* Significantly different from control (P 0

* Significantly different from control (P 0.05). Open in a separate window Figure 5 Activation of caspase 3/7 activity in main EGC cultures. of patients with CD expressing BDNF highly. The combination of TNF- and IFN- was able to induce apoptosis in main EGCs, whereas these factors alone did not. Brain derived neurotrophic factor (BDNF) attenuate glia cell apoptosis to a small extent, but neutralizing antibodies against BDNF dramatically increased apoptosis. Conclusions Mucosal EGC apoptosis is an important obtaining in the gut of patients with CD. Proinflammatory cytokines, which are highly increased in CD, induce EGC apoptosis, whereas the neurotrophin BDNF might be protective for EGC. Since EGCs are implicated in the maintenance of the enteric mucosal integrity, EGC apoptosis may contribute to the pathophysiological changes in CD. chronic inflammation of the gut prospects to EGC injury/apoptosis, likely mediated by cleavage of the effector caspases-3. Through their involvement in neurotransmitter uptake/release and production of neurotrophic factors [9], EGCs are crucial for maintaining a metabolically homeostatic environment for the mucosa environment. Hence, we investigated the effect of EGC injury around the mucosal neurochemical microenvironment by analyzing the BDNF secretion in the mucosa of CD patients and controls. BDNF could not be Erg detected in controls (Physique 1JCL) but was highly expressed in mucosal EGCs of patients with CD (Physique 1GCI). This obtaining underlined the effect of chronic gut inflammation EGC mucosal microenvironment. Furthermore in the inflamed intestines of patients with CD TrkB receptors could be detected in GFAP-positive EGCs (Physique 1MCO). Open in a separate window Physique 1 Biopsies of the inflamed colon of patients suffering from CD and controls were double immunolabelled with anti GFAP (A,D,G,J reddish) and cCaspase-3 (B,E green) or anti BDNF (H,K green) antibodies and were analyzed by optical sectioning using a confocal microscope. Both 1alpha-Hydroxy VD4 antigens, GFAP (A) and c-Caspase-3 (B) can be detected at high levels in the intestinal wall of the inflamed colon of CD (A,B). The merged images (C) reveal an almost total overlap of both immunoreactivities (yellow). Only few GFAP-positive cells (D) display no cCaspase-3 immunoreactivity (E) in the control section (F). Although a high immunoreactivity of BDNF (H) 1alpha-Hydroxy VD4 in GDNF-positive cells (G,I) in the biopsies of patients with CD can be detected, in control biopsies GFAP-positive EGCs (J), which are positioned in the mucosal plexus in close vicinity to the epithelium of the colon, show no BDNF secretion (K,L). GFAP-positive EGCs (M) express TrKB receptors (N) in the inflamed colon of patients with CD (MCO). (Level bars, 50 m). Semiquantitative Analysis of GFAP+, BDNF+, TrkB+ and cCaspase-3+ cells in the Mucosal Plexus The numbers of GFAP+, BDNF+, TrkB+ and cCaspase-3+ cells cells per square millimeter in colon sections of patients with CD and control sections were counted. In cross-sections of the colon of patients with CD we found 346 GFAP+ cells/mm2, 20 BDNF+ cells/mm2, 20 cCaspase-3+ cells/mm2, whereas only about one third GFAP+ cells, BDNF+ cells and nearly no cCaspase-3+ cells were counted in control sections (95/mm2; 53/mm2, 22/mm2, respectively; Physique 2ACC). Open in a separate window Physique 2 Semiquantification of GFAP-positive (A), BDNF-positive (B) and cCaspase-3-positive (C) cells in cross-sections of patients with CD and controls. Whereas 30 GFAP-positive cells/mm2, 20 BDNF-positive cells/mm2, 20 cCaspase-3-positive cells/mm2 are found in CD colon walls, only one third or less of this number is usually observed under control conditions. Significantly different from control (P 0.05). Neurotrophin receptor expression on EGCs Next we investigated if EGCs might also express BDNF receptors establishing an autocrine loop. Indirect immunofluorescence exhibited expression of the BDNF specific neurotrophin receptor TrkB (Physique 3A). Western blot analysis corroborated these findings (Physique 3B). Open in a separate window Physique 3 Detection of TrkB receptor in main EGC culture. Indirect immunofluorescence (A) and Western blot (B) of the TrkB receptor. The molecular excess weight (kDa) of the full length protein, as well as the truncated form is shown on the right of the western blot figures. Apoptosis in EGC and the influence of BDNF We then tested in cultured EGCs, if the addition of BDNF could attenuate apoptosis induced by cytokines. Incubation of cultured main EGCs with TNF- or IFN- alone did not led 1alpha-Hydroxy VD4 to significantly increased apoptosis in these cells. However, 1alpha-Hydroxy VD4 the combination of both factors increased the activation of caspase 3/7 by two fold. Addition of BDNF slightly attenuated the apoptosis in EGCs, however this difference was not statistically significant (Physique 4). If the cells had been preincubated using a neutralizing antibody against BDNF, the enteric glia was even more vunerable to apoptosis extremely,.