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A Priori Dietary -3 Lipid Supplementation Results in Local Pancreatic Macrophage and Pulmonary Inflammatory Response Attenuation in a Model of Experimental Acute Edematous Pancreatitis (AEP)

Suhail Sharif, MD, Michael Broman, BS^*, Tricia Babcock, MS^*, Evan Ong, MD^*, David Jho, BA^*, Marek Rudnicki, MD, W. Scott Helton, MD^* and N. Joseph Espat, MD^*

From the Department of Surgery, University of Illinois at Chicago, Chicago, Illinois; and the^* Department of Surgery, University of Illinois/Metropolitan Group Hospitals, Chicago, Illinois

Correspondence: N. Joseph Espat, MD, MS, FACS, Associate Professor of Surgery, University of Illinois at Chicago, Department of Surgery M/C 958, 840 S. Wood St, Room 435E, Chicago, IL 60612. Electronic mail may be sent to jespat{at}uic.edu.

Background: Acute pancreatitis is often complicated by multiorgan dysfunction, which is postulated to occur in part by macrophage infiltration into the pancreas. Eicosapentaenoic acid (EPA), an -3 fatty acid, is the principal biologic component of fish oil and has clinically and experimentally been demonstrated to be anti-inflammatory. We hypothesized that dietary EPA supplementation before the induction of pancreatitis would attenuate both M-mediated local pancreatic and systemic pulmonary inflammatory response in an in vivo model of acute edematous pancreatitis (AEP). Methods: Male Sprague-Dawley (SD) rats were pretreated 2 times per day with oral gavage with EPA (-3 fatty acid; 5 mg/kg/dose) or -6 fatty acid control (5 mg/kg/dose) or saline (equal volume) for 2 weeks. AEP was induced in -3, -6, and saline pretreated rats by 5 hourly subcutaneous (SC) injections of cerulein. Pancreas, lung, and serum were harvested 3 hours after the last cerulein injection. Severity of pancreatitis was confirmed by serum amylase and by histopathologic score. Pancreatic macrophage infiltration was assessed by confocal fluorescent microscopy, and pulmonary leukocyte respiratory burst (LRB) analysis was performed on mononuclear cells obtained from bronchioalveolar lavage (BAL). Results: All animals demonstrated acute pancreatitis through hyperamylasemia and histopathologic examination. Confocal analysis demonstrated significantly lower macrophage infiltration, and BAL analysis by flow cytometry demonstrated significantly lower (p < .05) LRB in the -3-treated group compared with the -6 and the saline pancreatitis group. Conclusions: Attenuation of both pancreatic M inflammatory response and pulmonary leukocyte respiratory burst in AEP by EPA supports further investigation into the potential role for EPA dietary supplementation in the progression of pancreatitis-associated sequelae.

Acute pancreatitis (AP) can range from mild edema to severe tissue necrosis characterized by acinar cell edema and inflammation.¹ AP is a devastating disease that in its severe form can lead to systemic complications and multiorgan failure.² The systemic lethal sequelae of AP are similar to those of sepsis and are mediated through a diffuse and progressive activation of the inflammatory cascade. Several mediators such as activated pancreatic enzymes, cytokines, endotoxins, superoxides, and arachidonate metabolites have been shown to play important roles in the pathogenesis of pancreatitis.³

In experimental AP, activated peritoneal macrophages infiltrate the pancreas and are postulated to be the key reason for pancreatic dysfunction and cytokine liberation. Evidence supports that pancreatitis-associated TNF- and IL-1 production is implicated in subsequent local inflammation and tissue destruction.⁴

Cytokines produced and released from the inflamed pancreas can potentially stimulate Kupffer cells and hepatocytes within the liver to produce additional inflammatory mediators. In fact, the liver was recently shown to contribute to the elevation in serum IL-6 during AP.⁵ Hepatic cytokine elaboration can also have downstream effects on alveolar macrophages that cause pulmonary dysfunction.⁵ This inflammatory cascade beginning at the level of the gastrointestinal tract and ending in the lung has been referred to as the "gut-hepatic-pulmonary axis" and may explain how pancreatitis rapidly progresses to pulmonary dysfunction. Leukocyte respiratory burst (LRB) is a common test used to measure the inflammatory response of the lungs to an inflammatory insult anywhere in the body. It not only gives a quantitative value for the number of leukocytes within the lung parenchyma but also quantifies the relative state of activation of these leukocytes. A crucial and very important gateway in the pathogenesis of pancreatitis and its associated sequelae is the initial macrophage activation within the pancreas. This gateway might provide a site of action for attenuation of pancreatitis and its inflammatory response.

