Kathy A. Schall

Adult zebrafish intestine resection: a novel model of short bowel syndrome, adaptation, and intestinal stem cell regeneration

Loss of significant intestinal length from congenital anomaly or disease may lead to short bowel syndrome (SBS); intestinal failure may be partially offset by a gain in epithelial surface area, termed adaptation. Current in vivo models of SBS are costly and technically challenging. Operative times and survival rates have slowed extension to transgenic models. We created a new reproducible in vivo model of SBS in zebrafish, a tractable vertebrate model, to facilitate investigation of the mechanisms of intestinal adaptation. Proximal intestinal diversion at segment 1 (S1, equivalent to jejunum) was performed in adult male zebrafish. SBS fish emptied distal intestinal contents via stoma as in the human disease. After 2 wk, S1 was dilated compared with controls and villus ridges had increased complexity, contributing to greater villus epithelial perimeter. The number of intervillus pockets, the intestinal stem cell zone of the zebrafish increased and contained a higher number of bromodeoxyuridine (BrdU)-labeled cells after 2 wk of SBS. Egf receptor and a subset of its ligands, also drivers of adaptation, were upregulated in SBS fish. Igf has been reported as a driver of intestinal adaptation in other animal models, and SBS fish exposed to a pharmacological inhibitor of the Igf receptor failed to demonstrate signs of intestinal adaptation, such as increased inner epithelial perimeter and BrdU incorporation. We describe a technically feasible model of human SBS in the zebrafish, a faster and less expensive tool to investigate intestinal stem cell plasticity as well as the mechanisms that drive intestinal adaptation.

Fibroblast growth factor 10 alters the balance between Goblet and Paneth cells in the adult mouse small intestine

Intestinal epithelial cell renewal relies on the right balance of epithelial cell migration, proliferation, differentiation, and apoptosis. Intestinal epithelial cells consist of absorptive and secretory lineage. The latter is comprised of goblet, Paneth, and enteroendocrine cells. Fibroblast growth factor 10 (FGF10) plays a central role in epithelial cell proliferation, survival, and differentiation in several organs. The expression pattern of FGF10 and its receptors in both human and mouse intestine and their role in small intestine have yet to be investigated. First, we analyzed the expression of FGF10, FGFR1, and FGFR2, in the human ileum and throughout the adult mouse small intestine. We found that FGF10, FGFR1b, and FGFR2b are expressed in the human ileum as well as in the mouse small intestine. We then used transgenic mouse models to overexpress Fgf10 and a soluble form of Fgfr2b, to study the impact of gain or loss of Fgf signaling in the adult small intestine. We demonstrated that overexpression of Fgf10 in vivo and in vitro induces goblet cell differentiation while decreasing Paneth cells. Moreover, FGF10 decreases stem cell markers such as Lgr5, Lrig1, Hopx, Ascl2, and Sox9. FGF10 inhibited Hes1 expression in vitro, suggesting that FGF10 induces goblet cell differentiation likely through the inhibition of Notch signaling. Interestingly, Fgf10 overexpression for 3 days in vivo and in vitro increased the number of Mmp7/Muc2 double-positive cells, suggesting that goblet cells replace Paneth cells. Further studies are needed to determine the mechanism by which Fgf10 alters cell differentiation in the small intestine.

Human and Murine Tissue-Engineered Colon Exhibit Diverse Neuronal Subtypes and Can Be Populated by Enteric Nervous System Progenitor Cells When Donor Colon Is Aganglionic.

PURPOSE Tissue-engineered colon (TEC) might potentially replace absent or injured large intestine, but the enteric nervous system (ENS), a key component, has not been investigated. In various enteric neuropathic diseases in which the TEC is derived from aganglionic donor colon, the resulting construct might also be aganglionic, limiting tissue engineering applications in conditions such as Hirschsprung disease (HD). We hypothesized that TEC might contain a diverse population of enteric neuronal subtypes, and that aganglionic TEC can be populated by neurons and glia when supplemented with ENS progenitor cells in the form of neurospheres. MATERIALS AND METHODS Human and murine organoid units (OU) and multicellular clusters containing epithelium and mesenchyme were isolated from both mouse and human donor tissues, including from normally innervated and aganglionic colon. The OU were seeded onto a biodegradable scaffold and implanted within a host mouse, resulting in the growth of TEC. Aganglionic murine and human OU were supplemented with cultured neurospheres to populate the absent ENS not provided by the OU to rescue the HD phenotype. RESULTS TEC demonstrated abundant smooth muscle and clusters of neurons and glia beneath the epithelium and deeper within the mesenchyme. Motor and afferent neuronal subtypes were identified in TEC. Aganglionic OU formed TEC with absent neural elements, but neurons and glia were abundant when aganglionic OU were supplemented with ENS progenitor cells. CONCLUSION Murine and human TEC contain key components of the ENS that were not previously identified, including glia, neurons, and fundamental neuronal subtypes. TEC derived from aganglionic colon can be populated with neurons and glia when supplemented with neurospheres. Combining tissue engineering and cellular replacement therapies represents a new strategy for treating enteric neuropathies, particularly HD.

