Table 1.4 Congenital anomalies found in association with HSCR
Chapter 2: Materials and Methods
2.1. Standard sample isolation techniques
2.1.1. Embryonic mouse
A Home Office project license was not required for the work detailed within this thesis as no regulated procedures were performed. Female CD1 mice (Charles River Laboratories, Kent, UK) were time mated and sacrificed under UK Home Office Schedule 1 conditions by CO2 asphyxiation at 11.5 days post coitum (dpc). Vaginal plug = 0.5dpc. A laparotomy was performed under sterile conditions, using 70% (v/v) ethanol as a disinfectant, at which time the peritoneal cavity was opened, uterus excised en-‐block and placed in warmed high glucose (4.5g/l) Dulbecco’s modified Eagle medium with L-‐glutamine and sodium pyruvate (DMEM, Life Technologies, Paisley, UK). 9cm plastic petri dishes prepared with 25ml Sylgard® 184 (Dow Corning, Midland MI, USA) were used as dissection plates and sterilised using 70% (v/v) ethanol and exposure to UV light for 30 minutes. Each embryo was removed from the uterus intact and the decapitated before proceeding further. Dissection was performed aseptically using a dissection microscope (M165FC, Leica Microsystems, Bucks, UK) to remove any attached mesentery and to isolate the desired region of bowel (Fig 2.1). Resected specimens were kept for a maximum of 2h in high glucose DMEM at 37°C supplemented with 10ng/ml gentamycin, 100 Units/ml penicillin, and 100 μg/ml streptomycin (Life Technologies) until required.
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2.1.2. Adult and neonatal mouse
Adult CD1 mice or postnatal day 1 pups from female CD1 mice (Charles River Laboratories) were sacrificed under Schedule 1 by CO2 asphyxiation and cervical dislocation. Dissection was performed under the same conditions as embryonic mice. A laparotomy was performed, the colon removed intact and kept as per embryonic bowel until required.
2.1.3. Postnatal human
Ethical approval was obtained from the North West 3 Research Ethics Committee (Ref:10/H1002/77) to collect human colonic samples during elective surgical procedures. Individual consent was obtained from the parents of each child recruited into the study and included samples from both patients with Hirschsprung’s disease (HSCR) and controls, i.e. those undergoing either the formation or closure of colostomy for pathology not thought to affect the innervation of the bowel (Table 2.1). Samples were anonymised before processing and all human tissue was both stored and destroyed according to guidelines set down in the Human Tissue Act 2004. 1cm2 specimens of bowel were taken from both the ganglionic and aganglionic regions of the resected colon – confirmed by an absence of ganglion cells on fresh frozen sections examined by a consultant paediatric pathologist intraoperatively. A small 2mm section was immediately fixed in 4% (w/v) paraformaldehyde, and the remaining specimen was transported back to the laboratory, wrapped in sterile saline soaked gauze on ice at 4°C within 4 hours.
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Figure 2.1 Dissection of Embryonic mouse bowel
Figure 2.1. (A) Embryonic murine colon +/-‐caecum were harvested at E11.5 at which time the wave front of migratory ENS progenitors will have just reached the caecum(Druckenbrod, et al. 2005). For generation of embryonic neurospheres the caecum was removed intact. Ganglionic specimens (D) for transplantation experiments were created by dividing the caecum at A1. Aganglionic samples were taken at A2, distal to the migratory wave front and either cultured alone (B) or transplanted with a neurosphere at the proximal end (C). Any mesentery attached to the bowel was removed.
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Table 2.1. Underlying pathology and age of study recruits.
Table 2.1. Successful progenitor cell isolation is taken as the formation of neurospheres within 28 days of culture. Term gestation is taken as birth a between 37 and 42 weeks gestation. The gestational age of all births outside this range are given.
Patient Pathology Age at Gestation Successful progenitor cell isolation
from:
ID Operation Ganglionic
Bowel Aganglionic Bowel
H001 Hirschsprung’s – Short segment 1 month Term Yes Not attempted H002 Hirschsprung’s – Short segment 1 month Term Yes Not attempted H003 Hirschsprung’s – Short segment 2 months Term Yes Not attempted H004 Hirschsprung’s – Short segment 2 months Term Yes Not attempted H005 Hirschsprung’s – Short segment 1 month Term Yes Not attempted H006 Hirschsprung’s – Short segment 4 months Term Yes Not attempted H007 Hirschsprung’s – Short segment 1 year 36 Yes Not attempted H008 Hirschsprung’s – Short segment 2 months Term Yes Not attempted H009 Hirschsprung’s – Short segment 1 month Term Yes Not attempted H010 Hirschsprung’s – Short segment 2 months Term Yes Not attempted H011 Hirschsprung’s – Short segment 2 months Term Yes Not attempted H012 Hirschsprung’s – Short segment 3 months Term Yes Not attempted H013 Hirschsprung’s – Short segment 6 months Term Yes Yes H014 Hirschsprung’s – Short segment 3 months Term Yes Yes H015 Hirschsprung’s – Short segment 3 months Term Yes Yes H016 Hirschsprung’s – Long segment 3 months Term Yes Yes H017 Hirschsprung’s – Short segment 3 months Term Yes Yes H018 Hirschsprung’s – Short segment 3 months Term Yes Yes H019 Hirschsprung’s – Short segment 1 year Term Yes Yes H020 Hirschsprung’s–Ileal/Total colonic 2 weeks Term Yes No H021 Hirschsprung’s – Short segment 5 weeks Term Yes Yes H022 Hirschsprung’s – Short segment 4 months Term Yes Yes H023 Hirschsprung’s – Total colonic 3 weeks Term Yes No C001 Anorectal Malformation 1 year Term Yes N/A C002 Gastroschisis 2 months 35 Yes N/A C003 Anorectal Malformation 1 day Term Yes N/A C004 Constipation – Sigmoid colectomy 5 years Term Failed N/A C005 Constipation – Sigmoid colectomy 16 years Term Failed N/A C006 Anorectal Malformation 6 months Term Yes N/A C007 NEC – Closure of stoma 3 months 29 Yes N/A C008 Anorectal Malformation 3 months Term Yes N/A C009 Isolated colonic perforation 6 months 28 Yes N/A
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