Alleviating intestinal ischemia-reperfusion injury in an in vivo large animal model: developing an organ-specific preservation solution.

INTRODUCTION: This study investigated the role of a novel nutrient-rich preservation solution in alleviating intestinal ischemia-reperfusion (IR) injury in a large animal model. MATERIALS AND METHODS: Porcine intestines were treated in vivo with the following intraluminal flush solutions: group 1, none; group 2, University of Wisconsin solution; group 3, an amino acid-based solution, previously shown to be effective in reducing IR injury in rodent models. Intestinal ischemia was induced in vivo for 60 min, followed by 180 min reperfusion. Key metabolic aspects were assessed in relation to two fundamental kinase mechanisms that govern cell fate, AMP kinase, and Jun kinase. RESULTS: After 180 min reperfusion, groups 1 and 2 exhibited clefting, denudation, and mucosal hemorrhage, whereas injury was markedly reduced in group 3 (median grades 4.5 and 5 vs. 0; P<0.05). In contrast to groups 1 and 2, group 3 tissues exhibited a full recovery of adenylates (ATP, total adenylates) and an effective control of oxidative stress throughout reperfusion. Neutrophil-mediated inflammation was abrogated in group 3. An up-regulation of two key enzymes (glutaminase and alanine aminotransferase) provided a mechanism for the superior recovery of energetics and the preservation of mucosal integrity in group 3. A strong activation of AMP-activated protein kinase resulting in the up-regulation of a primary proapoptotic kinase mechanism, Jun kinase, was evident in groups 1 and 2. DISCUSSION: A strategy of intraluminal administration of a nutrient-rich solution represents a potential therapy for alleviating intestinal IR injury; these findings suggest a more effective method for the ischemic storage of intestine.

Dose-response effects of milrinone on hemodynamics of newborn pigs with hypoxia-reoxygenation.

OBJECTIVE: Neonatal asphyxia causes cardiogenic shock and pulmonary hypertension with decreased brain perfusion. We examined the dose-response of milrinone on systemic, pulmonary, and carotid circulations in a model of neonatal hypoxia-reoxygenation. DESIGN AND SETTING: Controlled, block-randomized study in a university research laboratory. SUBJECTS: Mixed breed piglets (1-3 days, 1.5-2.3 kg). INTERVENTIONS: In acutely instrumented piglets normocapnic alveolar hypoxia (10-15% oxygen) was induced for 2 h followed by reoxygenation with 100% oxygen (1 h) then 21% oxygen (3 h). At 2 h of reoxygenation after a volume loading (10 ml/kg) either saline or milrinone (bolus and infusion at 0.25, 0.5, or 0.75 microg/kg per minute) was given for 2 h in a blinded-randomized fashion (n = 7/group). MEASUREMENTS AND RESULTS: All milrinone-treated groups had higher cardiac output and stroke volume than those of saline-treated hypoxic controls, which showed progressive decline in these measurements. At 2 h of infusion plasma milrinone levels were significantly correlated with cardiac output (r = 0.6), which increased from pretreatment value in the group receiving 0.75 microg/kg per minute. Milrinone maintained mean arterial pressure; heart rate and pulmonary arterial pressure did not differ between groups. Milrinone prevented continued increases in systemic and pulmonary vascular resistances after hypoxia-reoxygenation. Milrinone infusion at higher doses increased common carotid flow. Milrinone-treated piglets had increased systemic and carotid oxygen delivery, with no difference in plasma and myocardial lactate levels among groups. CONCLUSIONS: When used to treat shock in newborn piglets with hypoxia-reoxygenation, milrinone improved cardiac output and carotid flow while maintaining systemic blood pressure. Pulmonary hypertension was not aggravated. Further studies are needed to confirm these findings in asphyxiated neonates.

Cardio-renal recovery of hypoxic newborn pigs after 18%, 21% and 100% reoxygenation.

