ABSTRACT. Background: Gut ischemia-reperfusion (gut I/R) accompanying severe surgical insults leads to neutrophilmediated injury and is regarded as a triggering event in early multiple-organ failure. Our previous study demonstrated dietary restriction to down-regulate leukocyte activation. Therefore, we hypothesized dietary restriction might be beneficial in terms of surviving I/R. We also evaluated leukocyte activation and the level of organ glutathione, an antioxidative substance. Methods: Institute of Cancer Research mice received chow, 170 (ad libitum), 119 (MR: mild restriction) or 68 (SR: severe restriction) g/kg per day for 7 days. Exp. 1: The mice (n = 59) underwent 15 or 45 minutes of gut ischemia and survival was observed. Exp. 2: The mice (n = 73) were killed before or 60 or 120 minutes after 15-minute ischemia. Reactive oxygen intermediate (ROI) production by circulating myeloid cells and CD11b expression was determined. Some mice were assessed for nuclear factor κ B (NFκB) activation. Glutathione levels were measured in some of the small intestine and liver samples from each group. Results: Dietary restriction decreased survival. Circulating myeloid cell priming and activation, in terms of ROI production and CD11b expression, were enhanced in the ad libitum group but not in the restricted groups. NFκB was activated only in the ad libitum group. Gut and hepatic glutathione levels were lower in the SR than in the ad libitum group. Dietary restriction caused histologic damages in gut, liver, and lung 120 minutes after reperfusion. Conclusions: Dietary restriction blunts leukocyte priming and activation after gut ischemic insult but worsens the outcome by, at least in part, decreasing antioxidative activities. Clinically, nutrition replenishment may be required to improve the outcome of gut hypoperfusion. (Journal of Parenteral and Enteral Nutrition 29:345-352, 2005)
Recent advances in trauma research have resulted in the evolution of concepts possibly explaining multipleorgan failure (MOF) pathogenesis.1 The present hypothesis is that postinjury MOF results from an excessive proinflammatory response (early MOF) or delayed immunosuppression, causing severe infectious complications (late MOF).2-5 Numerous research efforts have demonstrated gut hypoperfusion accompanying severe surgical insults to be a key event leading to MOF.5-7
Gut hypoperfusion and subsequent recovery of gut perfusion (ie, gut ischemia-reperfusion [I/R]) primes and activates circulating leukocytes.8,9 These leukocytes accumulate in remote organs and injure tissues, causing an autodestructive response during the perioperative (early) stage.8 On the other hand, during the postsurgical (late) stage, gut I/R causes ileus and bacterial colonization in the intestinal lumen, leading to bacterial translocation and occasionally aspiration pneumonia.10,11 Therefore, appropriate modulation of a gut I/R-induced host response may improve the outcomes of injured patients.
We have explored nutrition therapy, modulating host responses to gut IfR, for several years.12-15 Consequently, we documented parenteral nutrition (PN) before ischemia to worsen survival after gut I/R compared with enterai nutrition.13 Addition of glutamine to PN before ischemia partly restored resistance to gut I/R.12 However, administration of glutamine or arginine during ischemia resulted in poor survival at early time points after gut I/R, possibly by triggering extreme leukocyte activation.14,15
In this study, we examined the effects of dietary restriction on survival after gut I/R. We also evaluated parameters of leukocyte activation and the organ level of antioxidative substance, glutathione (GSH). Because dietary restriction reduces leukocyte activation in peritonitis, we hypothesized that this type of nutrition modulation would improve the outcome of gut I/R with inhibition of inflammatory responses.
MATERIALS AND METHODS
The experiments reported herein conform to the guidelines for the care and use of laboratory animals established by the Animal Use and Care Committee of the National Defense Medical School. Male Institute of Cancer Research mice (Nippon SLC, Hamamatsu, Japan) were housed under controlled conditions of temperature and humidity with a 12-hour: 12-hour light-dark cycle. Mice were fed commercial mouse chow with water ad libitum for 1 week before protocol entry.
