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

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 24 h 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 12 h, 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. Keywords Intraluminal preservation solution Intestinal-specific Oncotic/osmotic agents Organ preservation Energetic stress Oxidative stress Osmotic stress Introduction During the last several years, our laboratory has developed an amino acid (AA)-based preservation solution tailored to the metabolic requirements of the small bowel. Numerous in vitro and in vivo studies have demonstrated that luminal administration of this solution results in improved morphology and metabolic status of the small bowel following cold storage, ischemia–reperfusion, and experimental transplantation [26,9,22,27,28] . Although the composition and route of administration differ between our solution and the UW solution, there exists a major commonality: the presence of osmotic impermeants to prevent tissue edema. At a molecular level, under normal circumstances, Na+/K+ ATPase ensures Na+ extrusion from the cell, which results in Na+ functioning as an osmotic agent outside the cell [2] . The extracellular Na+ counteracts the osmotic pressure developed from intracellular proteins and impermeant anions. However, the situation is much different during organ storage. Hypothermia and declining ATP levels collectively suppress Na+/K+ pump activity, causing Na+ and Cl− to follow concentration and electrochemical gradients into the cell. As these ions enter, water follows and the cell swells. To offset the flow of water into the cell, an impermeant agent is required extracellularly; 110–140 mmol/l has been established as the effective concentration range for an intravascular solution [2] . Hydroxyethylstarch (HES), a modified natural polymer of amylopectin [36] , is the colloid contained in UW solution for oncotic support [32] . Although the solution contains HES on the basis of preventing cold storage interstitial edema [32] , there exists some controversy over its effectiveness, especially for static organ storage. Some studies report that HES may be omitted from the UW solution without detrimental effects on organ preservation [38,14,4] , while others indicate a protective role of HES on stored tissues [2,32,1,6,7,20] . Since these studies deal solely with the intravascular delivery of preservation solution, they do not provide information about the role of colloids in solutions administered intraluminally for intestinal grafts. Although our AA solution contains low molecular weight osmotic agents, we suspect that an additional oncotic agent may also be necessary in order to maintain tissue water balance. In a recent study, we compared graft injury between tissues stored with AA solution containing either 70 kDa dextran or high molecular weight HES (2200 kDa) as the oncotic agent [17] . Results indicated that tissues in the HES group exhibited superior energetic status and morphology following cold storage. Nevertheless, it is evident that osmotic stress remains a significant contributing factor to storage injury at the concentration used. The current study tested the hypothesis that there is an optimal concentration of HES that will control osmotic stress during cold storage. Metabolic and morphological parameters of the intestine were assessed over a 24 h time-course in groups receiving a nutrient-rich intraluminal preservation solution supplemented with 0%, 2.5%, 5%, and 10% HES. Materials and methods Summary of experimental design Briefly, small intestine from rats was flushed intravascularly with University of Wisconsin (UW) solution, isolated and flushed intraluminally with a preservation solution which contained varying amounts (0–10%) of hydroxyethylstarch (HES). Intestines were stored at 4 °C and samples were taken over a 24 h time-course. Details Male Sprague–Dawley rats (200–300 g) were obtained from the University of Alberta and used as organ donors. All experiments were conducted in accordance with Canadian Council on Animal Care policies. Surgical procedure and organ procurement Rats were fasted 10–12 h and provided water ad libitum . Anesthesia was induced with pentobarbital (65 mg/250 g; IP), followed by inhalational isoflurane (0.5–2%) to maintain anesthesia. Following a midline laparotomy, the aorta was exposed infrarenally and at the celiac trunk. The supraceliac aorta was clamped and 2–4 ml modified University of Wisconsin (UW) solution was administered via the infrarenal aorta. The major modification was the inclusion of dextran 70 in place of the HES in the original UW solution. This modification was necessary to more closely approximate the original UW solution, as our access to the original product was limited. The vena cava was transected to facilitate the outflow of blood and perfusate. The entire jejunum and ileum was subsequently harvested. On the ‘back table’, a nutrient-rich preservation solution, termed the “AA solution” to denote the high content of amino acids, was used to flush the lumen (40 ml; ∼2.0 ml/g), allowing the effluent to exit uninhibited. Each end was ligated with 3–0 silk, leaving the bowel filled without