Justin P Wagner1, Veronica F Sullins1, Hassan A Khalil1, James C Y Dunn2. 1. Department of Surgery, Division of Pediatric Surgery, University of California, Los Angeles, Los Angeles, CA 90095-1749, USA. 2. Department of Surgery, Division of Pediatric Surgery, University of California, Los Angeles, Los Angeles, CA 90095-1749, USA; Department of Bioengineering, University of California, Los Angeles, Los Angeles, CA 90095-7098, USA. Electronic address: jdunn@mednet.ucla.edu.
Abstract
INTRODUCTION: Hirschsprung's disease is characterized by colonic aganglionosis, curable only by surgical correction. Stem cells may offer regenerative benefits while preventing surgical risks. Existing Hirschsprung's model systems are limited by alimentary compromise and spontaneous ganglionic reconstitution. We endeavored to generate a model of permanent colonic aganglionosis to support longitudinal cell therapy studies. METHODS: Among adult female Lewis rats (n=11), laparotomy was performed and one-centimeter segments of descending colon were isolated from continuity and denervated by trans-serosal benzalkonium chloride (BAC) exposure. Postoperative weights were plotted. The colon segments were retrieved after 50 or 100days. Immunohistochemical staining (IHC) for beta-III tubulin (TUJ1) and glial fibrillary acid protein (GFAP) revealed colonic ganglia. Muscle layer diameter and the presence of ganglia were contrasted between normal and denervated segments. RESULTS: All animals survived, experienced 5% weight loss after one week, and then consistently gained weight. Isolated segments had significantly hypertrophied smooth muscle layers compared to normal colon. Ganglia were identified by IHC in normal colonic segments, and denervated colonic segments had no IHC evidence of myenteric ganglia. CONCLUSION: Colonic segmental isolation and denervation result in an effective model of irreversible colonic aganglionosis. Animals retain alimentary function. Muscularis hypertrophy, myenteric denervation, and normal animal longevity are suitable for long-term studies of cell therapy.
INTRODUCTION:Hirschsprung's disease is characterized by colonic aganglionosis, curable only by surgical correction. Stem cells may offer regenerative benefits while preventing surgical risks. Existing Hirschsprung's model systems are limited by alimentary compromise and spontaneous ganglionic reconstitution. We endeavored to generate a model of permanent colonic aganglionosis to support longitudinal cell therapy studies. METHODS: Among adult female Lewis rats (n=11), laparotomy was performed and one-centimeter segments of descending colon were isolated from continuity and denervated by trans-serosal benzalkonium chloride (BAC) exposure. Postoperative weights were plotted. The colon segments were retrieved after 50 or 100days. Immunohistochemical staining (IHC) for beta-III tubulin (TUJ1) and glial fibrillary acid protein (GFAP) revealed colonic ganglia. Muscle layer diameter and the presence of ganglia were contrasted between normal and denervated segments. RESULTS: All animals survived, experienced 5% weight loss after one week, and then consistently gained weight. Isolated segments had significantly hypertrophied smooth muscle layers compared to normal colon. Ganglia were identified by IHC in normal colonic segments, and denervated colonic segments had no IHC evidence of myenteric ganglia. CONCLUSION:Colonic segmental isolation and denervation result in an effective model of irreversible colonic aganglionosis. Animals retain alimentary function. Muscularis hypertrophy, myenteric denervation, and normal animal longevity are suitable for long-term studies of cell therapy.
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