Eicosapentaenoic acid (EPA) alone or as an -3 fish oil emulsion has been clinically and experimentally demonstrated to have significant anti-inflammatory activities, resulting in decreased cytokine production, NFB inhibition, and macrophage-mediated systemic dysfunction.^6–9 We hope to use the anti-inflammatory properties of -3 fatty acid (FA) to show if it can attenuate the inflammatory response seen in pancreatitis. To date, no in vivo studies have been performed to investigate the potential effects of -3 FA therapy in a model of experimental AP.

In this pilot study, we hope to demonstrate the effects of -3 FA in cerulein-induced AP specifically at macrophage infiltration into the pancreas and at the LRB in the lungs.

	MATERIALS AND METHODS

Top MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION

 
Materials
The described experiments were approved by the University of Illinois at Chicago, Animal Care Committee. All animals were individually housed and maintained in accordance with University Animal Care policies. Cerulein was purchased from Bachem Chemical Co (Torrance, CA). -3 FA was purchased as EPA-DHA (600 mg:400 mg) extra strength dietary supplement softgel tablets from Metagenics. Commercially available corn oil was used as -6 FA supplement.

Meal Feeding
Male Sprague-Dawley rats (200–250 g body wt) were trained to "meal feed" before the beginning of the study. Meal training acclimates the animals to food presentation between 8 PM and 8 AM, which was scheduled to coincide with alternating light and dark cycles (light, 8 AM to 8 PM; dark, 8 PM to 8 AM). Rats were presented with standard lab chow and water ad libitum. This regimen has been used previously in our laboratory with good results.¹⁰

Rats were randomly divided into 5 groups: pure control (PCG; n = 2), experimental control (ECG; n = 3), treatment group (TG; n = 5), control fatty acid (CFG; n = 5) and pancreatitis group (PG; n = 6). Study days 0–6 were used for acclimation. Oral gavage was performed twice daily at 8 AM and at 5 PM on study days 7–21, with 5.0 g/kg/d of EPA of EPA-DHA capsules for the TG group, isovolemic 5.0 g/kg/d of corn oil (isocaloric) combined with 10 IU vitamin E/g of corn oil for the CFG group, or isovolemic but nonisocaloric normal saline (NS) to gavage animals from the PG and ECG groups. PCG group did not receive any gavage treatment. Vitamin E was administered to prevent the biologic oxidation of EPA and to prevent the depletion of vitamin E stores in the liver that occurs with fat supplementation.

Model of AP
On the eve of study day 21, AP was induced by subcutaneous (SC) injections of cerulein (50 µg/kg) at hourly intervals for 4 hours with the first injection 8 hours before killing and the last injection 3 hours before killing (total of 5 injections per animal). All animals from TG, CFG, ECG, and PG groups received cerulein injections. PCG animals received normal saline SC injections.

Rat Killing and Tissue Harvesting
Bronchioalveolar lavage (BAL) of all animals was performed before exsanguination. After receiving intraperitoneal anesthesia with ketamine HCl (100 mg/100 g body weight), a tracheostomy was performed. The trachea was then lavaged using 5 mL of PBS slowly in order to avoid any hemorrhage from alveolar rupture. This procedure was repeated four times in order to obtain adequate lavage for analysis. After BAL, a midline sternotomy was performed and blood collected from direct cardiac puncture. Immediately after exsanguination, the lungs, pancreas, and liver were harvested before complete cessation of circulation. The lungs were placed in aluminum foil and flash frozen in liquid nitrogen. The pancreas was split into 3 parts arbitrarily. The head of the pancreas was placed in 10% formalin for histology; the body of the pancreas was placed in aluminum foil and flash frozen for confocal analysis.

Biochemical Assays
Serum was collected, placed on ice, and prepared for subsequent analyses. Serum amylase levels were determined using the Phadebas amylase test, with amylase levels measured spectrophotometrically as described by the manufacturer.

Pancreas Histology
Pancreata were harvested and placed in 10% buffered formalin, as described earlier. The tissue was then embedded in paraffin, sectioned (4 µm), and stained with hematoxylin and eosin for histologic evaluation. For severity scoring, the pancreas (10 random fields on each slide) of all animals from each group was examined and scored for necrosis, vacuolization, inflammation, and edema by a pathologist who was blinded as to the treatment. The scoring was based on the scale as provided in Table I. Any score of 2 or above represents involvement of majority of the tissue and is considered a high score for a given category. Likewise, a score of <2 is considered a low score.

