Acute pancreatitis severity is exacerbated by intestinal ischemia-reperfusion conditioned mesenteric lymph

Acute pancreatitis severity is exacerbated by intestinal ischemia-reperfusion conditioned mesenteric lymph Richard S. Flint, MBChB,a Anthony R. J. Phillips, MBChB, PhD,a,b Sharleen E. Power, BSc,b P. Rod Dunbar, MBChB, PhD,b Caroline Brown, BSc,b Brett Delahunt, MBChB, MD,c Garth J. S. Cooper, MBChB, PhD,b and John A. Windsor, BSc, MBChB, PhD,a Auckland and Dunedin, New Zealand

Objective. To determine the effect of intestinal ischemia-reperfusion (IIR) on acute pancreatitis (AP) and the role of mesenteric lymph. Summary background data. Intestinal ischemia is an early feature of AP and is related to the severity of disease. It is not known whether this contributes to the severity of AP or is a consequence. Methods. Two experiments are reported here using intravital microscopy and a rodent model of mild acute pancreatitis (intraductal 2.5% sodium taurocholate). In the first, rats had an episode of IIR during AP that was produced by temporary occlusion of the superior mesenteric artery (30min or 3 3 10min) followed by 2h reperfusion. In a second study rats with AP had an intravenous infusion of mesenteric lymph collected from donor rats that had been subjected to IIR. In both experiments the pancreatic erythrocyte velocity (EV), functional capillary density (FCD), leukocyte adherence (LA), histology and edema index were measured. Results. The addition of IIR to AP caused a decline in the pancreatic microcirculation greater than that of AP alone (EV 42% of baseline vs. 73% of baseline AP alone, FCD 43% vs 72%, LA 7 fold increase vs 4 fold increase). This caused an increased severity of AP as evidenced by 1.4--1.8 fold increase of pancreatic edema index and histologic injury respectively. A very similar exacerbation of microvascular failure and increased pancreatitis severity was then demonstrated by the intravenous infusion of IIR conditioned mesenteric lymph from donor animals. Conclusions. Unidentified factors released into the mesenteric lymph following IIR injury are capable of exacerbating AP. This highlights an important role for the intestine in the pathophysiology of AP pathogenesis and identifies mesenteric lymph as a potential therapeutic target. (Surgery 2008;143:404-13.) From the Department of Surgery, Faculty of Medicine and Health Sciencesa and School of Biological Sciences,b University of Auckland, and Department of Pathology and Molecular Medicine,c Wellington School of Medicine, University of Otago, New Zealand

ACUTE PANCREATITIS (AP) is commonly encountered in clinical practice, affecting 4.5 per 100,000 people per year.1 Nearly a third will develop a severe form of the disease that is characterized by These studies were supported by the University of Auckland Research Committee, the Maurice & Phyllis Paykel Trust, and Lottery Health (New Zealand). R.F. acknowledges salary support by the Royal Australasian College of Surgeons. Accepted for publication October 11, 2007. Reprint requests: Prof. John A. Windsor, Department of Surgery, Faculty of Medical and Health Sciences, Park Rd, Grafton, Auckland, New Zealand. E-mail: [email protected]. 0039-6060/$ - see front matter Ó 2008 Mosby, Inc. All rights reserved. doi:10.1016/j.surg.2007.10.005

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pancreatic necrosis and multiple organ failure. The mechanisms that lead to severe AP remain unclear and, despite recent improvements in supportive care mortality remains high at 14–30%.2,3 The intestine, once considered a quiescent organ in severe AP, is being reassessed in the light of its significance in other critical illnesses.4 The concept of intestinal bacterial translocation is not new5 and is probably relevant to severe AP as intestinal flora are responsible for most of the late septic complications of pancreatic necrosis.6 A wider role for the intestine is suggested by experimental and clinical studies that demonstrate that intestinal changes are an early feature of AP,7 are proportional to the severity of AP,8-10 and recover with resolution of the illness.11

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Recently mesenteric lymph has become a focus for more intensive study with the reports that it appears implicated in distant organ damage during the evolution of multi-organ failure from shock.12 The similarity of severe AP to other major inflammatory diseases, the frequent association with shock and the known associated change in gut perfusion suggests that mesenteric lymph is worthy of consideration for a role in pancreatitis pathogenesis. Here we report on two studies that have examined the role of the intestine in acute pancreatitis. Using intravital fluorescence microscopy (IVM) and a rodent model of sodium taurocholate AP we first quantified the effect that intestinal ischemia-reperfusion (IIR) injury had on the pancreatic microcirculation and its impact on AP severity. We then determined that the peripheral administration of IIR conditioned mesenteric lymph from donor rats could cause an almost identical pancreatic injury in the recipient rats. MATERIAL AND METHODS Two experiments were performed: (1) investigation of the effect of IIR on the pancreatic microcirculation and histopathology during AP; (2) determination of whether there was any effect on the pancreas in rats with and without preexisting AP by injecting normal or ÔconditionedÕ mesenteric lymph from donor rats subjected to isolated IIR. All experiments were conducted with the appropriate approvals from the Animal Ethics Committee of the University of Auckland. Surgery. Adult male Wistar rats (400--460 g) were fed standard rat chow (Teklad 2018) ad libitum then fasted overnight for at least 10 h, with free access to water. Anesthesia (induction: sodium pentobarbitone (30 mg, i.p.), buprenorphine (40 mg, i.p.)) was maintained by propofol (10 mg/mL, 0.05--0.4 mL/h, i.v.). Ventilation was via a tracheostomy and a pressure controlled ventilator (40% O2/air v/v; Kent Scientific, Torrington, CT, USA). The respiratory rate was 60–80 bpm; peak inspiratory pressures 15–20 cmH2O; end-tidal CO2 35–45 mmHg monitored by a CO2SMO Plus capnograph and Analyse Plus software (Novametrix Medical Systems Inc, USA). The left femoral vein was cannulated (PE50) for intravenous infusions and fluorochrome administration; the left femoral artery (PE35) for blood pressure and heart rate (HR) monitoring, and the left jugular vein (PE50) for central venous pressure (CVP) monitoring. Mean arterial blood pressure (MAP) was

