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A Novel Histidine-Tryptophan-Ketoglutarate Formulation Ameliorates Intestinal Injury in a Cold Storage and ex vivo Warm Oxygenated Reperfusion Model in Rats

Aim : This study aims to evaluate protective effects of a novel histidine-tryptophan-ketoglutarate solution (HTK-N) and to investigate positive impacts of an additional luminal preservation route in cold storage-induced injury on rat small bowels. Methods : Male Lewis rats were utilized as donors of small bowel grafts. Vascular or vascular plus luminal preservation were conducted with HTK or HTK-N and grafts were stored at 4 °C for 8 h followed by ex vivo warm oxygenated reperfusion with Krebs-Henseleit buffer for 30 min. Afterwards intestinal tissue and portal vein effluent samples were collected for evaluation of morphological alterations, mucosal permeability and graft vitality. Results : The novel HTK-N decreased ultrastructural alterations but otherwise presented limited effect on protecting small bowel from ischemia-reperfusion injury in vascular route. However, the additional luminal preservation led to positive impacts on the integrity of intestinal mucosa and vitality of goblet cells. In addition, vascular plus luminal preservation route with HTK significantly protected the intestinal tissue from edema. Conclusion : HTK-N protected the intestinal mucosal structure and graft vitality as a luminal preservation solution. Additional luminal preservation route in cold storage was shown to be promising.


Introduction
Small bowel transplantation (SBT) is an established therapy for intestinal failure patients (1). During the course of SBT, ischemia-reperfusion injury (IRI) is inevitable throughout allograft procurement, preservation and subsequent transplantation with the intestinal mucosa being highly vulnerable to IRI (2). The consecutive severe tissue damage (3) leads to an inflammatory response including complement activation, endothelial activation, neutrophil sequestration (4) and consequently results in postoperative infection and rejection (5).
The current preservation strategy for intestinal grafts consisting of vascular perfusion followed by static cold storage (CS) with University of Wisconsin solution (UW) has not changed for decades and is still the gold standard (6,7). Histidine-tryptophan-ketoglutarate (HTK) solution, an extracellular type and low-viscosity solution with a relatively low potassium concentration was postulated superior in maintaining intestinal viability (2) and has been increasingly administered in clinical application (6,8,9). Yet, the difference of preservation outcome between UW and HTK is still controversial and no evidence has been found which hints significant advantage of HTK in terms of histological and functional assessment (6,7,10).
Cell culture studies examining hepatocytes and endothelial cells in different preservation solutions showed the occurrence of iron-dependent hypothermic injury mediated by reactive oxygen species (ROS) in all solutions (11)(12)(13). Furthermore, a toxic property of histidine also leading to the formation of ROS was observed (14,15). Histidine is an important ingredient of HTK due to the substantial buffer capacity for maintaining pH during preservation (16,17). To counteract hypothermic injury and the cytotoxicity of histidine in HTK, a novel preservation solution, HTK-N, modified on the basis of HTK, was developed with reduced histidine content. Instead, N-α-Acetyl-L-histidine, membrane-permeable iron chelators (deferoxamine and LK 614), glycine and other amino acids (AAs) were added (Table 1). N-α-Acetyl-L-histidine shares similar structure with histidine but shows almost no toxicity, partially because of its decreased cellular uptake (15). Functioning together with the iron chelators, N-α-Acetyl-Lhistidine markedly alleviates the iron-dependent injuries (11,15,18). Since sodium influx plays a critical role in hypoxic injury (19,20) glycine and alanine were added to ameliorate hypoxic injury by inhibiting ligand-gated chloride channels and non-specific leaks of small ions including sodium (21)(22)(23)(24). It was reported that aspartate could improve mitochondrial function during ischemia and reperfusion and has a considerable impact on overall cellular recovery (25). Moreover, reduced chloride content is required for mitigating hypothermic injury during preservation in some cell types (11). Arginine is the substrate of nitric oxide synthase (NOS) (26) and is reduced during ischemia and reperfusion by activity of arginase (27). Addition of arginine was proven to have a positive influence on IRI (28).
Compared to solid organs like liver and kidney, the unique features of the complex intestinal architecture determine that the sensitive enterocytes at the luminal surface cannot be reached by vascular preservation routes (7). This is important since the mucosal layer with the most abundant enterocytes is the initial site of intestinal impairment during preservation (29). Loss of intestinal barrier integrity induces bacterial translocation with the risk of local and systemic infection (30). A series of studies documented that additional luminal preservation could elicit protective effects and thus counteract CSinduced injury (31)(32)(33). Moreover, the addition of nutrients and AAs to normal solution and alteration of preservation parameters (e.g. addition of hydrogen and macromolecular compounds in the preservation solution) have also been proven to be beneficial (30, [34][35][36][37][38], but the optimal components for intestinal luminal preservation remain unknown (2,7,10,39,40). Based on these previous studies, HTK-N was now examined in intestinal luminal preservation route.
In this study we compared the protective impact of HTK and HTK-N on rat small bowel preservation, as well as strategies of vascular route (V) and vascular as well as luminal preservation routes (VL). For this purpose, a small bowel warm oxygenated reperfusion system was used to mimic early posttransplant conditions. This reperfusion system has already been certified technically accessible, stable and was widely used (41)(42)(43)(44)(45). Both structure and function relevant parameters proved to be easily accessible.

