sham operation (group 1) and unilateral hind limb arterial and venous occlusion for 1 h followed by no reflow for 2 h (group 2), arterial and venous reflow for 2 h (group 3), arterial reflow alone for 2 h (group 4), arterial reflow alone for 1 h followed by arterial and venous (delayed) reflow for a further 1 h (group 5), and pretreatment with an enteral combination antioxidant before occlusion of both artery and vein and delayed venous reflow (group 6). Plasma hydroperoxide (HPO) and glutathione peroxidase concentration, hind limb skeletal muscle and lung tissue wet : dry weight ratios and myeloperoxidase (MPO) concentration were measured. RESULTS: The plasma HPO level in the femoral vein effluent was significantly greater after delayed venous reflow (mean(s.e.m.) 2. 02(0.54) micromol/l) than in control animals (0.98(0.10) micromol/l) (P < 0.05). There was also a significantly greater tissue wet : dry weight ratio after delayed venous reflow than in controls, in skeletal muscle (mean(s.e.m.) 6.89(0.14) versus 5.34(0.54); P < 0. 05) and lung (9.20(1.14) versus 7.23(0.38); P < 0.05) tissue. Lung tissue MPO activity was significantly greater after delayed venous reflow compared with controls (3.20(0.28) versus 1.86(0.14) units/g; P < 0.005), and also in comparison to simultaneous arterial and venous reflow (2.40(0.24) units/g; P < 0.05). In the antioxidant pretreatment group there was no significant increase in plasma HPO concentration, tissue MPO level or tissue wet : dry weight ratio compared with the control group. CONCLUSION: In combined major arterial and venous injury of the limb, delayed restoration of venous drainage leads to significantly greater local skeletal muscle injury and remote neutrophil-mediated lung injury. These results support the clinical rationale for early restoration not only of arterial inflow but also venous drainage by means of intraluminal shunts.

Characterization of the Kupffer cell response to exogenous endotoxin in a rodent model of obstructive jaundice.

BACKGROUND: Sepsis and endotoxaemia occur frequently in biliary obstruction. Impaired Kupffer cell endocytosis is implicated in these events. Tumour necrosis factor and interleukin 6, secreted by Kupffer cells, are important mediators of sepsis. Kupffer cell clearance of endotoxin and secretion of cytokines in experimental obstructive jaundice were investigated. METHODS: Wistar rats were randomized to bile duct ligation, sham operation or control. Groups (n = 8) were studied 1 and 3 weeks after operation. Kupffer cell function was assessed using in situ hepatic perfusion. RESULTS: Clearance of endotoxin was significantly depressed 1 week (median (interquartile range) 20.3 (10.5-27.1) per cent) and 3 weeks (22.1 (20.2-23.2) per cent) after bile duct ligation compared with that in respective sham animals (35.5 (29.9-41.6) and 40.9 (37.7-47.0) per cent) and controls (39.5 (37.3-46.8) per cent). Secretion of tumour necrosis factor was significantly greater 1 week (1113.7 (706.5-1436. 8) pg/ml) and 3 weeks (1118.2 (775.7-1484.1) pg/ml) following bile duct ligation compared with that in respective sham animals (114.3 (0-178.5) and 107.6 (63.7-166.4) pg/ml) and controls (0 (0-20.7) pg/ml). Interleukin 6 was not secreted by sham or control animals but was present in the perfusate from jaundiced animals at 1 and 3 weeks (52.5 (9.9-89.5) and 66.2 (60.2-193.1) pg/ml). CONCLUSION: These data demonstrate simultaneous impairment of Kupffer cell clearance of endotoxin and increased secretion of proinflammatory cytokines in experimental obstructive jaundice. These diverse responses may contribute to the development of sepsis-related complications in biliary obstruction.

Lower limb ischaemia-reperfusion injury causes endotoxaemia and endogenous antiendotoxin antibody consumption but not bacterial translocation.