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maintained at 80–110 mmHg by titrating the propofol infusion and preserving the CVP within 10% of its starting value by intravenous 0.9% saline 1mL/h and intermittent 0.2–0.5 mL boluses of Gelofusine (B. Braun Medical, Bella Vista, NSW, Australia). A warming pad was used to keep rectal temperature at 37 ± 0.5°C. The MAP, HR, CVP, and temperature data were continuously monitored by a 16S PowerLab (ADInstruments, Bella Vista, NSW, Australia). A transverse laparotomy was followed by careful mobilization of the spleen and pancreatic body and tail to enable the IVM microscope access to the entire pancreas. The animal was placed on its left side and the pancreas placed on a custom designed, temperature controlled aluminum stage with the IVM microscope positioned above the pancreas. Plasticine was molded around the edge of the pancreas and the tissue was moistened with 0.9% saline before a glass cover slip was placed on top. The exposed spleen was covered in oxygenimpermeable polyvinyl chloride wrap. After the operation, the animal was allowed to reach a steady state for 15 min before any measurements were made. Induction of acute pancreatitis. This protocol was initially optimized in a pilot study (data not shown) and is based on published methods.13 The pancreatic duct was cannulated trans-duodenally via the Papilla of Vater (24 gauge angiocath). The tip of the angiocath was inserted 3 mm into the common biliary-pancreatic duct and bile was drained for 5 min, the last 2 min followed occlusion of the duct at the hilum of the liver (Biemer vascular clip, AESCULAP, Tuttlingen, Germany) and elevation of the head to 45 degrees. Sodium taurocholate (2.5% in 0.9% saline) was infused into the pancreatic duct with the bile duct still occluded (Genie Precision Infusion Pump, Kent Scientific Corporation) at a rate of 0.1 mL/min and a dose of 0.1 mL/ 100 g body weight. The cannula and Biemer clip on the bile duct were removed before further experimentation. Induction of intestinal ischemia. The SMA was encircled with a 3 mm diameter saline filled atraumatic vascular occluder (OC-3, In Vivo Metric, Healdsburg, CA, USA) to produce occlusion of the SMA distal to the pancreaticoduodenal artery. An immediate transient rise in MAP and macroscopic evidence of intestinal ischemia were used to confirm SMA occlusion. A duration of 30 min of SMA occlusion was chosen on the basis of other studies suggesting this to be a significant but survivable insult.14

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Mesenteric lymph collection. Donor mesenteric lymph for re-infusion into rats with and without AP (experiment 2) was collected from non-fasted male Wistar rats (weight 500--580 g). Through a transverse laparotomy the mesenteric lymph duct was cannulated aseptically with silastic tubing (o.d 1.1 mm, i.d. 0.64 mm, primed with heparinized saline, 50 U/mL) and secured with tissue glue (HistoacrylÒ, B. Braun, Bella Vista, NSW, Australia) before exteriorizing the distal end through the right posterolateral abdominal wall. The lymph was allowed to flow freely into heparinized pediatric blood tubes (BD MicrotainerÒ Blood Collection Tubes, Becton, Dickinson and Company, North Ryde, NSW, Australia) stored on ice. To augment lymph flow 0.9% saline (1 mL/100 g body weight/h) was infused into the small intestine through a duodenostomy (PE55 tubing). The pylorus was occluded with a 3/0 silk ligature to prevent saline reflux into the stomach. The ischemic conditioned lymph was thus collected from 5 animals during 30 min of intestinal ischemia (induced by SMA occlusion) and for 3 h of intestinal reperfusion. For control purposes, normal lymph was collected at corresponding time periods from 5 animals not subjected to IIR. Lymph collected from all the donor ischemic and control animal groups were pooled (47 mls and 27 mls respectively), aliquoted (2 mL), then stored at 80°C. The aliquots of lymph were thawed immediately prior to use and infused intravenously (2 mL over 30 min) into experimental animals undergoing IVM with and without AP (as described above). Intravital microscopy. Erythrocytes were labeled with FITC (Cat no. F250-2, Sigma-Aldrich Pty Ltd) in a procedure modified from that of Butcher and Weissman,15 and used as tracers of microcirculatory flow. Briefly, male Wistar donor whole blood was immediately mixed with heparin (100 u/ 1 mL blood), centrifuged (400 g, 10 min) then the plasma and buffy coat were removed. The remaining erythrocytes were washed (Alsever’s solution, A3551, Sigma at 3 mL/1mL of erythrocytes), centrifuged again (400 g, 10 min) before decanting the supernatant. This was repeated twice before washing with bicine buffer (B3876, Sigma). Then fresh bicine buffer was added in equal volume to that of the erythrocytes before adding FITC (4 mg/1mL of rbc) dissolved in 0.1 mL of n,n-dimethyl formamide (27054-7, Sigma). This was left at room temperature for 2–3 h on a rocker plate. The erythrocytes were again washed with bicine buffer until the supernatant was clear. Then 0.9% saline containing 14% citrate-

