Over the last decade, there has been a trend towards using organs from older donors in clinical transplantation. Livers and kidneys are frequently transplanted from donors over 70 years of age with acceptable results.1,2 The field of intestinal transplantation, however, is far more conservative, and donors over 50 years of age are rarely considered. Additionally, it is recommended to strive for an intestinal cold preservation time shorter than 9 hours.3,4 These criteria seriously limit the donor pool and raise significant logistical challenges.
It has previously been suggested that the small intestine develops age-related morphological changes. Older intestines seem to develop a progressive intestinal barrier dysfunction, which may be associated with mucosal atrophy, damage to tight junction (TJ) structure, and altered TJ protein composition.5 Altered TJ integrity was shown to be one of the main mechanisms behind the progression of intestinal preservation injury (IPI).6 Furthermore, intestinal epithelial barrier function and its resistance to IPI may decrease with advancing donor age. However, solid evidence documenting or refuting the increased susceptibility of intestines from older donors is lacking.
We have previously shown that grafts receiving additional luminal preservation (LP) with a macromolecular solution based on polyethylene glycol (PEG) maintained their histological and functional epithelial integrity over longer periods of cold storage compared with intestines undergoing vascular perfusion alone.6-8 Nothing is known about the effect of LP on grafts from older donors. We, therefore, performed a direct comparison assessing the development of IPI in intestines from donors of different ages. In addition, our aim was to investigate if LP could confer a benefit and counterbalance any potential detrimental effects of higher donor age.
MATERIALS AND METHODS
Female Sprague-Dawley rats were kept in 12-hour light-dark cycles, controlled temperature and pathogen-free environment, receiving rat chow and water ad libitum. Animals were used at 3, 14, and 20 months, respectively. The animals were not fasted before surgery. The study followed the regulations outlined by the European Union, and it was reviewed and approved by the local committee of the Swedish Animal Welfare Agency (no. 1040/2017).
Surgery and Sampling
The rats were anesthetized with a mixture of 2% isoflurane in air. The abdomen was opened through a midline incision and the infrarenal aorta was ligated above the bifurcation. Thereafter, a short aortic segment above the emergence of the superior mesenteric artery was isolated. After ligating the aorta above the superior mesenteric artery, the right atrium was cut to facilitate venous venting. The intestine was then slowly perfused retrogradely in situ through the infrarenal aorta with 5 mL/min ice-cold Custodiol solution until completely blood-free (10–12 mL in total). After perfusion, the distal part of the small intestine was cut out and divided into 2 parts. These parts were randomly assigned to receive a luminal PEG solution or to serve as controls (without LP solution). Graft ends were ligated with 3-0 silk ligature, and the intestines were stored in ice-chilled perfusion solution. After 4, 8, and 14 hours of cold storage graft segments were retrieved from the preservation solution, ligated ends removed, and placed in 4% buffered formalin or snap-frozen. The right kidney and the infraceliac aorta were resected and stored in formalin.
Solutions and Experimental Groups
Ice-cold Custodiol (Dr Franz Köhler Chemie, Alsbach-Hähnlein, Germany) was used for the intestinal perfusion in all groups. For intra-LP, we used a commercially available PEG-based solution (Movicol, Norgine, United Kingdom) having an osmolality of 240 mOsm/kg. Solutions’ content is detailed in Table 1. LP was performed as previously described by the Edmonton group.9 In brief, the cold PEG solution was gently flushed through the lumen, and the effluent was allowed to freely exit the distal end until clear (≈10 mL). The end of the intestinal segment was then ligated with 3-0 silk and further 2–3 mL of the solution were infused, leaving the bowel filled without turgor and allowing an even distribution of the solution throughout the entire length of the retrieved graft. The intestinal content was left in place in the control groups.
The animals were divided into 6 experimental groups:
- Group Y: Young, 3-month-old animals, no LP (n = 8)
- Group Y-L: Young, 3-month-old animals with LP (n = 8)
- A: Adult, 14-month-old animals, no LP (n = 8)
- A-L: Adult, 14-month-old animals with LP (n = 8)
- O: Old, 20-month-old animals, no LP (n = 8)
- O-L: Old, 20-month-old animals with LP (n = 8)
Full-thickness tissue samples were formalin-fixed and embedded in paraffin. Five-micron sections were stained with hematoxylin and eosin. At least 6 fields on 2 different sections were examined at medium power magnification (×100). The ischemic injury was graded using the Chiu/Park score (Table 2).10
Morphometric analysis was performed on hematoxylin and eosin-stained sections using an image analysis system (Image J, NIH, Bethesda, MD) on samples (n = 8) taken directly after graft retrieval from all 3 age groups. Parameters measured were villus length and mucosal thickness and included at least 25 villi per histological specimen. The number of villi per microscopic field (at ×100 magnification) was determined as a measure of villus density.