turgor. The bowel was stored in 30 ml of each solution and stored on ice at 4 °C. Tissue samples (1–2 g) were taken at 4, 8, 12 and 24 h post-flush. At the respective time point, the segment to be sampled was tied off while remaining in preservation solution. This ensured that equivalent amounts of tissue and luminal solution were removed and that the rest of the bowel remained undisturbed. To arrest metabolic activity, samples were snap frozen in liquid nitrogen, and stored at −65 °C until processed. Experimental groups All experimental groups ( n = 6) were pre-treated luminally with AA solution containing the following concentrations of HES: Group 1 – 0% Group 2 – 2.5% Group 3 – 5% Group 4 – 10%. Composition of solutions AA solution contained: 0–10% hydroxyethylstarch (HES), according to group designation, plus the following components: lactobionate (20 mM), adenosine (5 mM), allopurinol (1 mM), BES [ N , N -bis(2-hydroxyethyl)-2-aminoethanesulfonic acid] (15 mM), glutamine (35 mM), glucose/glutamate/aspartate (20 mM each), arginine/glycine/valine/asparagine/lysine/threonine/serine (10 mM each), methionine/ornithine/leucine/isoleucine/histidine/proline/cysteine (5 mM each), tyrosine/tryptophan (1 mM each), hydroxybutyrate (3 mM), pH 7.4. Hydroxyethylstarch was purified as previously described from 2-hydroxyethylstarch (HES), Aldrich #465143, 25,000 cps] [29] ; the final HES product had a M w = 2,200,000 Da. Modified UW solution contained: lactobionic acid (100 mM), raffinose (30 mM), KOH (100 mM), NaOH (15 mM), KH 2 PO 4 (25 mM), MgSO 4 (5 mM), adenosine (5 mM), allopurinol (1 mM), dextran (67.3 kDa; 5%), pH 7.4. Histology Full-thickness biopsies were formalin-fixed and processed to H&E sections. Ischemic injury was assessed using Park’s grading system modified for intestinal storage [23] ; Table 1 . Scanning electron microscopy Specimens were glutaraldehyde-fixed and post-fixed with osmium tetroxide. Samples were dehydrated in ethanol and dried in a CO 2 critical-point dryer and mounted. Samples were sputter-coated with gold and examined under the Hitachi SEM S-2500 scanning electron microscope [19] . Sample preparation and metabolite assay Frozen samples were extracted 1:5 weight/volume in perchloric acid containing 1 mM EDTA. Precipitated protein was removed by centrifugation (20 min, 20,000 g ). Acid extracts were neutralized with 3 M KOH/0.4 M Tris/0.3 M KCl and recentrifuged (20 min, 14,000 g ). Neutralized extracts were immediately processed via standard enzyme-linked metabolite assays [25] . Spectrophotometric analysis was performed to measure the absorbance of NADH at 340 nm, providing quantification of ATP, ATP/ADP, total adenylates, lactate, and alanine. Values are reported as μmol per gram protein [18] . Malondialdehyde was assessed from frozen tissue homogenized 1:10 in phosphate-buffered saline. The homogenate was then processed and fluorescence was compared to standard amounts of MDA [21] . Statistical analysis Metabolite data were reported as mean ± SE for each group. Statistical differences between groups were determined using Analysis of Variance (ANOVA), followed by Tukey’s post hoc test; P < 0.05 was reported. Differences in histology grades was assessed by a non-parametric Kruskal–Wallis test, P < 0.05 was reported. Results Histology ( Table 1 , Figs. 1 and 2 ) Assessment of H&E sections revealed there were no significant differences in histologic grades between any group after 4 h storage; median grades for Groups 1–4 were 3, 3, 1.5, and 0, respectively ( Table 1 ). However, after 8 h, Groups 2 (2.5% HES) and 3 (5% HES) showed significantly less tissue damage than Group 1 (0% HES); median grades for Groups 2 and 3 were 2.5 (indicating moderate subepithelial clefting), while Group 1 had a median grade = 7 (transmucosal injury), P < 0.05. Group 4 (10% HES) grades were highly variable, ranging from 0 to 8 with a median of 2.5. However, after 12 h the high degree of variability in Group 4 dropped dramatically, consistently revealing significant injury compared to Groups 2 and 3; both Groups 1 and 4 exhibited significantly greater injury with grades of 7 and 8 compared to grades of 3 for both Groups 2 and 3 ( P < 0.05). After 24 h storage, only Group 3 (median grade = 3) revealed significantly less tissue damage than Group 1 (median grade = 7.5, P < 0.05) or Group 4 (median grade = 7, P < 0.05). Group 2 had a median grade = 5, which was not significantly different than any of the other groups, including Group 3 due to high variability. Interestingly, the range of injury in Group 2 was grades 3–8 with the majority of specimens (4/6) having considerable villus damage including loss of villus tissue; whereas in Group 3, 4/6 specimens only had minor issues of subepithelial clefting. Scanning electron microscopy (SEM) is a powerful tool and can be useful as corroborative evidence in conjunction with standard light microscopy. In Fig. 2 , micrographs of representative injury were presented for tissues subjected to 12 h cold storage. Micrographs of Groups 1 and 4 showed a high degree of damage at the intestinal villi, with extensive epithelial villus denudation and injury to regenerative cryptal regions. Contrasting these observations, despite