Biochemical alterations in rat Thiry-Vella fistulas.

In order to obtain baseline information on the secretory function of normal rat bowel for our work on intestinal graft ischemia, we studied several biochemical parameters in rat Thiry-Vella fistulas (TVF). TVFs were created in 200-g male Lewis rats (n = 11) using the 25-cm segment of jejunum normally used as a graft in our intestinal transplant model. The stomas were matured primarily and the animals were allowed to recover. The TVFs were flushed at 0, 6, and 24 h and then daily for up to 21 days with 12 mL normal saline solution. The effluent was collected and analyzed for total protein (TP), secretory phospholipase A2 (sPLA2), intestinal fatty acid binding protein (I-FABP), lactate dehydrogenase (LDH), and N-acetylglucosamine (NAGA). TP content was 0.12 +/- 0.01 mg/mL up to 48 h, then gradually increased and stabilized at 0.39 +/- 0.05 mg/mL at day 21. By sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), one major protein band was identified in the low-molecular-mass range (15 kD), consistent with I-FABP and sPLA2. Secretory PLA2 levels decreased over the first 4 days to a low of 115 +/- 24.8% hydrolysis/min/fraction, then gradually rose to a plateau at approximately 529.76 +/- 88.36% hydrolysis/min/fraction by day 18. I-FABP levels rose rapidly from 0 ng/mL at 2 h to 900 +/- 250.0 ng/mL at 6 h and approximately 3000 +/- 304.9 ng/mL by day 14. LDH levels at 2 h and 48 h did not differ, with 0.03 +/- 0.004 and 0.03 +/- 0.005 optical density units (OD)/min/mL, respectively. NAGA levels were 0.07 +/- 0.05 OD/h/mL at 2 h and rose to 0.14 +/- 0.04 OD/h/mL at 48 h. These data suggest that after an early equilibration period, biochemical secretion into the lumen of normal rat bowel reaches a state of equilibrium, and therefore appears to reflect the baseline biochemical status of the bowel. Some of these levels are not negligible as one would expect in "normal" bowel. This information should prove extremely helpful as a baseline study of abnormal conditions of the intestine, such as ischemia or rejection.

Potentially lethal complications of central venous catheter placement.

BACKGROUND: Placement of central venous catheters, although often considered to be a relatively safe and "junior"-level procedure, may be associated with life-threatening complications. METHODS: A recent surgical death associated with placement of a central venous catheter at this Institution led to submission of a questionnaire to pediatric surgeons referenced through the American Pediatric Surgical Association directory regarding knowledge of similar incidents and information regarding catheter placement-related complications. RESULTS: Results to this response, although anecdotal, provided data regarding complications of an acute nature, which fell into the categories of pneumothorax, hydrothorax, cardiac tamponade, and hemothorax. Of 10 children with cardiac tamponade, 7 were infants, and most complications were associated with needle stick for access, with symptoms developing within minutes up to 12 hours after the procedure. Drainage of the tamponade was performed by aspiration alone in 3 cases; surgical drainage in 6 children resulted in survival in 9 of the 10 patients. Hemothorax was described in 19 patients and appeared to be more common in children in the 1- to 6-year age group, usually associated with percutaneous access techniques. Thoracotomy for hemothorax was performed in 16 children with 11 survivors. Vascular injury to subclavian artery, vein, or superior vena caval were noted in most at operation. CONCLUSIONS: Although data included in this review are entirely anecdotal and not subject to scientific scrutiny or analysis, certain conclusions appear evident. Inherent risks of central venous catheters are intrinsic and should be discussed with the family in obtaining preoperative consent, including life-threatening risks that may necessitate urgent surgical intervention (by thoracotomy or other means). Certain technical aspects of the procedure should be rigidly followed with an experienced surgeon in attendance throughout the procedure. Rapid evaluation should be performed for any unexplained problems that occur in the operating theatre or during the early postoperative period.

Modification of disaccharidase activity measurement in rat bowel.

This study describes a modification of the existing disaccharidase assay in rat small bowel in which whole bowel, rather than mucosa, is utilized. In addition, the use of total vs. specific activity as a more accurate unit of measurement of disaccharidase activity is discussed.

Plasma intestinal fatty acid binding protein in neonates with necrotizing enterocolitis: a pilot study.

BACKGROUND/PURPOSE: Intestinal fatty acid-binding protein (IFABP) is found within cells at the tip of the intestinal villi, an area commonly injured when necrotizing enterocolitis (NEC) occurs. This study was undertaken to determine if measuring IFABP concentrations in the bloodstream early in the course of NEC would differentiate patients by severity before clinical findings made it clear who had stage 3 NEC and who had milder stages. METHODS: Three plasma samples from newborn infants evaluated for NEC were obtained at symptom onset and after 8 and 24 hours. IFABP concentration was measured by radioimmunoassay. Infants were classified by the final and most severe stage of NEC, and IFABP levels were compared between groups at each sampling. RESULTS: IFABP was detectable in blood samples from all 7 infants with stage 3 NEC compared with 3 of 24 with stages 1 or 2 NEC. Elevated plasma IFABP concentrations were detectable before clinical staging could be made in 5 of the 7 subjects with stage 3 NEC. CONCLUSION: IFABP may be a specific marker for early identification of severe NEC.

