Albumin Infusion After Reperfusion Prevents Gut Ischemia-Reperfusion-Induced Gut-Associated Lymphoid Tissue Atrophy 
From the * Division of Basic Traumatology,
National Defense Medical College Research Institute, Tokorozawa, Japan; Correspondence: Kazuhiko Fukatsu, MD, Division of Basic Traumatology, National Defense Medical College Research Institute, 3–2 Namiki, Tokorozawa-City, Saitama 359-8513, Japan. Electronic mail may be sent to fukatsu{at}ndmc.ac.jp.
Background: Our recent study clarified that gut
ischemia-reperfusion (I/R) causes gut-associated lymphoid tissue (GALT) mass
atrophy, a possible mechanism for increased morbidity of infectious
complications after severe surgical insults. Because albumin administration
reportedly reduces hemorrhagic shock–induced lung injury, we
hypothesized that albumin treatment prevents GALT atrophy due to gut I/R.
Methods: Male mice (n = 37) were randomized to albumin, normal
saline, and sham groups. All groups underwent jugular vein catheter insertion.
The albumin and normal saline groups underwent 75-minute occlusion of the
superior mesenteric artery. During gut ischemia, all mice received normal
saline infusions at 1.0 mL/h. The albumin group was given 5% bovine serum
albumin in normal saline at 1.0 mL/h for 60 minutes after reperfusion, whereas
the normal saline group received 0.9% sodium chloride at 1.0 mL/h. The sham
group underwent laparotomy only. Mice were killed on day 1 or 7, and the
entire small intestine was harvested. GALT lymphocytes were isolated and
counted. Their phenotypes ( Despite recent advances in the management of severely injured patients, multiple-organ failure (MOF) may still develop, and the mortality rate remains unacceptably high.1–5 In recent decades, the underlying mechanisms have been extensively investigated. Consequently, gut hypoperfusion is now postulated to be an important mechanism leading to postinsult MOF.2,3 Gut ischemia-reperfusion (I/R) models the clinical phenomena of gut hypoperfusion and subsequent recovery of perfusion after severe surgical insults, including shock, trauma, and cardiac or aortic surgery. Using gut I/R models, researchers have obtained possible insights into the pathogenesis of MOF.2,6 First, the reperfused gut serves as a priming bed for circulating polymorphonuclear neutrophils (PMNs).3,7 These excessively primed and activated PMNs accumulate in remote organs and injure host tissues by releasing reactive oxygen intermediates and toxic enzymes.7 Second, gut I/R causes ileus and increases gut permeability, which may exacerbate bacterial translocation and microaspiration.7 These changes may be associated with susceptibility to severe infectious complications. Moreover, our recent study clarified another mechanism of impaired host defense after gut I/R.8 Gut I/R causes gut-associated lymphoid tissue (GALT) mass atrophy.8 Because GALT plays a central role in systemic mucosal immunity, GALT atrophy may allow pathogens and toxins to cross the mucosal barrier.9–11 Therefore, new therapeutic strategies aimed at preventing gut I/R-induced GALT changes are needed. Albumin is well known to have colloidal and scavenging properties.12,13 Albumin administration has been shown to reduce lung injury, to improve hemodynamics and the microcirculation, and to attenuate inflammatory responses in hemorrhagic shock models.12,14 Clinically, a meta-analysis of acutely ill cases found a reduction in complication rates with albumin therapy.15–17 We therefore hypothesized that albumin treatment prevents gut I/R-induced GALT atrophy. This study was designed to determine whether early resuscitation, using albumin, reverses gut I/R-induced GALT changes.
Animals Male Institute of Cancer Research (ICR) mice (Nippon SLC, Hamamatsu, Japan) were housed under controlled temperature and humidity conditions with 12-hour light/dark cycles and fed commercial mouse chow ad libitum for 1 week before protocol entry. The mice were maintained in accordance with the guidelines of the National Defense Medical College for the Care and Use of Laboratory Animals, and the studies were approved by the Animal Use and Care Committee of the National Defense Medical College.
