A possible mechanism for prevention of intestinal programmed cell death after ischemia-reperfusion injury by hepatocyte growth factor pretreatment

Background/Purpose: Intestinal ischemia-reperfusion (IR) injury produces necrosis and apoptosis resulting in tissue loss. The authors have observed previously that pretreatment with hepatocyte growth factor (HGF) attenuates enterocyte apoptosis after IR. This study investigated the effects of HGF on tissue levels of caspase-8, an apoptosis initiator, and caspase-3, an apoptosis effector, in intestinal mucosa after IR. Methods: Thirty rats underwent placement of jugular venous catheters connected to osmotic pumps; 15 rats received vehicle, and 15 rats received HGF (150 [mu ]g/kg/d). After a 48-hour infusion, 5 rats from each group underwent either 35 minutes of superior mesenteric artery occlusion alone, or ischemia followed by 2 or 6 hours of reperfusion. Mucosal protein was analyzed for caspase-8 and caspase-3 activity. DNA fragmentation was used to measure the presence of apoptosis. Statistical significance was determined using analysis of variance. Results: After 6 hours of reperfusion, caspase-3 activity was increased significantly in control animals (P [lt ] .05). In HGF-pretreated animals, caspase-8 and caspase-3 activities were significantly reduced at 6 hours compared with control animals (P [lt ] .05). Conclusion: By preventing the activation of enterocyte caspase enzymes, and, thus, reducing apoptosis, HGF may enhance preservation of the intestine after IR injury.     

Ischaemic preconditioning improves microvascular perfusion and oxygenation following reperfusion injury of the intestine

BACKGROUND: Ischaemia-reperfusion (IR) injury of the intestine occurs commonly during abdominal surgery. Ischaemic preconditioning (IPC) provides a way of protecting the organ from damage inflicted by IR. This study was designed to evaluate the beneficial effect of IPC, focusing on the intestinal microcirculation and oxygenation in intestinal IR injury. METHODS: Rats were allocated to three groups. Animals in the IR and IPC groups underwent 30 min of intestinal ischaemia followed by 2 h of reperfusion. In the IPC group this was preceded by 10 min of ischaemia and 10 min of reperfusion. Animals in the third group underwent laparotomy but no vascular occlusion. Intestinal microvascular perfusion, oxygenation and portal venous blood flow (PVF) were monitored continuously. At the end of the reperfusion period, blood samples were obtained for measurement of lactate dehydrogenase (LDH) and biopsies of ileum for histological evaluation. RESULTS:: IPC improved intestinal microvascular perfusion and tissue oxygenation significantly at the end of the reperfusion period (P < 0.001). PVF improved significantly in the IPC compared with the IR group (P = 0.005). The serum LDH concentration was significantly lower in the IPC than the IR group (mean(s.e.m.) 667.1(86.8) versus 1973.8(306.5) U/l; P < 0.001) Histological examination showed that ileal mucosa was significantly less injured in the IPC group. CONCLUSIONS: This study demonstrated that IPC improves intestinal microvascular perfusion and oxygenation.     

Effect of ischemic preconditioning on hepatic microcirculation and function in a rat model of ischemia reperfusion injury

Ischemic preconditioning (IPC) may protect the liver from ischemia reperfusion injury by nitric oxide formation. This study has investigated the effect of ischemic preconditioning on hepatic microcirculation (HM), and the relationship between nitric oxide metabolism and HM in preconditioning. Rats were allocated to 5 groups: 1. sham laparotomy; 2. 45 minutes lobar ischemia followed by 2-hour reperfusion (IR); 3. IPC with 5 minutes ischemia and 10 minutes reperfusion before IR; 4. L-arginine before IR; and 5. L-NAME + IPC before IR. HM was monitored by laser Doppler flowmeter. Liver transaminases, adenosine triphosphate, nitrites + nitrates, and guanosine 3'5'-cyclic monophosphate (cGMP) were measured. Nitric oxide synthase (NOS) distribution was studied using nicotinamide adeninine dinucleotide phosphate (NADPH) diaphorase histochemistry. At the end of reperfusion phase, in the IR group, flow in the HM recovered partially to 25.8% of baseline (P < .05 versus sham), whereas IPC improved HM to 49.5% of baseline (P < .01 versus IR). With L-arginine treatment, HM was 31.6% of baseline (NS versus IR), showing no attenuation of liver injury. In the preconditioned group treated with L-NAME, HM declined to 10.2% of baseline, suggesting not only a blockade of the preconditioning effect, but also an exacerbated liver injury. Hepatocellular injury was reduced by IPC, and L-arginine and was increased by NO inhibition with L-NAME. IPC also increased nitrate + nitrate (NOx) and cGMP concentrations. NOS detected by NADPH diaphorase staining was associated with hepatocytes and vascular endothelium, and was induced by IPC. IPC induced NOS and attenuated HM impairment and hepatocellular injury. These data strongly suggest a role for nitric oxide in IPC. (Liver Transpl 2002;8:1182-1191.)     

