Journal of Histochemistry and Cytochemistry, Vol. 47, 1201-1212, September 1999, Copyright © 1999, The Histochemical Society, Inc.


ARTICLE

Cerium-based Histochemical Demonstration of Oxidative Stress in Taurocholate-induced Acute Pancreatitis in Rats: A Confocal Laser Scanning Microscopic Study

Géza Teleka, Jean-Yves Scoazecb, Jacques Chariota, Robert Ducroca, Gérard Feldmannb, and Claude Rozéa
a INSERM U410, Faculté de Médecine Xavier Bichat, Université Paris 7, Paris, France
b INSERM U327, Faculté de Médecine Xavier Bichat, Université Paris 7, Paris, France

Correspondence to: Claude Rozé, INSERM U410, Faculté de Médecine Xavier Bichat, 16 Rue Henri Huchard, BP 416, 75870 Paris, France.


  Summary
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Materials and Methods
Results
Discussion
Literature Cited

Direct in vivo histological detection of oxygen-derived free radicals (OFRs) in inflammatory conditions is not fully resolved. We report an application of cerium histochemistry (in which capture of OFRs by Ce atoms results in laser-reflectant cerium-perhydroxide precipitates) combined with reflectance confocal laser scanning microscopy (CLSM) to demonstrate the evolution of oxidative stress in taurocholate-induced acute pancreatitis (AP) in rats. Animals were perfused with CeCl3 in vivo and cryostat sections of pancreata were studied by CLSM. Vascular endothelium was immunolabeled for PECAM-1. OFR production by isolated polymorphonuclear leukocytes (PMNs) incubated in vitro with CeCl3 was quantified by image analysis. In the pancreas, strong OFR-derived cerium reflectance signals were seen in acinar cells at 1–2 hr, capillaries and small venules were frequently engorged by cerium precipitates, and adherent PMNs presented weak intracellular reflectance signals. At 8–24 hr, acinar cell OFR production decreased, whereas adherent/transmigrated PMNs displayed abundant intra- and pericellular reflectance. PECAM-1 expression was unchanged. PMNs from ascites or blood showed significant (p<0.01) time-dependent OFR production, plateauing from 2 hr. The modified cerium capture/CLSM method allows the co-demonstration of in vivo oxidative stress and cellular structures labeled with fluorescent markers. In vivo oxidative stress was shown histologically for the first time in experimental AP. (J Histochem Cytochem 47:1201–1212, 1999)

Key Words: experimental acute pancreatitis, reactive oxygen species, cerium histochemistry, reflectance confocal, microscopy, polymorphonuclear leukocytes, PECAM, immunofluorescence


  Introduction
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Introduction
Materials and Methods
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Since the first report in the literature (Sanfey et al. 1984 ), a large body of evidence suggests that oxygen free radicals (OFRs) are involved in the pathogenesis of acute pancreatitis (AP) and its complications. The noxious effects of OFRs are numerous: cell membrane injury by peroxidation of polyunsaturated lipids (Levy et al. 1997 ) and proteins, DNA damage, inhibition of glycolysis (Hinshaw et al. 1990 ), and oxidative phosphorylation (Hyslop et al. 1988 ), resulting in depletion of intracellular ATP and alterations of ATP-dependent Ca2+ and Na+ pumps. Despite extensive research (reviewed by Schoenberg et al. 1994 ), the precise cellular sources of OFR production, their relative importance, and the time course of their generation remain unclear. Moreover, the different pancreatitis models and experimental procedures appear to present divergent results (Steer et al. 1991 ; Niederau et al. 1992 ; Nordback et al. 1995 ; Weber et al. 1995 ). One suggested source of OFRs is the pancreatic acinar xanthine oxido-reductase enzyme system, because under certain conditions it can be converted into the oxidase form, which generates superoxide anion and hydrogen peroxide. This transition may occur by pre- and post-translational protein modification in hypoxia (Terada et al. 1997b ) or by irreversible proteolytic cleavage by activated trypsin (Amaya et al. 1990 ; Nordback et al. 1993 ; Closa et al. 1994 ). The other potential source of OFRs is infiltration by inflammatory cells (Tsuji et al. 1994 ; Inoue et al. 1996 ; Ito et al. 1996 ), most notably the polymorphonuclear leukocytes (PMNs), which can produce large amounts of OFRs during the "respiratory burst" by NADPH oxidase and myeloperoxidase.

In vivo detection of OFRs, such as the superoxide anion, hydrogen peroxide, or hydroxyl radical, is difficult. Current methods include biochemical measurement of the byproducts of OFR-induced cell injury (e.g., conjugated dienes, malondialdehyde, protein carbonyls), electron paramagnetic resonance spectroscopy, chemiluminescence (Kishimoto et al. 1995 ; Peralta et al. 1996 ), certain fluorogenic probes (Suzuki et al. 1993 ), and administration of free radical scavengers to inhibit the oxidative stress. Most of these methods, however, provide little information about the precise site of OFR formation. To our knowledge, the production of OFRs in AP has never been demonstrated at a cellular/histological level in vivo.