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phosphate-dextrose (C7165, Sigma) was added to the FITC-erythrocytes to give a final hematocrit of 50% and stored in the dark at 4°C for up to 7 d. During experimentation 0.5 mL of the animals’ blood was withdrawn and replaced with the same volume of FITC- 50% hematocrit erythrocytes. Leukocytes were labeled with 0.1 mL 0.5% Rhodamine 6G (25243-3, Sigma) administered intravenously 15 min before the first IVM recording and repeated each hour of experimentation. The pancreatic microcirculation was analyzed with an epifluorescent microscope (Zeiss Axiotech Vario 100 and Plan-neofluar 10 3 0.30 objective (Carl Zeiss Ltd.) with blue filter block [excitation wavelength 450--490 nm, emission wavelength > 515 nm] and green filter block [excitation wavelength 546 nm, emission wavelength > 590 nm]). Images were recorded with a charge coupled device video camera (DXC-C33P, Sony) and a digital recorder (DSR-DU1, Sony). Recorded video was transferred to computer for off-line analysis. One venule and seven capillary bed observation sites distributed randomly throughout the entire exocrine pancreas (head, body, tail) were identified and grid referenced at the beginning of the experiment. Pancreatic microcirculation was repeatedly assessed at these sites (venule 1 min, capillary sites 10 s) at each experimental time point thus ensuring wide and repeated sampling coverage of the entire pancreas through the experiment. Microcirculatory analysis. Capillary erythrocyte velocity (EV) was measured off-line with computer image analysis and the line-shift diagram method (Cap-Image, H. Zeintl, Heidelberg, Germany). For each time period the velocities in 10--90 capillaries in each of the seven sites were calculated and the average was taken as the EV for that time period. The functional capillary density (FCD) was defined as the combined length of perfused capillaries within a given area for each observation site using the same image analysis software. This was averaged for each time period. To ensure there was no bias present in the data extraction process a blinded independent investigator re-analyzed the images in 10 random animals. Results for each site at each time period were then compared to the original and there were no significant differences found in any of the analyses. Leukocyte adherence. A leukocyte was considered to be adherent to the venular endothelial wall if it did not move for at least 30 s. Leukocyte adherence was expressed as the number per mm2 of endothelial surface.16 Histopathologic scoring. At the end of the experiment the pancreas was excised, divided

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into head and tail and separately fixed in neutral phosphate-buffered 10% formalin. They were then embedded in paraffin sliced into 5 mm sections and stained with hematoxylin and eosin. The slides were examined to determine the severity of pancreatitis using a light microscope and a standardized scoring system17 and was performed by a consultant histopathologist blinded to the slides’ group identity. This system resulted in maximum scores being calculated for the whole pancreas for each of the following variables: leukocyte infiltration, fat necrosis, acinar necrosis, pancreatic hemorrhage, and edema. The final total was generated as the numerical sum of each component measurement and this was used to compare pancreatitis severity between the groups. Edema index. Harvested pancreas was blotdried with tissue paper. A 3 3 3 mm piece of pancreatic head was excised and the wet weight was measured. The tissue was freeze dried until the weight did not change (5 d). The edema index was then calculated as the ratio of wet weight to dry weight of the tissue. Statistical analysis. Differences in the EV, FCD, and LA between multiple groups were determined at each measurement time-point using one-way ANOVA with Tukey’s multiple comparison tests. The same test was used to determine differences between multiple groups in the pancreatic edema index. Histopathologic scores of pancreatitis severity were analyzed with the modified analysis of variance for discrete data (Proc CATMOD). Differences in the MAP within each group over time were assessed by one-way repeated measures ANOVA. Descriptive analysis of data is expressed as means ± SEM. Statistical analysis was evaluated using InStat 3.0b for Apple (GraphPad Software Inc.) and SAS 8.2 (SAS/STAT Software). A P < .05 was accepted as significant in all study comparisons. Experimental protocol. In the first experiment rats were randomly allocated to 6 groups (I to VI, n = 10). Group I, control; group II, underwent an episode of intestinal ischemia (1 3 30 min superior mesenteric artery (SMA) occlusion) followed by 2 h of intestinal reperfusion; group III, had repeated short episodes of intestinal ischemia and reperfusion (3 3 10 min SMA occlusion each separated by 10 min of reperfusion) then followed by 2 h of continuous reperfusion; group IV, had AP alone; group V, AP with a single episode of 30 min intestinal ischemia (as in group II); group VI, AP and repeated short episodes of intestinal ischemia (as in group III). Eight IVM recordings were taken during the experiment: baseline; 15 min after induction of AP; during intestinal ischemia; and