Prognostic value of intraoperative renal tissue oxygenation measurement on early renal transplant function

Ischemia time is a prognostic factor in renal transplantation for postoperative graft function and survival. Kidney transplants from living donors have a higher survival rate than deceased donor kidneys probably because of shorter ischemia time. We hypothesized that measurement of intraoperative kidney oxygenation (μHbO₂) and microvascular perfusion predicts postoperative graft function. We measured microvascular hemoglobin oxygen saturation by reflectance spectrophotometry and microcirculatory kidney perfusion by laser Doppler flowmetry 5 and 30 min after kidney reperfusion on the organ surface in 53 renal transplant patients including 19 grafts from living donors. These values were related to systemic hemodynamics, cold ischemia time (cit), early postoperative graft function and length of hospital stay. μHbO₂ improved 30 min after reperfusion compared to 5 min (from 67% to 71%, P < 0.05). μHbO₂ correlated with mean arterial blood pressure and central venous pH (P < 0.01). Most importantly, μHbO₂ was significantly higher in kidneys from living compared with deceased donors (74% vs. 63%) and in kidneys without vs. with biopsy‐proven postoperative rejection (71% vs. 45%, P < 0.001). Finally, μHbO₂ correlated positively with cit and postoperative creatinine clearance and negatively with postoperative plasma creatinine, need for hemodialysis and length of hospital stay. Our results suggest higher oxygen extraction and thus oxygen demand of the grafts shortly after reperfusion. The intraoperative measurement of tissue oxygenation in kidney transplants is predictive of early postoperative graft function. Future studies should evaluate the potential effect of intraoperative therapeutic maneuvers to improve organ tissue oxygenation in renal transplantation.     

Different Intervals of Ischemic Preconditioning on Small Bowel Ischemia-Reperfusion Injury in Rats

Purpose

The purpose of this study was to establish morphologically the best time of vascular occlusion to induce ischemic preconditioning (IPC) for rat small bowel undergoing ischemia and reperfusion injury.

Methods

After approval by the Ethics Committee, 36 EPM-1 young adult Wistar rats from 300-350 g were distributed into 6 groups: sham (S); ischemia and reperfusion (IR), with 50 minutes of cranial mesenteric artery occlusion and 30 minutes of reperfusion; IPC with 1 cycle of 2 minutes (IPC-2), 5 minutes (IPC-5), 10 minutes (IPC-10), or 15 minutes (IPC-15), followed by sustained IR. The animals anesthetized with ketamine (60 mg/kg) and xylazine (10 mg/kg) intramuscular (IM), were maintained on mattress heat, hydrated with saline (80 mL/kg), and injected with 100 IU heparin. Samples of jejunum were stained with hematoxylin and eosin (HE) and classified according to Park et al. Statistical analysis of results was performed using Kruskal-Wallis tests (P < .05).

Results

The histological evaluation showed no difference between IR and IPC15 rats (5.2 and 5, respectively; P = .84). Greater jejunal injury was observed with IPC15 (5) compared with other groups (IPC2 = 3, P = .03; IPC5 = 3.2, P = .05; IPC10 = 2.8, P = .02, respectively). There was no difference between groups IPC2 × IPC5 × IPC10.

Conclusion

Morphologically, IPC with short times promoted greater intestinal protection against the IR lesion in rats.     

