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Background: Short bowel syndrome (SBS) is a condition that results from inadequate intestinal absorptive capacity, usually after the loss of functional intestine. We have previously developed a severe model of SBS in zebrafish that demonstrated increased intestinal adaptation (IA) and epithelial proliferation in SBS zebrafish. However, many children with SBS do not have this extreme intestinal loss. Therefore, in this study, we developed a variation of this model to evaluate the effects of increasing intestinal length on IA and the complications of SBS., Materials and Methods: After Institutional Animal Care and Use Committee approval, adult male zebrafish were assigned to three groups: sham (n = 30), S1-SBS (n = 30), and S3-SBS (n = 30). Sham surgery included ventral laparotomy alone. S1-SBS surgery consisted of laparotomy with creation of a proximal stoma at S1 (jejunostomy equivalent) and ligation at S4. S3-SBS surgery had stoma creation at S3 (ileostomy equivalent) and the same ligation. Fish were harvested at 14 d. Markers of IA were measured from proximal intestinal segments, and the liver was analyzed for development of hepatic steatosis., Results: At 14 d, S3-SBS fish lost less weight than S1-SBS and had increased markers of IA compared with sham fish, which were decreased compared with S1-SBS fish. S3-SBS fish had decreased proximal intestinal inflammation compared with S1-SBS fish. S1-SBS fish developed extensive hepatic steatosis. Although S3-SBS fish have increased hepatic steatosis compared with sham fish, it is decreased compared with S1-SBS., Conclusions: Longer remnant intestine decreases the extent of IA, inflammation, and hepatic steatosis in a zebrafish model of SBS., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Inaccurate assessment of injected drug delivery may increase cost and morbidity or reduce efficacy. Yet currently most injections are evaluated solely by the formation of a visible wheal that might not truly estimate the actual area of effect. We hypothesized that thermal injection measurement (TIM) might verify appropriate temperature at the time of injection, as required for some temperature-sensitive vaccines and provide more accurate information about the area of delivery., Methods: 0.1 mL of either iced (n = 11) or room temperature (n = 17) methylene blue solution was injected subcutaneously in mice under anesthesia and photos taken with an iPhone 7 built-in camera and Thermal Seek Camera phone plug-in. After 5 min, true values were determined at necropsy., Results: TIM was closer in value to the measured area at necropsy than the area of the visualized skin wheal at both ice temperature and room temperature. The difference between the true value and thermal area assessment of iced solution averaged 0.15 cm 2 as compared with the difference between the true value and wheal size, which averaged 0.27 cm 2 (P = 0.04). At room temperature, this was maintained for thermal and visible wheal differences, 0.23 cm 2 and 0.65 cm 2 , respectively (P = 0.0006)., Conclusions: TIM can assess temperature at the time of injection and is more accurate than visual inspection. TIM could be applied to colorless injections and areas that are hard to visualize such as scar. As a portable phone plug-in, it might be a useful adjunct to aid the evaluation of injected drug delivery including in resource-limited settings., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Improving treatment for short bowel syndrome requires a better understanding of how intestinal adaptation is affected by factors like mechanoluminal stimulation. We hypothesized that in mice, luminal diversion via an ileostomy would drive adaptive changes similar to those seen in human intestine after diversion while offering the opportunity to study the immediate events after resection that precede intestinal adaptation., Materials and Methods: With Institutional Animal Care and Use Committee approval, a distal ileostomy with a long distal Hartman's was created in 9- to 14-week-old C57/B6 mice (n = 8). Control mice only had a midline laparotomy without stoma formation (n = 5). A rim of tissue from the proximal stoma was resected as a historical control for the proximal segment. Postoperatively, mice received a high-protein liquid diet and water ad libitum. On day 3, tissue from both the proximal and distal limbs were collected for histologic and RNA analysis. Morphometric measures, immunofluorescent antigen detection, and RNA expression were compared with Student paired t-tests with a P value < 0.05 considered significant., Results: At 3 d, survival for mice with an ileostomy was 87% and average weight loss was 12.5% of initial weight compared to 6.05% for control mice. Compared to the distal limb, the proximal limb in mice with an ileostomy demonstrated significantly taller villi with deeper and wider crypts. The proximal limb also had decreased expression of intestinal stem cell markers