Secretory event in intestinal grafts during preservation ischemia.

BACKGROUND: Ischemia triggers secretion of proteins from the intestine, including type II secretory phospholipase A2 (sPLA2). This "secretory event" was studied in intestinal grafts during the first few hours of preservation by measuring total protein, sPLA2, and other enzymes in the UW preservation solution over time. The effect of PX-13, a PLA2 inhibitor, was also studied. MATERIALS AND METHODS: Twenty-five centimeter intestinal grafts were harvested from Lewis rats, flushed, and preserved in UW solution +/- PX-13 at 4 degrees C. UW samples from 0 to 48 h (n = 5 each) were analyzed for total protein, sPLA2, lactate dehydrogenase (LDH), N-acetylglucosamine (NAGA), and lysozyme. Nonpreserved grafts were homogenized in PBS as tissue controls. Standard biochemical methods were used for all assays. RESULTS: Total protein increased rapidly by 5 min, continued to rise more slowly until 30 min, and then stabilized. The most significant increase in sPLA2 activity occurred between 90 and 180 min. NAGA increased most markedly between 30 and 180 min, while LDH increased in the first 30 min, although the level of both enzymes was negligible compared to tissue enzyme. Lysozyme levels were minimal at all times. PX-13 decreased sPLA2 activity markedly at all time points. CONCLUSION: Total protein levels increased before sPLA2, suggesting that sPLA2 may be secreted in response to other proteins or enzymes released even earlier during preservation (e.g., cytokines). These elevations do not appear to be caused by cell death. Phospholipase A2 secretion may be blocked, and this may greatly improve the outcome of intestinal preservation.

Phospholipase A2 secretion during intestinal graft ischemia.

The time-dependent appearance of phospholipase A2 (PLA2) activity in the preservation media of ischemic rat intestinal grafts is described. In controls, Ca2+-dependent, secretory PLA2 activity accumulated rapidly during the first 6 hr of ischemia, followed by a linear increase for up to 48 hr. LDH levels, by contrast, increased linearly throughout the 48 hr of ischemia. Addition of inhibitors of PLA2, cyclooxygenase, and lipooxygenase blocked accumulation of PLA2, but not LDH. PX-13, a novel PLA2 inhibitor, was most effective: 40 microM inhibited release by 86%, while 25 microM indomethacin (cyclooxygenase blocker) or nordihydroguiaretic acid (lipooxygenase blocker) inhibited 41 and 36%, respectively. That appearance of PLA2 activity, but not LDH, is attenuated by inhibitors of the eicosanoid cascade suggests a secretory event rather than leakage from dying cells. The secreted PLA2 is most likely the proinflammatory sPLA2 that has been implicated as a stress-induced protein and priming agent in ischemia-reperfusion injury.

Secretory phospholipase A2 levels in rat small bowel.

Preliminary studies on ischemia/reperfusion injury in transplanted small bowel grafts showed that secretory phospholipase A2 (sPLA2) may play a substantial role by breaking down membrane phospholipids. This study sought to determine the normal values of sPLA2 in the rat small bowel as a function of site and length as a baseline for future studies. The entire small bowel of male Lewis rats (200 g) was flushed with normal saline to eliminate solid contents. In group 1, the entire small bowel was divided into 5-cm segments (numbered 1-9), which were snap frozen and processed the same day for sPLA2. In group 2, a 25-cm segment of bowel (corresponding to segments 2-6 in group 1) was harvested from each animal, snap frozen, and immediately processed for sPLA2. To assess the effect of bowel storage on enzyme content, group 3 and group 4 grafts were stored for 7 and 14 days, respectively, at -85 degrees C prior to processing. All samples were homogenized in buffer, extracted with H2SO4 and assayed for sPLA2 activity using [1-14C]oleate-labeled autoclaved Escherichia coli as substrate. Results were analyzed statistically by ANOVA. sPLA2 activity rose from 85.46 +/- 14.46% hydrolysis/min fraction-1 in segment 1, to 476.38 +/- 176.75% hydrolysis/min fraction-1 in segment 9. The increase was linear and statistically significant (p < .0001). There was no significant difference in enzymatic activity between groups 2, 3, and 4. Group 2 activity was 263.02 +/- 43.74% hydrolysis/min fraction-1. This value was not statistically different from the mathematically calculated mean of segments 2-6 in group 1 (237.75). The results show that (1) sPLA2 activity increases predictably with distance from the ligament of Treitz (2) storage at -85 degrees C does not affect sPLA2, activity, and (3) 25-cm grafts may be evaluated in toto with reproducible baseline enzyme activity. Given the variability of enzyme activity along the course of the rat small bowel, it is imperative that exact location be identified in any studies evaluating sPLA2 activity.