Interleukin-11 enhances intestinal absorptive function after ischemia-reperfusion injury

Background/Purpose: Interleukin-11 (IL-11) is a multifunctional cytokine that has been shown to improve small bowel adaptation and enhance cellular recovery after bowel ischemia. This study was designed to examine the effects of systemic IL-11 on small bowel absorptive function in a rat model of intestinal ischemia and reperfusion (IR) injury. Methods: Sprague-Dawley rats underwent placement of a venous catheter connected to an osmotic pump, which delivered its contents over 3 days. Rats were divided into 3 groups: sham operation/systemic saline; 30-minute superior mesenteric artery occlusion/systemic saline; superior mesenteric artery occlusion/systemic IL-11, 750 [mu ]g/kg/d. After the infusion, ¹⁴C-galactose or ¹⁴C-glycine absorption was measured using an in vivo, recirculation technique. Statistical significance was determined using analysis of variance. Results: In control rats, 30 minutes of IR decreased absorption of galactose from 2.62 to 2.02 [mu ]moles/cm² (P [lt ] .01), and glycine from 2.79 to 1.72 [mu ]moles/cm² (P [lt ] .01). Rats treated with systemic IL-11 showed improved absorption of galactose of 2.39 [mu ]moles/cm² (P [lt ] .05), and glycine at 2.21 [mu ]moles/cm² (P [lt ] .05). Mucosal DNA content was reduced significantly from 7.37 to 5.61 [mu ]g DNA/mg by IR (P [lt ] .01). IL-11 treatment did not significantly alter DNA content during this period. Conclusions: These data show that 30 minutes of intestinal IR significantly decreases intestinal absorptive function in this animal model. When compared with untreated control animals, administration of systemic IL-11 significantly increased the absorption of carbohydrate and amino acid in rats recovering from mesenteric IR.     

Hypothermia throughout intestinal ischaemia-reperfusion injury attenuates lung neutrophil infiltration

Background/Purpose: Secondary organ damage to the lungs is an important consequence of intestinal ischaemia reperfusion (IIR) injury. Moderate hypothermia ameliorates gut necrosis and liver energy failure after IIR but potential beneficial effects on lung neutrophil infiltration after reperfusion of ischaemic bowel have not been investigated. Methods: Adult Sprague-Dawley rats underwent 60 minutes intestinal ischaemia followed by 120 minutes of reperfusion. The animals were maintained at either normothermia (36[deg ] to 38[deg ]C) or moderate hypothermia (30[deg ] to 32[deg ]C). Four groups were studied: (A) sham normothermia; (B) IIR normothermia; (C) sham hypothermia; and (D) IIR hypothermia. Lungs and terminal ileum were removed for measurement of myeloperoxidase activity (a marker of neutrophil infiltration). Results are expressed as milliunits per milligrams protein, mean [plusmn] SEM, and one-way analysis of variance (ANOVA) with Tukey post-test was used for group comparisons. Results: Lungs: IIR at normothermia significantly increased lung neutrophil infiltration assessed by myeloperoxidase activity compared with sham-operated controls (normothermia sham 4.6 [plusmn] 1.0, n = 8; normothermia IIR 37.7 [plusmn] 13.8, n = 8; P = .011). Moderate hypothermia during IIR significantly attenuated lung neutrophil infiltration (7.2 [plusmn] 2.1, n = 9) compared with normothermia IIR (P = .016) such that myeloperoxidase activity was similar to that found in sham normothermia (4.6 [plusmn] 1.0, n = 8) and sham hypothermia (3.1 [plusmn] 1.3, n = 8). Intestine: Gut myeloperoxidase activity was 0.9 [plusmn] 0.5 in sham normothermia (n = 9) and 2.3 [plusmn] 0.6 after normothermic IIR (n = 8). After IIR at hypothermia gut myeloperoxidase activity (0.5 [plusmn] 0.2; n = 8) was significantly less than normothermic IIR (P = .035) and higher than sham hypothermia (0.2 [plusmn] 0.1, n = 9; P = .01). Conclusions: These results indicate that moderate hypothermia may prevent damage to another distant organ, ie the lungs, by preventing recruitment of neutrophils. This may be of benefit in decreasing distal organ damage in diseases in which intestinal ischaemia-reperfusion is implicated in the pathogenesis. J Pediatr Surg 38:88-91.     