Experimental Protocol Male ICR mice (n = 37) were randomized to albumin (n = 13), normal saline (NS; n = 14), and sham groups (n = 10; Figure 1). Animals were anesthetized with a ketamine (100 mg/kg) and xylazine (10 mg/kg) mixture. The right jugular vein catheter was inserted for resuscitation in all 3 groups. The albumin and NS groups underwent 1.5-cm midline laparotomy and occlusion of the superior mesenteric artery (SMA) with a microvascular clip for 75 minutes. The sham group underwent jugular vein catheter insertion and laparotomy with identification, but not occlusion, of the SMA. Thereafter, the laparotomy incision was immediately closed. During gut ischemia, all groups received 0.9% sodium chloride infusions at 1.0 mL per hour. After a 75-minute SMA occlusion, the laparotomy incision was reopened to remove the clip and then closed in the I/R groups. In the sham group, the laparotomy incision was only reopened and closed. The albumin group was given 5% bovine serum albumin (Sigma, St. Louis, MO) in 0.9% sodium chloride at 1.0 mL/h via the jugular vein catheter for 60 minutes after reperfusion, whereas the NS and sham groups received 0.9% sodium chloride at 1.0 mL/h. The catheter was subsequently removed and the mice were put in cages with free access to water and chow. At the end of the 1-day or the 7-day period, the mice were anesthetized as previously described and killed by cardiac puncture (Day 1: albumin group, n = 9; NS group, n = 8; sham group, n = 6; Day 7: albumin group, n = 4; NS group, n = 6; sham group, n = 4). The entire small intestine was harvested for GALT lymphocyte isolation. Nasal and bronchoalveolar washings were obtained by lavage with 2 mL of phosphate-buffered saline solution. Small-intestinal washings were also obtained, using a total of 20 mL of Hanks' balanced salt solution (HBSS; GIBCO, Auckland, New Zealand). Secretory immunoglobulin A (IgA) levels were measured in some of the mice (albumin group, n = 10; NS group, n = 11).
Cell Isolation PPs. PPs were dissected from the serosal side of the intestine and then chopped finely. The fragments were treated with collagenase (Sigma; 40 U/mL) in RPMI1640 for 60 minutes at 37°C with constant shaking. After collagenase digestion, the cell suspensions were passed through nylon filters. IE and LP cells. After PP excision, the intestine was turned inside out and cut into 4 segments. The segments were incubated with RPMI1640 containing 5% fetal bovine serum (FBS), 1% glutamine, and a 1% antibiotic mixture (penicillin and streptomycin; GIBCO) for 45 minutes at 37°C in a water shaker (150 rpm). Supernatants containing released sloughed epithelial cells and IE lymphocytes were stored on ice. The remaining tissue pieces were incubated 3 times for 45 minutes each time, with RPMI1640 containing collagenase (Sigma; 40 U/mL), 5% FBS, glutamine, and an antibiotic mixture at 37°C at 150 rpm in a water shaker. The supernatants containing LP cells were pooled on ice after a 45-minute incubation. Supernatants were filtered through a glass wool column. Suspensions were centrifuged, the pellets were resuspended in 40% Percoll (Pharmacia, Piscataway, NJ), and the cell suspensions were overlaid on 75% Percoll. After centrifugation for 20 minutes at 600 x g at 25°C, viable lymphocytes were recovered from the 40%/75% interface and washed in RPMI1640. The lymphocytes were resuspended in RPMI1640 with 5% FBS, 1% glutamine, and a 1% antibiotic mixture and then counted. This procedure yields a cell population that is 95%–100% viable by trypan blue exclusion.
Flow Cytometry
IgA Quantification
Statistical Analysis
Body Weight Change On day 1, all groups showed significant body weight loss (Table I). On day 7, only the NS group showed significant weight gain compared with preexperimental values. There were no significant differences in pre- and postexperimental weight or weight change among the 3 groups.
Total cell yields from GALT. On day 1, the cell yields from PPs in the gut I/R groups were significantly lower than that from the sham group (Figure 2). However, albumin treatment significantly increased PP lymphocytes compared with the NS group. On day 7, there were no significant differences in PP cell yields among the 3 groups (Figure 2). The IE lymphocyte numbers on days 1 and 7 did not differ significantly among the 3 groups (Figure 3). Gut I/R significantly reduced LP lymphocyte numbers on day 1 compared with the sham laparotomy (Figure 4). The albumin group showed moderate but statistically significant recovery of LP cell numbers on day 1 compared with the NS group. There were no differences among the groups on day 7.