BACKGROUND: It has been suggested that reperfusion of the acutely ischaemic lower limb alters gut permeability. The effect of lower limb ischaemia-reperfusion on systemic endotoxin and antiendotoxin antibody concentrations and the incidence of bacterial translocation was investigated. METHODS: Systemic endotoxin and antiendotoxin antibody concentrations were measured in five groups of male Wistar rats: control, after 3 h of bilateral hind limb ischaemia alone, and after 3 h of bilateral hind limb ischaemia followed by 1, 2 or 3 h of reperfusion. A second experiment examined translocation of indigenous bacteria following 2 h of reperfusion in a similar model. RESULTS: Ischaemia followed by reperfusion for 1, 2 or 3 h caused a significant increase in plasma endotoxin concentration to mean(s.e.m.) 10.0(3.0), 44.8(19.2) and 20.2(6.2) pg/ml compared with that in control animals (2.58(0.91) pg/ml) or animals in the ischaemia alone group (1.2(0.9) pg/ml) (P < 0.05). This was associated with a significant reduction in endogenous antiendotoxin antibody (immunoglobulin (Ig) G and IgM) concentration. No significant bacterial translocation was detected in any of the groups studied. CONCLUSION: These results demonstrate that a remote and isolated ischaemia-reperfusion injury to the lower limb, in the absence of infection or bacterial translocation, causes endotoxaemia. Further studies are needed to evaluate the role of endogenous antiendotoxin antibodies in this situation.

Conclusive evidence of endotoxaemia in biliary obstruction.

BACKGROUND: Endotoxaemia is implicated in the pathophysiology of obstructive jaundice. The EndoCab enzyme linked immunosorbent assay (ELISA) is a novel assay which measures endogenous antibody (IgG) to the inner core region of circulating endotoxins (ACGA). AIMS: To investigate the significance of endotoxaemia in biliary obstruction using the EndoCab assay and assess the specificity of the humoral response to endotoxin compared with an exogenous antigenic challenge (tetanus toxoid, TT). METHODS: Three groups of adult male Wistar rats were studied: no operation, sham operation, and bile duct ligation for 21 days (BDL). In the second study, rats rats received prior immunisation with TT. RESULTS: In the preliminary experiment, plasma ACGA was significantly increased in the BDL group (306.6 (18.3)% versus 119.9 (6.7)% and 105.2 (4.6)% in the sham and no operation groups, respectively; p < 0.001). Although the mean endotoxin concentration in the BDL group was greater than that in the control groups this was not significant. There was a strong positive correlation between ACGA and endotoxin concentrations (p = 0.0021). In the second study mean ACGA after 21 days of BDL was significantly elevated (267.1 (31.2)% versus 101.6 (21.2)% at baseline, p < 0.0001). ACGA was unaffected in the other two groups. TT antibody concentrations fell in all three groups; only in the BDL group was the fall significant (97.6 (5.3)% versus 78.8 (4.2)% at baseline, p < 0.05). CONCLUSIONS: The specific rise in ACGA supports the hypothesis that endotoxin has an integral role in the pathophysiology of obstructive jaundice. The production of anticore glycolipid antibodies specifically reflects systemic endotoxaemia in this model. The EndoCab assay provides a novel, sensitive, and specific method for endotoxin detection.

Lower limb ischaemia-reperfusion injury alters gastrointestinal structure and function.

BACKGROUND: It has been suggested that bowel permeability is altered following abdominal aortic aneurysm surgery. The effect of ischaemia-reperfusion injury to the lower limb on the morphological structure, neutrophil infiltration and permeability of the bowel was investigated. METHODS: Histological assessment of the bowel was undertaken in five groups of Wistar rats: control, 3 h of bilateral hind limb ischaemia and 3 h of bilateral hind limb ischaemia followed by 1, 2 or 3 h of reperfusion. Using an everted gut sac model and 14C-labelled polyethylene glycol, the effect of ischaemia-reperfusion on small bowel permeability was studied. RESULTS: The small bowel showed a significant decrease in mucosal thickness, villus height and crypt depth in animals subjected to ischaemia followed by 2-hr reperfusion (mean(s.e.m.) 420(15), 217(9) and 163(6) microns respectively) compared with controls (481(11), 245(6) and 195(6) microns) (P < 0.05). Neutrophil count within the lamina propria was similar in the different groups. A significant increase in mean(s.e.m.) 14C-labelled polyethylene glycol translocation was detected in animals subjected to ischaemia-reperfusion compared with controls (760(40) versus 560(27) c.p.m. per ml per h) (P < 0.05). CONCLUSION: These data suggest that reperfusion of acutely ischaemic extremities produces structural and functional changes in the small intestine, although these changes are not associated with increased neutrophil infiltration within the bowel wall.

