Articles in PresS. Am J Physiol Gastrointest Liver Physiol (December 23, 2003).10.1152/ajpgi.00407.2003
Soluble Complement Receptor 1 (sCR1) Preserves Endothelial Barrier Function and Microcirculation in Postischemic Pancreatitis in the Rat
E. von Dobschuetz1, O. Bleiziffer2, S. Pahernik2,3, M. Dellian4, T. Hoffmann5, K. Messmer2 1
Department of General- and Visceral- Surgery,
Albert-Ludwigs-University, Freiburg, Germany 2
Institute for Surgical Research,
Ludwig-Maximilians-University, Munich, Germany 3
Department of Urology, University of Mainz, Mainz, Germany 4
Department of Otorhinolaryngology,
Ludwig-Maximilians-University, Munich, Germany 5
Maria-Theresia Klinik, Munich, Germany
Short title: sCR1 and postischemic pancreatitis Keywords:
permeability, ischemia, reperfusion, acute pancreatitis, transplantation
Corresponding Author:
Ernst von Dobschuetz Department of General- and Visceral-Surgery Albert-Ludwigs-University Hugstetter Str. 55 79106 Freiburg Germany
Fax: +497661/988768 e-mail:
[email protected] Phone: +497661/988725
Copyright (c) 2003 by the American Physiological Society.
Abstract:
Components of the activated complement cascade are considered to play a pivotal role in ischemia reperfusion induced organ injury. Using intravital epifluorescence microscopy we investigated the effect of complement inhibition by the recombinant soluble complement receptor 1 (sCR1, TP10) on the effect of macromolecular microvascular permeability, functional capillary perfusion and leukocyte endothelium interaction in postischemic pancreatitis. Anaesthetised Sprague-Dawley rats were subjected to 60 min of normothermic pancreatic ischemia induced by microclipping of the blood supplying arteries of the organ. Rats who received intravenous sCR1 (15 mg/kg b.w.; n = 7) during reperfusion showed a significant reduction of permeability (1.77 ± 1.34 *10-8 cm/s.; n = 7) of TRITC-Rhodamin labelled albumin injected 90 min after the onset of reperfusion as compared to vehicle treated animals (6.95 ± 1.56 *10-8 cm/s) (n= 7). 120 min after the onset of reperfusion the length of red blood cell perfused capillaries (FCD) was significantly improved (from 279 ± 15.7 cm-1to 330 ± 3.7 cm-1.; n = 7) and the number of leukocytes adherent to postcapillary venules was significantly reduced (from 314 ± 87 mm-2 to 163 ± 71 mm-2.; n = 7) by sCR1 as compared to vehicle treatment. Complement inhibition by sCR1 ameliorates effectively pancreatic ischemiareperfusion induced microcirculatory disturbances and might be considered for treatment of postischemic pancreatitis.
2
Introduction: Ischemia-reperfusion is a causative factor in the pathogenesis of acute pancreatitis after mesenteric artery embolisation (20), cardiac bypass operation (5) and the early inflammatory response after pancreas transplantation (21). The stimulus of ischemiareperfusion induces generation of oxidative stress and following apoptosis of endothelial cells which are shown to activate the complement cascade by the classical (12) and Lectin (4) mediated pathway. Furthermore ischemia reperfusion in pancreas is considered to activate and release trypsin which is a well known activator of the complement cascade (5). Microcirculatory pancreatic damage showing impairment of functional capillary perfusion (7), activation and adhesion of leukocytes in postcapillary venules (7) and increase of endothelial macromolecular permeability (3) are the crucial hallmarks of early reperfusion. Recombinant soluble complement receptor 1 (TP10, sCR1) has proven its therapeutic usefulness in ischemia-reperfusion injury of myocardium (31), skeletal muscle (17) and small intestine (32) through blocking of the complement cascade during the early onset of reperfusion. Furthermore sCR1 ameliorated graft function after experimental kidney (18), lung (15) and liver transplantation (10). Due all these studies reduction of microcirculatory disturbances by inhibition of leukocyte adherence and preservation of the endothelial barrier function was considered as the decisive effect of sCR1 in reperfusion injury. Although graft pancreatitis is one of the major complications of pancreas transplantation up to now there is no reliable prevention of this early postischemic microcirculatory disturbance. The aim of this study was to investigate the effect of sCR1 on the early microcirculatory deterioration during ischemia-reperfusion-induced pancreatitis using a novel method of
3
measuring macromolecular permeability combined with analysed leukocyte adherence and functional capillary perfusion by means of intravital microscopic technique.
