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Basic and Experimental Research

Hydrogen-Enriched Preservation Protects the Isogeneic Intestinal Graft and Amends Recipient Gastric Function During Transplantation

Buchholz, Bettina M.1,2; Masutani, Kosuke3; Kawamura, Tomohiro4; Peng, Ximei5; Toyoda, Yoshiya5; Billiar, Timothy R.6; Bauer, Anthony J.1; Nakao, Atsunori4,6,7

Author Information
doi: 10.1097/TP.0b013e318230159d

Abstract

Through the advent of tolerogenic antirejection regimens during the last decade, small intestinal transplantation has become a life-saving option for irreversible alimentary tract dysfunction with parenteral nutrition failure (1, 2). However, early postoperative graft dysfunction and recipient morbidity remains high because of unavoidable ischemia-reperfusion (I/R) injury, intestinal manipulation, and intestine-derived endotoxemia, which synergistically trigger significant mucosal trauma and dysmotility in the immunocompetent and intraluminally colonized engrafted organ (3, 4).

Hemopoietic inflammatory mechanisms, such as the recently described postoperative Th1 memory cell-driven gastrointestinal field effect (5) and leukocyte-derived inducible nitric oxide synthase (iNOS) mediating postoperative- and transplant-induced dysmotility are attenuated by lymphoablative therapy and antirejection immunosuppression (6, 7). Unfortunately, these interventions also diminish the induction of beneficial anti-inflammatory mediators such as hemopoietic-derived interleukin (IL)-10 (8) and, importantly, would not efficiently abate the immunocompetence of nonhemopoietic cell populations. Nonhemopoietic immune mechanisms, which are activated by tissue trauma derived danger signals and lumenal-originating bacterial motifs, can dramatically compromise the mucosal barrier by inhibiting enterocyte proliferation/migration (9) and disrupting enterocyte tight junction microdomains (10) thereby perpetuating bacterial translocation, and generate an inflammatory response and disturb gastrointestinal motility (11). Because, currently no specific adjuvant attempts are clinically made to restrain this understudied arm of inflammation during the transplant procedure, the foremost objective during transplantation is to primarily minimize graft damage and thereby obviate graft injury in the hope of accelerating early restoration of intestinal graft morphology and function.

Current clinical preservation strategies are suboptimal since detrimental ischemia time-dependent inflammatory processes and injury to neuromuscular structures of the human intestinal graft still occur (4). Although extensively investigated experimentally (12), new intestine-tailored preservation solutions have not been introduced into the field of human transplantation, probably because of the practice of multiorgan donation. The investigation of graft protecting substances that do not differentially interfere with the individual physiologic needs of organs would therefore represent a helpful concept.

We have previously shown that combined donor and recipient inhalation of the novel signaling gas, hydrogen, efficiently protects intestinal isogeneic rodent grafts by demonstrating improved mucosal barrier function and graft contractility, and amelioration of remote organ inflammation (13, 14). Therefore, our objective was to determine whether the simplified approach of ex vivo hydrogen preloading of the graft by the use of a hydrogen-enriched solution for vascular washout, luminal irrigation, and cold storage would effectively blunt intestinal graft injury and recipient transplant-induced inflammation in rodents.