OBJECTIVES: We examined the effects of 18%, 21% or 100% oxygen on the recovery of the heart and kidneys in a short-term survival model of neonatal hypoxia-reoxygenation (HR). DESIGN: Controlled, block-randomized animal study. SETTING: University animal research laboratory. SUBJECT: Large White piglets (1-3 days, 1.7-2.5 kg). INTERVENTIONS: Piglets received normocapnic hypoxia (15% oxygen) (2 h) and were reoxygenated with 18%, 21% or 100% oxygen (1 h) (n=7 per group) then 21% oxygen (2 h). Sham-operated pigs (n=7) had no HR. MEASUREMENTS AND RESULTS: Seventeen of 21 HR piglets recovered from moderate hypoxemia (mean PaO(2) 27-33 mmHg and pH 7.20-7.22, associated with tachycardia and hypotension). Systemic arterial pressure, heart rate, left renal arterial flow, oxygen transport, plasma troponin-I and creatinine levels were monitored and recovered with no differences among HR groups over 4 days after resuscitation. The 100% group had increased myocardial oxidative stress (oxidized glutathione levels) and the most cardiac HR-induced injury. There were no differences in renal oxidative stress and HR-induced injury among groups. Early oxygenation at 1 h after resuscitation correlated with the plasma troponin-I level at 6 h (r = -0.51 and 0.64 for SaO(2) and systemic oxygen extraction ratio, p<0.05, respectively) and renal HR-induced injury at 4 days (r =0.61 for renal oxygen delivery, p<0.05). CONCLUSIONS: In hypoxic piglets, 18%, 21% and 100% reoxygenation caused similar systemic and renal hemodynamic and functional recovery. The indicators of oxidative stress and HR injury in myocardial and renal tissues suggest that the reoxygenation with 100% oxygen appears sub-optimal and the use of 18% oxygen offers no further benefit, when compared with 21% oxygen.

N-acetylcysteine improves the hemodynamics and oxidative stress in hypoxic newborn pigs reoxygenated with 100% oxygen.

Neonatal asphyxia may lead to cardiac and renal complications perhaps mediated by oxygen free radicals. Using a model of neonatal hypoxia-reoxygenation, we tested the hypothesis that N-acetylcysteine (NAC) would improve cardiac function and renal blood flow. Eighteen piglets (aged 1-4 days old, weighing 1.4-2.2 kg) were anesthetized and acutely instrumented for continuous monitoring of pulmonary and renal artery flow (cardiac index [CI] and renal artery flow index [RAFI], respectively) and mean blood pressure. Alveolar hypoxia was induced for 2 h, followed by resuscitation with 100% oxygen for 1 h and 21% oxygen for 3 h. Animals were randomized to sham-operated, hypoxic control, and NAC treatment (i.v. bolus of 150 mg/kg given at 10 min of reoxygenation followed by 100 mg/kg per h infusion) groups. Myocardial and renal tissue glutathione content and lipid hydroperoxide levels were assayed, and histology was examined. After 2 h of hypoxia, all animals were acidotic (pH 6.96 +/- 0.04) and in cardiogenic shock with depressed renal blood flow. Upon reoxygenation, CI and RAFI increased but gradually deteriorated later. The NAC treatment prevented the decreased CI, stroke volume, mean blood pressure, systemic oxygen delivery, RAFI, and renal oxygen delivery at 2 to 4 h of reoxygenation observed in hypoxic controls (versus shams, all P < 0.05). The myocardial and renal tissue glutathione content was significantly higher in the NAC treatment group (versus controls). The CI and RAFI at 4 h of reoxygenation correlated with the tissue glutathione redox ratio (r = 0.5 and 0.6, respectively, P < 0.05). There were no significant differences in heart rate, pulmonary artery pressure, systemic oxygen uptake, and tissue lipid hydroperoxide levels between groups. No histologic injury was found in the heart or kidney. In this porcine model of neonatal hypoxia and 100% reoxygenation, NAC improved cardiac function and renal perfusion, with improved tissue glutathione content.

Relationship between energetic stress and pro-apoptotic/cytoprotective kinase mechanisms in intestinal preservation.

BACKGROUND: A recent study from our laboratory documented significant improvements in post-transplant viability in an experimental model of intestinal transplantation when a novel, nutrient-rich preservation solution was used during cold storage. The current study investigated the relationship between energetic/oxidative stress responses and fundamental kinase signaling events during the period of organ storage. This relationship may be a key factor contributing to improved graft viability after storage in a nutrient-rich preservation solution. METHODS: Rat small intestine was harvested and flushed intraluminally with University of Wisconsin (UW) solution or an amino acid-rich (AA) solution as follows: Group 1, no luminal flush (clinical control); Group 2, luminal UW solution; Group 3, luminal AA solution. Energetics (ATP, total adenylates), oxidative stress (malondialdehyde), histology, and MAPK (P38, JNK, ERK)/AMPK/Caspase-3 were assessed throughout 12-hour cold storage. RESULTS: P38 and JNK were upregulated strongly in Group 2 after 1- and 12-hour storage. Group 3 exhibited a delayed activation and subsequent downregulation of these pre-apoptotic signals. Between 6 to 12 hours, a strong upregulation of ERK was observed in Group 3. AMPK downregulation correlated with a reduction in AMP/ATP ratio, ERK upregulation, and P38/JNK downregulation in Group 3. After 12-hour storage, histology indicated superior preservation of mucosal architecture in Group 3 tissues. CONCLUSIONS: A nutrient-rich preservation solution abrogates pre-apoptotic signaling (JNK and P38) and upregulates cytoprotective signals (ERK). Our data support the concept of a concerted effort facilitating cellular protection in response to ischemic stress.