Management of pediatric intramuscular venous malformations

BACKGROUND Intramuscular venous malformations (VMs) are rare, but can be highly symptomatic. There are few reports on outcomes, particularly pain, functional limitations, and muscle contractures. We aimed to compare results of medical management, sclerotherapy, and surgical resection. METHODS We retrospectively reviewed 45 patients with an extremity or truncal intramuscular VM between June 2005 and June 2015 at a single institution. Outcomes were compared between treatment modalities with ANOVA and χ2 tests. RESULTS Six patients (13%) were treated with medical management, 4 (9%) with surgical resection, 23 (51%) with sclerotherapy, and 12 (27%) with both surgery and sclerotherapy. Sclerotherapy alone decreased pain in 72%. Only 20% of patients presented with muscle contracture. For these patients, 33% resolved with sclerotherapy, physical therapy, and aspirin; 22% resolved with surgery, and 45% had persistent contracture. 40% of patients treated with sclerotherapy then surgery developed new muscle contractures, compared to 4% of sclerotherapy only patients and 0% of surgery only patients (p=0.04). CONCLUSIONS Medical management, surgery and sclerotherapy are effective treatments for intramuscular VMs. Observation and supportive care can be a primary treatment for patients with minimal symptomatology and no functional limitations. Sclerotherapy is more effective for treating pain than contractures and when used alone, rarely causes a new muscle contracture.

Inhibition of Fgf signaling in short bowel syndrome increases weight loss and epithelial proliferation

Background. Signaling by fibroblast growth factor is critical for epithelial proliferation, differentiation, and the development of many organs, including the intestine. Fibroblast growth factor 10 and fibroblast growth factor 2c are upregulated after massive bowel resection during intestinal adaptation. This pathway is conserved highly. We hypothesized that inhibition of fibroblast growth factor signaling would impair intestinal adaptation in the zebrafish model of short bowel syndrome and allow insight into the negative regulation of this pathway. Methods. Short bowel syndrome equivalent to a high jejunostomy was generated in adult male hsp70:dnfgfr1‐GFP zebrafish, wildtype fish exposed to tyrosine‐kinase inhibitor, and wildtype fish in absence of tyrosine‐kinase inhibitor. Heat shock in hsp70:dnfgfr1‐GFP fish decreases fgf 1 expression. Parameters including weight, proliferation, and differentiation were evaluated after harvest in experimental and control groups. Results. Although short bowel syndrome zebrafish lost more weight relative to sham zebrafish in both groups, heat shock fish with short bowel syndrome lost more weight compared with non‐heat shock fish with short bowel syndrome. In the non‐heat shock controls, the villus epithelial perimeter increased in short bowel syndrome compared with sham fish, but this did not occur in heat shock fish. Non‐heat shock fish with short bowel syndrome fish had significantly increased Bromodeoxyuridine(+) proliferative cells per hemivillus compared with non‐heat shock‐sham, while heat shock‐short bowel syndrome had a more substantial increase in Bromodeoxyuridine(+) cells compared with HS‐sham. Non‐heat shock‐short bowel syndrome demonstrated a significantly increased percentage of Alcian blue(+) goblet cells per hemivillus compared with non‐heat shock‐sham, while the heat shock‐short bowel syndrome demonstrated decreased Alcian blue(+) cells compared with non‐heat shock‐short bowel syndrome. In contrast, SU5402 inhibited epithelial proliferation while increasing weight loss. Conclusion. Inhibition of fibroblast growth factor‐1 signaling in short bowel syndrome decreases epithelial adaptation, increases Bromodeoxyuridine‐labeled cells at 2 weeks, and exacerbates weight loss while decreasing epithelial goblet cells.

Intestinal adaptation in proximal and distal segments: Two epithelial responses diverge after intestinal separation

Background. In short bowel syndrome, luminal factors influence adaptation in which the truncated intestine increases villus lengths and crypt depths to increase nutrient absorption. No study has evaluated the effect of adaptation within the distal intestine after intestinal separation. We evaluated multiple conditions, including Igf1r inhibition, in proximal and distal segments after intestinal resection to evaluate the epithelial effects of the absence of mechanoluminal stimulation. Methods. Short bowel syndrome was created in adult male zebrafish by performing a proximal stoma with ligation of the distal intestine. These zebrafish with short bowel syndrome were compared to sham‐operated zebrafish. Groups were treated with the Igf1r inhibitor NVP‐AEW541, DMSO, a vehicle control, or water for 2 weeks. Proximal and distal intestine were analyzed by hematoxylin and eosin for villus epithelial circumference, inner epithelial perimeter, and circumference. We evaluated BrdU+ cells, including costaining for &bgr;‐catenin, and the microbiome was evaluated for changes. Reverse transcription quantitative polymerase chain reaction was performed for &bgr;‐catenin, CyclinD1, Sox9a, Sox9b, and c‐Myc. Results. Proximal intestine demonstrated significantly increased adaptation compared to sham‐operated proximal intestine, whereas the distal intestine showed no adaptation in the absence of luminal flow. Addition of the Igf1r inhibitor resulted in decreased adaption in the distal intestine but an increase in distal proliferative cells and proximal &bgr;‐catenin expression. While some proximal proliferative cells in short bowel syndrome colocalized &bgr;‐catenin and BrdU, the distal proliferative cells did not co‐stain for &bgr;‐catenin. Sox9a increased in the distal limb after division but not after inhibition with the Igf1r inhibitor. There was no difference in alpha diversity or species richness of the microbiome between all groups. Conclusion. Luminal flow in conjunction with short bowel syndrome significantly increases intestinal adaption within the proximal intestine in which proliferative cells contain &bgr;‐catenin. Addition of an Igf1r inhibitor decreases adaptation in both proximal and distal limbs while increasing distal proliferative cells that do not colocalize &bgr;‐catenin. Igf1r inhibition abrogates the increase in distal Sox9a expression that otherwise occurs in short bowel syndrome. Mechanoluminal flow is an important stimulus for intestinal adaptation.