T1769 Disruption of the Hedgehog Signaling Pathway in Crohn's Disease: A Novel Role in Intestinal...
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T1769 Disruption of the Hedgehog Signaling Pathway in Crohn's Disease: A Novel Role in Intestinal Inflammation Agnes N. Yoshimoto, Morgana T. Castelo-Branco, Claudio Azevedo, Adriane R. Neves, Fernanda Buongusto, Luiza Moraes, Cesonia A. Martinusso, Alyson do Rosario, Antonio José V. Carneiro, Celeste C. Elia, Heitor Souza Background and aims: Mucosal immunity and epithelial integrity are interconnected factors involved in intestinal homeostasis. The hedgehog (Hh) family of morphogens is essential in embryogenesis, but it has been also associated with inflammation and tissue repair. We hypothesized that the Hh-pathway could affect the inflammatory response and cell survival, crucial factors in IBD pathogenesis. Methods: Endoscopic biopsies were obtained from the inflamed colon of 14 patients with Crohn's disease (CD), 12 ulcerative colitis (UC), and 15 controls. Frozen sections of mucosal samples were analyzed by immunohistochemistry using antibodies against Sonic hedgehog (Shh), Indian hedgehog (Ihh), and Gli-1. Double immunofluorescence was used for co-localization studies. RT-PCR was used for analyzing the expression of Gli-1 mRNA. Tissue cultures of colon samples were performed during 24h in the presence of Shh peptide, anti-Shh antibody, cyclopamine, a specific Hh inhibitor, or diluent. Supernatants were analyzed for cytokine production, by ELISA, and total protein was extracted from tissue for caspase-3 activity assay. Results: CD colonic mucosa showed a lower expression of Gli-1 mRNA compared to UC and controls (p=0.03). Hh-proteins were characteristically expressed at the superficial epithelium, and a marked reduction was observed in CD compared to UC (p=0.04), and controls (p=0.001). In the lamina propria, low densities of Hh-family members basically do not co-localize with infiltrating T-cells or macrophages. Baseline levels of cytokines in culture supernatants were higher in IBD com- pared to controls. In CD colon explants, treatment with Shh peptide resulted in a significant decrease in the levels of TNF-alpha (p=0.04), IL-17 (p=0.01), and TGF-beta (p=0.03). Caspase-3 activity was higher in CD and significantly decreased after blockade of Hh, either with anti-Shh antibody(p=0.04) or with cyclopamine (p=0.03), in contrast to UC and controls. Levels of Gli-1 negatively correlated to caspase-3 activity (CC -0.75, p=0.01). Conclusion: Normal colonic mucosa constitutively expresses Hh-proteins, which appear to constitute a key pathway for intestinal homeostasis. Hh-signaling affects cytokine production and the control of apoptosis within the colonic mucosa. Disruption of the intestinal Hh- pathway might be implicated in the pathogenesis of CD. T1770 Enteric Nematodes Regulates Host Proteolytic Pathway on Immune Cells: Influence on Host Immune Response Luigi Notari, Aiping Zhao, Jennifer A. Stiltz, Shu Yan, Jaladanki N. Rao, Joseph F. Urban, Terez Shea-Donohue Enteric nematodes generate proteases contributing to their ability to adapt and survive to host defenses. We showed previously that these proteases alter intestinal epithelial permeability facilitating then their ability to activate PAR1 and PAR2 on intestinal macrophages (MΦ). Two general phenotypes of MΦ are known: classically activated MΦ (CAMΦ), characterized by increased production of NO via NOS-2 and alternatively activated MΦ (AAMΦ) character- ized by arginase-1 expression. Aim: To investigate the role of activation of PARs on MΦ function and phenotype and their regulation in the context of immune and inflammatory responses. Methods: Nippostrongylus brasiliensis (Nb) were isolated from infected mice and cultured in antibiotic-containing medium. Worm-generated serine proteases were isolated from conditioned medium and tested for activity. HEK293T cells were transfected with vectors expressing PAR1 or PAR2. Purified serine proteases were tested for ability to cleave PARs by measuring calcium signaling using a Fura2 fluorescence imaging system. Bone marrow-derived monocytes were isolated from WT or TLR4 -/- mice, differentiated into MΦ with M-CSF, and treated with PAR1 or PAR2 activating peptides (TFLLR or SLIGRL, 10- 50 μM) or purified proteases. Gene expression was quantified by real-time qPCR. Results: Two components were isolated from the worm conditioned media, with serine protease activity and apparent molecular weights of 26- and 30-kDa (SP26 and SP30). SP26 induced a calcium