Intestinal decontamination using povidone-iodine compromises small bowel storage quality.

BACKGROUND: Povidone-iodine (PI) (Betadine, Purdue Fredrick Co., Norwalk, CT) is used during pancreas transplantation to provide duodenal decontamination without any intestinal complications. METHODS: This study assessed the combined effect of a luminal pretreatment with PI followed by a proven amino acid (AA)-rich preservation solution on small bowel (SB) storage quality. Rat SB was procured using intra-arterial University of Wisconsin solution followed by luminal administration with combinations of AA+/-PI pretreatment as follows: group 1, 20 mL AA solution; group 2, 20 mL PI and 20 mL AA solution; group 3, 100 mL AA solution; and group 4, 20 mL PI and 100 mL AA solution. Cellular energetics, permeability, and histology were analyzed throughout 10-hr cold storage. RESULTS: After 10 hr, adenosine triphosphate levels remained 2.9 to 3.2 micromol/g protein lower in the PI-treated groups than in groups 1 and 3. Functional parameters were also negatively affected by PI treatment. Mannitol permeability was 203 and 66 nmol/cm2 per hr in groups 2 and 4 versus 46 and 44 nmol/cm2 per hr in groups 1 and 3. Median grades of histologic injury (Park's) at 10 hr were 3, 5.5, 4, and 5 for groups 1 through 4, respectively. Luminal flushing with PI or AA solution resulted in an equivalent clearance greater than 95% of enteric bacteria present. CONCLUSIONS: hort-duration luminal PI exposure potentiates SB injury and provides no improved intestinal decontamination.

Human small bowel storage: the role for luminal preservation solutions.

BACKGROUND: Graft injury incurred during periods of cold storage remains a factor affecting the success of small bowel (SB) transplantation. No one preservation solution, including the gold standard University of Wisconsin (UW) solution, has been able to maintain graft integrity for storage periods paralleling that of other commonly transplanted intra-abdominal organs. We investigated the role for the luminal administration of preservation solutions in a small animal model, documenting significantly improved graft quality. The current study addresses direct clinical applicability using human SB. METHODS: Human SB was obtained at the time of standard multiviscera procurement. After a common intra-arterial UW flush, the SB was immediately removed from the abdomen, randomly divided into three segments, and treated as follows (n=6-9): group 1, no luminal flush; group 2, luminal flush with UW solution; and group 3, luminal flush with an amino acid- enriched solution. Analysis of cellular energetics, permeability, and histologic injury was performed throughout 24 hr of cold storage. RESULTS: Mucosal barrier function, measured by mannitol permeability, was significantly better overall in groups 2 and 3, with 24-hr values measuring 31 and 34 nmol/cm2/hr versus 57 nmol/cm2/hr, respectively (both P<0.05). Significantly less morphologic injury was also noted in the luminally treated specimens (groups 2 and 3) compared with the clinical standard (vascular flush with UW solution). Damage in group 1 reached gross villus denudation with an obvious elevated risk of villus tissue loss, whereas groups 2 and 3 only exhibited epithelial clefting to varying degrees. CONCLUSION: This study supports luminal administration of preservation solutions for improvement of human SB graft quality during clinically relevant periods of cold storage.

Potentiating the benefit of vascular-supplied glutamine during small bowel storage: importance of buffering agent.