Intestinal Mucosal Goblet Cells
Positive staining with Alcian blue indicated the presence of acidic mucins within goblet cells (GC). Five-micron intestinal sections were stained with Alcian blue (3%), and positive villus cells were counted in 7 different fields from at least 2 sections at high magnification (×400) by a single observer.
Age-related Changes in Kidneys and Aorta
Renal and aortic sections of 5-µm thickness were submitted to hematoxylin and eosin, elastin van Gieson and von Kossa staining (for aorta), and periodic acid Schiff staining (for kidneys) and examined blindly by an experienced pathologist.
Ussing Chamber Experiments
The Ussing Pulse Method was used to determine the tissue’s epithelial electrical resistance (Rep) and the epithelial ion current was received by using Ohm’s law, where I = U/R (current = voltage/resistance, ie, epithelial ion current = potential difference [PD]/Rep) as previously described.11 In brief, the method is based on the concept that the epithelium consists of a capacitor and resistor coupled in parallel. The whole-thickness rat small intestine was mounted in conventional Ussing chambers with an aperture area of 0.29 cm2 (Warner Instruments, Hamden, CT). After mounting, each half chamber was filled with 5 mL Krebs solution maintained at 37°C and continuously oxygenated with 95% O2 and 5% CO2 and stirred by the gas flow. The PD was measured with a pair of matched calomel electrodes (REF401, Radiometer analytical, Denmark). The data were collected using an amplifier and specially constructed software developed in LabView (National Instruments, Austin, TX). Up to 6 preparations were retrieved from each intestinal segment after 4, 8, and 14 hours of preservation. The electrical parameters were measured over a 20-minute experiment. Normal values were obtained using pristine intestines transported on ice to the laboratory and mounted in the Ussing chambers within 15 minutes from procurement (n = 4/group).
Fluorescein sodium salt (FSS; Mw-376kDa) (Sigma-Aldrich, Stockholm, Sweden) or fluorescein isothiocyanate-dextran (FD4; Mw-4000kDa) (Sigma-Aldrich) was added to the luminal chamber side to a final concentration of 1 and 2 mg/mL, respectively. Samples (0–2 mL) were then collected from the serosal chamber at baseline and after 20 minutes, and the concentration of each probe was measured by fluorescence at the excitation wavelength of 480 nm and the emission wavelength of 535 mm.
Western Blot Protein Analysis
Frozen specimens were homogenized in a PE buffer (10 mmol/L potassium phosphate buffer, pH 6.8, and 1 mmol/L EDTA) containing 10 mmol/L 3-[(3-cholamidopropyl) dimethylammonio]-1-propane sulphonate (Boehringer Mannheim, Mannheim, Germany) and protease inhibitors. Samples were then mixed with sodium dodecyl sulfate buffer before being loaded on a NuPage 10% Bis-Tris gel using MOPS buffer (Invitrogen AB, Lidingö, Sweden) (25 µg/well). After electrophoresis, the proteins were transferred to a polyvinyldifluoride transfer membrane (Hybond, 0.45 mm, RPN303F; Amersham, Buckinghamshire, United Kingdom) using the iBlot dry blotting system. Following repeated washing cycles and blocking in 0.2% (w/v) I-block reagent (Applied Biosystems, Bedford, MA), membranes were incubated overnight at 4°C with primary antibody against Claudin-3 (34-1700, Invitrogen AB, Lidingö, Sweden), Claudin-4 (32-9400, Invitrogen AB), Tricellulin (48-8400, Invitrogen AB), and Occludin (71-1500, Invitrogen AB). After repeated washings, a secondary antibody was applied for 1 hour at room temperature, and visualization was performed using the chemiluminescent enzyme substrate CDP-Star (Tropix, Bedford, MA). Each time the membrane was incubated with a new primary antibody, the previous antibody was removed with stripping buffer (Re-Blot Plus Mild Solution, Millipore, Temecula, CA). The gel was stained in 0.2% Coomassie blue and used to determine the efficiency of the protein transfer. The total amount of protein on each lane as revealed by the staining was also used for quantification (normalization).