response in PAR2 transfected HEK293T cells, while SP30 had the same effect on PAR1 transfected cells. Treatment of bone marrow-derived MΦ with SLIGRL or SP26 upregulated IL-13 (2.2 and 30 fold respectively) and downregulated NOS-2 (75%) and PAR1 (63 and 67%) expression. Similar effects were observed in macrophages isolated from TLR4 -/- mice. TFLLR or SP30 induced a concentration-dependent downregulation of IL-13 (89 and 30%) and NOS-2 (47%) and a strong upregulation of PAR2 expression (5 ± 2 and 80 ± 3 fold). SP30 did not alter IL-13 expression in TLR4 -/- mice-derived MΦ. Conclusions: Activation of PARs on intestinal MΦ by nematode-generated serine proteases modulates immune and inflammatory responses. PAR2 activation has primarily anti-inflammatory effects while PAR1 activation has both pro-and anti-inflammatory actions. Of equal importance is the ability of PAR2 activation to negatively influence the expression of PAR1, and PAR1 to positively affect the expression of PAR2 on MΦ, providing a unique mechanism of interaction between these two receptors. This timing of the activation of PARs by SP26 or SP30 may impact the nature of the macrophage response. T1771 Diabetes is Associated With Activation of MAP Kinase That Modulates Transient K + Current in Rat Dorsal Root Ganglia Neurons: Implication in Colonic Visceral Hypersensitivity in Diabetes Gintautas Grabauskas, Andrea Heldsinger, Xiaoyin Wu, Chung Owyang Patients with longstanding diabetes mellitus frequently demonstrate visceral hypersensitivity (VH) and anorectal dysfunction. Visceral sensory neuropathy is associated with abnormal Ca 2+ signaling in the dorsal root ganglia (DRG) neurons. In addition, it has been reported that K + channels play a pivotal role in neuronal excitability and their downregulation increases pain sensation. We hypothesize that diabetes is associated with activation of MAP kinase that modulates transient K + current in rat DRG neurons. The enhanced excitability of these neurons may be responsible for the VH observed in poorly controlled diabetics. To test this S-575 AGA Abstracts hypothesis, we performed whole-cell patch-clamp recordings and Western blots using DRG neurons from control and streptozotocin-induced diabetic rats. Colorectal distension (CRD) studies were performed to examine the pathophysiological significance of our In Vitro findings. Patch-clamp whole-cell recordings on isolated DRG neurons projecting to the distal colon showed that elevation of intracellular Ca 2+ concentrations using the Ca 2+ ionophore ionomycin (1 μM) inhibited I A currents by 82% (n=15, P<0.05). Administration of the PKC inhibitor Calphostin C (1 μM) or MAP kinase inhibitor U0126 (10 μM) markedly attenuated the inhibitory effect of ionomycin (1 μM), indicating that increased intracellular Ca 2+ activates the PKC-MAPK pathway which in turn inhibits I A current. DRG neurons from 12 wk diabetic rats showed a 60% reduction in I A current (405+95 pA control vs 147+45 pA STZ-diabetic, P<0.05). Superfusion of the MAP kinase inhibitor U0126 (10 μM) significantly restored the amplitude of the I A current (from 145+35 pA to 324+65 pA, P<0.05). Western blot analysis of DRG neurons revealed that the level of pMAPK and pKv4.2, a subunit that mediates the I A current, increased with the progression of diabetes. At 12 wks of diabetes, the levels of pMAPK and pKv4.2 were 320+45% and 370+32% of control respectively (P<0.05). Behavioral pain studies showed that CRD induced visceromotor response (VMR) was mark- edly enhanced in 12 wk diabetic rats. VMR to 20, 40 and 60 mmHg CRD increased from 3+0.5, 26+3, 41+4 muscle contractions/5 sec in nondiabetic rats to 39+4, 49+6 and 62+3 contractions/5 sec in 12 wk diabetic rats. Intrathecal administration of U0126 (5 μg) normal- ized the VMR to CRD in diabetic rats. In conclusion, we demonstrated that activation of MAP kinase that modulates transient K + current occurs in diabetes. This results in the reduction of I A current and enhanced excitability of DRG neurons. These changes may contribute to VH and abnormal colonic motility frequently observed in longstanding diabetes. T1772 Alteration in the mRNA Levels of Neurotrophins and High Affinity Receptors in the Distal Colon and Primary Afferent Neurons During Colitis Li-Ya Qiao, John R. Grider We have reported that trinitrobenzene sulfonic acid (TNBS) colitis increased the immunoreac- tivity of both TrkA and TrkB, high affinity receptor for nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) respectively, in the lumbar L1 and sacral S1 dorsal root