Comparison of the effects of normothermic machine perfusion and cold storage preservation on porcine intestinal allograft regenerative potential and viability.

Intestinal transplantation (IT) is the final treatment option for intestinal failure. Static cold storage (CS) is the standard preservation method used for intestinal allografts. However, CS and subsequent transplantation induce ischemia-reperfusion injury (IRI). Severe IRI impairs epithelial barrier function, including loss of intestinal stem cells (ISC), critical to epithelial regeneration. Normothermic machine perfusion (NMP) preservation of kidney and liver allografts minimizes CS-associated IRI; however, it has not been used clinically for IT. We hypothesized that intestine NMP would induce less epithelial injury and better protect the intestine's regenerative ability when compared with CS. Full-length porcine jejunum and ileum were procured, stored at 4 °C, or perfused at 34 °C for 6 hours (T6), and transplanted. Histology was assessed following procurement (T0), T6, and 1 hour after reperfusion. Real-time quantitative reverse transcription polymerase chain reaction, immunofluorescence, and crypt culture measured ISC viability and proliferative potential. A greater number of NMP-preserved intestine recipients survived posttransplant, which correlated with significantly decreased tissue injury following 1-hour reperfusion in NMP compared with CS samples. Additionally, ISC gene expression, spheroid area, and cellular proliferation were significantly increased in NMP-T6 compared with CS-T6 intestine. NMP appears to reduce IRI and improve graft regeneration with improved ISC viability and proliferation.

Changes in equine intestinal stem/progenitor cell number at resection margins in cases of small intestinal strangulation.

BACKGROUND: Intestinal epithelial stem cells (ISC) are responsible for epithelial regeneration and are critical to the intestine's ability to regain barrier function following injury. Evaluating ISC biomarker expression in cases of small intestinal strangulation (SIS) may provide insight into clinical progression. OBJECTIVES: Intestinal resection margins from cases of SIS were evaluated to determine if (1) evidence of injury could be identified using histomorphometry, (2) ISC biomarker expression was decreased in the proximal resection margin compared to control and distal resection margin, and (3) the ISC biomarker expression was associated with the number of preoperative risk factors negatively related to outcome, post-operative complications, or case outcome. STUDY DESIGN: Retrospective cohort study. METHODS: Intestinal samples were obtained intraoperatively from resection margins of adult horses with SIS and horses euthanised for reasons unrelated to colic. Preoperative risk factors negatively related to outcome, post-operative complications, and case outcome were obtained from medical records. Horses were grouped as euthanised intraoperatively, postoperatively, or survived to discharge. Histomorphometry and immunofluorescence were performed to evaluate tissue architecture and ISC and progenitor cell number. Groups were compared using one-way ANOVA. Associations between biomarker expression and the number of preoperative risk factors and post-operative complications negatively related to outcome were determined using linear regression modelling. RESULTS: Thirty-six cases of SIS were evaluated. Ki67+ cell counts were decreased in the proximal (mean = 15.45 cells; 95% CI = 10.27-20.63; SD = 4.17; p = 0.02) and distal resection margins (mean = 15.05; 95% CI = 8.46-21.64; SD = 4.141; p = 0.03) in horses euthanised postoperatively compared to control (mean = 23.62 cells; 95% CI = 19.42-27.83; SD = 5.883). In the distal resection margin, an increase in SOX9+ Ki67+ cells were associated with a decrease in the total number of preoperative risk factors negatively related to outcome (95% CI = 0.236-1.123; p = 0.008, SE = 0.1393). MAIN LIMITATIONS: Small population size. CONCLUSIONS: Proliferating cell and ISC numbers may be associated with case outcome.

Culture of equine intestinal epithelial stem cells after delayed tissue storage for future applications.