Bupropion Reduces the Inflammatory Response and Intestinal Injury Due to Ischemia-Reperfusion

Intestinal ischemia-reperfusion (I/R) causes severe organ failure and intense inflammatory responses, which are mediated in part by the cytokine tumor necrosis factor-alpha (TNF-alpha). Bupropion is an antidepressant known to inhibit TNF-alpha production. We sought to examine the protective effects of bupropion on intestinal I/R injury in 15 male Sprague-Dawley rats that were randomized to sham surgery, 45 minutes of intestinal ischemia followed by 180 minutes reperfusion, or bupropion (100 mg/kg) before the intestinal I/R injury. To evaluate the systemic inflammatory response induced by intestinal I/R, we measured serum levels of TNF-alpha, interleukins-1 and -6, lipid peroxidation, and transaminases. Histologic analysis evaluated intestinal injury using the Chiu muscosal injury score. After I/R, Chiu score in control animals was 3.6 ± 1.2 vs 2.6 ± 0.53 in animals that received bupropion (P < .05). Bupropion pretreatment reduced intestinal. I/R injury and blunted serum elevations of TNF-alpha (0.96 ± 1.1 ng/mL vs 0.09 ± 0.06 ng/mL, P < .05) and interleukin-1 (0.53 ± 0.24 ng/mL vs 0.2 ± 0.11 ng/mL, P < .05). Bupropion in reduced intestinal I/R injury through immunomodulatory machanisms that involve inflammatory cytokines such as TNF-alpha.     

Remote ischemic perconditioning protects the liver from ischemia–reperfusion injury

Background

Ischemia–reperfusion (IR)–induced injury is a frequent sequel of major liver resections. IR injury after prolonged surgical interventions could be the source of increased risk of postoperative morbidity and mortality. Hepatoprotective effects of this new feasible method called remote ischemic perconditioning (RIPER) were investigated in our rat model of IR injury.

Materials and methods

Male Wistar rats underwent ischemia for 60 min on two-thirds of their livers, followed by 1, 6, and 24 h of reperfusion (n = 72, 8 per group). During liver ischemia, but before reperfusion, rats in the treated groups received four cycles of brief infrarenal aortic clamping as perconditioning. Liver microcirculation was monitored by laser Doppler flowmeter parallel with mean arterial pressure measurements. Liver tissue injury and redox homeostasis were investigated. Furthermore, serum tumor necrosis factor alpha (TNF-α) levels were measured.

Results

In the RIPER group, compared with the IR group, serum transaminase levels were significantly lower after each reperfusion period (alanine aminotransferase: 1 h, P < 0.001; 6 h, P < 0.05; 24 h, P < 0.01 and aspartate aminotransferase: 1 h, P < 0.001; 6 h, P < 0.05; 24 h, P < 0.05). Reperfusion microcirculatory parameters significantly improved in the perconditioned group compared with those in the IR group (reperfusion area: P = 0.005; maximal plateau: P = 0.0002). Regarding TNF-α levels, significant differences were detected between the two IR injured groups (RIPER versus IR: 1 h, 34.3 ± 12.8 pg/mL versus 205.7 ± 60.9 pg/mL, P < 0.001; 6 h, 60.6 ± 11.7 pg/mL versus 110.4 ± 21.6 pg/mL, P < 0.05). Results of the histologic assessment and redox state measurements also showed favorable changes.

Conclusions

Our team firstly reported the protective effects of RIPER on liver morphology, redox homeostasis, and microcirculation and proposed the changes of TNF-α expression.     

Does preexisting mental illness influence outcome in pediatric trauma?

