Intestinal transplantation (ITx) is a life-saving procedure for patients with complicated intestinal failure. Despite improvements in donor selection and post-transplant management, outcomes lag behind those of other solid organ transplants. ITx recipients frequently suffer from persistent enteric dysfunction, for which no effective targeted therapy currently exists, while the underlying mechanisms remain insufficiently understood [1–4].

During the transplantation process, graft physiology is severely altered. Procurement requires transection of lymphatic vessels, raising concerns regarding post-ITx lipid absorption. Likewise, autonomic (sympathetic and parasympathetic) innervation is disrupted, leaving the graft reliant on hormonal regulation and intrinsic enteric nervous system activity (intramuscular and submucosal ganglia) without central control [5]. Moreover, long-term functional consequences of ischemia–reperfusion injury (IRI) on the transplanted graft remain unknown and might not be trivial considering the intestine’s high sensitivity to ischemia [5, 6]. These long-term physiological and histological modifications may impair ITx outcomes and remain poorly characterized [7].

Fluorescent dextrans are non-digestible fluorophore-labeled polysaccharides that enable quantitative assessment of intestinal permeability by measuring blood fluorescence following oral administration [8, 9]. Dextran type, defined by their molecular weight and the conjugated fluorescein, allows an independent evaluation of paracellular and transcellular transport pathways [8, 10, 11]. In addition, dextran fecal excretion provides a functional measure of gastrointestinal (GI) motility.

In this pilot study, we aimed to characterize long-term functional and morphological changes associated with intestinal transplantation in a syngeneic rat ITx model using fluorescent dextrans, following recovery from the acute post-transplant phase.

In this pilot study, we investigated intestinal graft permeability and motility 2 days before and 4 weeks after syngeneic ITx using orally administered fluorescent dextran. We observed a significant increase in paracellular absorption after ITx, whereas transcellular absorption was unchanged. This increase may be partly explained by an increased post-transplant VL and VW, which expands the absorptive surface area. Interestingly, these functional and morphological changes align with previous findings reported in the literature, using the same model [17, 18].

Molecular weight is a primary determinant of intestinal permeability, as demonstrated in Ussing chamber studies [19]. The paracellular pathway, regulated by tight-junction integrity, mainly mediates the transport of ions, water, and hydrophilic molecules with molecular weights between approximately 0.4 and 20 kDa [11]. In contrast, the transcellular pathway comprises three major mechanisms: (i) passive diffusion of small hydrophilic and lipophilic compounds (<0.4 kDa), (ii) energy-dependent active transport via specific transporters (e.g., sugars, amino acids, and vitamins), and (iii) endocytosis of larger peptides and proteins, which is the dominant route for high–molecular weight molecules (40–80 kDa) [11]. Fluorescent dextrans are validated tools for assessing intestinal permeability and enable discrimination between these pathways based on molecular weight [8]. Accordingly, we used 4 kDa dextran to evaluate paracellular permeability and 70 kDa dextran to assess transcellular transport, the latter being more selective for endocytosis [19, 20].

The timing of sampling helps to identify the intestinal site of dextran absorption. Based on GI transit kinetics, circulating dextran levels reflect sequential uptake from the jejunum, ileum, and colon [21, 22]. In rats, small intestinal transit occurs within approximately 2–6 h after gavage, with the 6-h time point already including partial colonic exposure [23]. In our model, post-ITx overall GI transit time was similar compare to Baseline, although we did not differentiate between small intestine and colonic transit.

Our results demonstrate an increased paracellular permeability after ITx, whereas transcellular permeability was unchanged and even reduced at 24 h. Because paracellular transport is driven by concentration gradients and tight-junction integrity, increased 4 kDa dextran uptake may primarily reflect structural alterations of the inter-epithelial compartment. The transcellular pathway, which represents a much larger absorptive surface, depends on epithelial metabolism and seems preserved in our experiment [24, 25]. We also observed increased VL and VW, indicating an expansion of the absorptive surface. This may also explain the heightened paracellular flux, whereas transcellular permeability remains self-limited by slower, energy-dependent transepithelial transport [26]. Increased occludin expression post-ITx appears to support this second hypothesis, although insufficient to discriminate between the two.

A global increase in absorption surface area would be expected to enhance both FITC4 and TRITC70 permeability. However, only paracellular permeability was increased. One potential explanation may be the graft hypercontractility. Accelerated intestinal transit could reduce mucosal contact time, thereby limiting absorption [25]. This effect may be more pronounced for transcellular than for passive paracellular transport, consistent with the findings of Yamamoto et al. They showed that small intestine absorption in Wistar rats decreases with increasing molecular weight, an effect that may be further exacerbated by accelerated transit. [27]. The subsequent increased delivery of dextran to the colon is unlikely to significantly affect plasma levels, given its poor colonic permeability, as suggested by PEG studies showing minimal colonic absorption of molecules ≥0.6 kDa [28]. Furthermore, accelerated small intestine transit may also contribute to the observed mucosal adaptations by functionally mimicking reduced small bowel length. This could stimulate compensatory mucosal remodeling, including increased VL and VW. Such adaptations have been described after ITx, although they may also reflect ongoing epithelial regeneration triggered by IRI [20, 29].