BACKGROUND: Equine intestinal epithelial stem cells (ISCs) serve as potential targets to treat horses with severe intestinal injury. The ability to isolate and store ISCs from intestinal biopsies creates an opportunity for both in vitro experiments to study ISC dynamics in a variety of intestinal diseases, and, in the future, utilize these cells as a possible therapy. If biopsies could be successfully stored prior to processing for ISCs, this would increase the availability of sample repositories for future experimental and therapeutic use. However, delayed culture of equine ISCs following prolonged sample storage has not been described. The objective of this study was to describe the isolation and culture of equine ISCs following delayed tissue storage. Small intestinal full thickness biopsies were collected post euthanasia. Fresh tissue was immediately processed or stored at 4 °C for 24, 48 and 72 h (H) before processing. Intestinal stem cells (crypts) were dissociated and cultured. Size, growth efficiency and proliferation potential were compared between resultant enteroids ("mini-guts") derived from each storage timepoint. In a separate study, growth efficiency of cryopreserved crypts was compared to cryopreserved enteroid fragments to investigate prolonged storage techniques. RESULTS: Intestinal crypts were successfully isolated and cultured from all timepoints. At 72H post initial collection, the intestine was friable with epithelial sloughing; resultant dissociation yielded more partial crypts. Enteroids grown from crypts isolated at 72H were smaller with less proliferative potential (bud units, (median 6.5, 3.75-14.25)) than control (median 25, 15-28, p < 0.0001). No statistical differences were noted from tissues stored for 24H compared to control. Following cryopreservation, growth efficiency improved when cells were stored as enteroid fragments (median 81.6%, 66.2-109) compared to crypts (median 21.2%, 20-21.5, p = 0.01). The main limitations included a small sample size and lack of additional functional assays on enteroids. CONCLUSIONS: Equine ISCs can be isolated and cultured after prolonged tissue storage. Resultant enteroids had minimal differences even after 24-48H of whole tissue storage. This suggests that ISCs could be isolated for several days from samples properly stored after procedures, including surgery or necropsy, and used to create ISC repositories for study or therapy of equine intestinal diseases.

Orthotopic Transplantation of the Full-length Porcine Intestine After Normothermic Machine Perfusion.

Successful intestinal transplantation is currently hindered by graft injury that occurs during procurement and storage, which contributes to postoperative sepsis and allograft rejection. Improved graft preservation may expand transplantable graft numbers and enhance posttransplant outcomes. Superior transplant outcomes have recently been demonstrated in clinical trials using machine perfusion to preserve the liver. We hypothesized that machine perfusion preservation of intestinal allografts could be achieved and allow for transplantation in a porcine model. Methods: Using a translational porcine model, we developed a device for intestinal perfusion. Intestinal samples were collected at the time of organ procurement, and after 6 h of machine perfusion for gross and histologic evaluation, hourly chemistry panels were performed on the perfusate and were used for protocol optimization. Following transplantation, porcine recipient physical activity, systemic blood parameters, and vital signs were monitored for 2 d before sacrifice. Results: In initial protocol development (generation 1, n = 8 grafts), multiple metabolic, electrolyte, and acid-base derangements were measured. These factors coincided with graft and mesenteric edema and luminal hemorrhage and were addressed with the addition of dialysis. In the subsequent protocol (generation 2, n = 9 grafts), differential jejunum and ileum perfusion were observed resulting in gross evidence of ileal ischemia. Modifications in vasodilating medications enhanced ileal perfusion (generation 3, n = 4 grafts). We report successful transplantation of 2 porcine intestinal allografts after machine perfusion with postoperative clinical and gross evidence of normal gut function. Conclusions: This study reports development and optimization of machine perfusion preservation of small intestine and successful transplantation of intestinal allografts in a porcine model.

Diagnostic imaging of a basihyoid bone fracture and partial avulsion of the medial pterygoid muscle in a horse.

A 3-year-old gelding was presented for further evaluation and treatment of a swelling over the left mandible and inability to eat and drink. Radiographs of the mandible were unremarkable. Computed tomography (CT) of the head demonstrated a fracture of the basihyoid bone and partial avulsion of the medial pterygoid muscle. Ultrasound examination was performed to establish a baseline and confirmed the fracture. The gelding was managed conservatively, recovered uneventfully, and was able to return to training after 4 months of rest. Key clinical message: Computed tomography and ultrasonography in the horse provided information about injury to the basihyoid bone and insertion of the medial pterygoid muscle.

Diagnostic par imagerie d'une fracture de l'os basihyoïde et avulsion partielle du muscle ptérygoïde médial chez un cheval. Un cheval hongre âgé de 3 ans fut présenté pour évaluation et traitement d'enflure du mandibule gauche et inhabilité à manger et à boire. Des radiographies du mandibule ne montraient rien de particulier. La tomodensitométrie (CT) de la tête a montré une fracture de l'os basihyoïde et une avulsion partielle du muscle ptérygoïde médial. Une échographie fut effectué afin d'établir un seuil de base et confirmer la fracture. L'hongre a été géré de manière conservatrice, a récupéré sans complication, et a pu retourner à l'entraînement après 4 mois de repos.Message clinique important :La tomodensitométrie et l'échographie chez un cheval ont fourni de l'information sur une blessure à l'os basihyoïde et à l'insertion du muscle ptérygoïde médial.(Traduit par Dr Serge Messier).