RESULTS

Hydrogen and Oxygen Solution Levels

As measured by gas chromatography, gas bubbling of lactated Ringer's (LR) solution, and University of Wisconsin (UW) solution (Viaspan; Du Pont, Wilmington, DE) resulted in similar average hydrogen concentrations in hydrogen-bubbled LR (1.08 ppm, equivalent to 1080 ng/mL or 0.54 mM) and hydrogen-bubbled UW (1.09 ppm, equivalent to 1090 ng/mL or 0.545 mM) with no detectable hydrogen levels in nitrogen- or air-bubbled LR or UW (n=3) (DHS-001; ABLE, Tokyo, Japan), which was similar to reports by Itoh et al. (15). Saturation of dissolved hydrogen declined similarly for both hydrogen-enriched solutions over time reaching 47% and 40% of the initial saturation at 6 hr after cessation of aeration, respectively (see Table, SDC 1,https://links.lww.com/TP/A504, which lists hydrogen concentrations, oxygen concentrations, and pH of the aerated preservation solution). Tissue concentration of hydrogen in intestinal grafts was not directly assessed, but the proof of principle of hydrogen's ability to rapidly diffuse across membranes and enrich the target tissue has been done by Hayashida et al. (16) and Oharazawa et al. (17) for both solvated and inhaled hydrogen. None of the solutions were rendered hypoxic by bubbling with air, nitrogen, or hydrogen with the exemption of hydrogen-enriched LR and UW during the first hour after hydrogen aerating (DM-1032; ABLE, Tokyo, Japan). However, the temporary hypoxia was only mild (85% of normal values). Importantly, no change in pH, which approximates the negative logarithm of the molar concentration of dissolved hydronium ions, was detectable (LRair 6.7, LRN2 6.7, LRH2 6.6; UWair 7.4, UWN2 7.4, UWH2 7.4).

Cytoprotective Effects in Intestinal Grafts by Ex Vivo Hydrogen Preservation

The mucosa's vulnerability to I/R injury is a well-known feature of intestinal transplantation. Therefore, we assessed mucosal morphology of the intestinal grafts 3 hr after reperfusion as a parameter of preservation efficiency. In sham-treated animals, no erosions, submucosal edema, or hemorrhage could be detected resulting in a histology score of 0.3±0.02. SITxN2 grafts demonstrated severe mucosal damage (7.1±0.61), which was significantly ameliorated by graft preservation in hydrogen-enriched LR (3.2±0.53) (n=5; Fig. 1A,B) similar to that seen historically with hydrogen inhalation (14). Serum levels of lactate dehydrogenase (LDH), an indicator of cell integrity, increased in SITxN2 (260.3±32.3) whereas the values in SITxH2 group were significantly lower (119.3±11.4) compared with those in SITxN2 measured at 24 hr after reperfusion (n=8; Fig. 1D).

FIGURE 1.
FIGURE 1.:
Graft morphology, graft oxidative stress, and cell integrity. Transplant-induced alterations in mucosal graft morphology (representative pictures (A), histological score (B)) and tissue malondialdehyde levels indicating lipid peroxidation (C) were significantly attenuated in intestinal grafts preserved with hydrogen-enriched lactated Ringer's solution (SITxH2) in contrast to nitrogen-treated grafts (SITxN2) at 3 hr after engraftment. Loss of cell integrity as measured by systemic lactate dehydrogenase (LDH) liberation at 24 hr after engraftment occurred to a significantly lesser extent in recipients of hydrogen-treated intestinal grafts (D) (*P<0.05 vs. sham, #P<0.05 vs. SITxH2).

Hydrogen-Enriched Solution Blunted Oxidative Graft Injury

Oxidative injury during the reperfusion period of the transplant procedure drives lipid peroxidation, which interferes with membrane integrity. Jejunal tissue malondialdehyde (MDA), a marker of lipid peroxidation, was significantly increased in full-thickness specimens of SITxN2 grafts (0.29±0.033) compared with sham jejunal MDA levels (0.12±0.013). Hydrogen treatment significantly diminished the lipid peroxidation marker in jejunal samples obtained from SITxH2 grafts (0.19±0.023) (n=6; Fig. 1C).

In Vivo Gastrointestinal Transit Was Delayed After Intestinal Transplantation

The coordinated propulsion of chyme is one of the major functions of the gastrointestinal tract rendering nutrient absorption and digestate excretion possible. The aboral movement of the orally fed liquid dye was significantly delayed in SITxN2 recipients exhibiting a geometric center (GC) of 6.5±0.3 compared with the GC of sham-treated rodents (9.2±0.26) (n=8; Fig. 2A) at 24 hr after reperfusion. Gastrointestinal transit was improved in SITxH2 recipients (GC 7.6±0.3). However, the segmental marker distribution did not normalize and the difference between the two transplanted groups did not reach statistical difference.

