j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 4 ( 2 0 1 5 ) 2 8 9 e2 9 6

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Luminal solutions protect mucosal barrier during extended preservation Mihai Oltean, MD, PhD,a,* Mats Hellstro¨m, PhD,a Catalin Ciuce, MD,a,b Changlian Zhu, MD, PhD,c and Anna Casselbrant, PhDd a

Department of Surgery/Laboratory for Transplantation and Regenerative Medicine, Institute of Clinical Sciences, Sahlgrenska Academy at the University of Gothenburg, Gothenburg, Sweden b First Surgical Clinic, University of Medicine and Pharmacy, Cluj-Napoca, Romania c Institute of Neuroscience and Physiology, Center for Brain Repair and Rehabilitation, University of Gothenburg, Gothenburg, Sweden d Department of Gastrosurgical research and Education, Institute of Clinical Sciences, Sahlgrenska Academy at the University of Gothenburg, Gothenburg, Sweden

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abstract

Article history:

Background: Mucosal barrier injury during intestinal preservation (IP) and transplantation

Received 17 July 2014

favors life-threatening infections. Luminal delivery of solutions containing amino acids or

Received in revised form

polyethylene glycols (PEGs) may improve preservation results and reduce this injury. We

26 September 2014

tested if solutions containing glutamine and PEG influence the mucosal injury.

Accepted 2 October 2014

Materials and methods: Rat intestines were perfused and stored in Viaspan-University of

Available online 7 October 2014

Wisconsin solution. Before IP, a PEG 3350 solution was introduced intraluminally alone (group 1) or supplemented with 40 mmol/L L-glutamine (group 2). Controls underwent

Keywords:

vascular flush alone (group 3). Preservation injury was evaluated after 8, 14, and 24 h by

Organ preservation

histology and goblet cell count. Tight-junction proteins zonula occludens-1, claudin-3,

Tight junctions

claudin-4, and caveolin-1 were studied by immunofluorescence. Maltase and caspase-3

Preservation solutions

activity were also analyzed.

Intestinal transplantation

Results: Group 1 showed mild edema at 8 h and mucosal disruption by 24 h; these features

Ischemia

were greatly improved in group 2 where continuous mucosa was found after 24 h of IP. Intestines in group 3 did worse at all time points with subepithelial edema (Park/Chiu grade 3) and marked goblet cell depletion; caspase-3 activity was lowest in group 2. Tightjunction proteins varied continuously during IP; zonula occludens-1 expression and colocalization with claudins decreased significantly in group 3 but not in other groups. Claudin-3 was distinctly localized in the membrane, but stained diffuse, cytoplasmic at later time-points. Claudin-4 changed to a cytoplasmic granular pattern. No caveolin-1 colocalization was observed. Conclusions: Luminal PEG and glutamine delay epithelial breakdown and preserve several important mucosal features during extended IP. ª 2015 Elsevier Inc. All rights reserved.

* Corresponding author. The Transplant Institute, Sahlgrenska University Hospital, Gothenburg 413 45, Sweden. Tel.: þ46 704 906 156; fax: þ46 31 41 34 40. E-mail address: [email protected] (M. Oltean). 0022-4804/$ e see front matter ª 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2014.10.001

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j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 4 ( 2 0 1 5 ) 2 8 9 e2 9 6

Introduction

Intestinal transplantation recorded major advancements over the past decade because of improved patient selection, novel surgical techniques, refined immunosuppression, and monitoring [1]. Nevertheless, intestinal preservation (IP) remained unchanged despite suboptimal results and is still based on an initial vascular perfusion followed by static cold storage in University of Wisconsin (UW)eViaspan (Bristol Myers Squibb, Solna, Sweden) or histidine-tryptophan-ketoglutarate solutions [2,3]. The current clinical approach allows for 10 h usually results in extensive mucosal breakdown, which may promote bacterial translocation and result in life-threatening sepsis. The approach presented herein may result in a prolongation of this period beyond 14 h. It is known that intestinal reperfusion on transplantation leads to an actual worsening of the preservation injury with one or two grades on the Park scale [17,18]. In this setting, this would result in extensive mucosal destruction in the control group, whereas it would still theoretically maintain the epithelial continuity in the groups receiving luminal macromolecular solutions after 14 h of cold storage. Furthermore, other benefits could be expected due to the effects of additional glutamine. Glutamine has multiple nonnutritional effects including modulation of intracellular signaling pathways and inhibiting apoptosis. Elevated concentrations (millimolarity) of glutamine have been shown to protect cell lines exposed to an environmental stress similar to intestinal cold storage settings (nutrient starvation, hyperosmolality), whereas