Purpose. According to national estimates, up to 10% of American children suffer from some type of mental illness. Despite this high incidence, few studies have examined the role of a preexisting psychiatric disorder on childhood trauma. We hypothesize that children with such a diagnosis will have more severe injuries and poorer outcomes then other children. Methods. A 5-year (1998-2002) review of the Pennsylvania Trauma Outcome Study database was performed and information regarding children less than or equal to 16 years was abstracted. We evaluated the effects of a preexisting psychiatric diagnosis (not otherwise defined) and attention deficit disorder (ADD) on the type and mechanism of injury, injury severity, and outcome. Statistical analysis was performed using Student’s t-test, chi square, and Fisher’s exact test. Results. Over the study period 19,825 children were admitted to a Trauma Center: 530 (2.7%) had a preexisting psychiatric diagnosis (PD) and 189 (1%) had ADD. Children with ADD and PD were older than their peers (12.1 ± 3.3 versus 9.6 ± 5.6; P < 0.0001 and 13.7 ± 3.2 versus 9.5 ± 5.6; P < 0.0001), but did not differ in injury severity or overall mortality. However, those with PD were more likely to be victims of penetrating trauma (9.6% versus 7%; P < 0.022), and have longer ICU (2.2 versus 1.5 days; P = 0.0003) and hospital (5.7 versus 4.1 days; P < .0001) lengths of stay than unaffected children. Furthermore, children with PD were more likely to be discharged to a location other than home (P = 0.0006). Both the ADD and the PD groups were less likely to use protective devices (P < 0.03; P = 0.0011). Conclusions. Despite similar injury severity, children with a preinjury psychiatric history have longer hospitalizations than their nonaffected peers. This may be due in part to difficulty in finding appropriate postdischarge facilities for these children. Injury prevention strategies in this population should address the importance of protective equipment. Finally, the relatively low percentage of children identified as having ADD or PD in this study could be the result of underreporting and thus potentially affect the results.     

Serial transverse enteroplasty (STEP): a novel bowel lengthening procedure

Background/Purpose: Bowel lengthening may be beneficial for children with short bowel syndrome. However, current techniques require at least one intestinal anastomosis and place the mesenteric blood supply at risk. This study seeks to establish the technical principles of a new, simple, and potentially safer bowel lengthening procedure. Methods: Young pigs (n = 6) underwent interposition of a reversed intestinal segment to produce proximal small bowel dilation. Five weeks later the reversed segment was resected. Lengthening of the dilated bowel then was performed by serial transverse applications of a GIA stapler, from opposite directions, to create a zig zag channel. A distal segment of equal length served as an in situ morphometric control. Contrast radiologic studies were performed 6 weeks later, and the animals were killed. Statistical comparisons were made by paired t test with P less than .05 considered significant. Results: After bowel lengthening, all animals gained weight (66.7 [plusmn] 3.0 [SD] kg v 42.5 [plusmn] 3.5 kg; P [lt ] .001) and showed no clinical or radiologic evidence of intestinal obstruction. Intraoperatively, immediately after serial transverse enteroplasty, the intestine was substantially elongated (82.8 [plusmn] 6.7 cm v 49.2 [plusmn] 2 cm; P [lt ] .01). Six weeks after surgery, the lengthened intestinal segment became practically straight and, compared with the in situ control, remained significantly longer (80.7 [plusmn] 13.1 cm v 57.2 [plusmn] 10.4 cm; P [lt ] .01). There was no difference in diameter between these segments (4.3 [plusmn] 0.7 cm v 3.8 [plusmn] 0.4 cm; P value, not significant). Conclusions: Serial transverse enteroplasty (STEP) significantly increases intestinal length without any evidence of obstruction. This procedure may be a safe and facile alternative for intestinal lengthening in children with short bowel syndrome. J Pediatr Surg 38:425-429.     

Ischemic preconditioning attenuates capillary no-reflow and leukocyte adherence in postischemic pancreatitis

Background and aims Ischemic preconditioning (IPC) has been shown to protect several organs from ischemia–reperfusion injury. Postischemic microvascular dysfunction is considered to be the key mechanism of early graft pancreatitis after transplantation. The aim of the study was to determine whether brief ischemia and reperfusion before prolonged ischemia followed by reperfusion is protective in respect to microcirculatory derangement in postischemic pancreatitis.Methods In an in-situ model of ischemia–reperfusion was induced in the isolated pancreatic tail segment. Wistar rats were randomized to one group (n=7/group) with 2-h ischemia and reperfusion (I/R) and another group with 10-min ischemia and 10-min reperfusion (IPC) before the prolonged ischemia time. Microcirculation was observed for 2 h by intravital-fluorescence microscopy that analyzed functional capillary density and leukocyte adherence in postcapillary venules. Histological damage was quantified by a semiquantitative score (edema, vacuolization, PMN infiltration, necrosis).Results IPC resulted in a significant improvement of functional capillary density (248±20 vs 372±8 cm^–1, P<0.001), a significant reduction in leukocyte adherence in postcapillary venules (476±79 vs 179±15 cells/mm², P<0.001) and in significantly lower histological damage (score 9±0.8 vs 5±1.4, P<0.001), when compared with the ischemia–reperfusion group.Conclusion IPC reduces pancreatic inflammatory reaction by preservation of postischemic microcirculation. Therefore, it might become a useful procedure before organ procurement in pancreas transplantation.     