FIGURE 2.
FIGURE 2.:
In vivo gastrointestinal function after intestinal transplantation. Gastrointestinal transit delay (A) and recipient gastroparesis (B) at 24 hr after reperfusion were improved in recipients receiving hydrogen preserved intestinal grafts but remained significantly different from sham-treated animals in both transplanted groups. Solid markers which emptied out of the stomach of SITxH2 rats were rapidly transported aborally within the grafted intestine (C) (*P<0.05 vs. sham).

Transplant-Induced Recipient Gastroparesis Showed Improvement by Hydrogen-Enriched Graft Preservation

Posttransplant gastroparesis after human intestinal transplantation requires postoperative feeding through a jejunostomy. In our rodent transplant model, solid gastric emptying of small chrome steel balls was severely impaired in recipients of SITxN2 grafts (6.2%±4.98%) at 24 hr after reperfusion in contrast to normal gastric emptying rates (97.5%±2.5%) (n=8; Fig. 2B). Gastroparesis was markedly although not significantly lessened in SITxH2 recipients (27.2%±9.5%). However, gastric-emptied chrome steel balls were transported more aborally in the SITxH2-grafted intestine (Fig. 2C).

Transplant-Induced Suppression of In Vitro Jejunal Graft Smooth Muscle Contractility Was Ameliorated by Hydrogen Treatment

A major determinant of graft motility is the functional integrity of the enteric neuromuscular unit with its effector arm the smooth muscle syncytium. In vitro muscarinic contractile activity of jejunal muscle strips of SITxN2 grafts 24 hr after reperfusion was significantly diminished (1.9±0.34 g/mm2/sec, 46% of sham values), indicating sustained functional impairment or persistent smooth muscle cell damage of the intestinal graft, in comparison with mechanical activity of sham-treated intestinal muscularis (4.1±0.43 g/mm2/sec) (n=8; Fig. 3A,B). The transplant-induced suppression of SITxH2 graft muscle contractility was markedly improved (3.2±0.41 g/mm2/sec, 78% of sham values) and no longer significantly different from sham values.

FIGURE 3.
FIGURE 3.:
In vitro intestinal contractile function after intestinal transplantation. At 24 hr after reperfusion, muscarinic jejunal smooth muscle contractility of SITxN2 grafts was significantly suppressed while ex vivo hydrogen treatment of intestinal grafts notably preserved smooth muscle function; representative traces (A), calculated contractile force (B) (*P<0.05 vs. sham).

Graft Proinflammatory Gene Expression Was Significantly Diminished by Hydrogen Treatment

The intestinal muscularis is a potent source of inflammatory mediators affecting gastrointestinal motility after various local and systemic insults including intestinal manipulation, I/R injury and endotoxin exposure (11, 14, 18), which are all characteristics of intestinal transplantation. Both the transcription factor early growth response gene-1 (EGR-1), centrally involved in mediating postoperative rodent ileus (18), and its downstream target gene IL-6, a predictor of mortality in human sepsis, were significantly upregulated in intestinal muscularis extracts of SITxN2 grafts 3 hr after reperfusion relative to sham-treated rodents and SITxH2 grafts (n=6; Fig. 4A). In addition, IL-1ß and iNOS, which mediates gastrointestinal motor dysfunction (19), followed the same induction pattern with significant differences between the two transplanted groups.

FIGURE 4.
FIGURE 4.:
Relationship of molecular inflammation in the intestinal graft muscularis externa, posttransplant systemic inflammatory protein level, and heme oxygenase-1 (HO-1) induction in intestinal grafts before reperfusion. The anti-inflammatory potential of hydrogen preloading is demonstrated by its ability to significantly diminish graft inflammation in SITxH2 rodents relative to SITxN2 animals at 3 hr after reperfusion (A). Correlating with the decreased inflammatory graft message and improved preserved morphology, systemic IL-6 levels were found to be significantly downregulated in the serum of rats 24 hr after engraftment of hydrogen-treated intestines compared with nitrogen preserved intestines (B). Hydrogen-triggered HO-1 protein expression in full-thickness intestinal specimens during cold storage for 3 hr (D), but detectable prereperfusion HO-1 mRNA induction was restricted to the muscularis compartment (C) (*P<0.05 vs. sham, #P<0.05 vs. SITxH2).