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at 10, 30, 60, 90, and 120 min respectively during the intestinal reperfusion phase. In the second experiment rats were randomly allocated to 4 groups (VII to X, n = 8). Group VII, had an intravenous infusion of ÔnormalÕ lymph; group VIII, an intravenous infusion of ÔIIR conditionedÕ lymph; group IX, AP with an intravenous infusion of normal lymph (as in group VII); group X, AP with an intravenous infusion of IIR conditioned lymph (as in group VIII). Five IVM recordings were taken: baseline; 15 min after induction of pancreatitis; and at 10, 30, and 60 min following lymph infusion. RESULTS All animals survived experiments 1 and 2. The MAP remained at baseline levels throughout the course of both experiments except during the initial phase of intestinal ischemia (Tables I and II). The induction of intestinal ischemia by occlusion of the SMA caused an 11–16% increase from baseline MAP (groups II, III, V, and VI) that resolved by the time of the next microcirculatory measurement. Individually tailored intravenous fluid treatment (1 mL/h normal saline and 0.2–0.5 mL boluses of gelofusine) resulted in no difference in the MAP of the groups with AP (V and VI) and without AP (II and III) that underwent SMA occlusion (Tables I and II). Microcirculatory analysis. As shown in Figure 1 A and B, the baseline recordings of the pancreatic exocrine microcirculation revealed uniform capillary perfusion in all groups. The induction of intestinal ischemia alone (single or multiple episodes) did not affect the capillary perfusion (groups II and III). Induction of AP alone (group IV) caused a moderate reduction in EV and the FCD that remained stable for the duration of the experiment (approximately 73% and 72% of baseline for EV and FCD respectively). However, the addition of an IIR insult to the acute pancreatitis (groups V and VI) caused a marked further deterioration in the pancreatic exocrine microcirculation once intestinal perfusion had been re-established. This continued to deteriorate until the end of the experiment when the effect was maximal (42% and 43% of baseline for EV and FCD respectively). At no stage during the experiment was there a significant difference in the EV or FCD between groups with single or multiple episodes of IIR (Groups II, III, V, and VI). The intravenous infusion of normal mesenteric lymph into animals, with or without AP (groups VII and IX) had no effect on the pancreatic microcirculation (Figure 1, D and E). Infusion of

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Table I. Mean arterial pressure taken during each of the intravital microscopy measurements for experiment 1 (acute pancreatitis with or without the addition of intestinal ischaemia – reperfusion) Intestinal reperfusion Group I II III IV V VI

Control 30min IIR 3x10min IIR AP AP+30min IIR AP+3x10min IIR Pz

Base 106 ± 107 ± 106 ± 110 ± 107 ± 106 ± NS

3 4 6 9 3 4

Post TC

Int Isch

106 ± 106 ± 107 ± 109 ± 106 ± 109 ± NS

108 ± 8 122 ± 9 123 ± 5 105 ± 8 119 ± 6 119 ± 3 < 0.01

9 4 5 8 3 9

10min 106 ± 105 ± 106 ± 106 ± 107 ± 105 ± NS

30min

4 3 7 6 4 7

104 ± 107 ± 106 ± 107 ± 105 ± 105 ± NS

60min

6 3 6 6 4 4

90min

103 ± 7 106 ± 5 103 ± 4 104 ± 8 103 ± 9 105 ±3 NS

106 ± 103 ± 105 ± 106 ± 106 ± 103 ± NS

5 4 6 3 7 7

120min 106 ± 101 ± 103 ± 104 ± 104 ± 104 ± NS

4 8 8 3 5 9

Py NS < 0.01 < 0.01 NS < 0.01 < 0.01

Base = baseline, Post TC = 15min after taurocholate infusion, Int Isch = intestinal ischaemia, AP = acute pancreatitis, IIR = intestinal ischaemia– reperfusion, N = no pancreatitis. Values are mean ± SEM. yOne way repeated measures ANOVA. zOne way ANOVA.

Table II. Mean arterial pressure taken during each of the intravital microscopy measurements for experiment 2 (acute pancreatitis with or without the infusion of intestinal ischemia–reperfusion conditioned mesenteric lymph) Post lymph infusion Group VII VIII IX X

Base N+NL N+IL AP+NL AP+IL Py

109 ± 109 ± 111 ± 110 ± NS

Post TC 3 6 4 5

109 ± 106 ± 108 ± 108 ± NS

5 5 10 4

10min 108 ± 109 ± 112 ± 108 ± NS

9 3 8 5

30min 105 ± 107 ± 111 ± 109 ± NS

7 4 7 7

60min 109 ± 106 ± 107 ± 105 ± NS

8 5 7 4

P* NS NS NS NS

Base = baseline, Post TC = 15min after taurocholate infusion, N = no pancreatitis, NL = normal mesenteric lymph, IL = IIR conditioned mesenteric lymph. Values are mean ± SEM. *One way repeated measures ANOVA. yOne way ANOVA.

IIR conditioned mesenteric lymph into normal animals (group VIII) caused a significant reduction in the EV measured at 10 min following infusion. The EV recovered to normal values at 60 min following infusion. These changes were not matched with the FCD, as there was no significant alteration following the infusion of IIR conditioned lymph. The infusion of IIR conditioned lymph into animals with AP (group X) caused an immediate and continuing deterioration in both the EV and FCD (33% and 27% of baseline for EV and FCD respectively). Leukocyte adherence. As shown in Figure 1, C, the induction of AP alone (group IV) caused an immediate and sustained increase in LA (approximately 4-fold increase from baseline LA). IIR alone (groups II and III) also caused a rise in LA albeit more gradual (5-fold increase from baseline LA). The addition of IIR to AP (groups V and VI) caused a combined effect of a 7-fold increase in the LA. This effect was replicated by an infusion of IIR conditioned lymph (group VIII) but not

normal lymph (group VII) as shown in Figure 1, F. Infusion of IIR conditioned lymph into animals with AP (group X) caused a significant increase in the LA (6.5-fold increase from baseline LA) that was greater than AP with normal lymph (group IX, 4-fold increase) or IIR lymph alone (group VIII, 5-fold increase). Pancreas histopathology. As shown in Table III, sodium taurocholate induced AP was confirmed by a significant increase in the pancreatitis severity score of group IV (AP alone) from group I (control) by 4-fold. The addition of IIR further exacerbated the severity of pancreatitis (by 1.8fold group IV vs group V) but there was no significant difference between the two different types of intestinal ischemia. The infusion of IIR conditioned lymph into animals with established AP (group X) significantly exacerbated the severity of pancreatitis (by 1.2-fold) a change comparable to the effect observed from IIR (group V) but the infusion of normal mesenteric lymph (group IX) had no effect.