ganglia (DRG) during colitis; the level of TrkA protein was up-regulated at 3 and 7 days while TrkB only at 7 days post colitis induction. We have also shown that BDNF mRNA and protein levels were increased in L1 and S1 DRG during 3 and 7 days of colitis. AIMS: 1) to characterize the changes in the mRNA levels of TrkA and TrkB in L1 and S1 DRG during colitis; 2) to characterize the changes in the mRNA levels of TrkA and TrkB in the inflamed distal colon; and 3) to characterize the changes in the mRNA levels of NGF and BDNF in the inflamed distal colon. METHODS: Colonic inflammation was induced in rats by intracolonic instillation of TNBS (1.5 mL/kg of 60 mg/mL solution in 50 % EtOH). Control animals received 50 % EtOH. Animals were killed on day 3 or 7 following the induction of colitis. Total RNA was extracted from the distal colon as well as the L1 and S1 DRG from all animals. Real-time PCR was performed with Taqman probes for TrkA, TrkB, NGF and BDNF with GAPDH as an internal control. RESULTS: The level of TrkA mRNA was not changed in either L1 or S1 DRG following both 3 days and 7 days of colitis. In the L1 DRG, TrkB mRNA level was increased at 7 days (1.3-fold, p<0.05) but not 3 days after colitis induction. In the S1 DRG, TrkB mRNA level was increased at both 3 days (1.4- fold, p<0.05) and 7 days (1.4-fold, p<0.05) after colitis induction. In the distal colon, TrkB mRNA level was increased at 7 days (1.5-fold, p<0.05) but not 3 days after colitis induction. TrkA mRNA level was increased at both 3 days (2-fold, p<0.05) and 7 days (4.8-fold, p<0.05) after colitis induction. The level of NGF mRNA was also increased in the inflamed distal colon at both 3 days (2.5-fold, p<0.05) and 7 days (3-fold, p<0.05) of colitis. We did not detect changes in the BDNF mRNA expression in the inflamed distal colon. CONCLU- SION: NGF and TrkA were produced in the inflamed distal colon and retrogradely transported to the DRG; BDNF and TrkB gene expression were up-regulated in the DRG during colitis. Colitis may promote BDNF anterograde transport from the DRG to the distal colon. T1773 Regional-and Tissue-Specific Variation in the Pattern of Expression of Trk Neurotrophins and Differential Changes in Response to Colonic Inflammation John R. Grider, Karnam S. Murthy, John F. Kuemmerle, Li-Ya Qiao The neurotrophins are neural growth factors that interact with specific Trk receptors: nerve growth factor (NGF) with TrkA, brain-derived neurotrophic factor (BDNF) with TrkB, and neurotrophin-3 (NT-3) with TrkC. Although they are present in gut, their regional and tissue distribution and response to colonic inflammation are not well characterized. The aim of this study was to identify the distribution of these neurotrophins and changes in expression in response to colonic inflammation. The method was to divide colons from naive and inflamed (TNBS-treated for 3 and 7 days) rats into 3 equal sections (proximal, middle, and distal) and to separate mucosa and muscle layers by blunt dissection. Following homogenization, neurotrophins were extracted and measured by specific ELISAs. The results demonstrate regional and tissue-specific patterns of expression of neurotrophins in colons from naive rats and differential changes in these patterns of mucosal and muscle expression of neurotrophins in response to inflammation. In naive animals, NGF levels increased from proximal to distal regions for mucosa but were equal in all regions for muscle. Inflammation caused a time-dependent increase in NGF in mucosa of proximal (66%) and distal (76%) but not mid-colon by day 7. There was no significant change in NGF in muscle from any region or at any time point. In contrast, in naive animals, BDNF levels decreased from proximal to distal regions for mucosa but increased from proximal to distal regions for muscle. In general, basal levels of BDNF were much lower than for the other neurotrophins (6 pg/mg for BDNF vs 540pg/mg for NGF and 410pg/mg for NT-3). Inflammation caused a time-dependent increase (2- to 9-fold) in BDNF in mucosa of all regions of colon by day 7. In contrast, muscle from only the proximal region showed a time-dependent increase (9- fold) by day 7. The pattern for NT-3 was also distinct. In naïve animals, NT-3 levels increased from proximal to distal regions for mucosa and decreased from proximal to distal regions for muscle. Inflammation caused a time-dependent increase (60%) in NT-3 in mucosal from AGA Abstracts
Transcript of T1769 Disruption of the Hedgehog Signaling Pathway in Crohn's Disease: A Novel Role in Intestinal...