equivalent grades = 3 in both Groups 2 and 3, only Group 2 exhibited consistent epithelial sloughing at villi apices (an early step in the process of villus denudation). No evidence of a loss of epithelial integrity was apparent in Group 3 tissues exhibiting a median injury grade = 3. Energetics ( Fig. 3 ) ATP levels exhibited a progressive decline in all experimental groups over the 24 h storage. However, ATP in Group 4 was generally lowest of the four experimental groups, followed by Group 1. Of particular relevance was the superior maintenance of ATP in Group 3; values were at least 0.6–1.0 μmol/g greater than in the other groups following 8–12 h, P < 0.05. ATP/ADP ratios are a reflection of the mitochondrial enzyme machinery to generate ATP via oxidative phosphorylation. ATP/ADP ratios exhibited trends similar to ATP, with Group 3 exhibiting the highest values of all groups over the first 12 h storage ( P < 0.05 after 8–12 h compared to all groups). Alterations in total adenylates (TA) levels were similar to ATP. Although Group 4 showed poorer maintenance of the TA pool ( P < 0.05), Group 3 consistently demonstrated superior maintenance of TA compared to Group 1; P < 0.05. Between 8 and 24 h, the optimal concentration of HES with respect to maintenance of TA was 5% (Group 3; P < 0.05). End-products ( Fig. 4 ) Evidence of metabolic stress was apparent in all experimental groups. There was an accumulation of lactate over the first 8 h storage (highest levels were detected in Groups 2–4 with increases of 84 ± 1 μmol/g, P < 0.05); Group 1 increased by 68 μmol/g (80% of the other groups). By 12–24 h levels exhibited a progressive decline, suggesting a loss of accumulated anaerobic end-product from the tissue. Amino acid metabolism plays a major role in intestinal metabolism and alanine is typically produced as a result of various transamination reactions as other amino acids enter the Kreb’s cycle. Thus, alanine accumulation in isolated intestine is a positive indicator of amino acid catabolism as a potential energy source during cold storage. At all time-points, Group 3 exhibited the highest amount of alanine production ( P < 0.05 compared to all groups at all time-points; this was particularly pronounced at 24 h. Oxidative stress ( Fig. 5 ) The generation of malondialdehyde (MDA), a by-product of lipid peroxidation, did not occur until after 4 h storage. By 8 h, Groups 1 and 2 exhibited significant increases compared to Groups 3 and 4 ( P < 0.05); these increases continued until 12 h at which point peak values of 200 ± 4 nmol/g had been reached. Although MDA levels did increase in Groups 3 and 4 following 12 h storage (to 163 μmol/g for both groups), levels were almost 20% lower than the control group (Group 1). Interestingly, values in Group 4 (containing the highest percentage of starch) dropped significantly lower than Group 3 (and the other groups) after 24 h, P < 0.05. Discussion Throughout the last three decades, the major clinical application of hydroxyethylstarch (HES) has been for plasma volume expansion. HES has documented safety for use in humans; compared to other colloids such as gelatins, dextrans, and albumin, the frequency of anaphylactic reactions due to HES is significantly lower [36] . Recognizing its colloidal properties, Belzer and Southard included HES in their UW organ preservation solution to control tissue edema [2] . The molecular weight of HES is a major determinate of its colloidal activity. Although the HES in UW solution has a weight average MW of 250,000 Da (JH Southard, personal communication) with a fraction of starch molecules as low as 100,000 Da, we selected HES with a MW of 2.2 million Da for our preservation solution in this study. The permeability characteristics between vascular endothelial cells and intestinal epithelial cells are necessarily different. Permeability of the intestinal lumen has a much greater MW limit than the vascular endothelium; hence, a significantly higher MW starch is needed for use in our luminally administered preservation solution than the starch used in the intravascularly delivered UW solution. Hydrostatic and impermeant forces govern the movement of fluids between intracellular and extracellular compartments. However, hydrostatic forces do not apply to the maintenance of fluid movement in this study. The model of graft storage presented in this report is that of static storage; thus, only impermeant forces contribute to net flux. To date, damaging shifts in net fluid flow during cold storage are suggested to originate from the intestinal vasculature and/or lumen [3,31] . By administering a common UW vascular flush to all treatment groups, cold storage fluid shifts of vascular origin were controlled for. Thus, solely altering HES concentration in the intraluminal AA solution allowed us to attribute any inter-group differences in mucosal morphology and biochemical parameters to fluid shifts of luminal origin. Presuming that the presence of an oncotic agent is a necessary determinant affecting static cold storage when employing an intraluminal preservation solution, one would expect that alterations in morphology of the mucosal epithelium, along with tissue energetics, are