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Fig 1. Pancreatic microcirculation in a sodium taurocholate (TC) model of acute pancreatitis with the addition of either intestinal ischaemia – reperfusion (IIR) (A, B, C) or infusion of IIR-conditioned mesenteric lymph (D, E, F). Measurement endpoints were erythrocyte velocity (EV) (A, D), functional capillary density (FCD) (B, E), and leukocyte adherence (C, F). Baseline = baseline recordings, post TC = 15 min after induction of pancreatitis, Int Isch = during intestinal ischaemia, Intestinal reperfusion measurements were from onset of reperfusion, Post lymph infusion = were from time of starting infusion of IIR-conditioned or normal lymph. AP = acute pancreatitis. 30 min-IIR = 30 min of continuous intestinal ischaemia. 3 3 10 min IIR = 3 episodes of 10 min intestinal ischaemia separated with 10 min of intestinal reperfusion. N = no pancreatitis. NL = normal lymph. IL = IIR-conditioned lymph. Group numbers are denoted as Roman numerals within parentheses. Values are means ± SEM. See text for statistical analysis methodology. P < .05: a; groups I, II, III vs IV, V, VI. b; IV vs V, VI. c; I vs II, III. d; VII, VIII vs IX, X. e; IX vs X. f; VII vs VIII. g; VII vs VIII, IX, X. h; VIII, IX vs X. i; VIII vs IX.

Edema index. As shown in Figure 2, A and B, the changes to the level of pancreatic edema were similar in distribution to that of the final histologic severity scores (Fig 2). The combined induction of AP and IIR (groups V and VI) caused a significant increase in pancreatic edema (1.4fold) when compared to AP alone (group IV). Likewise, the infusion of IIR conditioned lymph

into animals with AP (group X) caused a 1.8-fold increase in pancreatic edema when compared to AP and normal lymph (group IX). DISCUSSION These studies have shown that the microvascular perfusion and histologic severity of established AP can be exacerbated by a concomitant early IIR

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Table III. Pancreatitis combined head and tail histopathology severity scores for pancreatic necrosis, edema, parenchymal hemorrhage (Hem), fat necrosis, and inflammation (Inflam) Group

Necrosis

I II III IV V VI

Control 30min IIR 3x10min IIR AP AP+30min IIR AP+3x10min IIR

0 0.1 ± 0 4.9 ± 6.7 ± 6.9 ±

VII VIII IX X

N+NL N+IL AP+NL AP+IL

Edema

Hem

Fat necrosis

1.0 0.4 1.0

2.4 3.0 3.6 5.4 6.2 6.5

± ± ± ± ± ±

1.0 0.1 0.3 0.8 0.3 0.7

0 0 0 0.1 ± 0.1 0.1 ± 0.1 0.1 ± 0.1

0.1 ± 0.1 ± 0 1.6 ± 4.6 ± 4.5 ±

0 0 5.3 ± 1.0 6.9 ± 1.0

1.8 2.4 5.8 6.9

± ± ± ±

0.6 0.7 0.4 0.9

0 0 0.1 ± 0.1 0.1 ± 0.1

0.1

0.1 0.1

Inflam

Total Score

0.8 0.9 1.0

0.1 0.2 0.1 0.5 1.2 1.3

± ± ± ± ± ±

0.1 0.2 0.1 0.3 0.5 0.6

2.2 3.1 3.6 8.4 15.6 15.9

± ± ± ± ± ±

0.8 0.9 1.0 0.3a 0.5a,b 0.5a,b

0 0 4.4 ± 1.0 4.8 ± 1.1

0.1 0.2 1.0 1.7

± ± ± ±

0.1 0.1 0.1 0.7

1.6 ± 2.5± 14.2 ± 19.8 ±

0.7 1.5c 0.7c,d 1.2c,d

Total scores are expressed as mean ± SEM and group identities as per Fig 1. P < .05: a; groups I, II, III vs IV, V, VI. b; IV vs V, VI. c; VII, VIII vs IX, X. d; IX vs X. (Proc CATMOD).

injury. These effects could be replicated by peripheral intravenous administration of conditioned mesenteric lymph from donor rats that had undergone a comparable isolated IIR insult. It appears that the intestine plays an important emerging role in the pathogenesis of severe AP18,19 and other severe illness.20 Experimental studies have shown that AP causes intestinal ischemia21 and a loss of intestinal barrier integrity.9 These findings have been correlated in clinical studies where increased permeability of the intestinal mucosa occurs early and is related to the severity of the disease.10 Furthermore, the extent of intestinal intramucosal ischemia (as measured indirectly by nasogastric tonometry) is proportional to the severity of AP and can predict death.8 Whether these perfusion changes are a consequence of or a contributor to severe AP is unclear22 but these studies led us to hypothesize that intestinal ischemia during AP causes a reduction in pancreatic perfusion22 and increases the likelihood of developing pancreatic necrosis. Progressive and measurable impairment of pancreatic microcirculation with AP23 correlates with the severity of the disease and the degree of microcirculatory impairment.24 Previous studies with laser Doppler flowmetry show that pancreatic perfusion is particularly susceptible to sustained attenuation by an IIR injury.25 In this study we extend this understanding of how an ischemic insulted intestine and pancreas interact by using IVM to quantify the attenuation of the pancreatic perfusion (EV and FCD) as well as increase in LA. Both observations are key components of severe AP pathogenesis.26 It was of note that these microcirculatory changes, and the concomitant exacerbation in histologic severity, occurred without any significant or sustained systemic hypotension.