T1769
Disruption of the Hedgehog Signaling Pathway in Crohn's Disease: A NovelRole in Intestinal InflammationAgnes N. Yoshimoto, Morgana T. Castelo-Branco, Claudio Azevedo, Adriane R. Neves,Fernanda Buongusto, Luiza Moraes, Cesonia A. Martinusso, Alyson do Rosario, AntonioJosé V. Carneiro, Celeste C. Elia, Heitor Souza
Background and aims: Mucosal immunity and epithelial integrity are interconnected factorsinvolved in intestinal homeostasis. The hedgehog (Hh) family of morphogens is essential inembryogenesis, but it has been also associated with inflammation and tissue repair. Wehypothesized that the Hh-pathway could affect the inflammatory response and cell survival,crucial factors in IBD pathogenesis. Methods: Endoscopic biopsies were obtained from theinflamed colon of 14 patients with Crohn's disease (CD), 12 ulcerative colitis (UC), and 15controls. Frozen sections of mucosal samples were analyzed by immunohistochemistryusing antibodies against Sonic hedgehog (Shh), Indian hedgehog (Ihh), and Gli-1. Doubleimmunofluorescence was used for co-localization studies. RT-PCR was used for analyzingthe expression of Gli-1 mRNA. Tissue cultures of colon samples were performed during24h in the presence of Shh peptide, anti-Shh antibody, cyclopamine, a specific Hh inhibitor,or diluent. Supernatants were analyzed for cytokine production, by ELISA, and total proteinwas extracted from tissue for caspase-3 activity assay. Results: CD colonic mucosa showeda lower expression of Gli-1 mRNA compared to UC and controls (p=0.03). Hh-proteinswere characteristically expressed at the superficial epithelium, and a marked reduction wasobserved in CD compared to UC (p=0.04), and controls (p=0.001). In the lamina propria,low densities of Hh-family members basically do not co-localize with infiltrating T-cells ormacrophages. Baseline levels of cytokines in culture supernatants were higher in IBD com-pared to controls. In CD colon explants, treatment with Shh peptide resulted in a significantdecrease in the levels of TNF-alpha (p=0.04), IL-17 (p=0.01), and TGF-beta (p=0.03).Caspase-3 activity was higher in CD and significantly decreased after blockade of Hh, eitherwith anti-Shh antibody(p=0.04) or with cyclopamine (p=0.03), in contrast to UC andcontrols. Levels of Gli-1 negatively correlated to caspase-3 activity (CC -0.75, p=0.01).Conclusion: Normal colonic mucosa constitutively expresses Hh-proteins, which appear toconstitute a key pathway for intestinal homeostasis. Hh-signaling affects cytokine productionand the control of apoptosis within the colonic mucosa. Disruption of the intestinal Hh-pathway might be implicated in the pathogenesis of CD.