Systemic Inflammatory Response Is Blunted by Hydrogen Treatment

Both graft and remote organs contribute to the transplant-triggered systemic inflammatory response. Circulating protein levels of IL-6 measured at 24 hr after reperfusion were significantly elevated in recipients of nitrogen-treated grafts compared with sham-treated animals but were considerably reduced in SITxH2 rats relative to SITxN2 rats (n=8; Fig. 4B). However, systemic nitrite, a nitric oxide (NO) metabolite, was not detectable in the serum of neither sham-treated nor any of the transplanted animals at the time point of functional measurements (data not shown).

Hydrogen Induces Intestinal Graft Heme Oxygenase-1 Before Reperfusion

Based on preliminary gene-array analysis (data not shown), we assessed the expression of heme oxygenase-1 (HO-1) as a contributing mechanism for hydrogen's protective effects. Counteracting proinflammatory effects, the enzyme HO-1 has been described as a key anti-inflammatory brake (20, 21). HO-1 protein was significantly upregulated in prereperfusion full-thickness specimens after 3 hr of cold ischemia time in the intestines preserved in hydrogen-enriched LR relative to both sham specimens and intestines preserved with nitrogen-bubbled LR (Fig. 4D). Hydrogen-induced HO-1 mRNA upregulation was specifically detected within the intestinal muscularis subjected to cold ischemia but not the mucosa (n=5; Fig. 4C).

Solvated Hydrogen Improved Small Intestine Recipient Survival After Extended Cold Ischemia Time in UW Solution

Recipient survival is an ultimate evaluation for intestinal graft function. After using hydrogen-enriched LR for mechanistic investigations, we attempted to ensure that dissolved hydrogen has similar effects when used in a different solvent, a necessity before future translation into the clinical practice. Moreover, a prolonged cold ischemia time of 6 and more hours is reality in human isolated intestinal transplantation. Hence, we conducted additional experiments looking at survival after extended cold ischemia time with graft preservation in UW solution (Viaspan), the current clinical gold standard (1).

No mortality occurred in neither hydrogen nor nitrogen transplant group when recipients received intestinal grafts after a cold ischemia time of 3 hr in LR with the exception of technical failures (<5%). However, the extension of cold ischemia time to 6 hr resulted in high mortality of up to 59% in the groups receiving intestinal grafts preserved with air-bubbled UW or nitrogen-bubbled UW, which was significantly different to the survival rate of 80% in recipients of grafts treated with hydrogen-bubbled UW (n=12). The improved recipient survival was associated with diminished upregulation of muscularis IL-1ß and IL-6 mRNA in UWH2 grafts at 3 hr after reperfusion similar to preservation with hydrogen-enriched LR (n=5, Fig. 5B). Importantly, hydrogen's ability to induce prereperfusion HO-1 protein in intestinal to-be grafts did not differ between experimental short-term cold storage in LR and clinical extended cold storage in UW solution (Fig. 5C).

FIGURE 5.
FIGURE 5.:
Hydrogen mediates similar anti-inflammatory effects in intestinal grafts after prolonged cold storage in University of Wisconsin (UW) solution. Intestinal graft preservation for 6 hr in air-bubbled UW solution resulted in overall recipient survival of 41% (5/12). Nitrogen-aerated UW did not alter recipient survival, but ex vivo hydrogen preloading of grafts significantly improved animal survival to 71.4% (10/12). (A) Recipient animals were followed up for >14 days (*P<0.05 vs. SITxUW air, #P<0.05 vs. SITxUW H2). (B) Hydrogen significantly reduced upregulation of graft muscularis IL-1ß and IL6 (n=5, *P<0.05 vs. sham, #P<0.05 vs. SITxUW H2). Hydrogen's ability to induce prereperfusion heme oxygenase-1 (HO-1) protein in full-thickness specimens of intestinal to-be grafts (C) was also observed after extended cold storage in UW solution.