Unlike other related studies,27 this was not a global shock model but investigated an isolated intestinal injury. What was more surprising was that even a relatively benign series of intestinal insults (short ischemia/reperfusion periods) resulted in the same dramatic changes in the pancreatic microcirculation. This adds further weight to the accumulating evidence that the intestine may have an important role in the pathogenesis of severe acute pancreatitis.18 These observations led us to hypothesize that factor(s) derived from the intestine might be responsible for the observed effects and might be mediated by mesenteric lymph. In the current study the systemic administration of IIR conditioned lymphatic fluid was found to cause impairment of pancreatic perfusion and increased LA and to exacerbate the severity of AP, and to a similar extent to that seen when IIR occurred in the same rats pre-existing mild acute pancreatitis. Leukocyte activation is a well-recognized feature of both AP and IIR yet its role in the pathophysiology of AP remains to be fully elucidated. The severity of experimental AP can be ameliorated (but not prevented) by neutrophil depletion and anti-ICAM antibodies, and the combined effect of these two interventions is no greater than either alone.28 In the present study there were significant differences in LA within the pancreatic bed between normal lymph and IIR conditioned mesenteric lymph independent of the presence of AP. Even in the normal pancreas there was significant leucocyte adherence with the administration of IIR conditioned lymph, and in the absence of pancreatic microcirculation changes. This would be consistent with an increased expression of endothelial ICAM associated with leukocyte activation29 and due to factor(s) present within the mesenteric lymph originating

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Fig 2. Pancreatic oedema index in a sodium taurocholate (TC) model of rodent acute pancreatitis with the addition of either intestinal ischaemia – reperfusion (IIR) (A) or infusion of IIR-conditioned mesenteric lymph (B). Values are means ± SEM and group identities as per Figure 1. P < .05: a; groups I, II, III vs IV, V, VI. b; IV vs V, VI. c; VII, VIII vs IX, X. d; IX vs X.

from the injured intestine. This suggests that circulating conditioned mesenteric lymph is able to directly prime leukocytes without the need for them to pass through the injured intestine. Conversely acute pancreatitis alone can cause some LA when exposed to normal mesenteric lymph, presumably because the gland inflammation can itself upregulate endothelial binding proteins.28 Thus from our study we observe that LA in the pancreatic microcirculation can be induced by the local inflammation and by IIR conditioned mesenteric lymph and that together there is an even greater stimulus to leucoctye adherence. The proposition that AP severity might be exacerbated by lymphatic factors has been considered before and resulted in largely unsuccessful clinical attempts to treat AP by diverting thoracic duct lymph.30,31 More recently the focus has been in other critical illnesses where intestine derived lymph have been implicated as a source of factors involved in distant organ injury in the disease.32 It is of note that diversion of the mesenteric lymph in rat models of burns33 and hemorrhagic shock12 prevented distant organ damage and subsequent work has shown that mesenteric lymph collected following hemorrhagic shock is toxic to endothelial cells,34 up-regulates adhesion molecules,35 primes circulating neutrophils,36 and causes erythrocyte dysfunction.37 In our current study we refine the experimental injury to the isolated intestine and avoid the confounding effect of global

hypotension on non-intestine organs as occurs in shock models. In addition, this study examines the effect of IIR conditioned mesenteric lymph on the pancreas rather than on more traditional distant organ targets, such as the lung. The peripheral venous administration of donor lymph was done to mimic the normal lymphatic circulation where lymph enters the circulation in the subclavian vein. However, it is recognized that other routes of lymph uptake, such as direct peritoneal absorption, may be involved when a significant inflammatory process disrupts local lymphatic vessels or alters lymphatic flow due to valvular incompetence, and when direct peritoneal absorption occurs. An alternative route of lymph uptake may explain the lack of therapeutic success with thoracic duct ligation.30,38 It was not the aim of the current studies to identify the active factor(s) in mesenteric lymph responsible for the observed effects. However, our preliminary unpublished data suggests the effects are not due to bacteria, because the conditioned lymph was sterile. Furthermore the effects are not due to the common pro-inflammatory cytokines. There is a study investigating the effect of lymph after prolonged hypotension that has suggested that the lipid fraction of the lymph may be responsible by activating neutrophils, delaying neutrophil apoptosis, and priming of NADPH oxidase.39 It has been shown that the toxic effect of this post-shock lymph on endothelial cells varies

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over time and that the active factor(s) responsible is larger than 100kD.34,40 Subsequent work has identified phospholipase A2-derived lipids as capable of priming neutrophils41 and this is in keeping with earlier studies that found that inhibition of phospholipase A2 protected the lungs following IIR.42 Further studies will be needed to determine whether the active factors in conditioned mesenteric lymph are the same with an isolated intestinal model (current study) as that found with a global shock model. A number of limitations of the studies need to be discussed. The taurocholate model was used in these studies because it is widely reported in the literature and because it produces a dose dependent range of severity.13 We selected a dose to produce acute pancreatitis of moderate severity so that it could evolve in either direction; resolution or exacerbation. Although it might have been interesting to examine histology at later time points, it proved impractical because of the prolonged anesthetic times. Great care was taken to ensure that systemic hypotension did not develop and thus confound the explanation for the reduced pancreatic perfusion observed by the IVM. The MAP was carefully maintained throughout the experiments with the judicious use of intravenous fluids and constant monitoring of hemodynamic variables. The performance of intra-vital microscopy can itself cause some microcirculatory disturbance due to the reaction of fluorochromes (leukocyte activation) and warming of tissue with prolonged light illumination (vasoconstriction).43 To limit these effects we used multiple measurement sites across the pancreas, a low dose of cell-bound rather than tissuebound fluorochrome, and modest illumination times.43 Taurocholate induced pancreatitis produces a heterogenous pattern of disease which necessitated measuring the microcirculation at multiple and standardized sites across the pancreas. As high dose oxygen, hypercapnia, and inhalational anesthesia can affect the microcirculation44,45 we elected to use infusional propofol anesthesia and tight control of FiO2 (at 40%) and EtCO2 with continuous monitoring. Both forms of mild intestinal ischemia (30 minutes and 3 3 10 minutes) used in this study were selected to mimic what was considered to be common in patients with acute severe pancreatitis, where transient, partial, and intermittent ischemic insults are likely. This is due to a combination of third space fluid loss, hypotension and reflex splanchic vasconstriction, the effects of resuscitation fluids and the widespread use of non-selective