Examination of Protective Effect of Ischemic Postconditioning After Small Bowel Autotransplantation

Ischemia/reperfusion (I/R) injury is a serious condition that results from some surgical procedures, including intestinal transplantation. Ischemic postconditioning is defined as brief periods of reperfusion alternating with reocclusion applied during the early minutes after reperfusion. The objective of this study was to investigate the effect of ischemic postconditioning before small bowel autotransplantation. Total orthotopic intestinal autotransplantation was performed in 30 white domestic pigs. Grafts were stored in cold University of Wisconsin solution for 1, 3, or 6 hours. Duration of reperfusion was 3 hours in all grafts. Before reperfusion, the intestine was postconditioned via 3 cycles of ischemia for 30 seconds and reperfusion for 30 seconds (ischemic postconditioning protocol). Tissue from the small intestine was obtained after laparotomy (control group) and at the end of reperfusion periods. To monitor oxidative stress, tissue concentrations of malondialdehyde and reduced glutathione, and activity of superoxide dismutase were determined at spectrophotometry. Tissue damage on sections stained with hematoxylin- eosin was evaluated using a quantitative method (Scion Image software; Scion Corp, Frederick, Maryland). Our results demonstrated that ischemic postconditioning significantly decreased the reperfusion-ended lipid peroxidation value (mean ± SEM, 142.0 ± 7.1 μmol/g vs 125.0 ± 2.1 μmol/g; P < .05). Moreover, the capacity and activity of endogenous antioxidant protective systems (glutathione 789 ± 8.0 μmol/g vs 934 ± 5.7 μmol/g, and superoxide dismutase 110 ± 9 IU/g vs 126 ± 4 IU/g; P < .05) remained higher in the ischemic postconditioning groups compared with tissues without ischemic postconditioning. At quantitative analysis, tissue injury was increased by the duration of cold preservation. The greatest injury was observed in the mucosal and submucosal layers and in the depth of crypts after 6 hours of preservation. Ischemic postconditioning significantly decreased intestinal wall injury in each group (P < .05). It was concluded that ischemic postconditioning before reperfusion mitigated oxidative stress and histologic damage during small bowel autotransplantation.     

Effects of amnio-allantoic fluid exchange on bowel contractility in chick embryos with gastroschisis

Background/Purpose: Intestinal damage in patients with gastroschisis is characterized by bowel wall thickening, intestinal dilatation, mesenteric shortening, and a fibrous peel. The prevention of intestinal damage in gastroschisis by amnio-allantoic fluid (AAF) exchange has been reported using histologic and macroscopic evaluation of intestines, but the effects of this treatment on bowel contractility have not been investigated. The current study was performed to determine the effect of AAF exchange on the intestinal contractility in chick embryos with gastroschisis. Methods: Thirteen-day-old fertilized chick eggs were used. Gastroschisis was created through amnio-allantoic cavity. There were 3 study groups: control group, gastroschisis-only group, and gastroschisis-plus-exchange group. The bowels were evaluated by an in vitro muscle strip technique, and the response was expressed as a percentage of the maximum acetylcholine evoked contraction (E_max) in each tissue obtained. Additionally, parasympathetic ganglion cells per 10 plexus at the intestinal wall were counted. Differences between groups were analyzed by analysis of variance (ANOVA) followed by Tukey-Kramer. Probabilities of less than 5% were considered significant. Results: The intestines were thickened and covered by fibrous peel in the gastroschisis-only group when compared with the control group and the gastroschisis exchange group morphologically. There was a statistically significant decrease in contractility in the gastroschisis-only group compared with the control group (P [lt ] .05). It exerted 42.03 [plusmn] 46.73% contraction of control group's E_max. This decrease in contractility was significantly reversed in the exchange group (P [lt ] .05; E_max value of gastroschisis plus exchange group was 71.45 [plusmn] 23.54% of control group's E_max). Although the number of ganglia per 10 plexus was 76.7 [plusmn] 4.3 in the control group, it was measured 28% less in the gastroschisis-only group (P [lt ] .05). There was no significant difference between the ganglion numbers of control and exchange groups. Conclusions: Prenatal AAF exchange treatment prevents decreased bowel contractility in gastroschisis. Gastroschisis does not affect intestinal ganglia morphology, but the number of ganglion cells decreases. AAF exchange prevents these functional and morphologic adverse effects of disease. By these findings the expectancy of a better clinical result in gastroschisis with intrauterine pretreatment by amniotic fluid exchange increases.     

