Creatine-loading preserves intestinal barrier function during organ preservation.

We have developed a novel, intraluminal preservation solution that is tailored to the metabolic requirements of the intestine. This organ-specific solution addresses many of the problems associated with low temperature organ storage including energy, oxidative and osmotic stresses. However, conservation of energy levels remains one of the most difficult obstacles to overcome due to the inherent sensitivity of the mucosa to ischemia. Creatine-loading has become a popular and scientifically proven method of augmenting energy reserves in athletes performing anaerobic burst work activities. We hypothesized that if we could develop a method that was able to augment cellular energy levels, the structure and function of the mucosa would be more effectively preserved. The purpose of this study was to determine if creatine-loading is a feasible and effective strategy for preserving the intestine. Our data indicate that creatine loading has significant impact on energy levels during storage with corresponding improvements in mucosal structure and function. Both of our rodent models, a) continuous perfusion for 4 h and b) a single flush with our intraluminal preservation solution supplemented with 50 mM creatine, demonstrated significant improvements in creatine phosphate, ATP, Energy Charge and ATP/AMP following cold storage (P < 0.05). Notably, after 10 h creatine phosphate was 324% greater in Creatine-treated tissues compared to Controls (P < 0.05). Preferential utilization of glutathione in the Creatine group was effective at controlling oxidative injury after 10 h storage (P < 0.05). Improvements in barrier function and electrophysiology with creatine-treatment reflected superior mucosal integrity after 10 h storage; Permeability and Transepithelial resistance measurements remained at fresh tissue values. This was in stark contrast to Control tissues in which permeability rose to >300% of fresh tissue values (P < 0.005) and transepithelial resistance dropped by 95% (P < 0.005). After 10 h storage, Park's grading of histologic injury reflected extensive villus denudation (grade 4) in control tissues compared to healthy tissue (grade 0) in the Creatine group. This study demonstrates that a strategy of creatine supplementation of our intraluminal preservation solution facilitates the preservation of the intestinal mucosa during storage.

Redefining the properties of an osmotic agent in an intestinal-specific preservation solution.

AIM: To investigate the effects of dextrans of various molecular weights (Mw) during a 12 h cold storage time-course on energetics, histology and mucosal infiltration of fluorescein isothiocyanate (FITC)-dextran. METHODS: Rodent intestines were isolated and received a standard University of Wisconsin vascular flush followed by intraluminal administration of a nutrient-rich preservation solution containing dextrans of varying Mw: Group D1, 73 kdal; Group D2, 276 kdal; Group D3, 534 kdal; Group D4, 1185 kdal; Group D5, 2400 kdal. RESULTS: Using FITC-labeled dextrans, fluorescent micrographs demonstrated varying degrees of mucosal infiltration; lower Mw (groups D1-D3: 73-534 kdal) dextrans penetrated the mucosa as early as 2 h, whereas the largest dextran (D5: 2400 kdal) remained captive within the lumen and exhibited no permeability even after 12 h. After 12 h, median injury grades ranged from 6.5 to 7.5 in groups D1-D4 (73-1185 kdal) representing injury of the regenerative cryptal regions and submucosa; this was in contrast to group D5 (2400 kdal) which exhibited villus denudation (with intact crypts) corresponding to a median injury grade of 4 (P < 0.05). Analysis of tissue energetics reflected a strong positive correlation between Mw and adenosine triphosphate (r(2) = 0.809), total adenylates (r(2) = 0.865) and energy charge (r(2) = 0.667). CONCLUSION: Our data indicate that dextrans of Mw > 2400 kdal act as true impermeant agents during 12 h ischemic storage when incorporated into an intraluminal preservation solution.

Comparing the effects of Dextran 70 and Hydroxyethyl starch in an intestinal storage solution.

INTRODUCTION: Our lab has developed a novel strategy for intestinal preservation involving the intraluminal delivery of a nutrient-rich preservation solution. The aim of this study was to compare the effectiveness of two impermeant agents for use in our solution: Dextran 70 (D70; Mw=70 kDa) and Hydroxyethyl starch (HES; Mw=2200 kDa). METHODS: Rat intestines were procured, including an intravascular flush with University of Wisconsin solution followed by a 'backtable' intraluminal flush with: UW solution (group 1, UW), or an amino acid-based nutrient-rich preservation solution (AA solution) containing either 5% D70 (group 2, AA-D70) or HES (group 3, AA-HES). Tissue samples (n=6) were taken at 2, 4, 8, and 12 h cold storage; histology, energetic, end-product, and oxidative parameters were assessed. In separate groups (n=4), D70 and HES were fluorescently labeled with fluorescein isothiocyanate (FITC) in order to directly observe mucosal penetration of the starch and dextran. RESULTS: Over the 12 h storage time-course, direct visualization of the fluorescently labeled D70 showed penetration of the mucosal layer as early as 2 h and progressively continued to do so throughout the 12 h period. In contrast, HES did not cross the mucosal barrier and remained captive within the lumen. As time of storage progressed, grade of injury increased in all groups, however, at 4 and 12 h the AA-HES treated tissues exhibited significantly less injury compared to UW and AA-D70, P < 0.05. AA-HES group showed on moderate villus clefting (median grade 2; P < 0.05) while the AA-D70 group exhibited complete villus denudation (grade 4) and the UW group had extensive injury into the regenerative cryptal regions (grade 6). Metabolic parameters revealed a preferential maintenance of ATP and Energy Charge; increases in lactate, alanine and ammonium supported the involvement of aerobic and anaerobic pathways for energy production. CONCLUSION: The results of this study challenge the idea that oncotic support is not a fundamental requirement of static organ storage. Furthermore, our data suggests that HES is an effective oncotic agent for use in our intraluminal nutrient-rich preservation solution, while Dextran 70 is not.

Optimizing the concentration of hydroxyethylstarch in a novel intestinal-specific preservation solution.

INTRODUCTION: Our lab has developed an effective nutrient-rich solution that facilitates energy production and control of oxidative stress during static cold storage of the intestine; however, the requirement for oncotic agents, such as hydroxyethylstarch (HES), has not been evaluated. This study investigated the effectiveness and requirement for HES in an intraluminal preservation solution during a clinically relevant period of cold storage. METHODS: Rat intestines were procured, including an intravascular flush with University of Wisconsin solution followed by a 'back table' intraluminal flush with a nutrient-rich preservation solution containing varying amounts of HES (n=6 per group): Group 1, 0%; Group 2, 2.5%; Group 3, 5%; Group 4, 10%. Energetics, oxidative stress, and morphology were assessed over a 24h time-course of cold storage. RESULTS: Overall, the 5% HES solution, Group 3, demonstrated superior energetic status (ATP and total adenylates) compared to all groups, P<0.05. Malondialdehyde levels indicated a reduction in oxidative stress in Groups 3 and 4 (P<0.05). After 12h, median modified Parks' grades for Groups 2 and 3 were significantly lower than Groups 1 and 4, P<0.05. CONCLUSION: Our data suggests that when employing an intraluminal preservation solution for static organ storage, oncotic support is a fundamental requirement; 5% HES is optimal.