To detect locally the in vivo formation of OFRs, we have used a modified method based on cerium cytochemistry (reviewed by Van Noorden and Frederiks 1993 ). Reaction of cerium ions with OFRs forms stable, insoluble cerium perhydroxide (CeIII[OH]2OOH or CeIV[OH]3OOH) precipitates. Robinson and Batten 1990 , as well as Halbhuber et al. 1996 , have demonstrated that although these transparent deposits are hardly visible using normal optical microscopy, they can be visualized by taking advantage of their laser reflective properties employing the reflectance mode of confocal laser scanning microscopy (CLSM).

In this study, acute necrotizing pancreatitis was induced in rats using the sodium taurocholate method, a model that has been extensively documented previously by others (for review see Schoenberg et al. 1994 ). OFR production was detected at different time points by in vivo intravascular infusion of CeCl3 just before sacrifice. This was followed by CLSM demonstration of cerium perhydroxide deposits in cryostat sections of pancreata. PMNs isolated from blood and ascites and treated in vitro with CeCl3 were also evaluated. We focused our attention on the evolution of oxidative stress in acinar cells, in pancreatic vascular spaces (endothelial surfaces were histologically identified by immunofluorescence for platelet endothelial cell adhesion molecule, PECAM-1), and in OFR production by PMN leukocytes in the pancreas, blood, and ascites.


  Materials and Methods
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The Sodium Taurocholate Pancreatitis Model and Cerium Chloride Perfusion
Male Wistar rats (280–320 g) were fasted for 12 hr before the experiments but had free access to water. The animals were treated in accordance with the European Community Standards concerning the care and use of laboratory animals (INSERM and Ministère de l'Agriculture et de la Forêt, France, Authorization No. 02249). The animals were anesthetized by IP injection of 50 mg/kg sodium pentobarbital (Sanofi; Libourne, France) and a small (<1.5-cm) right paramedian laparotomy was performed. After exteriorization of the pancreas, the pancreatobiliary duct (PBD) was gently dissected at the hilum of the liver and temporarily closed with a soft microvascular clamp to prevent reflux of the infused material into the liver. The distal PBD was cannulated in a transpapillary fashion through a puncture of the duodenum, and the cannula was exteriorly fixed by a temporary ligature around the PBD. This was followed by a slow, retrograde injection (100 µl/100 g body weight, pressure 40 cm H2O) of lactated Ringer solution (Fresenius; Louviers, France) containing 5% sodium taurocholate (Sigma Chemical; St Louis, MO) into the PBD. All reagents were filtered through a 0.2-µm filter (Schleicher & Schuell France SARL; Ecquevilly, France) to remove particles that might cause artifactual reflectance signals. After this, all devices were removed and the laparotomy was closed in two layers.

At different time points (1, 2, 8, or 24 hr) after induction of AP, the animals (n = 5 at each time point) underwent re-laparotomy under pentobarbital anesthesia. Samples of the ascites were collected and then the distal segment of the abdominal aorta was cannulated. Two ml of blood was removed, and then 50 ml ceriumIII chloride solution (Sigma; 20 mmol/liter, dissolved in lactated Ringer) was slowly (over 5 min) injected into the aorta. The inferior vena cava was readily punctured for the release of the perfusate–blood mixture. In such a manner the entire aortic vascular bed was "washed through" with CeCl3 solution. Samples of the pancreas, lungs, heart, liver, and kidneys were rapidly collected, immediately snap-frozen in liquid nitrogen, and stored at -80C until processing. Control animals (n = 5) underwent laparotomy and collection of peritoneal fluid and abdominal aortic blood, followed by CeCl3 perfusion and tissue sampling in the same fashion as animals with AP. In all experiments, sampling and freezing of pancreata were performed within 1 min after the end of the cerium infusion.

Demonstration of OFR Production in PMNs Isolated from Ascites and Blood
Leukocytes were harvested from the ascites and blood by the method of Robinson (Robinson and Batten 1990 ), with slight modifications. Briefly, large drops of blood or ascites fluid were immediately placed on pre-cleaned multispot microscopy slides and incubated in a moist chamber at 36C for 2 min. The resulting coagulum was removed and the slides were rinsed free of nonadherent cells. More than 90% of the adherent cells were PMNs, with a viability index of >90% using the Trypan Blue exclusion test. The cells were incubated for 10 min in a CeCl3 solution (20 mmol/liter CeCl3 in lactated Ringer solution supplemented with 5 mmol/liter glucose), and then the cytochemical reaction was stopped by methyl alcohol fixation (2 min). The nuclei were stained with propidium iodide (PI, Sigma; 50 µg/ml) and, after washing, the preparation was mounted in glycerol gelatin (Sigma) supplemented with the anti-fading agent 1,4-diazabicyclo-[2.2.2.]-octane (DABCO, Sigma; 50 mg/ml). Control preparations included PMNs without cerium treatment.