4
Materials and Methods: Anaesthesia and Monitoring The experimental protocol of this study was approved by the local ethical governmental committee and conforms the Guiding principles in the Care and Use of Animals as approved by the Council of the American Physiologic Society. Male Sprague-Dawley rats (Charles River, Sulzfeld, Germany) weighing 180-260g were anaesthetised with ether and pentobarbital (50mg/kg body weight intraperitoneally) after an overnight fast with free access to tap water. After tracheotomy, respiration was volume controlled (frequency: 57-65 breaths/min; tidal volume: 2-2.5ml; FIO2: 0.25-0.40, Harvard Rodent Ventilator 683, Harvard Apparatus, South Natick, USA). The right carotid artery and the right jugular vein were canulated with a polyethylene catheter (PE-50, 0.58 mm ID, Portex, Hythe, Kent, UK) for continuous monitoring of mean arterial pressure (MAP) and heart rate. Rectal temperature was kept between 36.5°C and 37.5°C by means of a heating pad (Fa. Effenberger, Pfaffing, Germany). Adequate anaesthesia was maintained by intravenous infusion of pentobarbital (12mg /kg body wt/hr) and N2O admixture (0.65-0.75) to the inspired air. Arterial blood gases, acid/base status and blood lactate concentration were measured intermittently by a blood gas analyser (Ciba Corning 860, Chiron diagnostics GmbH, Fernwald, Germany) and were adjusted to the following values for baseline conditions by means of ventilator adjustment and i. v. injection of Na-bicarbonate 8.4%: pO2 =100-120 mmHg, pCO2=30-40 mmHg, ph=7.39+ 0.02 and base excess=0+2. During the experiment, arterial pO2 was kept above 90 mmHg by adjustment of FiO2, pCO2 was kept below 45 mmHg by alteration of the breathing frequency. Hematocrit in arterial blood was measured by a Coultercounter T540 (Coulter Electronics, Hialeah, Florida, USA).
5
Animal Model and Experimental Protocol After transverse laparatomy complete ischemia of the pancreas was induced by clipping the four blood supplying arteries (left gastric artery, gastroduodenal artery, splenic artery and caudal pancreaticoduodenal artery) to the pancreas by means of microvascular clips (closing force 70g, Aesculap, Tuttlingen, Germany). The complete microsurgical technique was described previously (7). Sham operated animals underwent the surgical procedure without induction of ischemia. After a stabilisation period of 15 min animals were randomly assigned to three groups: (a) sham operated group without ischemia (sham control, n=7); (b) 1h ischemia group receiving i. v. 3ml/kg body weight vehicle (ischemia control, n=7); (c) 1h ischemia group receiving i. v. 15 mg/kg body weight sCR1 (ischemia sCR1, n=7). SCR1 was further injected into three sham operated animals (15 mg/kg body weight); they showed no significant differences as compared to animals of the sham control group (data not shown). The dose (15 mg/kg body weight) of sCR1 used has proven its efficacy in other experiments in rats (10). Arterial blood samples (1ml) were taken for assay of serum total hemolytic complement activity before induction of ischemia, at the end of the experiment and were immediately replaced by aliquots of saline. 15 min after administration of the solutions the pancreas and spleen were exteriorised on an adjustable microscope stage and covered by a thin transparent plastic membrane to prevent drying. To avoid cooling of the organs, the whole animal and the stage were covered by swabs between the microscopy measurements. Arterial pressure (MAP) was continuously determined and registered on a recorder (Siemens XT Kompensograph, Siemens, Munich, Germany). Arterial blood gases from the carotid artery and hematocrit were measured at baseline conditions and 15, 60, and 120 mins after administration of the solutions. The
6
experiments were discontinued by an intravenous injection of an overdose of pentobarbital. Drugs Lyophilized soluble complement receptor 1 (TP10) was kindly provided by Avant Immunotheraputics, Inc. (Needham, U. S. A.). The vials contained mannitol (80 mg), monobasic potassium phosphate (4.36 mg), dibasic potassium phosphate (5.76 mg) and sodium chloride (5.48 mg) as buffer salt. Using water for injection as reconstitution fluid a solution containing 5mg/ml sCR1 was prepared, aliquoted into sterile syringes and frozen at –20° Centigrade until use. Vehicle was prepared containing the same buffer salt concentration. Intravital Microscopy and Quantification of Microvascular Parameters Intravital fluorescence microscopy of the pancreas was performed using a modified Leitz-Orthoplan microscope (Leitz, Wetzlar. Germany) with a mercury lamp (100W, HBO) attached to a Ploemo-Pak illuminator with I2/3 (excitation 450-490 nm, emission greater than 515 nm, used for permeability and leukocyte-adherence) and N2 (excitation 530-560nm, emission greater than 580nm, used for functional capillary density) filterblocks
(Leitz,
Wetzlar,
Germany)
for
epi-illumination.