lgr5, bmi1, sox9, and ascl2. Fewer goblet and enteroendocrine cells per hemivillus were also noted in the proximal limb. In control mice, none of these measures were significant between proximal and distal ileum except for villus height., Conclusions: This new murine ileostomy model allows study of intestinal adaptation without intestinal anastomosis, which can be technically challenging and morbid., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Short bowel syndrome causes significant morbidity and mortality. Tissue-engineered intestine may serve as a viable replacement. Tissue-engineered small intestine (TESI) has previously been generated in the mouse model from donor cells that were harvested and immediately reimplanted; however, this technique may prove impossible in children who are critically ill, hemodynamically unstable, or septic. We hypothesized that organoid units (OU), multicellular clusters containing epithelium and mesenchyme, could be cryopreserved for delayed production of TESI., Methods: OU were isolated from <3 wk-old mouse or human ileum. OU were then cryopreserved by either standard snap freezing or vitrification. In the snap freezing protocol, OU were suspended in cryoprotectant and transferred directly to -80°C for storage. The vitrification protocol began with a stepwise increase in cryoprotectant concentration followed by liquid supercooling of the OU solution to -13°C and nucleation with a metal rod to induce vitrification. Samples were then cooled to -80°C at a controlled rate of -1°C/min and subsequently plunged into liquid nitrogen for long-term storage. OU from both groups were maintained in cryostorage for at least 72 h and thawed in a 37°C water bath. Cryoprotectant was removed with serial sucrose dilutions and OU were assessed by Trypan blue assay for post-cryopreservation viability. Via techniques previously described by our laboratory, the thawed murine or human OU were either cultured in vitro or implanted on a scaffold into the omentum of a syngeneic or irradiated Nonobese Diabetic/Severe Combined Immunodeficiency, gamma chain deficient adult mouse. The resultant TESI was analyzed by histology and immunofluorescence., Results: After cryopreservation, the viability of murine OU was significantly higher in the vitrification group (93 ± 2%, mean ± standard error of the mean) compared with standard freezing (56 ± 6%) (P < 0.001, unpaired t-test, n = 25). Human OU demonstrated similar viability after vitrification (89 ± 2%). In vitro culture of thawed OU produced expanding epithelial spheres supported by a layer of mesenchyme. TESI was successfully generated from the preserved OU. Hematoxylin and eosin staining demonstrated a mucosa composed of a simple columnar epithelium whereas immunofluorescence staining confirmed the presence of both progenitor and differentiated epithelial cells. Furthermore, beta-2-microglobulin confirmed that the human TESI epithelium originated from human cells., Conclusions: We demonstrated improved multicellular viability after vitrification over conventional cryopreservation techniques and the first successful vitrification of murine and human OU with subsequent TESI generation. Clinical application of this method may allow for delayed autologous implantation of TESI for children in extremis., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Disasters occur randomly and can severely tax the health care delivery system of affected and surrounding regions. A significant proportion of disaster survivors are children, who have unique medical, psychosocial, and logistical needs after a mass casualty event. Children are often transported to specialty centers after disasters for a higher level of pediatric care, but this can also lead to separation of these survivors from their families. In a recent theoretical article, we showed that the availability of a pediatric trauma center after a mass casualty event would decrease the time needed to definitively treat the pediatric survivor cohort and decrease pediatric mortality. However, we also found that if the pediatric center was too slow in admitting and discharging patients, these benefits were at risk of being lost as children became "trapped" in the slow center. We hypothesized that this effect could result in further increased mortality and greater costs., Methods: Here, we expand on these ideas to test this hypothesis via mathematical simulation. We examine how a delay in discharge of part of the pediatric cohort is predicted to affect mortality and the cost of inpatient care in the setting of our model., Results: We find that mortality would increase slightly (from 14.2%-16.1%), and the cost of inpatient care increases dramatically (by a factor of 21) if children are discharged at rates consistent with reported delays to reunification after a disaster from the literature., Conclusions: Our results argue for the ongoing improvement of identification technology and logistics for rapid reunification of pediatric