Confocal Laser Scanning Microscopy of Leukocytes
The imaging and semiquantification of leukocyte OFR production, represented by the laser reflectance signals of cerium perhydroxide deposits, was performed by CLSM (Leica TCS 4D; Leica Lasertechnik, Heidelberg, Germany) using multichannel detection. The reflectance mode (laser wavelengths 488 and 568 nm) was used to detect cerium precipitates, and images of this channel were pseudocolored in "glow" (see Figure 2, color scale 1). The PI-stained nuclei were imaged by fluorescence. However, in the PMN imaging studies this channel was pseudo-colored in green to allow better visual differentiation from the reflectance signals (Figure 2, color scale 2). The transmission channel (in gray scale) was used to show cellular outlines (Figure 2, color scale 3). For morphological studies, high-resolution (final magnification x5000, and x2500, objective x63) images were taken (9 optic sections/cell), followed by pseudo-3D object volume reconstruction (SFP-Simulated Fluorescence Process; Leica built-in software), and digital superposition to give a three-layer composite image using Adobe Photoshop 5.0 software (Adobe Systems; San Jose, CA).



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Figure 1. In vitro comparison of reflectance signals obtained from cerium perhydroxide (A) and cerium phosphate (B) compounds, prepared as detailed in Materials and Methods and examined by reflectance CLSM. Cerium–perhydroxide is considerably more reflectant than the phosphate-containing derivatives.



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Figure 2. In these images, signals of the different CLSM detection channels were pseudo-colored using the system's predefined color tables (shown at right), as detailed in Materials and Methods. The stronger signals correspond to the brighter colors on each scale. (A–J) OFR production by PMN leukocytes treated in vitro with cerium chloride and imaged live by confocal microscopy. The cells were identified as PMNs by their nuclear outlines on transmission images. (A) Native image of a normal PMN isolated from peritoneal fluid, showing negligible amounts of reflectance ("glow"). (B–E) Images (taken at 1-min intervals) of the same PMN after stimulation by PMA in presence of CeCl3. There is a sharp time-dependent increase in the reflectance of intracytoplasmic membranes, suggesting OFR production. (F–J) PMN leukocyte isolated from ascites 2 hr after AP induction. (F) Native image. (G–J) Images taken at 1-min intervals after addition of cerium chloride. Rapid appearance of cytoplasmic reflectance signals suggests OFR production by the activated PMN. (K–N,P) Cerium-treated and then fixed PMNs (nuclei color-coded in green). (K) PMNs isolated from normal peritoneal fluid, showing sparse intracellular reflectance (L). PMNs from ascites 1 hr after AP induction show strong intracellular OFR production. The strong reflectance signals are limited to the cytoplasm, as can be appreciated on the xz vertical image series. (M) Quiescent PMN isolated from normal blood; (N). Blood-derived PMNs 8 hr after AP induction. (P) PMN from 8-hr pancreatitic ascites, presenting abundant extracellular cerium–perhydroxide deposits in contact with the plasma membrane. (O) Confocal microscopy image of a cerium-perfused normal pancreas tissue section, (reflectance in glow; PECAM-positive endothelial cells in green; nuclei in red; background in gray scale). Only small foci of cerium depositions are present (arrows).

Validation of the Cerium Method
Cerium ions can react with OFRs, but also with phosphates, to produce insoluble precipitations. Therefore, we wished to examine the specificity of CLSM reflectance signals to cerium perhydroxide. Halbhuber et al. 1996 have shown that cerium perhydroxide was considerably more reflectant than the cerium–phosphate compounds [CeIV(OH)PO4, CeIV(OOH)PO4, CeIII(OH)HPO4]. We compared the reflectance signals obtained from phosphate vs perhydroxide derivatives of cerium in our particular experimental setup. To mimic the in vivo events taking place during our experimental procedure, the above-described phosphate-free, cerium-containing injection fluid was mixed either 1:1 with Dulbecco's modified Eagle's medium (DMEM; Gibco Life Technologies, Rockville, MD) representing the same phosphate content as extracellular fluid (0.906 mmol/liter NaH2PO4) or with H2O2 (1% final concentration) or with the superoxide-producing mixture of xanthine oxidase (Sigma; 20 mU/ml, pH 8.5) and hypoxanthine (Sigma; 0.1 mmol/liter). The resulting insoluble precipitates were centrifuged (2000 rpm for 2 min), washed twice with normal saline, and after a final centrifugation they were dried on microscopic slides, rehydrated, and mounted. The preparations were imaged by CLSM using our standardized reflectance setting.