Animals and Study Design
All handling procedures and operations were performed in accordance with the German Animal Welfare Law and with approval by the administrative authority of North Rhine-Westphalia (Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen, reference number 84/02.04.2013.A050). All animal work took place in the central animal facility of the medical faculty at the University of Münster, Münster, Germany. All animals received humane care and had an acclimatization period for more than one week. Animals were allowed to have free access to normal chew food (Altromin, Lage, Germany) and water. The room was in 12 h light/dark cycles and the temperature was kept at 24 2°C. Animals were fasted 12 h before surgery but had access to water ad libitum.
Male Lewis rats (Charles River, Sulzfeld, Germany) weighing 200-270 g were randomly assigned into The study was performed in a randomized and double blinded way with unblinding by external laboratory personal after completion of all analyses.

Small Bowel Procurement and Preservation
All operations were conducted under sterile conditions. Under inhalation anesthesia with isoflurane (AbbVie, Ludwigshafen, Germany), a five centimeter abdominal midline incision was performed and jejunum and ileum were isolated with a vascular pedicle as described in detail by Minor et al. (45).

Goblet Cells Abundance
To evaluate the intestinal mucus-gel layer and vitality of mucosal goblet cells, four micrometers sections of formalin fixed, paraffin embedded (FFPE) samples were stained by 3% Alcian Blue solution (Merck, Darmstadt, Germany) followed by nuclear fast red (Waldeck, Muenster, Germany) counterstaining.
Photographs of five random fields from each slide were taken with a light microscope (Nikon, Tokyo, Japan) and goblet cells were identified based on mucopolysaccharide binding of the Alcian blue stain.
The results are presented as mean value of blue-stained goblet cells per high power field (HPF).

Apoptosis
Four micrometer sections of FFPE samples were stained with a commercially available In Situ Cell Death Detection Kit (Sigma-Aldrich, Munich, Germany) according to the manufacturer's instruction. Photographs of five random fields from each slide were taken with a light microscopy (Nikon) and results are shown as mean value of apoptotic cells per HPF.

Tissue Wet-to-Dry Ratio
Immediately after the 30 min reperfusion 10 cm segments from jejunum and ileum, respectively, were weighed in a standardized fashion for the value of wet weight. Afterwards, grafts were dried in an oven (Memmert, Büchenbach, Germany) at 80 °C for three days and weighed again to determine the dryweight value. The wet-to-dry ratio was calculated by dividing wet-weight by dry-weight.

Electron Microscopy
Two centimeter segments were fixed in 2% (v/v) formaldehyde and 2.5% (v/v) glutaraldehyde in 100 mM cacodylate buffer, pH 7.4, at 4 °C. After being washed with phosphate buffered saline (PBS), specimens were post-fixed in 0.5% (v/v) osmium tetroxide and 1% (w/v) potassium hexacyanoferrate (III) in 0.1 M cacodylate buffer for 2 h at 4 °C, followed by washing with distilled water. After dehydration in an ascending ethanol series from 30 to 100% ethanol, specimens were incubated twice in propylenoxide for 15 min. Next, small pieces of intestinal tissue were embedded in Epon using flat embedding molds. Ultrathin sections were cut with an ultramicrotome, collected on copper grids, and negatively stained with 2% uranyl acetate for 15 min. Electron micrographs were taken with a Phillips EM-410 electron microscope using imaging plates (Ditabis, Pforzheim, Germany).
The ultrastructure of intestinal epithelia was examined in a blinded fashion. Evaluation was performed focusing on the microvilli and organelle integrity including the endoplasmic reticulum (ER) and mitochondria swelling, electron density, nucleus integrity and cell necrosis or apoptosis. The evaluation results were presented in scores: 1: mild damage, 2: moderate damage, 3: severe damage.