BACKGROUND: Glutamine (gln)-supplemented University of Wisconsin (UW) solution improves overall small bowel (SB) preservation. Sustained gln metabolism in a system devoid of hepatic detoxification will necessarily result in the accumulation of pH active end products leading to nonphysiologic pH shifts. We hypothesized that simultaneous addition of N,N-bis[2-hydroxyethyl]-2-aminoethane sulfonic acid (BES), a known buffering agent, would potentiate the beneficial effect of gln supplementation by addressing the fundamental metabolic principle of pH homeostasis. METHODS: Sprague-Dawley SB rats were administered a vascular flush with one of four solutions: UW; UW+90 mM BES (UWB); UW+2% gln (UWG); or UW+2% gln+90 mM BES (UWBG). Indices of energetics, barrier function, gln catabolism, and histology (light and electron microscopy) were assessed over a 10-hr cold storage time course. RESULTS: Superior gln utilization in the UWBG group was indicated by elevated levels of key catabolites (glutamate, aspartate, glycine, ammonia). The addition of BES and gln resulted in significantly higher levels of all energetic parameters (ATP, total adenylates) at 10 hr compared with UW, UWB, and/or UWG. Barrier function was markedly improved after 10 hr storage in the UWBG group; mannitol permeability was 169 nmol/cm2/hr versus 572 and 445 nmol/cm(2)/hr (for UW and UWG, respectively). Histologic injury at 10 hr was 5.5, 7.5, and 8 (Park's grade) for UWBG, UWG, and UW. Ultrastructural damage was markedly reduced with UWBG, as assessed by grade of mitochondria damage. CONCLUSION: This study strongly supports that the beneficial effects of gln-enriched UW solution can be amplified when combined with an effective buffering agent such as BES.

Milrinone, dobutamine or epinephrine use in asphyxiated newborn pigs resuscitated with 100% oxygen.

BACKGROUND: After resuscitation, asphyxiated neonates often develop poor cardiac function with hypotension, pulmonary hypertension and multiorgan ischemia. In a swine model of neonatal hypoxia-reoxygenation, effects of epinephrine, dobutamine and milrinone on systemic, pulmonary and regional hemodynamics and oxygen transport were compared. DESIGN: Controlled, block-randomized study. SETTING: University research laboratory. SUBJECT: Mixed breed piglets (1-3 days, 1.5-2.3 kg). INTERVENTIONS: In acutely instrumented piglets, normocapnic alveolar hypoxia (10-15% oxygen) was induced for 2 h followed by reoxygenation with 100% oxygen (1 h) then 21% oxygen (3 h). At 2 h of reoxygenation, after volume loading (Ringer's lactate 10 ml/kg), either saline (placebo), epinephrine (0.5 microg/kg/min), dobutamine (20 microg/kg/min) or milrinone (0.75 microg/kg/min) were infused for 2 h in a blinded, block-randomized fashion (n = 6/group). MAIN RESULTS: All medications similarly improved cardiac output, stroke volume and systemic oxygen delivery (vs. placebo-controls, p < 0.05). Epinephrine and dobutamine significantly increased, while milrinone maintained, mean arterial pressure over pretreatment values while placebo-treated piglets developed hypotension and shock. The mean arterial to pulmonary arterial pressures ratio was not different among groups. All medications significantly increased carotid and intestinal, but not renal, arterial blood flows and oxygen delivery, whereas milrinone caused lower renal vascular resistance than epinephrine and dobutamine-treated groups. Plasma troponin I, plasma and myocardial lactate levels, and histologic ischemic features were not different among groups. CONCLUSIONS: In newborn piglets with hypoxia-reoxygenation, epinephrine, dobutamine and milrinone are effective inotropes to improve cardiac output, carotid and intestinal perfusion, without aggravating pulmonary hypertension. Milrinone may also improve renal perfusion.

Intestinal hemodynamic effects of milrinone in asphyxiated newborn pigs after reoxygenation with 100% oxygen: a dose-response study.

Neonatal asphyxia can result in poor perfusion, vasoconstriction, and decreased oxygen delivery in the intestine. Milrinone increases myocardial contractility and causes peripheral vasodilatation. We examined the dose-response of milrinone on the intestinal circulation, oxygen metabolism, and injury in a newborn piglet model of asphyxia-reoxygenation. Piglets (aged 1-3 days, weighing 1.5-2.3 kg) were acutely instrumented to measure superior mesenteric artery (SMA) flow and oxygen delivery. After stabilization, hypoxia (inspired oxygen concentration, 0.08-0.15) was induced for 2 h followed by reoxygenation with 100% O2 for 1 h then 21% O2 for 3 h. At 2 h of reoxygenation, saline or milrinone infusion at doses of 0.25, 0.5, or 0.75 microg/kg per min was given for 2 h in a blinded randomized fashion (n = 7 per group). Hemodynamic and oxygen transport parameters were analyzed at predefined time points. Intestinal tissue lactate concentrations, plasma milrinone levels, and intestinal glutathione redox status were determined at the end of the experiment. In the intestinal tract, milrinone significantly increased SMA flow and oxygen delivery while decreasing vascular resistance at a dose of 0.75 microg/kg per min (P < 0.05, ANOVA). A modest increase in SMA flow and oxygen delivery was found with milrinone at 0.5 microg/kg per min. Plasma milrinone levels correlated with SMA flow and vascular resistance (r = 0.5 and r = -0.6, respectively, P < 0.05). Intestinal lactate concentrations and histopathology were not significantly different among groups. Oxidized glutathione correlated with SMA vascular resistance and negatively with milrinone levels (r = 0.6 and r = -0.5, P < 0.05). When used to treat shock in a newborn model of asphyxia-reoxygenation, milrinone dose-dependently increases SMA flow and oxygen delivery with a significantly decreased SMA vascular resistance at higher doses.