Control experiments aimed to study the evolution of reflectance signals after the addition of cerium to activated leukocytes. PMNs were isolated from the blood of normal control animals, incubated in cerium-free lactated Ringer solution supplemented with glucose, and a series of images (five optical sections) were immediately recorded by CLSM. The cells were then activated with 1 µmol/liter phorbol 12-myristate 13-acetate (PMA; Sigma) and CeCl3 (20 mmol/liter final concentration) was added to the incubation fluid. The reaction was followed by CLSM, and similar image series were taken sequentially at 1-min intervals. Leukocytes freshly isolated from the blood and ascites of animals with AP were incubated without cerium, followed by addition of CeCl3 but omitting PMA stimulation. CLSM image series were recorded in an identical fashion. To test if cerium reflectance signals were indeed related to OFR production, pancreatitis-derived PMNs were incubated for 5 min with the NADPH oxidase inhibitor diphenylene-iodonium chloride (DPI; Calbiochem, La Jolla, CA) 20 µmol/liter, or the OFR scavengers superoxide dismutase (SOD, from bovine erythrocytes; Sigma, 100 U/ml) or catalase (Sigma, 1000 U/ml), or the mixture of SOD and catalase. Then CeCl3 was added for 10 min, after which the preparation was fixed, stained with PI, and mounted. Semiquantitative image analysis was performed as detailed below.

Semiquantitative Studies of Leukocyte OFR Production
For quantification purposes, 10 randomly chosen intact PMNs per slide preparation were imaged (5 optical sections/cell, final magnification x5000, objective x63) under precisely standardized acquisition parameters. The integral pixel intensities of the reflectance detection channel images were quantified for each optical section using the microscope's built-in image analysis software.

To achieve constant quantification circumstances, the slight fluctuations of laser power were counteracted by the following method. One particular normal, blood-derived, cerium-treated PMN leukocyte was designated as internal standard. The localization of this cell was marked and its reflectance was measured at half-maximal laser power (arbitrary value 100). To calibrate the laser before each further series of measurements, the control cell was revisited and analyzed. On the basis of a comparison with the original recordings, the laser power and the photomultiplier sensitivities were fine-tuned to achieve intensity values similar to those obtained from original internal standard images. The resulting quantification error of image acquisition parameters was thus minimized to less than 5%.

After this, the acquisition and measurement of PMN series were completed, and in the next phase the reflectance values were corrected in relation to the cell size. The cell surface areas were measured on each optical section using Image Tool 2.00 image analysis software (Univ. of Texas Health Science Center in San Antonio, TX; public domain), the integral reflectance signal intensity values were divided by the corresponding cell surface areas (µm2), and the average of five optical sections was calculated. Ten cells were evaluated for each blood or ascites/peritoneal fluid sample. Statistical differences were tested by analysis of variance (Statview software; SAS Institute, Cary, NC) after logarithmic transformation to minimize variance differences among groups.

Processing and Imaging of Pancreatic Tissue
For routine histology, formalin fixed and frozen sections of pancreata were stained with hematoxylin–eosin. For CLSM studies, high-precision (8 and 16 µm) cryostat sections were cut from the cerium-perfused pancreas samples on pre-cleaned, 3-aminopropyltriethoxy-silane (Sigma)-coated glass slides, then fixed for 5 min at -20C in methyl alcohol, supplemented with the protease inhibitor phenyl-methane-sulphonyl fluoride (PMSF; Sigma, 1 mmol/liter). Indirect immunofluorescence was performed to label PECAM-1 molecules on the endothelial surfaces of blood vessels. The following wash fluid was used during all staining procedures: lactated Ringer solution (Fresenius) supplemented with 1% bovine serum albumin (Sigma), protease inhibitors PMSF (0.1 mmol/liter), aprotinin (Trasylol; Bayer, Zurich, Switzerland, 200 µg/ml), filtered through a 0.2-µm single-use filter. Briefly, the sections were rehydrated with the wash fluid, then incubated with anti-PECAM-1 primary antibody (mouse monoclonal anti-rat CD 31; Serotec, Oxford, UK, 1:10 dilution) for 60 min. After repeated washing, the sections were incubated with FITC-conjugated secondary antibody, (goat anti-mouse; Jackson ImmunoResearch, West Grove, PA, 1:100 dilution) for 60 min. Nuclei were colored with 50 µg/ml PI and the preparations were mounted as above. Omission of primary antibody served as control. Observations were performed with CLSM using the parameters described above. Cerium reflectance signals were pseudo-colored in "glow," PECAM-1-associated FITC fluorescence in green, whereas nuclei were pseudo-colored in red on these images. The tissue structure background obtained by transmission mode was represented in gray scale. In each image series, nine optical sections were recorded per channel (magnification x800), the object volume reconstruction was performed using the system's built-in SFP software, and the images of each channel were digitally superposed and Gauss-filtered (if necessary) using Adobe Photoshop software to give a four-layer composite picture. No digital enhancements were made that would alter the information content of the original image files.


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Induction of Pancreatitis: Macroscopic and Routine Histological Examination of Pancreata
Control pancreata had a normal macroscopic and histological appearance. In contrast, all animals treated with sodium taurocholate developed acute necrotizing pancreatitis which showed increasing severity in a time-dependent manner and was characterized by the classical lesions, such as pancreatic hyperemia, edema, ascites, and peritoneal cytosteatonecrosis, acinar cell vacuolization, and heterogeneous necrosis with leukocyte infiltration. No mortality was observed in animals sacrificed within the 1–24-hr observation period.