T1770
Enteric Nematodes Regulates Host Proteolytic Pathway on Immune Cells:Influence on Host Immune ResponseLuigi Notari, Aiping Zhao, Jennifer A. Stiltz, Shu Yan, Jaladanki N. Rao, Joseph F. Urban,Terez Shea-Donohue
Enteric nematodes generate proteases contributing to their ability to adapt and survive to hostdefenses. We showed previously that these proteases alter intestinal epithelial permeabilityfacilitating then their ability to activate PAR1 and PAR2 on intestinal macrophages (MΦ).Two general phenotypes of MΦ are known: classically activated MΦ (CAMΦ), characterizedby increased production of NO via NOS-2 and alternatively activated MΦ (AAMΦ) character-ized by arginase-1 expression. Aim: To investigate the role of activation of PARs on MΦfunction and phenotype and their regulation in the context of immune and inflammatoryresponses. Methods: Nippostrongylus brasiliensis (Nb) were isolated from infected mice andcultured in antibiotic-containing medium. Worm-generated serine proteases were isolatedfrom conditioned medium and tested for activity. HEK293T cells were transfected withvectors expressing PAR1 or PAR2. Purified serine proteases were tested for ability to cleavePARs by measuring calcium signaling using a Fura2 fluorescence imaging system. Bonemarrow-derived monocytes were isolated from WT or TLR4-/- mice, differentiated into MΦwith M-CSF, and treated with PAR1 or PAR2 activating peptides (TFLLR or SLIGRL, 10-50 μM) or purified proteases. Gene expression was quantified by real-time qPCR. Results:Two components were isolated from the worm conditioned media, with serine proteaseactivity and apparent molecular weights of 26- and 30-kDa (SP26 and SP30). SP26 induceda calcium response in PAR2 transfected HEK293T cells, while SP30 had the same effecton PAR1 transfected cells. Treatment of bone marrow-derived MΦ with SLIGRL or SP26upregulated IL-13 (2.2 and 30 fold respectively) and downregulated NOS-2 (75%) andPAR1 (63 and 67%) expression. Similar effects were observed in macrophages isolated fromTLR4-/- mice. TFLLR or SP30 induced a concentration-dependent downregulation of IL-13(89 and 30%) and NOS-2 (47%) and a strong upregulation of PAR2 expression (5 ± 2 and80 ± 3 fold). SP30 did not alter IL-13 expression in TLR4-/- mice-derived MΦ. Conclusions:Activation of PARs on intestinal MΦ by nematode-generated serine proteases modulatesimmune and inflammatory responses. PAR2 activation has primarily anti-inflammatory effectswhile PAR1 activation has both pro-and anti-inflammatory actions. Of equal importance isthe ability of PAR2 activation to negatively influence the expression of PAR1, and PAR1 topositively affect the expression of PAR2 on MΦ, providing a unique mechanism of interactionbetween these two receptors. This timing of the activation of PARs by SP26 or SP30 mayimpact the nature of the macrophage response.
T1771
Diabetes is Associated With Activation of MAP Kinase That ModulatesTransient K+ Current in Rat Dorsal Root Ganglia Neurons: Implication inColonic Visceral Hypersensitivity in DiabetesGintautas Grabauskas, Andrea Heldsinger, Xiaoyin Wu, Chung Owyang
Patients with longstanding diabetes mellitus frequently demonstrate visceral hypersensitivity(VH) and anorectal dysfunction. Visceral sensory neuropathy is associated with abnormalCa2+ signaling in the dorsal root ganglia (DRG) neurons. In addition, it has been reportedthat K+ channels play a pivotal role in neuronal excitability and their downregulation increasespain sensation. We hypothesize that diabetes is associated with activation of MAP kinasethat modulates transient K+ current in rat DRG neurons. The enhanced excitability of theseneurons may be responsible for the VH observed in poorly controlled diabetics. To test this
S-575 AGA Abstracts
hypothesis, we performed whole-cell patch-clamp recordings and Western blots using DRGneurons from control and streptozotocin-induced diabetic rats. Colorectal distension (CRD)studies were performed to examine the pathophysiological significance of our In Vitrofindings. Patch-clamp whole-cell recordings on isolated DRG neurons projecting to the distalcolon showed that elevation of intracellular Ca2+ concentrations using the Ca2+ ionophoreionomycin (1 μM) inhibited IA currents by 82% (n=15, P<0.05). Administration of the PKCinhibitor Calphostin C (1 μM) or MAP kinase inhibitor U0126 (10 μM) markedly attenuatedthe inhibitory effect of ionomycin (1 μM), indicating that increased intracellular Ca2+ activatesthe PKC-MAPK pathway which in turn inhibits IA current. DRG neurons from 12 wk diabeticrats showed a 60% reduction in IA current (405+95 pA control vs 147+45 pA STZ-diabetic,P<0.05). Superfusion of the MAP kinase inhibitor U0126 (10 μM) significantly restored theamplitude of the IA current (from 145+35 pA to 324+65 pA, P<0.05). Western blot analysisof DRG neurons revealed that the level of pMAPK and pKv4.2, a subunit that mediates theIA current, increased with the progression of diabetes. At 12 wks of diabetes, the levels ofpMAPK and pKv4.2 were 320+45% and 370+32% of control respectively (P<0.05).Behavioral pain studies showed that CRD induced visceromotor response (VMR) was mark-edly enhanced in 12 wk diabetic rats. VMR to 20, 40 and 60 mmHg CRD increased from3+0.5, 26+3, 41+4 muscle contractions/5 sec in nondiabetic rats to 39+4, 49+6 and 62+3contractions/5 sec in 12 wk diabetic rats. Intrathecal administration of U0126 (5 μg) normal-ized the VMR to CRD in diabetic rats. In conclusion, we demonstrated that activation ofMAP kinase that modulates transient K+ current occurs in diabetes. This results in thereduction of IA current and enhanced excitability of DRG neurons. These changes maycontribute to VH and abnormal colonic motility frequently observed in longstanding diabetes.