Modulation of TNF alpha and IL-6 in a peritonitis model using pentoxifylline.

Sepsis leads to release of reactants that play an important role in the development of multiple organ failure. The kinetics of two early mediators of the response to sepsis, tumour necrosis factor (TNF alpha) and interleukin 6 (IL-6), and their modulation with pentoxifylline (PTF), were investigated. An established and clinically relevant animal model was employed, and sepsis was induced by cecal ligation and puncture (CLP) in Wistar rats. Six hours after the operation, there was an increase in IL-6 in all animals, which declined toward normal by 18 hours. This early phase of IL-6 production was not influenced by PTF. TNF alpha and IL-6 were significantly higher in the CLP group than in the animals treated with PTF at 24 hours. The blood pressure of the CLP group at 24 hours was significantly lower than that of the shams, and this decrease was not influenced by PTF. This decline in blood pressure may have been the stimulus to TNF production and the second phase of IL-6 production, which appeared to be inhibited by PTF. Pentoxifylline appears to attenuate systemic cytokine production in this model and may have a role in the management of clinical sepsis.

Biliary decompression promotes Kupffer cell recovery in obstructive jaundice.

BACKGROUND: Jaundiced patients undergoing surgical procedures have an increased risk of Gram negative sepsis with potential morbidity and mortality. Depressed Kupffer cell clearance capacity (KCCC) predisposes jaundiced patients to endotoxaemia and its sequelae. Biliary decompression remains the main therapeutic strategy in obstructive jaundice. AIMS: This study investigates the efficacy of internal (ID) and external biliary drainage (ED) on KCCC in an experimental model of extrahepatic biliary obstruction. METHODS: Adult male Wistar rats (250-300 g) were assigned to one of six groups: sham operated, where the bile duct was mobilised but not divided; bile duct ligation (BDL) for three weeks, and sham operated or BDL for three weeks followed by a second laparotomy and further 21 days of ID or ED, by way of choledochoduodenostomy or choledochovesical fistula respectively. KCCC was measured using an isolated hepatic perfusion technique with FITC labelled latex particles (0.75 mu) as the test probe. Plasma was assayed for bilirubin, endotoxin, and anticore glycolipid antibody (ACGA) concentrations. RESULTS: Jaundiced rats had reduced KCCC (p < 0.001), increased concentrations of ACGA (p < 0.001), and endotoxin (p < 0.001) compared with controls. Biliary drainage for three weeks produced a recovery in KCCC and normalisation of endotoxin and ACGA concentrations, however, external drainage was less effective than ID (p < 0.01). CONCLUSIONS: These data support the hypothesis that endotoxaemia and its mediated effects are integral in the pathophysiology of jaundice. Furthermore, a short period of internal biliary drainage is a useful therapeutic strategy in restoring Kupffer cell function and negating systemic endotoxaemia and consequent complications in biliary obstruction.

Mortality, endotoxaemia and cytokine expression after intermittent and continuous hepatic ischaemia.

This study compared mortality rates, endotoxaemia, systemic tumour necrosis factor (TNF) and interleukin (IL)-6 concentrations after continuous and intermittent hepatic ischaemia. Two groups of rats were subjected to continuous or intermittent left hepatic inflow occlusion for a total period of 120 min in each group. Intermittent ischaemia was associated with significantly lower mortality rates than continuous ischaemia (four of 20 versus 15 of 20; P = 0.0015). In a separate study, again following 120 min continuous or intermittent ischaemia, systemic blood was sampled at 0 min, 1 h, 3 h and 5 h after final clamp release for measurement of endotoxin, TNF and IL-6 concentrations. Endotoxin concentrations were significantly lower at 1 h, as were TNF and IL-6 concentrations at 3 and 5 h, after final clamp release in the group having intermittent ischaemia (P < 0.05). Intermittent ischaemia is associated therefore with significantly reduced mortality rates and lower systemic endotoxin, TNF and IL-6 concentrations when compared with continuous ischaemia.

Lactulose as an antiendotoxin in experimental colitis.