concentration-dependent. In the current communication, we were able to determine the net consequences of varying starch concentrations in our nutrient-rich preservation solution with standard light microscopy, corroborated with scanning electron micrographs; although, we did not directly assess real-time osmotically-induced alterations in morphology. Interestingly, the two extremes in AA solution HES concentration (0% and 10%) had the most detrimental effects on tissue morphology out of all concentrations tested. Clearly, the requirement for osmotic or oncotic agents has an optimal level as with many biological processes and deviation from this optimum results in hypotonic or hypertonic stresses. In this study, the negative effects of 10% HES were likely attributable to a generated hypertonic environment, thereby drawing cellular fluids into the intestinal lumen and resulting in osmotic stress with secondary effects on energy-producing processes. The consequence of 0% HES was similar, except that the initiating event was a hypotonic luminal environment that caused the epithelial cells to swell and possibly lyse; again, secondary negative effects on energy production exacerbated the insult. Both 2.5% and 5% HES exhibited favourable morphology throughout most of the storage time-course. HES-treated tissue (2.5%) exhibited good maintenance of mucosal morphology, with morphological grades equivalent to 5% HES-treated tissues after 8 and 12 h; however, electron micrographs showed evidence of apical epithelial sloughing potentially suggesting the initiation of villus denudation, the result of a non-optimal HES concentration. Overall, both H&E sections and electron micrographs indicated that 5% HES-treated tissue displayed superior mucosal morphology throughout the 24 h storage time-course. In this treatment group, no evidence of irreversible damage to the epithelium or to the mucosa (i.e. crypt damage) was evident. This concentration was the only one to have a significantly lower histology grade median than 0% (control group) after 8, 12, and 24 h storage times. Moreover, the 5% group had the least variation in grading, reflecting a consistent and reproducible effect. As expected, groups containing the highest (10%) and lowest (0%) concentration of HES incurred the most mucosal damage during storage. This trend was seen after 8 h storage, and most evident after 12 h. In the 0% and 10% groups after 12 h, scanning electron micrographs showed direct visual evidence of complete villus denudation with consistent infarction of the regenerative cryptal regions. The ability of mucosal epithelial cells to maintain their barrier function directly depends on energy levels [8] . Tight junctions between cells, which are responsible for the barrier, consist of dynamic, energy-requiring proteins [26] . Cellular energy levels are quickly exhausted during ischemic storage because many energy-consuming processes are still functioning even at hypothermia [10] . As graft energetics decline, the mucosal epithelium becomes less capable of maintaining its barrier function [16] . More than any other solid organ, intestinal graft quality reflects energy levels such as, ATP and TA. Tight junction dilation is linked to increases in transepithelial flux of enteric bacteria, ultimately leading to sepsis [37,5,39] . Fortunately, tight junction perturbation is reversible; repletion of ATP levels results in tight junction re-assembly and restored epithelial barrier function [9] . In order for ATP levels to efficiently regenerate upon blood flow reestablishment and reperfusion, purine levels must be maintained in a phosphorylated form (ATP, ADP, or AMP). Also, ATP–ADP ratios are usually used as a direct measure of the integrity of mitochondrial oxidative phosphorylation [26] . Hence, maintenance of cellular energetics typically indicates superior overall preservation of graft morphology and function. In the present study, increasing concentrations of HES in our luminal AA preservation solution had variable effects on tissue energetics. Although at each concentration tested (0–10% HES) all groups experienced a progressive decline in ATP over increasing storage times, 5% HES was the most effective concentration, maintaining higher ATP levels at each storage time point. Furthermore, this improvement maintained significance at the more clinically relevant storage times of 8 and 12 h; there was superior preservation of the TA pool (after 8 h, 12 h, and 24 h). In addition to a greater potential for ATP regeneration following storage, higher TA levels reflect lower rates of purine catabolism, potentially yielding hypoxanthine. This is particularly beneficial since hypoxanthine oxidation results in the production of a reactive oxygen species (ROS) in the form of superoxide. ATP-to-ADP ratios also declined in all groups throughout cold storage. However, at 8–12 h storage, 5% HES in AA solution demonstrated improved maintenance of this energy parameter. Evidence of increased amino acid metabolism with 5% HES was observed in heightened levels of alanine, the primary by-product of intestinal amino acid metabolism; conversely, in 0%, 2.5% and 10% HES-treated tissues, lower end-product levels were reflective