DISCUSSION

Recently, inhalation of 2% hydrogen gas has been therapeutically used in rodent lung, cardiac, and intestinal transplantation studies demonstrating great potential to protect the graft and the host by its potent antioxidant, antiapoptotic and anti-inflammatory properties (13, 14, 22, 23). From a practical standpoint of effectively translating bench science to clinical practice, solvated hydrogen is safer to use and transport compared with hydrogen gas. Moreover, ex vivo treatment of the isolated harvested organ gives rise to immensely different ethical and safety considerations compared with whole body donor/recipient treatments. Importantly, we herein demonstrate that simple ex vivo hydrogen preloading of intestinal grafts was significantly protective. Observations during the early phase of I/R injury including the anti-inflammatory mechanism of prereperfusion HO-1 induction, reduced oxidative stress with protection of mucosal morphology and blunted inflammation in the intestinal graft were reflected by improved graft motility and recipient gastric emptying as well as diminished circulating markers of inflammation and cell damage during the late phase of I/R injury when intestinal grafts were treated ex vivo with solvated hydrogen, ultimately resulting in significantly improved recipient survival.

Graft reoxygenation during reperfusion generates a burst of cytotoxic reactive oxygen species, which indiscriminately cause peroxidation of membrane lipids, oxidation of DNA, and denaturation of proteins detrimentally impacting short- and long-term graft function (24, 25). Mechanistically, hydrogen is known to diminish graft oxidative damage as indicated by lower tissue MDA levels, a marker of lipid peroxidation, in hydrogen-preserved grafts (14, 26). Yet, recent evidence indicates that hydrogen's cytoprotection is not only restricted to selective hydroxyl radical neutralization but also targets signaling elements in both the extrinsic and intrinsic apoptotic pathways resulting in antiapoptotic properties (22).

Importantly, along with nitric oxide, carbon monoxide, and hydrogen sulfide, hydrogen is now considered to be the fourth endogenous gaseous signaling molecule based on its ability to modulate signal transduction of the FcεRI (high affinity IgE receptor)-associated Lyn pathway, which prevents a degranulated mast cell immediate-type allergic reaction (13, 15). Mast cells are recruited into the gut muscularis in response to gut manipulation (27), and numerous studies have addressed the causative role of mast cells in postoperative ileus and the beneficial effect of mast cell stabilization on postoperative dysmotility and gastroparesis (28, 29). Hence, hydrogen-mediated prevention of mast cell degranulation could be a particularly beneficial mechanism in intestinal transplantation.

In this study, we focused on the anti-inflammatory effects of hydrogen and additionally demonstrate that HO-1 upregulation might mechanistically aid in protecting gastrointestinal motility. Inducible by hemin, HO-1 degrades heme to free iron, biliverdin, and carbon monoxide, with the latter two molecules known to play a significant role in protection against I/R generated oxidative stress in the small bowel (30–33). In the current study, HO-1 protein levels were significantly increased in hydrogen-treated intestinal full-thickness specimens before reperfusion independent of hydrogen's carrier solution and cold ischemia time, but the upregulation seemed to be limited to the muscularis compartment as indicated by the molecular studies, probably due to cell-type specific contributions. Additional assessment of the early proinflammatory molecular response within the reperfused graft muscularis strengthened the concept for a beneficial role of exogenous hydrogen supplementation in intestinal transplantation. Specifically, we observed a significant amelioration of the proinflammatory genes EGR-1, IL6, IL-1ß, and iNOS indicating hydrogen's ability to maintain immune homeostasis. Interestingly, innate immunity can drive the prorejection capacity of the host, for example, IL-6 regulates differentiation of naïve T cells toward the Th17 phenotype whereas simultaneously decreasing regulatory T-cell differentiation/expansion (34). Hence, amelioration of inflammation by improved graft preservation may additionally decrease allograft antigenicity.