The efficacy of lactulose as an antiendotoxin was studied and the effect of lactulose or colistin on faecal flora was investigated in a hapten-induced rat model of colitis. Enteral administration of lactulose to rats with colitis was associated with a significant reduction in the systemic concentration of endotoxin (median (range) 5.4 (0-19.9) versus 23.7 (0-145.0) pg/ml in colitic rats treated with water; 4.6 (0-10.8) pg/ml in healthy animals). Enteral administration of colistin significantly reduced the faecal count of aerobic Gram-negative bacilli (median (range) 2.84 (1.40-8.43) versus 8.26 (4.50-10.40) log10 colony-forming units per g faeces after treatment with water) but not the faecal load of endotoxin. Patients with inflammatory bowel disease may benefit from enteral treatment with lactulose to prevent systemic endotoxaemia and/or with colistin to modify enteric bacteria.

Enteral and parenteral anti-endotoxin treatment in experimental colitis.

Systemic endotoxemia has been described in ulcerative colitis and Crohn's disease and shown to correlate positively with disease activity and the extent of intestinal ulceration. This study evaluated the efficacy of antibiotic and anti-endotoxic treatment in reducing systemic endotoxemia in a hapten-induced rat model of colitis. Enteral administration of paromomycin was associated with a significant reduction in systemic endotoxin concentrations (7.4 +/- 1.2 pg/ml) when compared with controls (39.8 +/- 12.6 pg/ml; p = 0.032). Intravenous injection of taurolidine was also found to significantly reduce systemic endotoxemia (3.1 +/- 1.3 pg/ml) in comparison with controls receiving saline injection (17.5 +/- 4.2 pg/ml; p = 0.008). Enteral neomycin, parenteral polymyxin or metronidazole and cefuroxime were ineffective anti-endotoxin treatments in this model. Enteral paromomycin or parenteral tauro-lidine therapy are potential methods of preventing and treating systemic endotoxemia in patients with inflammatory bowel disease.

Bowel ischaemia and organ impairment in elective abdominal aortic aneurysm repair.

In 30 patients undergoing elective repair of abdominal aortic aneurysm the intramucosal pH (pHi) of the sigmoid colon was measured. Blood for endotoxin assay was taken at intervals before, during and after surgery. Daily measurements were made of liver transaminase activity and of arterial partial pressure of oxygen (PaO2). The mean (s.e.m.) peak systemic endotoxin concentration in those who developed intramucosal acidosis (pHi below 7.00) was 90(14) pg/ml, compared with 42(5) pg/ml in those who did not (P < 0.01). In the 14 patients whose pHi fell below 7.00, the mean (s.e.m.) postoperative rise in aspartate transaminase activity was 346(74) per cent, compared with 181(20) per cent in those whose pHi remained above this level (P < 0.05). The mean (s.e.m.) postoperative ratio of PaO2 to the fraction of inspired oxygen was 177(11) mmHg in those with intramucosal acidosis, compared with 260(24) mmHg in those whose pHi remained above 7.00 (P < 0.01). These results demonstrate a relationship between bowel ischaemia, endotoxaemia and organ impairment following elective aneurysm repair.

Endotoxaemia, the generation of the cytokines and their relationship to intramucosal acidosis of the sigmoid colon in elective abdominal aortic aneurysm repair.

Ischaemia of the large bowel occasionally occurs following abdominal aortic aneurysm repair and may lead to multiple system organ failure (MSOF). Intramucosal acidosis of the sigmoid colon is a good indicator of sigmoid colonic ischaemia. Intramucosal pH of the sigmoid colon was measured using the silicone tonometer in 21 patients undergoing abdominal aortic aneurysmectomy. Samples were taken for plasma endotoxin, tumour necrosis factor (TNF) and interleukin-6 (IL-6) measurements preoperatively, half-hourly during the operation, 2-hourly for the next 12 h, 4-hourly for a further 48 h and 8-hourly thereafter until the fifth day. The intramucosal pH of the sigmoid colon fell to less than 7.00 peri-operatively in 10 patients, four of whom developed diarrhoea; in comparison, this did not occur in any of the 11 whose pH remained greater than 7.00 (p = 0.036). Higher peak concentrations of endotoxin, TNF and IL-6 were found in those patients whose intramucosal pH fell to less than 7.00 compared to those whose pH remained greater than 7.00 (mean +/- S.E.M. pg/ml, endotoxin = 112 +/- 24 vs. 58 +/- 6, p < 0.05; TNF = 26 +/- 8 vs. 7 +/- 2, p < 0.05; IL-6 = 213 +/- 59 vs. 87 +/- 12, p = 0.09). In the two patients who died, both from the group with pH level less than 7.00, concentrations of IL-6 were considerably higher than that in most of the other patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of extrahepatic obstructive jaundice on Kupffer cell clearance capacity.