Absolute number of GALT lymphocytes with each phenotype. On day 1,
the B220+ cell numbers from PPs in the gut I/R groups were significantly
decreased compared with those in the sham group. The albumin group showed
significantly higher B220+ lymphocyte numbers in PPs and the LP than the NS
group (Tables II,
III and
IV). On day 7, there were no
significant differences in any cell yields. However, compared with day 1, the
numbers of PP
Respiratory tract and intestinal IgA levels. There were no significant differences in secretory IgA levels at any mucosal sites on day 1 or day 7 between the albumin and NS groups (Table V).
GALT works as the primary immunologic mucosal defense against intraluminal bacteria and toxins and provides immunologic protection for distant mucosal sites, such as the nasopharynx, breast, salivary glands, and lung.19–21 GALT consists of 2 components, inductive and effector sites.22 PPs are inductive sites initiating contact between naïve B and T cells and intraluminal antigens that have been taken up by M cells and processed by antigen-presenting cells.19 Sensitized lymphocytes leave PPs and home to intestinal and extraintestinal effector sites.23,24 The LP is the major mucosal effector site and is enriched with IgA-producing cells.25 The IE also functions as an effector site and regulates the proliferation and differentiation of epithelial cells, thereby providing the first line of host defense in the intestine.26,27 IgA produced by GALT cells plays an important role in intestinal immune defense because it prevents bacteria from adhering to epithelial cells.28,29 Thus, we examined major parameters for evaluating mucosal immunity, ie, lymphocyte numbers in PPs, IE spaces, and the LP, as well as secretory IgA levels.
We timed the GALT analysis, using our previous data as a reference.8 In our earlier study, GALT cell numbers reached their lowest levels on days 1 and 2 after 60-minute gut ischemia.8 IE and the LP lymphocyte numbers had been restored to those of the sham group by day 7, whereas PP cell numbers remained low. In other words, day 1 and day 7 seem to represent acute and recovery phases of GALT atrophy, respectively. In the present study, remarkable cell number reductions in PPs and the LP were observed in the I/R groups on day 1, as in our prior experiment. However, as expected, albumin treatment significantly restored both PP and LP cell numbers compared with the NS group. With regard to phenotypes, recovery of B220+ cell numbers in PPs and the LP was associated with albumin treatment. Thus, albumin resuscitation after gut I/R moderately reversed atrophy of both inductive and effector GALT sites. The findings obtained in this study may have clinical relevance. Albumin was administered after the insult, with the timing paralleling the clinical situation. Early resuscitation with albumin may reverse GALT loss induced by severe surgical insults, thereby possibly reducing infectious complication rates. We can speculate as to the mechanisms possibly underlying GALT atrophy after gut I/R. Because gut I/R injures the small intestine via free radicals and toxic enzymes released by PMNs and other mediators independent of PMNs,2 GALT cell death may occur. Reduced perfusion and the resultant hypoxia during gut I/R may damage GALT cells directly. Albumin has been shown to work as an antioxidant and a scavenger of free radicals in hemorrhagic shock models.12,14 Albumin resuscitation reportedly contributed to the prevention of hypotension and low tissue blood flow in a hemorrhagic shock model due to its colloidal properties.12 Albumin may also inhibit PMN spreading and hydrogen peroxide release.30 By reducing oxidative stress and simultaneously improving the oxygen supply to GALT, albumin might maintain GALT cell numbers, although we did not examine these parameters in the present study. Interestingly, in the current study, 75-minute gut ischemia did not decrease the IE lymphocyte number on day 1 in comparison to the sham laparotomy group. Furthermore, on day 7, GALT cell loss appeared to be reversed in the NS and albumin groups. These findings are inconsistent with our previous data (60-minute ischemia).8 It is possible that continuous IV infusion of saline or albumin during gut I/R contributed to the reduction in the magnitude of the insult. We injected only NS subcutaneously at the time of reperfusion in our previous study. Indeed, the survival rate on day 7 in the NS group in our current study was 77%, whereas survival rate in the previous 60-minute gut I/R study was only 30%.8 We measured secretory IgA levels in the NS and albumin groups in the present study. Despite GALT cell recovery in response to albumin therapy, no significant differences were recognized in IgA levels. In our previous experiment, there were no reductions in IgA levels with gut I/R compared with sham mice.8 Inflammatory stimulation and serum leakage due to gut I/R31,32 might have compensated for the reduced IgA levels in washings even in animals with severe GALT atrophy. In the present study, we determined the dose of albumin by referring to published reports and have not yet examined effects of albumin with different doses and durations. High dose or longer duration of albumin may improve GALT cell restoration more. However, it is also possible that too much albumin worsens tissue edema and hemorrhage because of its leakage to extravascular spaces and anticoagulant activity.12,14,33 In future studies, the optimal dose and duration of albumin therapy should be examined in gut I/R models. The mechanisms underlying the recovery of gut I/R-induced GALT cell loss, in response to albumin administration, await clarification. Although the benefits of albumin administration remain controversial,15–17,33–35 we believe that the present results raise the possibility of new clinical applications of albumin in patients with severe surgical insults. We conclude that early albumin administration after gut ischemia may maintain gut immunity by preventing GALT mass atrophy.