Experiment 1 (Figure 1-A) was performed to assess the influence of moderate and severe dietary restriction before gut ischemia on survival after reperfusion. Seven- to 8-week-old mice (n = 59) were randomly assigned to 3 dietary groups. The ad libitum (n = 19), moderate (n = 20), and severe restriction (n = 20) groups received mouse chow ad libitum, 170 g/kg (578 kcal/kg), 119 g/kg (405 kcal/kg), and 68 g/kg (231 kcal/kg) daily for 7 days, respectively. Our preliminary experiment revealed that the average chow consumption by mice with free access to chow was 170 g/kg per day.
Seven days after starting the respective diets, a 2.5-cm midline laparotomy was performed, and the superior mesenteric artery (SMA) was identified under general anesthesia (ketamine 100 mg/kg and xylazine 10 mg/kg mixture). We occluded the SMA with a microvascular clip for 15 or 45 minutes. The laparotomy incision was immediately closed and then reopened to remove the clip. All mice were resuscitated with a subcutaneous 1-mL saline injection just after reperfusion and had free access to chow and water during the observation of survival up to 120 hours.
In experiment 2, mice were randomized into 3 groups (ad libitum: n = 24, moderate restriction: n = 24, severe restriction: n = 25) and fed for 7 days as in experiment 1. The mice were killed before or 60 or 120 minutes after 15 minute gut I/R to examine the influences of dietary restriction on circulating myeloid cell (PMN + monocyte) priming and activation by measuring reactive oxygen intermediate (ROI) production, CD11b expression, and nuclear factor κ B (NFκB) activation. GSH levels were also examined in some of the small intestine and liver samples from each group (n = 9 in each group).
Measurement of ROI Production by Circulating Myeloid Cells
Reactive oxygen intermediate (ROI) production by circulating myeloid cells (PMNs + monocytes) was determined with a modification of previously described protocols.16 Blood samples were incubated with or without 500 ng/mL of phorbol myristate acetate (PMA) at 37°C for 15 minutes, and the reaction was then stopped by placing the samples on ice. The samples were incubated with 7.5 mmol/L dihydrorhodamine 123 for 10 minutes at 37°C, and the reaction was stopped by placing the samples on ice. Red blood cells were lysed with 2 mL of lysing solution for 3 minutes at room temperature. The samples were washed twice with ice-cold PBS and then fixed with 1% paraformaldehyde (PFA) before flow cytometric analysis (Epics XL; Coulter, Hileah, IL).
Measurement of Adhesion Molecule Expression on Circulating Myeloid Cells
One hundred microliters of the whole blood samples were incubated with saturating amounts of anti-CD 11 b monoclonal antibodies (fluorescein isothiocyanateconjugated rat antimouse CD 11 b; Pharmingen, San Diego, CA) for 30 minutes at 4°C. At the end of incubation, blood samples underwent erythrocyte lysis with 2 mL of FACS lysing solution for 3 minutes at room temperature. Then, the cells were washed twice with cold PBS followed by fixation with 1% PFA. The samples were stored at 4°C until the flow cytometric analysis.
Measurement of Gut and Liver GSH Levels
The liver and the entire small intestine were harvested, rinsed with normal saline, blotted dry, weighed, and frozen at -80°C. The samples were thawed and homogenized in 10 volumes of 5% sulfosalicylic acid. The homogenates of the liver and gut were centrifuged at 8000 × g for 10 minutes at 4°C. The supernatant glutathione (GSH) level was assayed using a commercially available Total GSH Quantification Kit (Dojindo Laboratories, Kumamoto, Japan). The absorbance was measured at 412 nm.
Measurement of Intranuclear NFκB Levels in Circulating Myeloid Cells
One hundred microliters of the whole blood samples underwent erythrocyte lysis with 2 mL of lysing solution for 5 minutes at room temperature. Next, the cells were washed twice with cold PBS, followed by fixation with 1% PFA. After centrifugation at 1600 rpm for 10 minutes, the leukocytes were resuspended in RPMI 1640 containing 1% fetal calf serum (FCS). The leukocytes were washed with ice-cold phosphate-buffered saline (PBS without Ca^sup 2+^: Sigma, St. Louis, MO) containing 0.1% bovine serum albumin (BSA) and fixed using 2% formaldehyde on ice for 10 minutes. After fixation, the leukocytes were washed and resuspended in PBS to a concentration of 1 × 10^sup 6^ cells/mL. Next, the leukocytes were cytocentrifuged (500 rpm for 5 minutes) onto microscope slides and permeabilized in 100% methanol for 3 minutes.