of reduced ATP-generating catabolism. Only 0% HES treatment resulted in a differential reduction in lactate, reflecting a negative influence of hyposmotic stress on glycolytic energy production. Previously, our laboratory showed that oxidative stress is an important component of static organ storage as a result of increased oxidative metabolism in the context of an intraluminal preservation strategy [26,40] . Malondialdehyde (MDA) is a by-product of lipid peroxidation and levels are a reliable index of oxidative stress. Cell membranes contain polyunsaturated fatty acids that are vulnerable to oxidative attack and broken down to yield the peroxidation product, MDA [30] . Membrane lipid peroxidation and declining ATP levels, are both key factors responsible for the disruption of mucosal epithelial cell tight-junctions [24] , contributing to loss of barrier integrity and subsequent enteric bacterial translocation and sepsis. In our study, 5% HES had the lowest amounts of MDA after 8 and 12 h storage. This effect was most striking at 8 h, as MDA levels with 5% HES were significantly lower than with 0% and 2.5% HES. Interestingly, after 24 h MDA was significantly lower with 10% HES, suggesting a potential concentration-dependant anti-oxidant effect due to HES. However, direct assessment of anti-oxidant capacity of HES is required for definitive proof of any proposed anti-oxidant properties. In this study, we focused on events pertaining directly to the static cold storage of intestinal tissue; intestinal transplantation itself can provide an additional level of complexity in that reperfusion can exacerbate any injurious events incurred during storage. Although it is beyond the scope of the present study, several starch-related effects relating to the inhibition of inflammatory processes will clearly become important following reperfusion after transplantation of the graft. HES may exert a protective effect on the intestinal mucosa by influencing deleterious inflammatory processes activated by oxidative stress incurred during ischemia and subsequent reperfusion. Superoxide molecules, a consequence of the xanthine oxidase-mediated oxidation of hypoxanthine, result in the accumulation and activation of neutrophils [15] . Both recruited neutrophils and oxygen free radicals activate the proinflammatory transcription factor NF-κB [33] . In turn, NF-κB may lead to the expression of specific genes involved in the production of mediator synthesis and amplification of the inflammatory response [11] . Upon adherence, activated neutrophils release multiple degradative enzymes (myeloperoxidase, elastase, collagenase) which potentiate the cell damage initiated by ischemia [12,13] . Therefore, any event reducing the involvement of neutrophils in I/R injury would likely improve functioning of the transplanted graft. Several investigators have focused on the relationship between HES and NF-κB. In a model of lung capillary permeability in endotoxic rats, Tian et al. [34] proposed a mechanism for HES inhibition of NF-κB. In their study, HES reduced LPS-induced activation of NF-κB in neutrophils in the lungs, leading to decreased microvascular endothelial permeability. Other reports have also demonstrated that HES exerts an anti-inflammatory effect via the inhibition of NF-κB activation [35] . In the intestine, NF-κB activation triggers endogenous proinflammatory mediators during I/R injury [33] . When NF-κB activation is inhibited, the degree of tissue injury diminished, which can be accompanied by a reduction in neutrophil recruitment [33] . In the current study, it is plausible that a significant amount of HES could penetrate the intestinal mucosa and cross the basolateral membrane into the capillaries. Although high MW HES (2.2 million Da) was used in our study, it is an average weight, containing a small number of molecules in the low MW range [36] , as does the UW solution [24] . Moreover, due to the low degree of substitution, large HES molecules may be hydrolyzed into smaller fragments by endogenous amylases, thereby permitting small amounts of HES leaking from the lumen into the intestinal capillaries; this may be beneficial in an ischemia–reperfusion situation such as that of stored organs following transplantation. However, this causal relationship remains to be clarified in the realm of intestinal preservation and transplantation. In conclusion, varied concentrations of HES in our nutrient-rich preservation solution had variable effects on ischemic cold storage of small bowel in rodents. Tissue energetics, oxidative stress, and histology supported the concept that in our nutrient-rich intraluminal preservation solution, an HES concentration of 5% is optimal for superior graft preservation during static cold storage. The use of oncotic rich intraluminal solutions for the graft preservation provides a novel approach to preventing the short and long term morbidity and mortality related to intestinal transplantation. Acknowledgments Special thanks to Mr. Jacek Studzinski and Ms. Geneva Hurd for excellent technical assistance. References [1] R. Adam A. Settaf B. Fabiani L. Bonhomme I. Astarcioglu N.K. Lahlou H. 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