Presented in the Premier Paper Session at the American Society for Parenteral and Enteral Nutrition Clinical Nutrition Week, February 12–15, 2006. Received for publication February 27, 2006. Accepted for publication April 20, 2006.
DiscussantDr Ikezawa, we commend you on a well-conducted, well-described experiment with a lovely animal model. In fact, there are 2 other gastrointestinal disease states with established animal models that you might choose to examine using your T-cell assays in order to clarify the disease processes and perhaps elucidate effective treatments. Inflammatory bowel disease is associated with altered immune function, particularly in the setting of pouchitis. An animal model of pouchitis was developed by Dr John Rombeau's group in 2002.1 It would be elegant to apply your current assay techniques to this model, in order to clarify the disease process as well as any benefit of antibiotic therapy. In addition, a few authors have described an inflammatory state in home parenteral nutrition (PN) patients, most of them with short bowel syndrome,2–4 though the mechanisms are not understood. One proposed mechanism is altered GALT function in the shortened length of intestine. Your measures of GALT function would certainly add clarity to this issue. My questions are:
Author's ResponseThank you for your comments and questions. With regard to the first question, the precise mechanisms are not clear from the present study. Because gut flow is reportedly more reduced in the tip of mucosa than in the lamina propria, it is possible that albumin did not reach the intraepithelial space and showed less effect on the intraepithelial than on the lamina propria cells.As to the second question, we consider that albumin may reduce oxidative stress and improve the oxygen supply, thereby restoring gut I/R-induced GALT atrophy. In hemorrhagic shock models, albumin has been shown to work as an antioxidant and a scavenger of free radicals, and contribute to the prevention of hypotension due to its colloidal properties. We have been examining the effects of albumin on circulating neutrophil activation. Albumin seemingly reduces neutrophil activation, but we have not yet reached statistically significant differences. There are some reports demonstrating that albumin protects against gut-induced lung injury and improves hemodynamics in hemorrhagic shock. We determined the dose of albumin referring to these reports and have not yet examined effects of albumin with different dose and duration. Higher dose or longer duration of albumin may more improve GALT cell restoration. However, it is also possible that too much albumin worsens tissue edema and hemorrhage because of its leakage to extravascular spaces and anticoagulant activity. Finally, we would like to apply the current method to other models for development of new therapeutic strategies to various disease conditions. As you suggested to us, it would be very interesting to measure GALT cell numbers and function in an inflammatory bowel disease model and a short bowel syndrome model.
Journal of Parenteral and Enteral Nutrition, Vol. 30, No. 5,
380-387 (2006)
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βTCR, 
TCR, CD4, CD8,
B220) were determined by flow cytometry. Results: On day 1, the gut
I/R groups showed significantly lower total lymphocyte and B cell numbers in
Peyer's patches and the lamina propria than the sham group. However, the
albumin infusion partially but significantly restored these cell numbers. On
day 7, there were no significant differences in any of the parameters measured
among the 3 groups. Conclusions: Albumin infusion after a gut
ischemic insult may maintain gut immunity by preventing GALT atrophy. 
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MATERIAL AND METHODS
RESULTS