Immunofluorescence (IF) Staining of Tight Junction Proteins
Four micrometer sections of FFPE samples were deparaffinized with xylene and rehydrated in graded alcohols. Afterwards, heat-induced epitope retrieval was conducted at 95 °C and slides were placed in 10 mM Tris-EDTA (pH 9.0) with 0.05% Tween-20 (Sigma-Aldrich) for 30 min and cooled down on ice for 15 min. After rinsing with PBS, slides were blocked in 5% bovine serum albumin for 1 h.
Presence of tight junction proteins were assessed by incubating slides with 1:400 dilution of Claudin-1 primary antibody (503-3414) or 1:500 dilution of Claudin-3 primary antibody (503-3834) and Claudin-temperature for 30 min. All antibodies were diluted with antibody diluent (Dako North America, Carpinteria, USA). Two sections as negative controls were incubated with pure antibody diluent without primary antibodies. After washing with PBS, Alexa Fluor 488 goat-anti-rabbit secondary antibody (659082, Invitrogen, Camarillo, USA) was applied and slides were incubated at room temperature for 1h in a humidity chamber. Afterwards, slides were washed again with PBS and incubated in 1 μg/ml 4', 6-Diamidino-2-phenylindole dihydrochloride (DAPI, Sigma-Aldrich) at room temperature for 4 min.
Slides were then rinsed again with PBS, coverslipped and assessed with a fluorescence microscope (Nikon). Images captured under the same conditions were analyzed by Image J and maximal signal intensity in each sample was measured.

Lactate Dehydrogenase (LDH) Release
During reperfusion, effluent was collected from the portal vein and a commercially available Quantichrom LDH kit (BioAssay Systems, Hayward, USA) was applied to determine LDH activity in effluent as an index of tissue damage according to the manufacturer's instruction.

Energy Metabolism
Segments of intestinal grafts were snap frozen in liquid nitrogen and stored at -80 °C for later assays.
The ATP content retained in intestinal tissue after 8 h cold storage and 30 min warm oxygenated reperfusion was determined as an indicator of ATP regeneration and graft vitality. A commercially available ATP Colorimetric/Fluorometric assay kit (BioVision, Milpitas, USA) was used according to the manufacturer's instruction. The results were corrected with the respective wet-to-dry ratio and shown in nanomoles per gram of dry-weight (nmol/g-dry tissue).

Carbohydrate Absorption
The intestinal lumen was filled with 3 ml of 5% galactose solution (Sigma-Aldrich) dissolved in 0.

Histology
Grafts with HTK-N vascular preservation presented the most serious histological alteration with highest Park/Chiu score (6.667 ± 0.211) and the HTK vascular / luminal preservation group presented the lowest score (5.333 ± 0.333), although differences were small. Interestingly, statistically significant difference was found in comparison between groups of HTK-N vascular with or without luminal preservation but not between groups of HTK solution (Figure 1 A-D, I).

Goblet Cells Staining
To evaluate the capability to produce mucus, goblet cell abundance in tissue samples was determined

Apoptosis
As presented in Figure 1 (K), small bowels with HTK-N vascular preservation showed the lowest apoptosis severity (74 ± 11 cells/HPF) while HTK vascular with or without luminal preservation groups were comparable and had the most apoptotic cells in mucosa (83 ±15 cells/HPF, 83 ±12 cells/HPF, respectively), although there was no significant difference between groups.

Wet-to-Dry Ratio
As presented in Figure 1 (L), samples with HTK vascular preservation presented the highest wet-to-dry ratio (7.375 ± 0.277) while samples with HTK vascular plus luminal preservation showed the lowest ratio (6.049 ± 0.237) and had significant difference with the group of HTK vascular preservation (* p < 0.05). No significant difference was found in comparisons between other groups.

Immunofluorescence
Claudin-1 signal intensity of the group HTK vascular and luminal preservation was the lowest (49.5 ± 5.258) among four groups but had no significant difference compared with other groups (Figure 3 D). there was no statistically significant difference (Figure 3 E). Grafts with HTK-N vascular plus luminal preservation had relatively higher signal intensity (52.5 ± 6.647) of Claudin-5 than the other three groups, which showed similar results, although no significant difference was found (Figure 3 F).

LDH Release
In the group of HTK vascular preservation LDH activity in effluent was higher (0.154 ± 0.55 IU/L) than in the other groups while HTK-N vascularly preserved grafts showed the least LDH release (0.058 ± 0.037 IU/L). No significant difference was found between groups (Figure 4 A).

Energy Metabolism
The tissue with HTK-N vascular preservation retained the lowest (12.03 ± 1.85 nmol/g-dry tissue), grafts preserved vascularly plus luminally with HTK the highest ATP content (14.44 ± 2.666 nmol/g-dry tissue), although values were relatively low under all conditions and differences were both, small and not significant (Figure 4 B).