survivors with their families after mass casualty events., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: Vascular endothelial growth factor (VEGF) is a key mediator of angiogenesis and vasculogenesis. However, the role of VEGF in the regulation of neonatal mouse development is not completely defined. We sought to determine the effect of VEGF inhibition on the development of the neonatal mouse using a transgenic approach., Materials and Methods: We generated triple transgenic mice that express the soluble VEGF receptor, (sFlt-1), specifically in the mesenchyme (dermo-1(Cre)- tetracycline reverse transcriptional activator (rtTA)(flox/flox)-tet(0)-sflt-1). Mothers of the pups (transgenic and littermate controls) were fed doxycycline chow at birth for transgene activation via breast milk, and the pups were sacrificed at various time points. To test reversibility of the phenotype, mice from both groups (n = 6) were switched to normal chow at P50 and monthly weights were measured for 9 mo., Results: Dermo-1(Cre)-rtTA(flox/flox)-tet(0)-sflt-1 mice were smaller compared with littermate controls at P21. There was a significant reduction in tissue VEGF levels following sFlt-1 expression. The rate of growth was reduced but did not impact overall survival after 1 y. A significant reduction in organ size as a percentage body weight was seen in the kidney and stomach, whereas the weight of the colon and spleen were relatively increased; however, no gross histologic difference was observed. After 6 mo on normal diet, the dermo-1(Cre)-rtTA(flox/flox)-tet(0)-sflt-1 mouse's weight doubled, indicating reversibility of phenotype., Conclusion: Mesenchymal-specific inhibition of VEGF in neonatal mice results in a severe but reversible arrest in somatic growth that does not affect overall survival at 1 yr. This mouse is a useful tool to test the function of VEGF in somatic growth., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Gastric cancer remains the second largest cause of cancer-related mortality worldwide. Postgastrectomy morbidity is considerable and quality of life is poor. Tissue-engineered stomach is a potential replacement solution to restore adequate food reservoir and gastric physiology. In this study, we performed a detailed investigation of the development of tissue-engineered stomach in a mouse model, specifically evaluating epithelial differentiation, proliferation, and the presence of putative stem cell markers., Materials and Methods: Organoid units were isolated from <3 wk-old mouse glandular stomach and seeded onto biodegradable scaffolds. The constructs were implanted into the omentum of adult mice. Implants were harvested at designated time points and analyzed with histology and immunohistochemistry., Results: Tissue-engineered stomach grows as an expanding sphere with a simple columnar epithelium organized into gastric glands and an adjacent muscularis. The regenerated gastric epithelium demonstrates differentiation of all four cell types: mucous, enteroendocrine, chief, and parietal cells. Tissue-engineered stomach epithelium proliferates at a rate comparable to native glandular stomach and expresses two putative stem cell markers: DCAMKL-1 and Lgr5., Conclusions: This study demonstrates the successful generation of tissue-engineered stomach in a mouse model for the first time. Regenerated gastric epithelium is able to appropriately proliferate and differentiate. The generation of murine tissue-engineered stomach is a necessary advance as it provides the transgenic tools required to investigate the molecular and cellular mechanisms of this regenerative process. Delineating the mechanism of how tissue-engineered stomach develops in vivo is an important precursor to its use as a human stomach replacement therapy., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Anorectal malformations (ARM) represent a variety of congenital disorders that involve abnormal termination of the anorectum. Mutations in Shh signaling and Fgf10 produce a variety of ARM phenotypes. Wnt signaling has been shown to be crucial during gastrointestinal development. We therefore hypothesized that Wnt5a may play a role in anorectal development., Methods: Wild type (WT), Wnt5a(+/-) and Wnt5a(-/-) embryos were harvested from timed pregnant mice from E15.5 to E18.5, and analyzed for anorectal phenotype. Tissues were processed for whole-mount in situ hybridization and histology., Results: Wnt5a is expressed in the embryonic WT colon and rectum. Wnt5a(-/-) mutants exhibit multiple deformities including anorectal malformation. A fistula between the urinary and intestinal tracts can be identified as early as E15.5. By E18.5, the majority of the Wnt5a(-/-) mutants display a blind-ending pouch of the distal gut., Conclusions: The expression pattern of Wnt5a and the ARM phenotype seen in Wnt5a(-/-) mutants demonstrate the critical role of Wnt5a during anorectal development. This study establishes a new model of ARM involving the Wnt5a pathway.