Immunocytochemical Detection NFκB
After permeabilization, the leukocytes on the slides were washed in PBS containing 0.1% BSA and incubated in blocking buffer (20% normal goat serum in PBS with 0.1% BSA) for 45 minutes at room temperature. The slides were then rinsed and the leukocytes were incubated with anti-Rel A(p65) rabbit polyclonal antibody (1:50; Santa Cruz Biotechnology, Santa Cruz, CA) in PBS with 0.1% BSA for 60 minutes at room temperature. The samples were washed in PBS with 0.1% BSA. The leukocytes were then incubated with an Alexa 488-conjugated secondary antibody (goat antirabbit IgG; Molecular Probes, Eugene, OR) diluted 1:500 in PBS with 0.1% BSA for 45 minutes at room temperature. After washing in PBS with 0.1% BSA, nuclei were counterstained with 25 µm/mL propidium iodide (PI, Sigma) and 100 µg/mL ribonuclease A (Sigma) for 25 minutes at room temperature. After the final washing, the slides were coverslipped with glycerol (25%) in PBS and analyzed by LSC (Laser Scanning Cytometer, Olympus, Tokyo, Japan).
After killing, small intestines, livers, and lungs were removed, fixed with 10% formalin, and embedded in paraffin. They were cut at a thickness of 4 µm and stained with hematoxylin-eosin.
The log-rank test was used for survival time comparisons. Data are presented as means ± SEM. Differences in ROI production, adhesion molecule expression, and GSH levels were analyzed by using ANOVA, followed by the post hoc test of Fisher's protected least significant difference. Differences were defined as statistically significant when the p value was
Survival curves after 15 and 45 minutes of SMA occlusion are shown in Figure 2. Survival time after 15 minutes' gut ischemia was significantly reduced in the severe diet restriction group compared with the ad libitum group. After 45-minute gut ischemia, survival was significantly lower in the moderate and severe diet restriction groups relative to the ad libitum group.
ROI production without PMA stimulation at 60 minutes after reperfusion was significantly higher in the ad libitum group relative to the diet restriction groups (Figure 3). ROI production with PMA stimulation at 60 minutes after reperfusion was significantly enhanced in the ad libitum group compared with that before ischemia (Figure 3). The ad libitum group showed significantly higher ROI production with PMA at 60 minutes than the severe restriction group (Figure 3).
Expression at 60 minutes was significantly higher in the ad libitum group than in the moderate restriction group (Figure 4). CD11b expression at 60 minutes tended to be higher in the ad libitum group relative to severe restriction group (p = .071). Increased intranuclear translocation of NFκB was observed only in the ad libitum, group, not in the restricted groups (Figure 5).
Gut GSH levels were significantly lower in the severe diet restriction group relative to the moderate restriction and ad libitum groups before and 120 minutes after gut I/R (Figure 6). Liver GSH levels were essentially the same in the 3 groups before and 60 minutes after gut I/R (Figure 7). However, at 120 minutes, the severe diet restriction group showed significantly lower liver GSH levels than the ad libitum group (Figure 7).
Representative photomicrographs of organs at 120 minutes after reperfusion are shown in Figure 8. Dietary restriction caused small intestinal, hepatic, and pulmonary damage. However, marked neutrophil accumulation was not observed in any organs of dietrestricted mice. The ad libitum groups did not show organ damages.
Malnutrition is an important risk factor of complications after surgical insults. To elucidate the mechanisms underlying susceptibility, various models of malnutrition have been used experimentally.17,18 Dietary restriction as applied in the present study results in protein-calorie malnutrition, including deficiency of micronutrient delivery.19 Using this model, we previously demonstrated that malnutrition decreases β2-integrin expression on circulating and exudative neutrophils and cytokine responses in peritonitis.18 We also clarified that dietary restriction blunts intracellular signaling in peritoneal leukocytes in response to fMLP stimulation.20
These findings prompted us to examine effects of dietary restriction on survival after gut I/R. Because early MOF after gut I/R is triggered by excessive inflammatory responses, blunting of leukocyte responsiveness to stimulation with dietary restriction could improve survival. Contrary to our hypothesis, dietary restriction reduced survival time after reperfusion compared with the ad libitum group in this study. The degree of the survival disadvantage correlated with the degree of dietary restriction.