Hydrogen preloading of the graft also had beneficial functional consequences analogous to inhaled hydrogen gas (14) by preventing the significant transplantation-induced suppression of in vitro muscarinic contractile activity at 24 hr after reperfusion. However, in vivo gastrointestinal transit was delayed in both transplanted groups in this study. On one hand, secretory diarrhea during mucosal regeneration with villi shedding and goblet cell hyperplasia might have resulted in a faster intestinal transit time in recipients of nitrogen-treated grafts despite severe graft muscularis dysfunction. On the other hand, exogenous supplementation of hydrogen showed favorable outcome but was less powerful in preserving in vivo gastrointestinal transit compared with the previously shown significantly improved transit after hydrogen inhalation (14). Coordinated in vivo motility of the extrinsically denervated intestinal graft is not only determined by smooth muscle contractile function but also by the intrinsic enteric nervous system and the network of interstitial cells of Cajal. The neuromuscular junction is exquisitely sensitive to ischemia with functional impairment observed in the human intestinal transplant (4). It is known that hydrogen is optimally effective when it is present during the reperfusion period (26), and the intestinal graft neurons might be more sufficiently and homogenously reached by sustained hydrogen inhalation that is distributed by the microvasculature rather than by ex vivo hydrogen preloading.

Solid gastric emptying is particularly vulnerable to injury because it requires complex coordinated antral peristalsis and pyloric relaxation, as opposed to the less complex tonic mechanisms of liquid emptying. In nitrogen-treated intestinal graft recipients, solid gastric emptying was strikingly almost completely halted during the late phase of I/R injury. Interestingly, exogenous IL-6 has been shown to exhibit both anorexigenic and gastroparetic effects (35). In the present study, the high systemic levels of IL-6 did correlate with delayed gastric emptying, and both parameters were improved by hydrogen treatment.

Preservation of both motility and mucosal barrier function are critical because they bidirectionally impact each other. SIRS-associated gastrointestinal dysmotility was shown to be associated with a higher incidence of gut-derived bacteremia and a high frequency of septic mortality (36). Specifically, in the immunocompromised intestinal transplant patient, scenarios associated with severe mucosal barrier failure like a prolonged cold ischemia time are reported to correlate with an increased prevalence of bacterial translocation (37). Consequently, the septic inflammatory response drives nonobstructive ileus (38). We have not specifically assessed bacterial translocation here but hydrogen effectively diminished cell damage as indicated by lessened graft tissue lipid peroxidation 3 hr after reperfusion and reduced systemic LDH liberation, reflecting the cumulative cell damage over the 24 hr posttransplant period. Furthermore, morphology of the mucosal monolayer, which forms an integral structural part of the mucosal barrier together along with the secretion of defensins and IgA-rich mucous, was favorably preserved by hydrogen treatment which might result from both reduced cytotoxicity and maintained epithelial cell proliferation. Importantly, mucosal morphology of intestinal grafts correlates with barrier function (14).

In summary, the utilization of a hydrogen-enriched preservation solution proved to be an effective tool to significantly ameliorate graft damage with preservation of graft function and protect the recipient from the systemic effects of transplantation, both ultimately facilitating recipient survival. Mechanistically, hydrogen appreciably reduced graft oxidative stress, maintained immune homeostasis by HO-1 induction and limited proinflammatory molecular responses. Hence, hydrogen-enriched LR or UW is a reductive and anti-inflammatory solution, and its clinical utilization for organ preservation seems feasible.

MATERIALS AND METHODS

Animals

Inbred male LEW (RT.1l) rats weighing 200 to 250 g were supplied from Harlan Sprague Dawley, Inc. (Indianapolis, IN) and housed in a laminar flow animal facility at the University of Pittsburgh on a standard diet and water supplied ad libitum. All procedures were performed in accordance with IACUC guidelines at the University of Pittsburgh.

Transplant Procedure and Hydrogen Treatment

Isolated orthotopic isogeneic small bowel transplantation with caval venous drainage was performed as detailed elsewhere (39). LR was bubbled for 5 min with 100% nitrogen (SITxN2) or 100% hydrogen (SITxH2) (Praxair, Danbury, CT) through an aerator and used for ex vivo graft vascular flush (5 mL), luminal irrigation (20 mL, additionally containing 0.5% neomycin-sulfate [Sigma]) and extraluminal preservation solution in a hermetically sealed sterile container during 3-hr cold ischemia time. Sham treatment consisted of laparotomy only.