Systemic endotoxemia consistently occurs in jaundiced patients undergoing surgery. Kupffer cell dysfunction is implicated in the development of endotoxemia and its postoperative complications. A novel in situ single-pass hepatic perfusion technique using a fluorescein isothiocyanate-labeled latex probe was developed for measuring Kupffer cell clearance capacity and was applied in an animal model of biliary obstruction. Control rats and rats jaundiced for 1, 2, 3, and 4 weeks' duration were studied. Kupffer cell clearance capacity, plasma bilirubin, endotoxin, and anticore glycolipid concentrations were measured. Maximal hyperbilirubinemia preceded reduced Kupffer cell clearance capacity. Rats jaundiced for greater than 2 weeks had a significantly decreased Kupffer cell clearance capacity but significantly higher endotoxin and anticore glycolipid concentrations. Anticore glycolipid concentrations correlated strongly with systemic endotoxemia and both were inversely correlated with duration of jaundice. Impairment of Kupffer cell clearance capacity may contribute to endotoxemia associated with cholestasis.

Adsorbents as antiendotoxin agents in experimental colitis.

The intestinal mucosa protects the body from a large reservoir of intraluminal pathogenic bacteria and endotoxins. This mucosal barrier is disrupted by the inflammation and ulceration of inflammatory bowel disease and may permit the absorption of toxic bacterial products. Systemic endotoxaemia has been demonstrated in ulcerative colitis and Crohn's disease and correlates with the extent and activity of disease. In this study the efficacy of absorbents as antiendotoxin agents in a hapten induced rat model of colitis is investigated. Induction of colitis was associated with systemic endotoxaemia. Enteral administration of terra fullonica and kaolin, but not of charcoal, significantly reduced systemic endotoxaemia (terra fullonica 4.2 (1.40) pg/ml; kaolin 5.29 (1.86) pg/ml; charcoal 32.7 (16.6) pg/ml; water 39.8 (12.6) pg/ml). Data expressed as mean (SE). With increasing severity of colitis, there was a decreasing ability of adsorbent therapy (terra fullonica) to control systemic endotoxaemia. Enteral administration of adsorbents controls gut derived systemic endotoxaemia in experimental colitis in animals and may be a useful antiendotoxin treatment in patients with inflammatory bowel disease.

Heredofamilial deficiency of monocyte esterase in patients with rheumatoid arthritis.

Deficiency of the monocyte ectoenzyme non-specific esterase is described in a heredofamilial pattern in four patients with rheumatoid arthritis. No association with HLA status or rheumatoid factor seropositivity was found.

A possible role for superoxide production in the pathogenesis of contact dermatitis.

Superoxide generation by blood monocytes was examined in patients with irritant, nickel, and chromate hand dermatitis. Phorbol myristate acetate stimulated monocytes generated significantly more superoxide in patients with nickel dermatitis, as did patients with hand eczema generally. No significant stimulation of monocyte superoxide generation occurred with either opsonized zymosan or PMA in the presence of excess superoxide dismutase in any of the groups of hand dermatitis. The results indicate a biochemical stimulation of superoxide which may accentuate the immunological damage in the skin that is observed in nickel and perhaps chromate dermatitis.

Diclofenac inhibits monocyte superoxide production ex vivo in rheumatoid arthritis.

Effects of the nonsteroidal anti-inflammatory drug, diclofenac, on stimulated monocyte superoxide production were assessed directly in vitro and following treatment of patients with rheumatoid arthritis ex vivo. Diclofenac inhibited superoxide generation provoked by serum treated zymosan (STZ) and fluoride anion (F) but not by phorbol myristate acetate (PMA) in vitro. Following patient therapy, inhibition of superoxide production occurred when STZ and PMA, but not F were used as stimuli. No changes were seen in control subjects. The contrasting profiles of inhibition seen in vitro and ex vivo suggest an indirect effect on superoxide production during clinical use of the agent. These data are consistent with the hypothesis that anti-inflammatory drugs may act in rheumatoid arthritis by inhibiting phagocyte superoxide anion production.