Rat Pancreas Frozen-Sectioning for Confocal Microscopy
Rat pancreata were removed surgically from the animals via the abdominal cavity, flash-frozen in liquid nitrogen, and stored at –80°C. Pancreas tissue was then embedded in optimal cutting temperature (OCT) aqueous mounting medium and sectioned at 7-µm thickness in a cryostat (Microm HM 505 N, Richard-Allan Scientific, Kalamazoo, MI). Tissue sections were mounted on coated glass slides (Fisher) for CD68 immunostaining in order to identify macrophage infiltration.

Rat Pancreas Immunostaining and Confocal Imaging
Lungs were also sectioned and mounted in a similar fashion to pancreatic tissue, as previously mentioned, and then fixed in 3.7% formaldehyde/PBS for 20 minutes and permeabilized in 0.4% Triton X-100 in PBS for 10 minutes. Sections were blocked with blocking buffer (Hanks' balanced salt solution [HBSS], 0.2% BSA, 0.01% NaN₃, 0.1% Triton X-100) for 30 minutes and then exposed to primary antibodies (anti-ICAM [H-108], rabbit, Santa Cruz, 1:50, and anti-CD68 [ED1], mouse, Serotec [Raleigh, NC], 1:50) in blocking buffer at RT for 1 hour. The sections were then washed 3 times in PBS, blocked again for 30 minutes, and exposed to the secondary antibodies (Alexa594-anti-rabbit, Alexa488-antimouse, Molecular Probes) at 1:100 concentration along with DAPI (4,6-diamindino-2-phenylindole) at 1:1000 (Molecular Probes) in blocking buffer for 30 minutes. Sections were then washed as before and mounted using ProLong mounting medium (Molecular Probes) and #1.5 glass coverslips overnight. Sections were imaged at 1000 x magnification using a Zeiss LSM 510 confocal microscope (Thornwood, NY). Ten separate high-power fields were examined from each of 9 different animals in each experimental group, using the presence of CD68-positive cells to indicate macrophage infiltration. The average number of macrophages per high-power field was quantified for each experimental group.

LRB Analysis
BAL fluid was collected as previously described and spun at 1000 rpm for 10 minutes. The resulting pellet was incubated at 37°C for 30 minutes in 100 µl of PBS +25 µl of DHR (dihydrorhodamine-123) solution and then centrifuged at 14,000 rpm for 5 minutes, dumping the supernatant, once again. The pellet was resuspended in 500 µL of 1% paraformaldehyde and filtered into conical tubes. Neutrophil population was measured using flow cytometer by measuring the fluorescence at 530 nm (FL1 green channel). Photomultiplier gain was adjusted so that fluorescence of the reagent blank was confined to the first decade of FL1 histogram.

Statistical Analysis
The data were analyzed using the SPSS for Windows package (SPSS Inc, Chicago, IL). Each variable was expressed as the mean with the accompanying means ±SEM. The data were analyzed by 1-way analysis of variance (ANOVA) or t-test with Tukey and Sheffe post hoc tests as appropriate, with significance defined at p .05.

	RESULTS

Top MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION

 
Serum Amylase
Serum amylase levels (Table II) were 7 times higher in cerulein-treated groups compared with control. However, there was no difference between the -3 group and the -6 or saline groups.

View this table: [in this window] [in a new window]   Table II The effect of -3 fatty acid on serum amylase in rats with cerulein pancreatitis*

Histopathology
Pancreatic histopathological grade documents the presence of predominantly edematous pancreatitis (Table III), as evidenced by a high grading score for edema formation and a low grading score for necrosis, vacuolization, and inflammation.

Local Macrophage Infiltration Into the Pancreas
-3 FA–treated rats demonstrated significantly fewer macrophage infiltration into the pancreatic parenchyma compared with -6 FA– and saline–treated rats, as shown in Figures 1 and 2.

View larger version (83K): [in this window] [in a new window]   FIGURE 1. Sections were imaged at 1000 x magnification using a Zeiss LSM 510 confocal electron microscope to document the presence of macrophages, which are represented by the luminance spots on each picture, showing the presence of CD68-positive cells to indicate macrophage presence.