Statistical differences between independent groups were calculated using the Kruskal-Wallis test followed by the Mann-Whitney U test. Data are presented as median ± range unless otherwise stated. GraphPad Prism6 (GraphPad Software, La Jolla, CA) was used, and the significance level was set at 0.05.
Tissue Injury and Morphometric Analyses
Light microscopy examination of normal intestines was unremarkable in the 3 age groups. There was no significant difference in villus length, villus width, or mucosal thickness. There was, however, a significant decrease in villus density with increasing age.
Cold storage generated a progressive epithelial injury in all groups in a time-dependent fashion (Figure 1). No significant difference was found at any time point based on the injury score between the grafts from young, adult, or old rats undergoing vascular perfusion alone. However, the addition of LP significantly lowered the injury score after 4 hours of cold storage for old rats. After 8 and 14 hours of cold storage, the addition of LP significantly reduced the injury score in all 3 age groups (P < 0.01). Histological changes induced by the various preservation times are summarized in Figure 1A.
Intestinal Mucosal GC
No significant difference in GC count was found between age groups at any time point. However, when a paired analysis was conducted to compare intestines with or without LP, we found that LP significantly delayed mucosal GC depletion (Figure 1B).
Age-related Changes in Kidneys and Aorta
Compared with the aortas of young animals (3 mo), aortas from old animals (20 mo) displayed reduced thickness of the vascular medial layer (Figure 2). The number of medial elastic fibers, as well as the number of vascular smooth muscle cells, was reduced in aortas at 20 months. Particularly, the presence of microcalcifications, resembling Mönckeberg’s arteriosclerosis, was observed in the media layer of all aortas at 20 months. The kidneys of old (20 mo) rats revealed histological signs of both glomerulosclerosis and tubulosclerosis as visualized by periodic acid Schiff stain.
Ussing Chamber Experiments
The PD decreased with increasing preservation time in all groups (Figure 3A). The PD was significantly higher in all the groups receiving LP at 4 and 8 hours (P < 0.01). After 14 hours the significance disappeared but the trend towards higher PD in the LP groups was still noted for all age categories (P < 0.1).
Rep mirrored PD findings and decreased with longer preservation time (Figure 3B). After 14 hours, the Rep was close to zero in the untreated intestines. The Rep was significantly higher than controls in young and old (P < 0.05) intestines with LP at 4 hours.
Intestinal permeability increased with time in all groups regardless of age. The permeability for the FSS probe increased with longer preservation time (P < 0.05) (Figure 3C). FSS-probe permeation tended to be highest in untreated old intestines when compared with old intestines with LP at all time points but this only reached statistical significance at 4 hours (P < 0.01). The FD4-probe permeability increased with longer preservation time in both young and old intestines (P < 0.05) (Figure 3D). Detailed results are presented in Table S1 (SDC, http://links.lww.com/TP/B825).
Western Blot Protein Analysis
Several age-related differences in TJ protein expression were found in normal intestines. Adult intestines had significantly higher tricellulin expression than young and old intestines. Old intestines revealed lower occludin expression compared with young intestines. Generally, the expression of all 4 TJ proteins studied decreased over the 14 hours of preservation, irrespective of donor age or luminal treatment. A significant decrease in tricellulin, claudin-3, and claudin-4 was noted in adult and old intestines already after 4 hours of cold storage. An effect of the luminal treatment was seen only sporadically (Figure 4).