Surgery March 2008 inotropes.8 Both forms of intestinal ischemia produced the same effects on pancreatic perfusion (Figure 1). This suggests that only a relatively minor ischemic insult is required to condition the mesenteric lymph and exacerbate pancreatitis severity. In conclusion, this study has shown that the severity of AP can be exacerbated by IIR and that this effect can be reproduced by systemic delivery of IIR conditioned donor mesenteric lymph. The identification of the active factor(s) in the conditioned mesenteric lymph is now a research priority as the role of the intestine in the pathophysiology of severe AP is becoming better understood.18 Once the active factor(s) are identified, then the manipulation of mesenteric lymph composition may become an another therapeutic strategy in the treatment of acute pancreatitis. Until such time the intestine must be protected by reducing the effects of splanchnic vasoconstrition by effective goal-directed resuscitation46 and of intestinal barrier failure by the nutritional support of enterocytes.47 We thank Vernon Tintinger for his administrative and logistical help with this study. REFERENCES 1. McKay C, Evans S, Sinclair M, et al. High early mortality rate from acute pancreatitis in Scotland. Br J Surg 1999; 86:1302-5. 2. Ashley S, Perez A, Pierce E, et al. Necrotizing pancreatitis: contemporary analysis of 99 consecutive cases. Ann Surg 2001;234:572-9. 3. Flint R, Windsor J, Bonham M. Trends in the intensive care management of severe acute pancreatitis: interventions and outcomes. Aust N Z J Surg 2004;74:335-42. 4. Marshall J, Christou N, Meakins J. The gastrointestinal tract: the ‘‘undrained abscess’’ of multiple organ failure. Ann Surg 1993;218:111-9. 5. Wolochow H, Hildebrand G, Lamanna C. Translocation of microorganisms across the intestinal wall in rats: effect of microbial size and concentration. J Infect Dis 1966;116:523-8. 6. Beger H, Bittner R, Block S, Buchler M. Bacterial contamination of pancreatic necrosis: a prospective clinical study. Gastroenterology 1986;91:433-8. 7. Wang X, Sun Z, Borjesson A, Andersson R. Inhibition of platelet-activating factor, intercellular adhesion molecule 1 and platelet endothelial cell adhesion molecule 1 reduces experimental pancreatitis-associated gut endothelial barrier dysfunction. Br J Surg 1999;86:411-6. 8. Bonham M, Abu-Zidan F, Simovic M, Windsor J. Gastric intramucosal pH predicts death in severe acute pancreatitis. B J Surg 1997;84:1670-4. 9. Ryan C, Schmidt J, Lewandrowski K, et al. Gut macromolecular permeability in pancreatitis correlates with severity of disease in rats. Gastroenterology 1993;104:890-5. 10. Ammori B, Leeder P, King R, et al. Early increase in intestinal permeability in patients with severe acute pancreatitis: correlation with endotoxaemia, organ failure, and mortality. J Gastrointest Surg 1999;3:252-62.