Graft hypercontractility have been suggested by several reports in literature. Ex vivo electrical stimulation studies in syngeneic ITx models demonstrate increased jejunal contractility with a shift in neurotransmission within the first 4 weeks after transplantation, from hypercholinergic to non-adrenergic-non-cholinergic pathways [30, 31]. In contrast, studies evaluating the impact of IRI on interstitial cells of Cajal report no change in the strength or frequency of smooth-muscle contraction [3]. Finally, Hirose et al. observed preserved neuronal, glial, and peptidergic markers after isogeneic jejunal transplantation, with evidence of partial re-innervation up to 400 days post-ITx [5]. Our results support these findings, showing no differences in intramuscular and submucosal ganglia and ganglion cells, although graft-specific transit time could not be assessed due to methodological limitations. Together, these data highlight the complexity of graft motility regulation after ITx, which complicates its pharmacological control despite preserved anatomical structure [30, 32].

Intestinal regeneration following injury is initiated by intestinal stem cells located at the base of the crypts. These cells differentiate into absorptive progenitors (e.g., enterocytes) or secretory lineages, including goblet cells, enteroendocrine cells, and Paneth cells [33]. Among the pathways regulating regeneration, Paneth cells play a central role by supporting and stimulating intestinal stem cell activity. Located within the crypts, they contribute to stem cell maintenance, modulation of the microbiota, phagocytosis, heavy metal handling, and preservation of epithelial barrier integrity through the secretion of antimicrobial peptides [34]. In humans, Paneth cell depletion has been reported in several inflammatory intestinal conditions, including celiac disease, active Crohn’s disease, necrotizing enterocolitis, and graft-versus-host disease [34–36]. Similarly, IRI is associated with reduced Paneth cell numbers, as demonstrated by Grootjans et al [37]. In the context of ITx, Kip et al. reported an early post-transplant decrease in Paneth cell numbers, although no direct association with IRI was established as Paneth cell apoptosis was not observed immediately after reperfusion [38]. Our findings are consistent with these observations and suggest that reduced Paneth cell numbers persist at 4 weeks post-transplantation.

This study has several limitations. First, the limited sample size may have precluded the detection of small differences in the studied parameters. Second, a 4-week follow-up may not adequately reflect long-term graft adaptation [39]. Reinnervation of the intestinal graft has been reported up to 400 days after ITx, and lymphatic reconnection typically occurs within 4–10 weeks post-transplantation [30]. Nevertheless, intestinal function appeared recovered, as indicated by normal recipient weight gain at 4 weeks in most rat ITx protocols [40]. In addition, normal long-chain triglyceride absorption, primarily mediated by lymphatic vessels, has been reported as early as 15 days after transplantation [41]. Third, intestinal permeability was assessed according to dextran molecular weight. Although this approach provides insight into graft adaptation, it does not fully predict nutrient or drug absorption, which primarily occurs via transcellular pathways and depends on multiple additional factors, thereby limiting the translational applicability of our findings [24]. Forth, despite known strain-dependent variability, overall GI transit was faster than previously reported [23, 42]. A more detailed assessment of motility would have required electrophysiological studies or graft-specific tracers. Fifth, a deeper analysis of intestinal barrier integrity and tight junctions may help determine whether changes in permeability reflect impaired barrier function or increased absorption surface. Moreover, the inclusion of a sham group would help distinguish these changes from those induced by the surgical stress. Finally, our experimental design did not account for the impact of immunosuppression on post-transplant intestinal adaptation, despite its central role in clinical ITx and its potential influence on graft physiology.

This pilot study provides novel insights into intestinal isograft physiology and histology. Using fluorescent dextran, we observed increased paracellular permeability with preserved transcellular absorption 4 weeks after ITx. Concurrently, villus height and width were increased, resulting in expansion of the absorptive surface. Overall GI motility was unchanged post-ITx, although small intestinal and colonic transit were not differentiated. In addition, this study demonstrates the feasibility of using fluorescent dextran to evaluate in vivo intestinal permeability and global transit. Further research is required to assess long-term adaptations beyond 4 weeks post-transplant and the impact of immunosuppression on graft function. Additionally, more comprehensive assessments of intestinal barrier integrity and electrophysiology may help elucidate the mechanisms underlying the observed changes.