Validation of the Cerium Method
As indicated in Figure 1, in comparison with perhydroxide precipitations (image series A), those containing cerium phosphate derivatives (image series B) showed negligible levels of laser reflectance using our CLSM image acquisition parameters. This was in agreement with the results of Halbhuber et al. 1996 .

Native images of normal living PMNs showed barely detectable reflectance (Figure 2A), whereas PMA-induced OFR production resulted in a time-dependent increase of reflectance signals in the presence of cerium. The changes are demonstrated in Figure 2B–2E, in which images of the same cell were taken at 1-min intervals. The corresponding integral pixel intensity values (reaching a plateau at 3–4 min) are shown in Figure 3.



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Figure 3. Quantification of reflectance signals (i.e., OFR production) in normal peritoneal fluid-derived PMN leukocyte shown in Figure 1A–1E. The reflectance was measured in the living cell by confocal microscopy, first unstimulated at time 0 (corresponding image, Figure 1A), then stimulated with PMA for 1 min, followed by the addition of cerium to the incubation fluid. Asterisks mark the time points corresponding to images shown in Figure 2A–2E.

Live PMNs from AP blood and ascites presented a rapid increase in their reflectance characteristics after addition of cerium during these dynamic online imaging experiments. A representative PMN isolated from ascites 2 hr after AP induction is shown in Figure 2F–2J (Figure 2F, no cerium; Figure 2G–2J, images of the same cell taken at 1-min intervals after addition of cerium to the incubation fluid). Figure 4 shows the changes in corresponding integral pixel intensity values. The integral intensity values measured in living PMNs showed good correlation with those obtained by the measurement series of cells fixed after cerium treatment.



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Figure 4. Quantification of reflectance signals (i.e., OFR production) in a PMN leukocyte isolated from ascites 2 hr after AP induction, as shown in Figure 1F–1J. The cell was imaged live by confocal microscopy, first without cerium at time 0 (image Figure 1F), then after addition of cerium to the incubation fluid (images Figure 1G–1J). A steep elevation of reflectance signal intensities follows the cytochemical reaction of cerium ions with OFRs produced by the activated leukocyte. Asterisks represent time points corresponding to images in Figure 2F–2J).

Similarly to live cells, fixed control PMNs prepared without cerium treatment showed virtually no reflectance signals at our standard acquisition settings. The specificity of CLSM reflectance signals for the reaction of cerium with OFRs was tested in activated PMNs using various inhibitors of oxidative stress.

Pretreatment of PMA-stimulated normal PMNs and pancreatitis-derived PMNs with the NADPH oxidase inhibitor DPI, as well as the OFR scavengers SOD, catalase, or the mixture of the two, before cerium addition resulted in a significant diminution of reflectance signals. A representative example is shown in Figure 5, in which PMNs isolated from the same animal 24 hr after AP induction were treated with the above inhibitors. The data representing the mean (±SEM) of 10 morphologically intact PMNs indicate that decreased OFR production by NADPH oxidase inhibition or enzymatic OFR scavenging correlates with the reduction of reflectance signals. DPI, SOD, or catalase treatment of pancreatitis-derived leukocytes abolished the pericellular reflectant signals and decreased the intracellular signals.



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Figure 5. Reflectance signals measured in PMN leukocytes incubated with various inhibitors of OFR formation. Cells were isolated from the blood (black columns) and ascites (white columns) of the same animal 24 hr after AP induction. The NADPH oxidase inhibitor DPI and the OFR scavengers SOD and/or catalase significantly inhibited the cerium-derived CLSM reflectance signals. (a, p<0.01 vs control; b, p<0.01 vs the other inhibitors).

OFR Production in Normal and Pancreatitis-derived Leukocytes
Normal PMNs isolated from control peritoneal fluid (Figure 2K) and blood (Figure 2M) were quiescent, presenting occasional weak and focal intracellular reflectance signals after cerium treatment. In contrast to normal PMNs from control animals, the PMNs isolated from pancreatitis showed a time-dependent increase of OFR production. The evolution of the cellular reflectance signals measured in blood- and ascites-derived PMNs (n = 50 per time point) is shown in Figure 6.



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Figure 6. Quantification of OFR production in normal and AP-derived PMNs. Confocal microscopy images were analyzed as described in Materials and Methods. In controls and at each time point of AP, 10 randomly chosen cells were imaged and analyzed per animal. Each point represents a cell. There is a steep elevation of intracellular reflectance values after 1 hr of AP, with a plateau phase after 2 hr.