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11. Juvonen P, Alhava E, Takala J. Gut permeability in patients with acute pancreatitis. Scand J Gastroenterol 2000;35: 1314-8. 12. Magnotti L, Upperman J, Xu D, et al. Gut-derived mesenteric lymph but not portal blood increases endothelial cell permeability and promotes lung injury after hemorrhagic shock. Ann Surg 1998;228:518-27. 13. Aho H, Nevalainen T, Aho A. Experimental pancreatitis in the rat. Development of pancreatic necrosis, ischaemia and edema after intraductal sodium taurocholate injection. Eur Surg Res 1983;15:28-36. 14. Beuk R, Heineman E, Tangelder G, et al. Effects of different duration of total warm ischemia of the gut on rat mesenteric microcirculation. J Surg Res 1997;73:14-23. 15. Butcher E, Weissman I. Direct fluorescent labeling of cells with fluorescein or rhodamine isothiocyanate. I: Technical aspects. J Immunol Meth 1980;37:97-108. 16. Massberg S, Enders G, Leiderer R, et al. Platelet-endothelial cell interactions during ischaemia/reperfusion: the role of P-selectin. Blood 1998;92:507-15. 17. Schmidt J, Rattner D, Lewandrowski K, et al. A better model of acute pancreatitis for evaluating therapy. Ann Surg 1992; 215:44-56. 18. Flint R, Windsor J. The role of the intestine in the pathophysiology and management of acute pancreatitis. HPB 2003;5:1-17. 19. Foitzik T. The enteral factor in pancreatic infection. Pancreatology 2001;1:217-23. 20. Carrico C, Meakins J, Marshall J, et al. Multiple-organ-failure syndrome. The gastrointestinal tract: the ‘‘motor’’ of MOF. Arch Surg 1986;121:196-208. 21. Juvonen P, Tenhunen J, Heino A, et al. Splanchnic tissue perfusion in acute experimental pancreatitis. Scand J Gastroenterol 1999;34:308-14. 22. Farrant G, Abu-Zidan F, Liu X, et al. The impact of intestinal ischaemia-reperfusion on caerulein-induced oedematous experimental pancreatitis. Eur Surg Res 2003;35: 395-400. 23. Kusterer K, Enghofer M, Zendler S, et al. Microcirculatory changes in sodium taurocholate-induced pancreatitis in rats. Am J Physiol 1991;260(2 Pt 1):G346-51. 24. Schmidt J, Ebeling D, Ryschich E, et al. Pancreatic capillary blood flow in an improved model of necrotizing pancreatitis in the rat. J Surg Res 2002;106:335-41. 25. Phillips A, Farrant G, Abu-Zidan F, et al. A method using laser Doppler flowmetry to study intestinal and pancreatic perfusion during acute intestinal ischaemic injury in rats with pancreatitis. Eur Surg Res 2001;35:361-9. 26. Menger M, Plusczyk T, Vollmar B. Microcirculatory derangements in acute pancreatitis. J Hepatobiliary Pancreat Surg 2001;8:187-94. 27. Kyogoku T, Manabe T, Tobe T. Role of ischemia in acute pancreatitis. Hemorrhagic shock converts edematous pancreatitis to hemorrhagic pancreatitis in rats. Dig Dis Sci 1992;37:1409-17. 28. Frossard J, Saluja A, Bhagat L, et al. The role of intercellular adhesion molecule 1 and neutrophils in acute pancreatitis and pancreatitis-associated lung injury. Gastroenterology 1999;116:694-701. 29. Wautier J, Setiadi H, Vilette D, et al. Leukocyte adhesion to endothelial cells. Biorheology 1990;27:425-32.

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30. Stone H, Fabian T, Morris E. Failure of thoracic duct drainage to ameliorate life-threatening physiologic derangements of acute alcoholic pancreatitis. South Med J 1983;76:613-4. 31. Dugernier T, Reynaert M, Deby-Dupont G, et al. Prospective evaluation of thoracic-duct drainage in the treatment of respiratory failure complicating severe acute pancreatitis. Intensive Care Med 1989;15:372-8. 32. Deitch E. Role of the gut lymphatic system in multiple organ failure. Crit Care Med 2001;7:92-8. 33. Magnotti L, Xu D, Lu Q, Deitch E. Gut-derived mesenteric lymph: a link between burn and lung injury. Arch Surg 1999;134:1333-40. 34. Adams C, Xu D, Lu Q, Deitch E. Factors larger than 100kD in post-hemorrhagic shock mesenteric lymph are toxic for endothelial cells. Surgery 2001;129:351-63. 35. Adams C, Sambol J, Xu D, et al. Hemorrhagic shock induced up-regulation of P-selectin expression is mediated by factors in mesenteric lymph and blunted by mesenteric lymph duct interruption. J Trauma 2000;51:625-31. 36. Zallen G, Moore E, Johnson J, et al. Circulating postinjury neutrophils are primed for the release of proinflammatory cytokines. J Trauma 1999;46:42-8. 37. Zaets S, Berezina T, Caruso J, et al. Mesenteric lymph duct ligation prevents shock-induced RBC deformability and shape changes. J Surg Res 2003;109:51-6. 38. Montravers P, Chollet-Martin S, Marmuse J, et al. Lymphatic release of cytokines during acute lung injury complicating severe pancreatitis. Am J Respir Crit Care Med 1995; 152(5 Pt 1):1527-33. 39. Gonzalez R, Moore E, Biffl W, et al. The lipid fraction of post-hemorrhagic shock mesenteric lymph inhibits neutrophil apoptosis and enhances cytotoxic potential. Shock 2001;14:404-8. 40. Deitch E, Adams C, Lu Q, Xu D. A time course study of the protective effect of mesenteric lymph duct ligation on hemorrhagic shock-induced pulmonary injury and the toxic effects of lymph from shocked rats on endothelial cell monolayer permeability. Surgery 2001;129:39-47. 41. Gonzalez R, Moore E, Ciesla D, et al. Phospholipase A(2)derived neutral lipids from posthemorrhagic shock mesenteric lymph prime the neutrophil oxidative burst. Surgery 2001;130:198-203. 42. Koike K, Moore E, Moore F, et al. Phospholipase A2 inhibition decouples lung injury from gut ischemia-reperfusion. Surgery 1992;112:173-80. 43. Steinbauer M, Harris A, Abels C, Messmer K. Characterization and prevention of phototoxic effects in intravital fluorescence microscopy in the hamster dorsal skinfold model. Langenbecks Arch Surg 2000;385:290-8. 44. Janssen G, Tangelder G, oude Egbrink M, Reneman R. Different effects of anaesthetics on spontaneous leucocyte rolling in rat skin. Int J Microcirc Clin Exp 1997;17:305-13. 45. Hudetz A, Biswal B, Feher G, Kampine J. Effects of hypoxia and hypercapnia on capillary flow velocity in the rat cerebral cortex. Microvasc Res 1997;54:35-42. 46. Chiara O, Pelosi P, Segala M, et al. Mesenteric and renal oxygen transport during hemorrhage and reperfusion: evaluation of optimal goals for resuscitation. J Trauma 2001;51:356-62. 47. Marik P, Zaloga G. Meta-analysis of parenteral nutrition versus enteral nutrition in patients with acute pancreatitis. Br Med J 2004;328:1407-12.