We examined several parameters for assessment of leukocyte priming and activation using 15 minutes gut IfR model in this study. Because most diet-restricted mice died at very early time points after 45 minutes' gut IfR, we chose a milder degree of ischemic insult for the measurement. ROI production by circulating myeloid cells without PMA may be a marker of cell activation, whereas ROI production with PMA reflects leukocyte priming status. Regardless of PMA stimulation, the ad libitum mice showed higher ROI production than the diet-restricted mice, especially the severely restricted mice.
Expression of CD11b, a p2-integrin, is increased after various stimuli and is also a marker of leukocyte activation.21 Interaction between CD11b and ICAM-1 expressed on endothelial cells is an important process of leukocyte-mediated tissue injury.2 Koike et al8 reported that blocking of CD11b prevents lung injury subsequent to gut I/R. In the present study, only the ad libitum group showed transiently increased CD11b expression, whereas the diet-restricted groups showed no remarkable change.
NFκB is a key ubiquitous transcription factor and plays a prominent role in the inflammatory cascade.23 Upon activation, NFκB translocates from the cytoplasm into the nucleus, where it binds to the promoter regions of a number of proinflammatory genes.23 We observed marked translocation of NFκB into nuclei only in the ad libitum mice.
Thus, dietary restriction blunted leukocyte activation in terms of ROI production, CD11b expression, and NFκB translocation. These findings are consistent with our previous data and do not contradict our hypothesis. Nonetheless, survival of the dietary restriction groups was significantly reduced compared with that of the ad libitum group. Other factors separate from leukocyte priming and activation may have strongly affected survival in this model. According to the present data, organ GSH levels are likely to influence the outcome.
GSH is one of the most important antioxidants involved in the stress response and is considered to act protectively against gut I/R-induced tissue damage. The mice with severe dietary restriction showed low gut GSH levels before ischemia and at 120 minutes after reperfusion. Severe dietary restriction also reduced hepatic GSH levels at 120 minutes after reperfusion compared with the ad libitum group. Interestingly, hepatic GSH levels before ischemia were not reduced in the dietary restricted groups. The mechanism for different pattern of GSH levels in the small intestine and liver is not clear from the present study. However, Grattagliano et al25 reported that 18-hour food deprivation did not affect hepatic GSH level. GSH level might be more preserved in liver compared with small intestine.
The present study may have important clinical relevance. Although it would not be feasible to give nutrition therapy before insults to trauma patients, there are many malnourished patients undergoing elective major abdominal, cardiac, or aortic surgery. Such surgery may cause gut I/R secondary to splanchnic hypoperfusion. Preoperative nutrition therapy for malnourished patients has been recognized as being beneficial in terms of recovery of host immunity. In addition, the results of the current study suggest that preoperative nutrition replenishment is beneficial for preventing gut hypoperfusion-induced organ failure.
In conclusion, moderate to severe dietary restriction blunts leukocyte priming and activation after gut ischemic insults but reduces organ antioxidative activities and worsens survival.