PMNs sampled 1 hr after induction of AP showed reflectance signals outlining intracellular cytoplasmic membranes (Figure 2L), whereas the areas corresponding to nuclei remained free of reflectance, as can be appreciated on the vertical "xz" image series (Figure 2L, xz). This reflectance pattern was found to be more intense at 2 hr after AP induction. From a quantitative point of view, the reflectance signal values were significantly higher at 1 hr than in controls (p<0.01), with a trend towards a moderate further increase at 2 hr (Figure 7). At 8 hr, large numbers of PMNs were present on the microscopic slides and the cells showed reflectant intracellular membrane outlines (Figure 2N), often with reflectant intracellular vacuoles. An apparent release of OFRs into the incubation medium was frequently observed at this time point, resulting in surprisingly large reflectant deposits around the cell. This phenomenon was largely unidirectional, and the extracellular reflectant "splash" precipitates remained in close contact with a focal area of the cell membrane, suggesting that this precipitation was indeed the result of active OFR release by the leukocytes (Figure 2P). Because their irregularity, the extracellular reflectant deposits were not included in the semiquantitative intensity measurements. At 24 hr after the induction of AP, almost all the blood- and ascites-derived PMNs produced large amounts of OFRs (Figure 6). Macroscopically, fine white precipitates were observable in the incubation medium, and strong intra- and pericellular deposits were seen on CLSM images. However, the steep elevation of intracellular OFR seen at 1 and 2 hr after AP induction did not increase further but reached a plateau phase. The correlation between the reflectance signals measured in blood- vs ascites-derived PMNs is shown in Figure 7. OFR production in the PMNs collected from blood or from ascites in the same animal was not significantly different.



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Figure 7. Comparison of average OFR production in PMNs derived from blood and ascites in controls and at different time points in animals with AP. OFR production is represented by the reflectance image intensity values. *p<0.01 with respect to the control group. No significant differences were found in OFR production between ascites- and blood-derived PMNs at the particular time points examined during the course of AP.

Immunohistological Analysis of PECAM Expression and Reflectance CLSM Detection of OFRs in Pancreatic Tissue
PECAM-1 was found to be constitutively present on endothelial surfaces, and we could not observe any apparent changes in its expression by immunofluorescence in AP samples vs normal controls. Samples from control pancreata had a normal histological appearance, no leukocyte adherence or infiltration was seen, and the acini showed only weak focal reflectance signals (Figure 2O, arrows). Control sections of PECAM-1 immunofluorescence prepared without the primary antibody were consistently negative.

At 1 hr after AP induction, focal necrotic areas were seen, mostly on the sections prepared from the head of the pancreas, whereas the histological structure of acini was relatively well preserved in the body and tail. Strong cytoplasmic reflectance signals were detected over the acini, and occasionally intravascularly in the periacinar PECAM-1-positive capillaries (Figure 8A and Figure 8B; arrow, adherent leukocytes). Langerhans islets and pancreatic ducts were free of cerium deposits. This strongly suggests that at this early time point OFR production was mainly localized within the acinar cells. At 2 hr the acinar cerium reflectance persisted, but the small capillaries and postcapillary venules were frequently engorged with cerium perhydroxide deposits (Figure 8C and Figure 8D). In certain images, a thin layer of reflectant precipitates on PECAM-positive surfaces suggested OFR production by endothelial cells. The arterioles were free of reflectant deposits. The observed images may be consistent with a phase when the OFRs produced by the acini are released into the blood and may eventually damage distant organ systems. Similar deposits were observed at this time point in the kidney glomeruli, in the sinusoids of the liver, and in the microvasculature of the lungs (unpublished observations). Leukocytes adhering to PECAM-1-positive pancreatic capillary walls were frequently seen, but their OFR production was found to be scarce and weak (Figure 8D; arrow, adherent leukocyte). The histological picture of pancreas samples 8 and 24 hr after induction was quite similar: destruction of the pancreas increased, which was represented by a heterogeneous distribution of necrosis with strong peri- and intrapancreatic leukocyte infiltration. In general, acinar OFR production was reduced, although some of the acini (mostly in non- or perinecrotic areas) still presented strong reflectance signals. In contrast to the earlier phases, at this time point the major sources of oxidative stress appeared to be the adherent leukocytes (morphologically identified as mostly PMNs on the separate confocal optical sections). The majority of these cells and their close surrounds were bright with cerium reflectance, especially after their transmigration through the capillary walls (Figure 8E and Figure 8F; arrows point to PMNs).



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Figure 8. Confocal microscopic images of cryostat sections of cerium chloride-perfused pancreata (color coding of the detection channels was performed using predefined color tables shown at right of figure. Reflectance: "glow," PECAM-1-positive vascular endothelium; green, nuclei; red, tissue background; gray scale). (A,B) Pancreata 1 hr after AP induction. Strong cerium deposit-derived reflectance signals are apparent intracellularly in the pancreatic acinar cells, representing active OFR production. Few adherent PMNs are present in the periacinar capillaries. However, the lack of reflectance signals in these PMNs suggests low levels of activation. (C,D) Acute pancreatitis at 2 hr. The OFR production in acinar cells persists, but, with a lower intensity. The PECAM-1-positive small postcapillary venules are engorged with cerium–perhydroxide deposits, suggesting release of OFRs into the vascular space. The arterioles are free of cerium deposits. Few adherent PMNs are visible in the venules (arrow). (E,F) Acute pancreatitis at 24 hr. The reflectance signals are diminished over the acini; many adherent and transmigrating PMNs (arrows) are present in the postcapillary venules, surrounded by cerium deposits. The images suggest that at this time point the PMNs are responsible for the OFR production in acute pancreatitis.