Effects of N-acetylcysteine on intestinal reoxygenation injury in hypoxic newborn piglets resuscitated with 100% oxygen.

BACKGROUND: Neonatal asphyxia may lead to the development of ischemia-reperfusion induced intestinal injury, which is related to oxygen-derived free radical production. N-Acetylcysteine (NAC) is a thiol-containing antioxidant which increases intracellular stores of glutathione. OBJECTIVES: Using a swine model of neonatal hypoxia-reoxygenation, we examined whether administration of NAC after resuscitation improved intestinal perfusion and reduced intestinal damage. METHODS: Twenty-four piglets (1-4 days old, 1.4-2.2 kg) were anesthetized and acutely instrumented for continuous monitoring of superior mesenteric arterial flow and oxygen delivery. Alveolar hypoxia was induced for 2 h, followed by resuscitation with 100% oxygen for 1 h and 21% oxygen for 3 h. Animals were randomized to sham-operated, hypoxic control and NAC treatment (150 mg/kg i.v. at 0 or 10 min of reoxygenation followed by infusion 100 mg/kg/h) groups. During hypoxia-reoxygenation, intestinal tissue glutathione content, caspase-3 activity and reoxygenation injury were examined. RESULTS: After 2 h of hypoxia, piglets were acidotic and hypotensive, with significantly depressed blood flow and oxygen delivery to the small intestine. Upon reoxygenation, hemodynamics recovered as did oxygen supply to the small intestine. After 4 h of reoxygenation, the NAC treatment improved mesenteric flow and oxygen delivery. Despite reducing the increase in caspase-3 activities after hypoxia-reoxygenation by NAC treatment, no significant differences in the glutathione content and histological grading of ileal injury were found among the experimental groups. CONCLUSIONS: In newborn piglets with hypoxia-reoxygenation, NAC may improve mesenteric blood flow and oxygen delivery without significant effect on tissue glutathione content. The protective role of NAC in the reoxygenated intestine after severe hypoxia warrants further investigation.

The isolation of nucleic acids from fixed, paraffin-embedded tissues-which methods are useful when?

Museums and pathology collections around the world represent an archive of genetic material to study populations and diseases. For preservation purposes, a large portion of these collections has been fixed in formalin-containing solutions, a treatment that results in cross-linking of biomolecules. Cross-linking not only complicates isolation of nucleic acid but also introduces polymerase "blocks" during PCR. A wide variety of methods exists for the recovery of DNA and RNA from archival tissues, and although a number of previous studies have qualitatively compared the relative merits of the different techniques, very few have undertaken wide scale quantitative comparisons. To help address this issue, we have undertaken a study that investigates the quality of nucleic acids recovered from a test panel of fixed specimens that have been manipulated following a number of the published protocols. These include methods of pre-treating the samples prior to extraction, extraction and nucleic acid purification methods themselves, and a post-extraction enzymatic repair technique. We find that although many of the published methods have distinct positive effects on some characteristics of the nucleic acids, the benefits often come at a cost. In addition, a number of the previously published techniques appear to have no effect at all. Our findings recommend that the extraction methodology adopted should be chosen carefully. Here we provide a quick reference table that can be used to determine appropriate protocols for particular aims.

Multiplex PCR with minisequencing as an effective high-throughput SNP typing method for formalin-fixed tissue.