Intestinal epithelial cells exposed to heat shock stress release a soluble compound that activates the virulence of P. aeruginosa

Introduction. Although there is a significant body of evidence that bacteria activate a variety of pro-inflammatory signals following their interaction with human epithelial cells, there is virtually no information on signaling in the opposite direction. In this study we sought to determine whether host cell factors released by intestinal epithelial cells subjected to stress could signal the opportunistic pathogen, P. aeruginosa, to express a key virulence related protein, the PA-I lectin. Methods. Human intestinal epithelia (Caco-2_bbe) were grown to confluence in HDMEM media. To expose cells to a clinically relevant stress, hyperthermic conditions were created by immersing sealed culture dishes in a 42°C water bath for 23 min, followed by 2 h of recovery at 37°C and 5% CO₂. Control cells were maintained in 37°C at 5% CO_2.. Media were then filtered through a 0.22-μm filter before serial centrifugal membrane filtration using 100, 50, 30, 10, and 3 kDa filters and resuspended in HDMEM. GFP reporter strains of P. aeruginosa carrying a fusion construct for the PA-I lectin were then exposed to the epithelial cell derived filtrates from heat shock stressed Caco-2 cells. Dynamic fluorimetry was used to measure fluorescence (i.e. PA-I activity) over time. To determine if inducing compounds were proteins, fractions capable of activating PA-I were then heat inactivated by heating to 70°C for 30 min and tested against active fractions by the same method. Results. A 30- to 50-kDa media fraction from heat-stressed cells induced a 10-fold increase in PA-I promoter activity over controls, as measured by fluorescence (1082 ± 216%, P < 0.01), while all other fractions showed no effect. Heat inactivation completely eliminated this effect (−156% of control ± 293%, P = NS). Conclusion. Human intestinal epithelial cells release a soluble 30- to 50-kDa compound(s) that can activate a key virulence protein in P. aeruginosa. We speculate that the molecular dialogue between a pathogen and the intestinal epithelium is bidirectional and can be initiated by host stress conditions such as hyperthermia.     

Revisiting ischemia and reperfusion injury as a possible cause of necrotizing enterocolitis: Role of nitric oxide and superoxide dismutase

Background/Purpose: The pathogenesis of necrotizing enterocolitis (NEC) is unknown. Ischemia and reperfusion (I/R) injury has been considered a major contributing factor. Nitric oxide (NO) and superoxide dismutases (SODs) have been shown to protect bowel from I/R injury. This study aims to assess (1) the ability of premature intestine to resist I/R injury compared with mature intestine and (2) the possible role of NO and SODs in modulating such response. Methods: Intestines from 5 groups of rats (n = 6 for each study group) were studied: (1) premature, gestational age 20 days; (2) premature, gestational age 22 days; (3) full-term, newborn; (4) infant, day 15; (5) infant, day 30. Experiments: (1) The degrees of I/R injury after 0, 30, 60, 90 and 120 minutes, respectively, of ischemia and 25 minutes of I/R were assessed histologically by a pathologist who was unaware of the operative details. (2) Tissue NO and copper levels were measured by electroparamagnetic resonance (EPR) study; and nitric oxide synthases, copper zinc (CuZn) SODs and manganese (Mn) SODs were examined immunohistochemically. (3) and (4) I/R injury was assessed in rats that had received intraperitoneal injections of L-arginine (NO donor) and L-NAME (NO antagonist), respectively. Results: For premature (1,2), newborn (3) and mature (4,5) intestines, grades of injury at maximum I/R period studied (120 minutes of ischemia, 25 minutes of reperfusion) were 0, 0, and 3, respectively (P [lt ] .05); NO levels were 1 u [plusmn] 1.5, 3 [plusmn] 2.5, and 22 u [plusmn] 3.5, respectively (P [lt ] .05); Cu levels were 150 u [plusmn] 15, 200 u [plusmn] 41 and 12 u [plusmn] 2, respectively (P [lt ] .05); NOS in intestines were +, +, +++ and CuZnSODs were ++, +++, +, respectively; and MnSODs were +++, ++, [minus ], respectively. No change in NO levels was detected in groups (1), (2), or (3) after L-arginine and L-NAME injections. Conclusions: Premature rat intestine is highly resistant to I/R injury, which may indicate that I/R alone, in the absence of other predisposing factors (eg, bacterial colonization) may not be sufficient in causing NEC. Nitric oxide does not have a protective role for immature and newborn intestines in I/R as in mature intestine. The high level of SODs of the immature and newborn intestine may play an important role in its high resistance to I/R injury. J Pediatr Surg 37:828-834.     