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Discussion
Literature Cited

In recent years, new light has been shed on the pathophysiology of AP by uncovering details of the local and systemic inflammatory explosion (for reviews see Neoptolemos et al. 1998 ; Norman 1998 ), and the acinar intracellular events (for reviews see Ward et al. 1995 ; Steer 1997 ). Local and systemic oxidative stress has been studied. However, the methods used were not suitable to detect the in vivo activity of OFRs in histological detail. This might be one reason for the divergent opinions concerning the exact cellular sources of these radicals. In this study we demonstrate for the first time the evolution of oxidative stress in AP at the cellular and tissue levels by introducing a novel modification to the previously described cerium cyto/histochemistry procedures. Cerium-based methods have been extensively used in oxidase enzyme research, pioneered by the groups of Karnovsky 1994 , Van Noorden and Frederiks 1993 , and Angermuller and Fahimi 1988 , in which tissue sections are incubated in vitro with cerium in the presence of the particular enzyme's specific substrate. The cerium–perhydroxide precipitates are visualized with the help of a second coloration step using diamino-benzidine (DAB) and cobalt. Alternatively, these (electron-dense) precipitates are detectable in cerium-perfused tissues by electron microscopy (Soares et al. 1994 ; Motoyama et al. 1998 ). Using CLSM in the reflectance mode, Robinson and Batten 1990 and Halbhuber et al. 1996 have demonstrated Ce–perhydroxide deposits directly by exploiting the laser-reflecting properties of these precipitates. We have combined this idea with an intra-aortic infusion of CeCl3 in a physiological solution at the time of sacrifice and have improved the technical details of CLSM detection. This methodology suffers from the potential limitation that a small fraction of reflectance signals may be derived from cerium phosphate derivatives. It has been stated by Halbhuber et al. 1996 that the cerium–perhydroxide precipitates are far more reflectant than the phosphates. Furthermore, they have found that among the cerium phosphate compounds the perhydroxy-phosphate (reaction product of cerium-hydroxyphosphates and H2O2) was the only precipitate that showed considerable reflectance. Because in our control studies the reflectance signals obtained from phosphate derivatives were substantially weaker compared to the perhydroxide derivatives, we concluded that the increased reflectance observed in AP samples correlates with oxidative stress, without discriminating among the particular OFR molecules. We believe that with our modifications the Ce–CLSM method is a useful and sensitive adjunct to the in vivo free radical research tool repertory, because it allows the co-demonstration of several other histological structures labeled with fluorescent markers. The suitability of reflectance signal quantification has been studied before (Cornelese-ten Velde et al. 1989 ). In agreement with Halbhuber et al. 1996 , we have found that quantification of cerium deposits is possible by image analysis under well-defined circumstances and if appropriate controls are used (the laser reflectance signals are related only to the microscope parameters and are not exposed to photobleaching as are most fluorescent molecules). When the intracellular cerium reflectance signals were quantified in PMNs, DPI, SOD, and catalase significantly and reproducibly decreased the formation of reflectant precipitates around PMNs and also in the cells, confirming their inhibitory action on oxidative stress. Because SOD catalyzes the dismutation of superoxide anions to H2O2 and because H2O2 is known to produce cerium–perhydroxide, one might have expected SOD to increase the formation of cerium–perhydroxide deposits, whereas the opposite was observed. We are not aware of published data detailing the characteristics of the potential reaction of cerium exclusively with superoxide anion, but a plausible hypothesis is that in an aqueous milieu cerium may form reflectant precipitates with superoxide directly, and more rapidly, than with the less reactive H2O2. The observed effect of SOD treatment may have, in fact, been due to the inhibition of this reaction. This is supported in part by our in vitro model experiments in which xanthine oxidase + hypoxanthine produced reflectant precipitates in the presence of cerium (not shown). At basic pH, which was maintained during these experiments, this enzyme–substrate system forms mostly superoxide anions.

Catalase and SOD also decreased intracellular reflectant signals, although these large molecules are generally believed not to enter into living cells. Little is known about the membrane-permeability characteristics of activated PMNs, and active uptake of these scavengers by pinocytosis cannot be excluded. Alternatively, these enzymes may decrease the intracellular availability of the freely diffusible H2O2 by eliminating it from the extracellular space.

OFR production was found to be significantly increased in AP (blood/ascites)-derived PMNs vs control in a time-dependent manner, reaching a plateau phase 2 hr after AP induction. The high-resolution images provided information about the intracellular sites and extracellular release of OFRs, findings that are in concordance with the high-resolution electron microscopic observations by Kobayashi et al. 1998 .