Table 2 e The amino-acid content in the luminal solution after different intervals of preservation. Amino acid

Group 1 8 h (n ¼ 3)

Glutamine (mmol/L) Glutamate (mmol/L) Glycine (mmol/L) Alanine (mmol/L) Citrulline (mmol/L) Valine (mmol/L) Methionine (mmol/L) Isoleucine (mmol/L) Leucine (mmol/L) Histidine (mmol/L) Lysine (mmol/L) Tryptophan (mmol/L) Arginine (mmol/L)

48 339 364 312 14 150 44 105 88 52 137 20 46

(0e126) (232e782) (232e763) (206e485) (0e25) (38e401) (10e115) (27e257) (0e273) (22e94) (51e227) (8e35) (14e160)

Group 2

14 h (n ¼ 3)

24 h (n ¼ 2)

37 472 414 322 10 81 22 58 109 35 92 16 64

752 599 406 73 110 33 75 147 36 114 19 84

(0e88) (218e487) (402e524) (273e438) (0e35) (75e131) (6e40) (26e89) (66e162) (33e48) (18e139) (0e19) (0e103)

0 (716e788) (589e610) (343e469) (51e96) (84e137) (24e43) (58e93) (113e182) (24e48) (87e141) (16e22) (61e107)

8 h (n ¼ 3) 70 330 340 344 18 104 33 70 137 37 124 14 97

(53e131) (228e412) (228e420) (291e432) (18e19) (58e184) (17e44) (43e135) (83e243) (25e64) (56e179) (7e29) (39e109)

14 h (n ¼ 5) 75 483 471 415 23 125 39 94 179 38 133 20 86

(61e109) (431e599) (381e566) (332e497) (16e28) (91e183) (22e52) (65e130) (120e241) (36e77) (92e199) (14e27) (61e118)

24 h (n ¼ 3) 0 (0e166) 860 (481e1067) 890 (444e890) 510 (287e693) 59 (33e91) 159 (54e354) 52 (17e90) 113 (35e236) 221 (72e453) 52 (21e130) 182 (64e352) 27 (8e59) 131 (45e225)

j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 4 ( 2 0 1 5 ) 2 8 9 e2 9 6

Fig. 3 e Caspase-3 activity (A) and maltase-glucoamylase activity (B) in group 1 (light gray bars), group 2 (dark gray bars), and group 3 (white bars). The horizontal line in the middle of each box indicates the median; the top and bottom orders of the box mark the 75th and 25th percentiles, respectively, and the whiskers mark the 5th and 95th percentiles; * P < 0.05. AMC [ aminomethyl coumarin.

glutamine deprivation decreases Hsp70 messenger RNA halflife, leads to cell shrinkage, and lowers intracellular adenosine triphosphate content [19,20]. We found less active caspase-3 in glutamine-treated intestines suggesting an upstream involvement of glutamine on the intrinsic apoptotic pathway. Glutamine was the sole amino acid decreasing in the luminal solution suggesting its use by the enterocytes. Conversion from glutamine to glutamate is known to occur spontaneously; however, we believe actual glutamine depletion occurred because glutamate levels were in the micromole range and much lower than the initial levels of glutamine. Glutamine-using cells react in a specific manner in the case of glutamine deprivation. Glutamine-deprived Caco-2 monolayers markedly revealed decreased claudin-1 expression and worsened several electrophysiological parameters, supposedly through the involvement of the phosphatidylinositol 3-kinase/Akt pathway [21]. Depending on the luminal treatment we found different patterns of claudin-3 expression, ranging from near-normal immunofluorescence signal in intestines receiving luminal glutamine to TJ disruptiondecreased claudin expression and internalization in the control group. However, the maintained ZO-1 and/or claudin3 colocalization after 14 and 24 h is not necessarily a glutamine-dependent phenomenon because it was also observed in the group receiving glutamine-free luminal treatment.

