Low or no expression of BMP2 and GDNF in the spasm segment of Hirschsprung disease. (A) Western blot analysis of BMP2 and GDNF expression in normal colon and different segments of HSCR. (B) The density of BMP2/GDNF in different HSCR segments and normal colon. The protein expression of BMP2 in the spasm segment of HSCR is approximately 1/3 fold the density of distension segment of HSCR and normal colon (n = 3, t-test, ∗P < 0.05, compared with normal colon and distension segment of HSCR); The protein expression of GDNF in the spasm segment of HSCR is approximately 1/4 fold the density of distension segment of HSCR and normal colon (n = 3, t-test, ∗P < 0.05, compared with normal colon and distension segment of HSCR). (C) Immunohistochemistry was done to detect the expression of BMP2 in normal colon and different segments of HSCR. BMP2 was highly expressed in myenteric nerve plexuses of the normal colon (a) and distension segment of HSCR (b), but minimally expressed in the transitional segment (c), and spasm segment of HSCR (d) (black arrow: positive expression of BMP2 in ganglion cells). (D) Immunohistochemistry was done to detect the expression of GDNF in normal colon and different segment of HSCR. GDNF was strongly expressed in the endoderm but minimally expressed or absent in the nerve plexus (a,b) and the ectoderm of spasm segment (d). (E) Real-time PCR analysis of BMP2 and GDNF expression in normal colon and different segments of HSCR. The mRNA expression of BMP2 in the spasm segment of HSCR is approximately 1/3 fold of distension segment of HSCR and normal colon (n = 3, t-test, ∗P < 0.05, compared with normal colon and distension segment of HSCR); The mRNA expression of GDNF in the spasm segment of HSCR is approximately 1/4 fold of distension segment of HSCR and normal colon (n = 3, t-test, ∗P < 0.05, compared with normal colon and distension segment of HSCR). (F) PCR analysis of BMP2, GDNF, NGF, and BDNF expression in normal colon and different segments of HSCR. The expression of BMP2 and GDNF was decreased in the spasm segment of HSCR. The mRNA expression of BMP2 or GDNF in the spasm segment of HSCR is approximately 1/4 or 1/5 fold the density of distension segment of HSCR and normal colon (n = 3, t-test, ∗P < 0.05, compared with normal colon and distension segment of HSCR); There is no significant difference in the density of mRNA expression in NGF and BDNF between the spasm segment of HSCR and the distension segment of HSCR and normal colon (n = 3, t-test, #P > 0.05, compared with normal colon and distension segment of HSCR); (black arrow: low or no expression of GDNF in ganglion cells; green arrow: positive expression of GDNF in the endoderm of normal colon and distension segment of HSCR; blue arrow: low expression of GDNF in the endoderm of spasm segment of HSCR).
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