We did not quantify reflectance signals in tissue sections because we believed that the spatial heterogeneity of the oxidative stress in the pancreas and its different cellular sources (acinar cells, leukocytes, and perhaps the endothelium) render a mechanistic quantification relatively meaningless. Therefore, we evaluated a large series of images in a histopathological fashion.

We have used an improved model of the classic taurocholate-induced AP (reduced operative stress, low pressure intraductal injection) and, as a result, the animals survived the crucial time period of observation without perioperative (first 24 hr) mortality. Although the mechanism of this model is complex, it is believed that the additive effects of retrograde intraductal fluid injection, the detergent properties of taurocholate and the potential presence of bacteria result in a severe inflammation comparable to that observed in human biliary pancreatitis. Classical control groups of this model include sham-operated animals with retrograde intraductal injection of saline instead of taurocholate (Closa et al. 1996 ), a procedure that does not induce the gross features of necrotizing AP. However, it does provoke pancreatic damage, characterized by significant serum amylase, lipase, and {gamma}-glutamyl transferase elevation, as well as reduced intrapancreatic glutathione content, which is an indirect sign of oxidative stress (Luthen et al. 1998 ). We have observed in our preliminary experiments that such treatment of control animals rapidly increases the intrapancreatic oxidative stress detected by the sensitive cerium method. Consequently, animals undergoing this treatment can not be considered as "normal controls." Even the nonspecific stress represented by a more than 10-min hypoxia from the start of CeCl3 injection until the freezing of the sampled pancreas activated OFR production. In addition, it is well documented that intraoperative hypoxia, excessive manipulation of the gland, and intraductal injection of X-ray contrast material during endoscopic retrograde cholangio-pancreatography can indeed provoke AP in humans, whereas in the rat pancreas experimental ischemia–reperfusion injury causes morphological damage and leukocyte adherence (Hoffmann et al. 1997 ). A detailed evaluation of particular forms of "non-pancreatitogenic" local and systemic damage in relation to histologically detectable intrapancreatic oxidative stress would indeed be interesting, but because of the complexity of the histological and CLSM techniques used, the comparison of all necessary control groups was beyond the scope of our study. Because during our preliminary experiments we found that low-stress sterile laparotomy did not cause generalized PMN activation, we decided as a reasonable compromise to use controls with single laparotomy and cerium infusion. Further work will be required for histological evaluation of the oxidative stress of mild edematous pancreatitis (cerulein model).

We have found that PECAM-1, which plays a role in the transmigration of leukocytes, was localized uniformly on the luminal plasma membrane of vascular endothelial cells (not only at cell-to-cell contact sites), supporting the observations by Scholz and Schaper 1997 . Moreover, we did not detect apparent changes in the tissue immunoexpression of PECAM-1 in AP. This is in concordance with the literature suggesting that inflammatory cytokines might alter this molecule functionally. However, its constitutive immunoexpression remains unchanged (Henninger et al. 1997 ).

In our experiments we have found that at the very early stage (1 hr) of AP it is indeed the acinar cell (most likely by means of xanthine oxidase) that is responsible for the oxidative stress. Later (2 hr), in addition to a sustained acinar OFR production, the high prevalence of intracapillary and intravenular cerium deposits suggests that the release of OFRs into the bloodstream may precipitate systematization of the oxidative stress. At a later stage (8 and 24 hr), generalized adherence, activation, and transmigration of leukocytes (mostly PMNs) occur with concomitant OFR production. Because OFRs have been shown to induce leukocyte adherence (Kusterer et al. 1993 ) and to upregulate cellular adhesion molecules, such as P-selectin (Terada et al. 1997a ) or ICAM (Baeuml et al. 1997 ), and because these molecules play a role in leukocyte activation, the later events observed by us are in concordance with previously published data. Conversely, it has been suggested that PMNs may be able to transform xanthine dehydrogenase to the oxidase form by a CD11a/CD18 and ICAM-1 adhesion molecule-dependent mechanism (Wakabayashi et al. 1995 ). We have detected sustained P-selectin and de novo ICAM overexpression on pancreatic endothelial surfaces at this time point, and this is now under investigation in our laboratory. At this later phase of AP, a number of mediators (e.g., cytokines, PAF) are upregulated, thereby contributing to increased leukocyte adherence, activation, and OFR production.

Many experimental and clinical studies have reported at best partial improvement with antioxidant therapies. However, our results indicate that the complexity and the multifactorial, interdependent sequence of pathophysiological events should not discourage further experimental and clinical treatment efforts to medically counteract the harmful oxidative stress caused by acute pancreatitis.


  Acknowledgments

Supported by grants from the IRMAD Foundation (France), Solvay Pharmaceuticals (France), and the Charles Debray Foundation (France).

We gratefully thank Dr Catherine Pasquier for generous assistance in leukocyte control experiments and Dr Germain Trugnan for technical advice concerning confocal microscopy.

Received for publication December 28, 1998; accepted April 17, 1999.


  Literature Cited
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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