Gordon Sacks, PharmD
University of Wisconsin, Madison
I would first like to commend Dr Ueno and his group for their innovative work in the area of dietary restriction and its effects on neutrophil-mediated immunity. This paper is a natural extension of their previous work using dietary restriction as a model for investigating the effects of malnutrition on the expression of adhesion molecules on circulating neutrophils and on chemokine production at inflammatory sites. PMNs represent the first line of defense, acting to eliminate invading bacteria at the site of infection. Adhesion molecules, such as CD11b and CD18, and chemokines, including IL-6, TNF-α, and macrophage inflammatory protein 2 (MIP-2), play important roles in PMN recruitment during local inflammatory processes. In response to an inflammatory stimulus, such as infection, activated PMNs marginate, adhere to endothelium, and migrate to local inflammatory sites early in the inflammatory process. Exudative rather than circulating PMNs play the most important role in host defense at local sites. Malnutrition, on the other hand, has been reported to impair PMN-mediated immunity by reducing PMN exudation, adhesion molecule expression on PMNs, and chemokine production. Using a glycogeninduced peritonitis model in mice, Dr Ueno's group previously showed that dietary restriction decreased CD11b/CD18 expression on circulating PMNs, MIP-2 levels in peritoneal fluid, and subsequent PMN exudation into the peritoneal cavity early in peritonitis. Seven days of dietary restriction also impaired phagocytosis while up-regulating ROI production by exudative PMNs. Only 1 day of ad libitum refeeding normalized CD11b/CD18 expression along with PMN exudation into the peritoneal cavity.
At the same time, Dr Ueno's group has examined the effects of different nutrition therapies on modulating host responses to gut hypoperfusion and gut ischemiareperfusion. Data from Dr Fred Moore's lab have demonstrated that gut I/R primes and activates circulating leukocytes, which go on to accumulate in remote organs and lead to MOF. Because dietary restriction reduced leukocyte activation in peritonitis, it was natural for Dr Ueno's group to investigate whether this type of nutrition modulation would improve the outcome of gut I/R with inhibition of inflammatory responses. Unexpectedly, survival was worse with dietary restriction, despite blunting of leukocyte activation after I/R in terms of ROI production, CD11b expression, and intranuclear transolocation of NFκB. This poor survival was in association with reduced gut and hepatic glutathione levels.
1. The gut GSH level was already low before I/R, but liver GSH was not. Can you explain in more detail what would account for this discrepancy?
2. The severely restricted mice showed low gut GSH before ischemia and at 120 minutes after reperfusion. Severe dietary restriction also reduced hepatic GSH levels at 120 minutes after reperfusion compared with the ad libitum group. However, the difference appeared to be due to hepatic GSH increasing in the ad libitum group over baseline, not necessarily decreasing in the severely restricted group from baseline as what occurred with gut GSH levels. Why did the ad libitum hepatic GSH levels increase over baseline?
With regard to the first question, the mechanism for different pattern of GSH levels in the small intestine and liver is not clear from the present study. It was reported that 18 hours' food deprivation did not affect hepatic GRH level in a rat model. Thus, GSH level might be more preserved in liver compared with small intestine.
As for the second question, it appears that ad libitum hepatic GSH levels at 120 minutes after reperfusion increased over baseline. However, there was no statistically significant increase in hepatic GSH levels of the ad libitum mice. Otherwise, it might be possible that GSH production was increased in the ad libitum group at 120 minutes. Reportedly, hepatic protein synthesis rate recovered quickly in a rat liver ischemia-reperfusion model.
Thus, hepatic GSH production could also be preserved after gut ischemia reperfusion. Mild inflammation due to short duration of gut ischemia (15 minutes) might have enhanced hepatic GSH production in the ad libitum group.
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Chikara Ueno, MD*; Kazuhiko Fukatsu, MD[dagger]; Yoshinori Maeshima, MD*; Tomoyuki Moriya, MD[double dagger]; Eiji Shinto, MD§; Etsuko Kara, MT[dagger]; Hidetoshi Nagayoshi, MD*; Hoshio Hiraide, MD[dagger]; and Hidetaka Mochizuki, MD*
From the * Department of Surgery I, National Defense Medical College, Saitama, Japan; [dagger] Division of Basic Traumatology, National Defense Medical College Research Institute, Saitama, Japan; [double dagger] Department of Surgery I, Chiba University Graduate School of Medicine, Chiba, Japan; and the § Department of Pathology II, National Defense Medical College, Saitama, Japan
Received for publication January 27, 2005.
Accepted for publication May 24, 2005.
Correspondence: Kazuhiko Fukatsu, MD, Division of Basic Traumatology, National Defense Medical College Research Institute, 3-2 Namiki, Tokorozawa, Saitama, Japan 359-8513. Electronic mail may be sent to email@example.com.
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