Background: Tissue-engineered small intestine, stomach, large intestine, esophagus, and gastroesophageal (GE) junction have been successfully formed from syngeneic cells, and employed as a rescue therapy in a small animal model. The purpose of this study is to determine if engineered intestine and stomach could be generated in an autologous, preclinical large animal model, and to identify if the tissue-engineered intestine retained features of an intact stem cell niche., Methods: A short segment of jejunum or stomach was resected from 6-wk-old Yorkshire swine. Organoid units, multicellular clusters with predominantly epithelial content, were generated and loaded onto biodegradable scaffold tubes. The constructs were then implanted intraperitoneally in the autologous host. Seven wk later, all implants were harvested and analyzed using histology and immunohistochemistry techniques., Results: Autologous engineered small intestine and stomach formed. Tissue-engineered intestinal architecture replicated that of native intestine. Histology revealed tissue-engineered small intestinal mucosa composed of a columnar epithelium with all differentiated intestinal cell types adjacent to an innervated muscularis mucosae. Intestinal subepithelial myofibroblasts, specialized cells that participate in the stem cell niche formation, were identified. Moreover, cells positive for the putative intestinal stem cell marker, doublecortin and CaM kinase-like-1 (DCAMKL-1) expression were identified at the base of the crypts. Finally, tissue-engineered stomach also formed with antral-type mucosa (mucus cells and surface foveolar cells) and a muscularis., Conclusion: We successfully generated tissue-engineered intestine with correct architecture, including features of an intact stem cell niche, in the pig model. To our knowledge, this is the first demonstration in which tissue-engineered intestine was successfully generated in an autologous manner in an animal model, which may better emulate a human host and the intended therapeutic pathway for humans.

Background/purpose: Solid organs production is an ultimate goal of tissue engineering. After refining a technique for intestinal engineering, we applied it to a solid organ, the spleen. Overwhelming postsplenectomy sepsis results in death in nearly half of all cases. This risk is pronounced in children. Necrosis of autotransplanted spleen slices occurs prior to regeneration. We postulate that tissue engineering techniques might be superior., Methods: Four groups of Lewis rats were compared: sham laparotomy, tissue-engineered spleen (TES), traditional spleen slices, and splenectomy. TES was generated from splenic units, multicellular components of juvenile spleen implanted on a biodegradable polymer scaffold, and spleen slices were derived from age-matched juveniles. Pneumococcal sepsis was induced at wk 16, and survival curves were constructed., Results: Tissue-engineered spleen protected against pneumococcal septicemia with a survival proportion of 85.7% compared with 41.17% of splenectomized animals. Spleen slice was also protective with 71.43% survival. Compared with splenectomy, control and TES groups were statistically significant (P = 0.0002, P = 0.0087; hazard ratio of splenectomy = 5.493) and the Slice group was nearly statistically significant (P = 0.0642, hazard ratio of splenectomy = 2.673)., Conclusions: TES is a novel application of tissue engineering to splenic regeneration and creates a functional spleen. This approach could be advantageous in severe pediatric trauma.