Extensive collections of formalin-fixed paraffin-embedded (FFPE) tissues exist that could be exploited for genetic analyses in order to provide important insights into the genetic basis of disease or host/pathogen cointeractions. We report here an evaluation of a 44 SNP multiplex genotyping method, multiplex PCR with minisequencing (MPMS), on 92 DNA extractions performed on six archival FFPE samples of variable DNA quality, which date between 9 and 25 years old. On the three extracts with highest quality, we found the assay efficiency to be near 100%. However, the efficiency of the lowest quality extracts varied significantly. In this study, we demonstrate that although direct measures of DNA concentration in the extracts provide no useful information with regard to subsequent MPMS success, the success of the assay can be determined to some degree a priori, through initial screening of the DNA quality using a simple quantitative real-time PCR (qPCR) assay for nuclear DNA, and/or an assay of the maximum PCR amplifiable size of nuclear DNA. MPMS promises to be of significant use in future genetic studies on FFPE material. It provides a streamlined approach for retrieving a large amount of genetic information using simple, single reactions and minute amounts of archival tissue/DNA. In the light of this evidence, we suggest that the systematic screening of FFPE collections may in the future provide valuable insights into the past.

Resuscitation with 100% oxygen causes intestinal glutathione oxidation and reoxygenation injury in asphyxiated newborn piglets.

OBJECTIVE: To compare mesenteric blood flow, oxidative stress, and mucosal injury in piglet small intestine during hypoxemia and reoxygenation with 21%, 50%, or 100% oxygen. SUMMARY BACKGROUND DATA: Necrotizing enterocolitis is a disease whose pathogenesis likely involves hypoxia-reoxygenation and the generation of oxygen-free radicals, which are known to cause intestinal injury. Resuscitation of asphyxiated newborns with 100% oxygen has been shown to increase oxidative stress, as measured by the glutathione redox ratio, and thus may predispose to free radical-mediated tissue injury. METHODS: Newborn piglets subjected to severe hypoxemia for 2 hours were resuscitated with 21%, 50%, or 100% oxygen while superior mesenteric artery (SMA) flow and hemodynamic parameters were continuously measured. Small intestinal tissue samples were analyzed for histologic injury and levels of oxidized and reduced glutathione. RESULTS: SMA blood flow decreased to 34% and mesenteric oxygen delivery decreased to 9% in hypoxemic piglets compared with sham-operated controls. With reoxygenation, SMA blood flow increased to 177%, 157%, and 145% of baseline values in piglets resuscitated with 21%, 50%, and 100% oxygen, respectively. Mesenteric oxygen delivery increased to more than 150% of baseline values in piglets resuscitated with 50% or 100% oxygen, and this correlated significantly with the degree of oxidative stress, as measured by the oxidized-to-reduced glutathione ratio. Two of eight piglets resuscitated with 100% oxygen developed gross and microscopic evidence of pneumatosis intestinalis and severe mucosal injury, while all other piglets were grossly normal. CONCLUSIONS: Resuscitation of hypoxemic newborn piglets with 100% oxygen is associated with an increase in oxygen delivery and oxidative stress, and may be associated with the development of small intestinal hypoxia-reoxygenation injury. Resuscitation of asphyxiated newborns with lower oxygen concentrations may help to decrease the risk of necrotizing enterocolitis.

Defining the role of a tailored luminal solution for small bowel preservation.

The mucosal layer is the initial site of small bowel (SB) graft injury sustained during cold storage. Vascular administration of preservation solutions alone is unable to prevent ischemic injury of this layer during clinically relevant storage periods. The SB is unique in that it possesses both a vascular and a luminal route by which preservation solutions can be administered. We hypothesized that addition of a luminal-delivered solution, formulated on amino acid requirements for energy- and non-energy-related reactions, would provide site-specific preservation of mucosal energetics, barrier function and morphology throughout an extended period of cold storage. Of the three luminal solutions containing amino acids which were tested (UWG, AA1, AA2), only the two groups (AA1, AA2), containing glutamine plus 18 other amino acids, +/- osmotic agent (lactobionate) and buffer (BES), exhibited significant improvements in energetics, barrier function, and histology compared to the clinical standard of isolated vascular University of Wisconsin (UW) solution. Although the AA1 and AA2 groups preserved barrier function and morphology up to 24h better than all other solutions tested, AA2 proved to be the only luminal solution with values of permeability, conductance, and short-circuit current not significantly different from freshly isolated tissues. Furthermore, the greatest reduction in histologic injury was effected by AA2 treatment (median grade 2 compared to control, UW(v), grade 8). This study documents that a luminal-delivered solution, formulated on physiologic SB requirements, provides targeted preservation of the SB mucosa.