In separate survival experiments, air-, nitrogen-, or hydrogen-bubbled UW was used for vascular flush, luminal irrigation, and storage of intestinal grafts with an extended cold ischemia time of 6 hr before isogeneic transplantation. In these animals, approximately 2 cm of graft terminal ileum was taken 3 hr after reperfusion for molecular analysis and alimentary continuation was restored thereafter. Containers with bubbled LR or UW but without grafts were used for serial measurements of hydrogen saturation, oxygen concentration, and pH.

Histopathological Analysis

Three hours after reperfusion, three intestinal segments of each individual recipient were fixed in 10% buffered formalin, embedded in paraffin, and 4-μm thick sections were stained with hematoxylin-eosin. The degree of mucosal injury was microscopically evaluated in a blinded fashion by a 0 to 9 summary score at 200× magnification: grade 0 equaled to healthy mucosa whereas erosions, submucosal edema, and hemorrhage each were rated by grades 1 to 3.

Assessment of Graft Lipid Peroxidation

Tissue MDA levels of lumen flushed jejunal full-thickness graft segments harvested 3 hr after reperfusion were determined according to the manufacturer's recommendations (Kit MDA-586; Oxidresearch, Portland, OR) (14).

Assessment of Systemic Inflammation and Cell Integrity

Serum was obtained from heparinized caval blood drawn 24 hr after reperfusion by cold centrifugation. Serum IL-6 was detected by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's recommendations (R&D systems, Minneapolis, MN). Serum NO levels were indirectly quantified using the Griess reagent (18) and serum LDH was measured using an autoanalyzer (Beckman Instruments, Fullerton, CA).

Gastrointestinal Transit, Recipient Gastric Emptying, and Graft Jejunal Smooth Muscle Contractility

Functional analysis was based on three concerted methods within the same rodent providing information about the severity and site of dysmotility at 24 hr after reperfusion. In vivo gastrointestinal transit of orally administered liquid FITC-dextran was assessed by its gastrointestinal distribution after 90 min transit time, and in vitro contractility was measured from bethanechol-elicited jejunal circular smooth muscle mechanical activity as described previously (14). Additionally, recipients were orally administered 10 chrome steel balls (1-mm diameter, Small Parts Inc.) by gavage 4 hr before organ harvest. The recipient stomach and the donor intestine were visually inspected for the distribution of the chrome steel balls and gastric emptying was calculated by [1−(balls within stomach/10)]×100.

Quantitative Real-Time RT-PCR

Total RNA was isolated from small bowel mucosa/submucosa or muscularis externa of sham animals and intestinal grafts before reperfusion or 3 hr after graft reperfusion. mRNAs for glyceraldehyde 3-phosphate dehydrogenase, EGR-1, HO-1, iNOS, IL-1β, and IL-6 were quantified in duplicate using SYBR Green two-step, real-time RT-PCR (see Table, SDC 2,https://links.lww.com/TP/A504, which lists the target-specific primer sequences) (40).

Western Blot Analysis

Western blot analysis was performed on 30 μg of whole cell protein from full-thickness intestinal tissue, obtained from intestinal grafts to-be before reperfusion. Nonspecific binding was blocked with nonfat dry milk before overnight incubation with the primary antibodies anti-HO-1 (Assay Designs, Plymouth Meeting, PA) and anti-ß-actin (Sigma-Aldrich, St. Louis, MO). Membranes were developed with the SuperSignal detection system (Pierce Chemical, Rockford, IL) after incubation with horseradish-peroxidase conjugated goat anti-mouse secondary antibody (Pierce Chemical).

Data Analysis

The results are expressed as mean±standard error of the mean (SEM). Statistical analysis was performed using the nonparametric Kruskal-Wallis test or repeated measures of one-way analysis of variance where appropriate. EZAnalyze add-in for Microsoft Excel was used to perform the F-test with Bonferroni post hoc group comparisons. For the survival study, Kaplan-Meier curves and log-rank test were performed. A probability level of P less than 0.05 was considered statistically significant.

ACKNOWLEDGMENT

The authors appreciate the technical assistance from Lisa Chedwick for histopathological staining.

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Keywords:

Oxidative stress; Inflammation; Preservation solution; Heme oxygenase-1; Gastrointestinal motility

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