Effect of flushing pressure on rat small bowel transplants.

Excessive flushing pressure in rat small bowel transplants may cause graft failure, but the optimal pressure for manual flushing has not been established. The goal of this study was to determine a safe yet effective pressure for intravascular graft flushing and to evaluate the consequences of higher pressures. The "usual" manual flush was found to be at a pressure of approximately 40 mm Hg. After harvest, grafts were flushed manually with heparinized normal saline solution while connected to a transducer. Two groups of 25-cm jejunal grafts (n = 5 each) were harvested from Lewis rats (200 g) on a pedicle of superior mesenteric artery and vein. Group 1 grafts underwent 3 successive flushes at 50, 100, 200 mm Hg, respectively. After each flush, full-thickness biopsies were obtained for light and transmission electron microscopy. Two grafts underwent syngeneic small bowel transplantation (SBTx), and biopsies were taken after reperfusion. Group 2 grafts were flushed at 50 mm Hg and biopsies were obtained. All grafts underwent SBTx and biopsies as in group 1. After flushing at 50 mm Hg. biopsies were essentially normal. When flushed at 100 mm Hg, there was pronounced epithelial separation with dilatation of the core of the villus. Flushing at 200 mm Hg resulted in complete separation of the villi, with occasional disruption in the crypts. All group 1 transplanted grafts were grossly nonviable immediately after reperfusion, while transplanted grafts in group 2 were grossly normal. The findings were consistent within each group and the difference between groups was statistically significant (0 vs. 100%). The results suggest that flushing pressures of 50 mm Hg in rat SBTx provide optimal evacuation of blood with minimal, reversible tissue injury. Higher pressures cause progressive histologic damage and may produce nonviable grafts. Flushing pressures in the rat model of SBTx should therefore be maintained at approximately 50 mm Hg.

Ethical issues: impact of the animal rights movement on surgical research.

The aggressive militancy of many animal rights or "antivivisectionist" groups is causing great consternation but little action on the part of medical and surgical researchers. Pediatric surgeons are particularly affected, since issues of tissue healing, growth and development, and organ or total-body responses to surgical insults must be established in the live organism, usually in animal models that cannot be replaced by other methods. Investigators have been threatened physically; laboratories have been vandalized and valuable data destroyed. Biomedical researchers have been called "animal-Nazis." The proliferation of animal rights groups such as the Animal Liberation Front (ALF) and People for the Ethical Treatment of Animals (PETA) have prompted the birth of pro-research organizations such as "Putting People First" and the "incurably ill For Animal Research" (iiFAR). The result of this pro and con activity is an extraordinary amount of time and expense devoted to cover the cost of new regulations and laboratory security (approximately $ 1.5 billion in the U. S. alone) at the expense of research budgets, adding to the increasing shortage of research funding. This situation has created dilemmas for the surgeon involved in basic animal research: is it worth taking personal risks to develop new techniques? Is it ethical to allow these fears to hinder progress in surgery? Should we do away with animal research entirely and test new techniques directly on children? Would that be ethical? These questions are difficult to answer, but must be addressed if we expect medicine to progress.

Impaired survival and growth in immunosuppressed young rats with lethal short gut syndrome and a small bowel transplant: an effect of cyclosporine.

Neonates and growing individuals have increased nutritional demands as compared with adults. To determine the functional ability of an intestinal graft to allow survival and growth, an otherwise lethal short gut model should be used (resection of both the entire small bowel and the cecum). In this study the authors investigated the survival and growth in young rats (80 to 125 g) with this lethal short gut syndrome (SGS) and either syngeneic or allogeneic segmental small bowel transplantation (SBTx). Additionally they sought to determine the effect of therapeutical doses of cyclosporine (CyA) in young, growing rats. To avoid total parenteral nutrition in rats undergoing SBTx, surgery was carried out in two steps: after segmental SBTx of a 25-cm jejunal graft, SGS was created 2 weeks later. Lewis rats underwent 1: Syngeneic segmental SBTx + SGS (n = 7); 2: Allogeneic segmental SBTx (donor: Lewis Brown Norway F1) + SGS + CyA (15 mg/kg/d for 7 days, then every other day for 21 days) (n = 9); 3: Syngeneic segmental SBTx + SGS + CyA as in group 2 (n = 5); 4: SGS alone (n = 5): 5: small bowel resection alone (n = 5); 6: sham laparotomy twice (n = 5); 7: sham laparotomy twice + CyA as in group 2 (n = 6). Weight, general condition, and nutritional serum variables were followed up regularly for 4 months. Rats with resection of small bowel survived but did not grow. Rats with small bowel resection + cecectomy died within 5 days.(ABSTRACT TRUNCATED AT 250 WORDS)