Microvascular
macromolecular permeability was measured by the following setup: To enable substraction of tissue autofluorescence a randomly selected capillary field (ROI) of the exocrine pancreas was acquired using a light immersion objective (L x32/0.4; Leitz, Wetzlar, Germany) and a silicon-intensified target (SIT) video camera (C2400-08, Hamamatsu, Herrsching, Germany). The picture was recorded on video tape (video recorder: AG-Panasonic, Munich, Germany) for off-line evaluation. Rhodamine labelled bovine serum albumin (40 mg/kg body weight, Molecular Probes, Leiden, The
7
Netherlands) was intravenously injected and the identical capillary field was recorded during the first 6 minutes after injection every 30s and up to 20 mins all 60s. Fluorescence illumination was reduced to 2s per timepoint to avoid photoxic effects and bleaching. Recorded pictures were digitized by an image analysis system (IBAS 2000, Kontron, Eching, Germany) and the fluorescence intensity of the capillary field was measured off-line after subtraction of tissue autofluorescence obtained before the injection by densitometrical measurement. Effective macromolecular permeability of albumin was calculated as described previously(29) using the following formula of Yuan et al. (34): P= (1-HTm) VVes/S (1/Im0 dIm/dt + 1/k) P as the vascular permeability is based on Im0 which represents the average fluorescence intensity Im of the capillary area recorded immediately after the vessels were filled with tracer molecules. In separate experiments (n=3) the time constant k of plasma clearance of Rhodamin labelled BSA was quantified through collecting arterial blood samples at t= 1, 2, 3, 4, 5, 10, 15, 20, 30, 45 and 60 mins following injection and fitting these data on an exponential function. Vessel volume Vves pro surface area S ratio in a pancreatic capillary field was analysed in three animals measuring the length of capillaries and the diameter by an CAMAs image analysing system (Dr. Zeintl, Heidelberg, Gemany) (9). Dynamic microhematocrit HTm of the pancreatic capillary field was measured in a separate experiment by the method described by Sarelius and Duling using the following formula(22): HTM = (FRBC / LF x MCV x 100) / [vRBC x Π (D/2)²] Briefly rat erythrocytes were labelled with Fluoresceinisothiocyanate (Sigma. St Louis, Missouri, USA). The erythrocytes were injected into the animal and cell flux FRBC of
8
labelled cells, the average cell velocity vRBC , and diameter of the vessel were measured in the pancreatic capillary fields. Labelled cell fraction LF of the whole erythrocyte volume was determined using a FACS analyser (Becton Dickinson, Heidelberg, Germany). MCV was calculated from a full blood cell count (Coulter Electronics, Hialeah, Florida, USA). Functional capillary density and leukocyte adherence were measured by the following set-up: 0.2 ml of 2.5% bovine albumin labelled with the fluorochrome fluorescein isothiocyanate (Sigma. St Louis, Missouri, USA) for contrast enhancement of microvessels and 0.1 ml 0.2% Rhodamin 6G (molecular weight 497; Sigma. St Louis, Missouri, USA) for in vivo staining of cytochrome c containing cells (leukocytes) was injected intravenously into the right jugular vein before the measurement time point. A saltwater immersion objective (SW X25/0.6; Leitz) allowed magnification of approximately 800x. The observations were recorded by means of a charge–coupled device (CCD) video camera (FK 6990; Cohu, Prospective Measurments, San Diego, California, USA) and stored on video tape (video recorder: AG-Panasonic, Munich, Germany) for off-line evaluation. Quantitative assessment of the microcirculation included determination of the functional capillary density (FCD), and the number of adherent leukocytes in the postcapillary venules. These parameters were measured at 120 mins after the start of reperfusion. FCD is defined as the length of red blood cell perfused capillaries (cm) per observation area (cm2) (16). The FCD was determined by analysis of the video tapes using CAMAs image analysing system (Dr. Zeintl, Heidelberg, Gemany) (9). Ten randomly selected regions of interest (400X300 µm) of the pancreas were evaluated. For quantification of leukocyte-endothelial interaction, at least three postcapillary venules (diameter