Interferon gamma inhibits inter-enterocyte communication by reducing connexin 43 phosphorylation

Introduction. Restitution of the intestinal barrier requires inter-enterocyte communication via the phosphorylated gap junction protein connexin 43. Experimental necrotizing enterocolitis (NEC) is characterized by elevated interferon gamma (IFN) and impaired restitution. We therefore hypothesized that IFN impairs gap junction (GJ) function and restitution by inhibiting phosphorylation of connexin43. We further sought to determine whether connexin 43 phosphorylation was altered in human NEC. Methods. Connexin 43 expression and phosphorylation was assessed in IEC-6 cells ± IFN (1000 u/ml) by SDS--PAGE and confocal microscopy. GJ function was detected by microinjecting IEC-6 cells ± IFN with the GJ tracer lucifer yellow (LY) and impermeant rhodamine dextran. GJ function was inhibited with oleamide (10 μM). In vitro restitution was assessed using time lapsed video microscopy of IEC-6 cells ± IFN migrating across a scraped wound. Small bowel mucosal scrapings were acquired from infants undergoing bowel resection for NEC and compared to specimens taken from infants without NEC. Data are mean ± SEM. Results. IFN significantly reduced phosphorylation of connexin 43 (band density relative to actin, Ctrl: 0.86 ± 0.3 versus IFNγ 0.37 ± 0.1, P < 0.05) and decreased membrane localization of the phosphorylated gap junctions. This correlated with decreased gap junction function as detected by loss of LY transfer (levels of intercellular transfer: Ctrl: 3.2 ± 0.2, IFN 2.4 ± 0.3, oleamide 0, P < 0.05). Oleamide and IFN significantly reduced enterocyte restitution (ctrl: 5 μm/h, oleam: 1.5 μm/h, IFN: 1.6 μm/h, P < 0.05), correlating with a lack of intercellular communication. Strikingly, phosphorylation of connexin 43 in the mucosa of infants with NEC was significantly reduced as compared to control bowel, which correlated with the extent of mucosal disease. Conclusion. IFN inhibits phosphorylation of connexin 43, leading to impaired inter-enterocyte communication and reduced restitution. The finding of decreased phosphoconnexin 43 in human NEC suggests that impaired inter-enterocyte communication may underlie the barrier dysfunction in this disease.     

Epidermal growth factor improves nutritional outcome in a rat model of short bowel syndrome

Background/Purpose: This study investigates the effect of epidermal growth factor (EGF) on nutrient absorption in a rat model of short bowel syndrome (SBS). Methods: Male juvenile rats underwent either transection (Sham) or ileocecal resection leaving a 20-cm jejunal remnant. Animals underwent follow-up for 10 days, and resected animals were treated with placebo or recombinant human EGF (1-53). Animals were pair fed; in vivo nutrient absorption, intestinal permeability, morphology, and total intestinal DNA and protein content were measured. Results: Resected EGF-treated animals lost significantly less weight than those in the placebo group ([minus ]4.2 [plusmn] 3 v [minus ]13.7 [plusmn] 6.9%), absorbed significantly more 3-0 methylglucose (76.8 [plusmn] 6.6 v 64.9 [plusmn] 10.1%), and had reduced permeability (lactulose/mannitol ratio, 0.35 [plusmn] 0.19 v 0.60 [plusmn] 0.20; P [lt ] .05 for all comparisons). Conclusions: These findings show that treatment of short bowel syndrome animals with EGF reduced weight loss and improved carbohydrate absorption and intestinal permeability. These findings suggest that enteral EGF may be a useful therapy for short bowel syndrome; further studies are indicated.