Carbohydrate Absorption
Small bowels with only HTK vascular preservation presented the lowest galactose concentration (0.307 ± 0.099 μg/ml) in the effluent drained from the portal vein during warm oxygenated reperfusion among these four groups while grafts with HTK-N vascular plus luminal preservation showed the highest results (0.428 ± 0.048 μg/ml), but there was no statistical significance (Figure 4 C).

Oxygen Consumption
Oxygen consumed by small bowels with HTK or HTK-N vascular preservation presented similar results (10.5 ± 2.446 ml/min/g dry tissue, 10.67 ± 2.187 ml/min/g dry tissue, respectively) and less than the other groups with additional luminal preservation, but no significant difference was found between groups ( Figure 4D).

Discussion
This study applied the novel preservation solution HTK-N in rat small bowel CS and evaluated its capacity to protect the integrity of intestinal structure and function. In addition, this study firstly compared HTK and HTK-N in two different preservation routes to assess whether vascular or vascular plus luminal preservation strategies are superior in protecting intestinal grafts from IRI.
With partial substitution of histidine with N-α-acetyl-L-histidine and the complement of iron chelators and AAs, HTK-N was expected to be a promising alternative to the currently used standard preservation solutions UW and HTK. Previous studies documented nontoxic property of HTK-N in a variety of organs and grafts including liver, kidney, lung, pancreas, intestine and heart (50)(51)(52)(53)(54)(55)(56). In addition, animal models showed protective effects of HTK-N on different organs including liver (18,50,51,57), lung (52) and heart (53,58,59). On the contrary, no noticeable superiority of HTK-N to HTK was found in studies on kidney (54), pancreas (55) and vascularized tissue isograft (60).
With the special focus on the small bowel, HTK-N was recently used in a rat small bowel transplantation model (56). It was shown that HTK-N elicited a significant improvement in intestinal microcirculation during the early posttransplant stage following 24h CS, an effect which was attributed to the addition of iron chelators. In line with this, we conducted this study to further discover whether HTK-N is superior to the conventional HTK regarding the protection on the structural and functional integrity of small bowels, but only limited evidence was achieved indicating the advantage of HTK-N compared to HTK in protecting intestinal grafts in both vascular and vascular plus luminal routes. One might speculate that the extreme susceptibility of small bowel to ischemia and reperfusion restricts the protective effect of this novel preservation solution, leading to unsatisfactory outcome despite the significant improvement on the microcirculation presented in the previous study. However, the study by Lautenschläger et al. preservation effects. Of note, wet-to-dry weight ratio, a marker of edema formation, was decreased by almost 50% with vascular HTK-N compared to vascular HTK (Fig. 1 L), suggesting improved vascular integrity after preservation with HTK-N. formation after preservation with HTK-N appeared to be decreased (Fig. 1 L).
Another purpose of this study was to investigate the impact of additional luminal perfusion and preservation with HTK or novel HTK-N. In the present study, the most noticeable and interesting result is the abundance of goblet cells in mucosa which still possessed the capacity of mucus secretion. Mucus plays a critical role in the establishment of intestinal mucosal barrier by separating the epithelial cells from the luminal content, especially bacteria (62,63). In addition, mucus is also a part of the innate immune defense, contributing to concentrate the antibacterial peptides secreted by Paneth cells (64). We found that samples with HTK-N vascular plus luminal preservation retained significantly more goblet cells than samples with only HTK-N vascular preservation; besides, similar situation happened on the HTK-preserved grafts, although no significant difference was found. On the other hand, morphological parameters were analogous. Park/Chiu score is a systemic measurement which focuses on the epithelial/subepithelial damage and mucosal barrier integrity (47,48). It was proven that mucosal injury worsens upon the reperfusion after CS, which causes further impairment of mucosal barrier integrity (65). Hence, the experimental design with a sequence of CS and reperfusion is more reasonable to evaluate the protective effects of organ preservation strategies than a process of only CS. In the present study, the intestinal tissue histological and ultrastructural assessment as well as wet-to-dry ratio, which reflects the tissue edema severity (44, 61), elucidated the superiority of HTK-N in luminal route.
In conclusion, this study applied a novel solution HTK-N in small bowel CS and evaluated its effects on protecting the morphological integrity, mucosal permeability and graft vitality, but no significantly different result was found between samples with HTK or HTK-N. This solution might need further additives to meet the unique requirement of intestine (nutrients supply, colloid complement, etc.). In  All concentrations are given in mmol/l, calculated osmolarity is given in mosmol/l, pH at 20°C.