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TJs act as barriers that selectively control the passage of solutes through the intercellular space, and claudins are the main determinants of this regulation [22,23]. This intercellular complex is composed of several transmembrane proteins of which claudin-4 regulates the selective permeability for cations (particularly sodium) [24,25]. The current results provide some indirect evidence of an early and significant TJ dysfunction during the intestinal cold storage, which is reflected by the sodium absorption and luminal leak of potassium and several amino acids of all polarities and sizes. The dysfunction does not seem related to claudin-3 or 4 because different inter- and intra-cellular staining patterns were associated with similar contents in the luminal compartment among the different groups. Furthermore, this dysfunction does not seem to have been influenced by the additional luminal glutamine because the spectrum of changes did not differ from the group receiving PEG alone. Consequently, this may imply the involvement of other TJ proteins found in the selective pores. Several mechanisms leading to TJ dysfunction have been identified including TJ disruption by actin cytoskeleton contraction and subsequent widening of the TJ or caveolin-1-mediated depolymerization during oxidative stress [26]. We found evidence of claudin-3 and 4 in the cytoplasm but no signs of its colocalization with caveolin-1 and thus we speculate that TJ dysfunction and disruption during intestinal cold storage is caveolin-1 independent. GCs produce a glycoprotein-rich mucous layer, which prevents bacteria from reaching the mucosa and protects the intestinal mucosal epithelium from luminal aggression [14]. A discharge of mucins may, however, occur in response to various stimuli and stressors (ischemia, inflammatory mediators, and lipopolysaccharide) as it was recently reported after warm ischemia in the ischemic colon [27]. In the present study, we show that both luminal preservation solutions significantly reduced this event and preserved GC morphology. Mucus swelling inside the GC and the subsequent mucosal disruption were reduced by the luminal macromolecules. Swelling is a critical determinant of mucus rheology because its protein concentration regulates its rheological properties on the mucosa (such as the diffusivity of newly released mucins). The most plausible explanation for our findings is that an improved osmotic balance and a GibbseDonnan equilibrium along the epithelial interface was established in the presence of PEG. Brush-border disaccharidase activity was little affected by the storage, indicating that the damage to the enterocyte itself is less pronounced than during warm ischemia. Luminal PEG did not seem to impair the ability of maltase to cleave its substrate. A major limitation of our study is the absence of reperfusion that would have introduced further valuable end points such as the reperfusion injury, mucosal repair, or animal survival. However, from a mechanistic point of view reperfusion is not necessarily advisable because of the extensive tissue destruction or the associated tissue inflammation and cellular infiltration [16]. Therefore, the current setting allowed for detailed observations and provides a wealth of valuable information for designing and evaluating novel strategies and solutions for luminal preservation.

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The use of a single glutamine concentration could also be regarded as another limitation. This design was chosen based on previous studies reporting structural and functional improvements using similar concentrations [5] and on own unpublished observations that yielded similar results using lower (20 mM) or higher glutamine (100 mM) concentrations. Thus, at this stage we believe the presence rather than concentration is important for the outcome of the study. Graft injury sustained during storage consistently begins with loss of mucosal barrier function and morphologic injury, leading to failure of this innate immunity barrier to isolate the sterile internal from the nonsterile external environment. Based on the in vitro preservation data presented here, our results strongly support the addition of glutamine in a PEGbased solution for luminal preservation to minimize structural and functional impairment of the intestinal mucosa incurred during storage.

Acknowledgment The present study has been supported by funds from Professor Lars-Erik Gelin Memorial Foundation for Transplant Research and Sahlgrenska University Hospital. Authors’ contribution: M.O. and A.C. contributed to the study design. M.O., M.H., C.C., and C.Z. did the data acquisition. M.O. and M.H. did the analysis and drafting of the article. C.C., C.Z., and A.C. reviewed the article.

Disclosure The authors reported no proprietary or commercial interest in any product mentioned or concept discussed in the article.

references

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Luminal solutions protect mucosal barrier during extended preservation.

Mucosal barrier injury during intestinal preservation (IP) and transplantation favors life-threatening infections. Luminal delivery of solutions conta...
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