The ever-increasing disparity between the demand for transplantable organs and donor organ availability has forced the reconsideration and expansion of the previously accepted criteria for organ donation. One of the first revisited donation criteria was donor age, which has constantly increased over the past 2 decades, despite well-documented age-related tissue changes and potentially impaired repair mechanisms.1,2,12 The current results indicate that aging intestines are not predisposed to more severe injury on the tissue or molecular level when compared with young intestines. This may imply that donor age alone should not disqualify from intestinal donation when other more stringent requirements such as cardiocirculatory stability, size, and immunologic matching are met. Eventually, increased utilization of potential intestinal donors may shorten the time on waiting list, which in about 25% of the US transplants exceeds 4 years.13 The intestinal mucosal barrier provides the most important line of defense against enteric bacteria, and its proper functioning depends on several factors, including the integrity of TJs. A significant body of evidence indicates several gastrointestinal, functional, and molecular changes that are induced by aging.14-16 Histological changes include shortened and scattered villi, decreased mucosal thickness, decreased villus height/width, and a reduced villus density in aging rats,5 but the literature is inconsistent.17 On the molecular level, aging is associated with remodeling of intestinal epithelial TJs, changes in TJ strands and wider paracellular spaces, decreased levels of mRNA, and decreased ZO-1 and occludin protein expression, among others. While we did not find any significant change in villus height, width, or mucosal thickness in the current study, we found that there was a significant reduction in villus density with increasing age. Similarly to other reports, the present study also found a decreasing expression of occludin with increasing age.5
The defensive role of the mucus layer is well documented and ranges from preventing bacteria or luminal chemical aggressors from gaining direct access to the epithelium18 to modulating inflammation and repair.19,20 In line with previous reports, the present study showed that luminal PEG solutions improved the overall mucosal morphology and preserved the mucin stores in the mucosal GCs. Interestingly, luminal treatment did not affect TJ protein alterations drastically, and the rather limited effect of LP seems to have been extended only to tricellulin, a protein located at the luminal, outermost part the junctional complex. Rep and mucosal permeability estimate the overall functional health of the epithelium, and the method is dependent on the interplay between several proteins within the junctional complex, the apical membrane, and the intercellular space.21 LP consistently improved the transepithelial PD and the intraepithelial current. Surprisingly, it did not result in a consistent pattern of protection as far as the permeability for FSS or FD4 was concerned. Whether this finding is due to high intragroup sample variation, because of the use of whole-thickness intestinal wall, which may have limited marker diffusion across the tissue from the mucosal to the serosal side, or technical factors remains unclear. Similarly, the corresponding findings at the TJ protein level were less consistent, suggesting that the protective mechanisms of LP are only partly dependent on shielding the TJ proteins assessed herein. This speculation is supported by the fact that improvements in the electrophysiological parameters were matched by a superior morphology.
The current study further confirms that LP appears to be an appropriate rescue strategy for donor intestines running a higher risk of developing advanced ischemia-reperfusion injury.22,23 LP significantly delayed the development of tissue injury and resulted in a mild preservation injury at a time when control intestines lost the mucosa entirely. Events surrounding organ procurement such as cardiac arrest, hemorrhage, or vasopressor use as well as unexpected logistical or medical issues prolonging cold ischemia time may compromise intestinal microcirculation and potentially affect the mucosa. While these results await independent confirmation, preferably using large animals or human intestines, recent evidence indicates that findings from intestinal preservation studies in rats may be extrapolated to clinical perspective to a larger extent than results using porcine intestines. This is due to the higher resilience of the latter to preservation injury compared with rats and humans.24
Experimental studies do often show discordant results compared with the outcomes noted in clinical practice. Part of these differences leading to frustrating delays in the clinical translation of various experimental concepts was ascribed to variations in age, sex, and comorbidities encountered in the clinical setting. These circumstances are usually overlooked in most experiments, which most often use young healthy animals. To our knowledge, no study has thus far addressed systematically the impact of age on the development of IPI. A report using human intestines from a large but rather heterogenous cohort of organ donors (in terms of donor type, duration of cold ischemia, or donor hemodynamic stability) did not find inferior histology in older donors nor any difference in graft histology between donors below or above 50.25 Age has been recognized as a potential source of misinterpretation along with other variables, such as rat strain,26 feeding status (ie, fasting),27 or the segment of small intestine studied,25,28 all appearing to be of significance for IPI. It is important to note that an accurate matching of human and rat age is challenging and somewhat controversial since several age conversion formulae are available.29 The advanced microscopic age-related changes found in the aortas and kidneys of old rats confirm the validity of the aging model used in this study and the progression towards senescence in the old rats. However, different organ systems in the rat may exhibit different aging velocities compared with human tissues and the development and fulfillment of histologic criteria matching the aging process in humans would be necessary to further validate any findings.17
A major limitation to our study is the absence of reperfusion that would have introduced further valuable end points, for example, reperfusion injury, mucosal repair, and animal survival. Aging leads to significant changes within the stem cell microenvironment, and age-related changes in intestinal stem cells could contribute to epithelial dysfunction.30 The subtle age-related differences in proliferation signals may, however, become overshadowed by the vigorous inflammatory activation, in particular through the JAK-STAT signaling.31,32 Hence, from a mechanistic point of view, reperfusion is not mandatory because of the subsequent tissue damage and inflammation.33 We believe that the current experimental setting provided enough valuable information in delineating and comparing the mechanisms behind the development of IPI and the positive effects of LP on all donor age groups.
Intestines from old donors did not convincingly demonstrate a more rapid development of IPI compared with the intestines from adult and young donors. The small differences seen were nullified by the use of LP that delayed IPI in all age groups. LP may allow longer preservation periods without an increased risk of mucosal damage.
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