View larger version (9K): [in this window] [in a new window]   FIGURE 2. Bar graph comparing the quantitative amount of macrophages/1000 x field present among each group. Ten sections per each animal were performed and compared with each group. The data were analyzed by t-test with Tukey and Sheffe post hoc tests as appropriate, with significance defined at p < .05.

View larger version (11K): [in this window] [in a new window]   FIGURE 3. Neutrophil population as measured by flow cytometry at 530 nm (FL1 green channel) is plotted using bar graph with the y axis representing the magnitude and the x axis representing the different groups. The data were analyzed by t-test with Tukey and Sheffe post hoc tests as appropriate, with significance defined at p < .05.

	DISCUSSION

Top MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION

Histopathologic evaluation further confirmed the presence of edematous pancreatitis (Table III). The severity score for edema formation in the pancreatic parenchyma was high (>2: 0–4), and the severity score for necrosis and vacuolization was low (<2: 0–4). Although the degree of edematous pancreatitis was not found to be statistically significant between TGs at 6 hours post–cerulein induction, we recognize that altering the time course may result in demonstrable differences and that the histologic examination as performed may not be sufficiently sensitive to detect potential differences.

In the present study, we have shown that certain aspects of the local and systemic inflammatory response in AP can be significantly attenuated with -3 FA. Despite the fact that our model was limited in severity, nonetheless a dramatic decrease in the amount of macrophage infiltration into the pancreas within 6 hours in -3-treated animals compared with -6 and pancreatitis alone animals was observed (Figure 2). As far as we know, this is the first experimental demonstration of attenuated macrophage infiltration into pancreatic parenchyma by -3 FA pretreatment dietary supplementation in experimental AP. Because the pancreas is devoid of inflammatory cells normally, the presence of macrophage in the pancreas represents one of the earliest indications of an inflammatory response to an insult. Attenuation of this local macrophage infiltration should translate into decreased systemic effects.

The present study was also designed to explore the possible affect of -3 FA on the pulmonary sequelae of AP. We hypothesized that attenuation of any local inflammatory response in the pancreas should result in attenuation of the systemic consequences of AP. We chose to evaluate the LRB in the lungs because it is a good measure of the amount of recruited inflammatory cell population and its relative activated state. In addition, pulmonary organ dysfunction is the key contributor to the morbidity and mortality associated with multiple-organ failure. As shown in Figure 3, we found that -3 FA results in statistically significant attenuation of LRB in the lungs compared with -6 FA and pancreatitis-alone groups.

The ability to inhibit specific steps of inflammation may provide clinicians and scientists with a number of ways to potentially prevent or ameliorate multiple-organ dysfunction after pancreatitis. If the migration of mononuclear cells into pancreatic parenchyma can be attenuated, this would ameliorate some of the local and systemic deleterious effects of pancreatitis. Fish oil emulsions rich in -3 FA have consistently demonstrated anti-inflammatory properties primarily through their effects on the macrophage component of the inflammatory response. We were also able to show such anti-inflammatory properties in our experiment.

In the present study, we were able to demonstrate attenuation of an early (macrophage infiltration) and delayed (pulmonary inflammation) event in the inflammatory response occurring during acute experimental pancreatitis. Future studies will encompass the exact mechanism of inhibition of pancreatitis by -3 FA and its effect on other organ systems (ie, liver, intestine).

	CONCLUSION

Top MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION

 
Cerulein induction results in mild AP. In the time point studied, we demonstrated statistically significant attenuation of pancreatic macrophage infiltration and pulmonary LRB with -3 FA pretreatment. This experiment is a gateway to future studies that will examine the cellular inhibition of inflammation in AP by -3 FA.

The study was supported by NIDDK-1 K08 DK DK60778–01 (Espat).

Top MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION

 
Presented in part at: Pancreas Club, SSAT 2004.

Received for publication March 29, 2006. Accepted for publication April 3, 2006.

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Journal of Parenteral and Enteral Nutrition, Vol. 30, No. 4, 271-276 (2006)
DOI: 10.1177/0148607106030004271


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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Saved Citations Download to citation manager Request Permissions Request Reprints Add to My Marked Citations Citing Articles Citing Articles via Google Scholar Citing Articles via Scopus Google Scholar Articles by Sharif, S. Articles by Espat, N. J. Search for Related Content PubMed PubMed Citation Articles by Sharif, S. Articles by Espat, N. J. Social Bookmarking                   What's this?