T1772
Alteration in the mRNA Levels of Neurotrophins and High Affinity Receptorsin the Distal Colon and Primary Afferent Neurons During ColitisLi-Ya Qiao, John R. Grider
We have reported that trinitrobenzene sulfonic acid (TNBS) colitis increased the immunoreac-tivity of both TrkA and TrkB, high affinity receptor for nerve growth factor (NGF) andbrain-derived neurotrophic factor (BDNF) respectively, in the lumbar L1 and sacral S1 dorsalroot ganglia (DRG) during colitis; the level of TrkA protein was up-regulated at 3 and 7days while TrkB only at 7 days post colitis induction. We have also shown that BDNFmRNA and protein levels were increased in L1 and S1 DRG during 3 and 7 days of colitis.AIMS: 1) to characterize the changes in the mRNA levels of TrkA and TrkB in L1 and S1DRG during colitis; 2) to characterize the changes in the mRNA levels of TrkA and TrkBin the inflamed distal colon; and 3) to characterize the changes in the mRNA levels of NGFand BDNF in the inflamed distal colon. METHODS: Colonic inflammation was induced inrats by intracolonic instillation of TNBS (1.5 mL/kg of 60 mg/mL solution in 50 % EtOH).Control animals received 50 % EtOH. Animals were killed on day 3 or 7 following theinduction of colitis. Total RNA was extracted from the distal colon as well as the L1 andS1 DRG from all animals. Real-time PCR was performed with Taqman probes for TrkA,TrkB, NGF and BDNF with GAPDH as an internal control. RESULTS: The level of TrkAmRNA was not changed in either L1 or S1 DRG following both 3 days and 7 days of colitis.In the L1 DRG, TrkB mRNA level was increased at 7 days (1.3-fold, p<0.05) but not 3 daysafter colitis induction. In the S1 DRG, TrkB mRNA level was increased at both 3 days (1.4-fold, p<0.05) and 7 days (1.4-fold, p<0.05) after colitis induction. In the distal colon, TrkBmRNA level was increased at 7 days (1.5-fold, p<0.05) but not 3 days after colitis induction.TrkA mRNA level was increased at both 3 days (2-fold, p<0.05) and 7 days (4.8-fold,p<0.05) after colitis induction. The level of NGF mRNA was also increased in the inflameddistal colon at both 3 days (2.5-fold, p<0.05) and 7 days (3-fold, p<0.05) of colitis. Wedid not detect changes in the BDNFmRNA expression in the inflamed distal colon. CONCLU-SION:NGF and TrkAwere produced in the inflamed distal colon and retrogradely transportedto the DRG; BDNF and TrkB gene expression were up-regulated in the DRG during colitis.Colitis may promote BDNF anterograde transport from the DRG to the distal colon.
T1773
Regional-and Tissue-Specific Variation in the Pattern of Expression of TrkNeurotrophins and Differential Changes in Response to Colonic InflammationJohn R. Grider, Karnam S. Murthy, John F. Kuemmerle, Li-Ya Qiao
The neurotrophins are neural growth factors that interact with specific Trk receptors: nervegrowth factor (NGF) with TrkA, brain-derived neurotrophic factor (BDNF) with TrkB, andneurotrophin-3 (NT-3) with TrkC. Although they are present in gut, their regional andtissue distribution and response to colonic inflammation are not well characterized. Theaim of this study was to identify the distribution of these neurotrophins and changes inexpression in response to colonic inflammation. The method was to divide colons fromnaive and inflamed (TNBS-treated for 3 and 7 days) rats into 3 equal sections (proximal,middle, and distal) and to separate mucosa and muscle layers by blunt dissection. Followinghomogenization, neurotrophins were extracted and measured by specific ELISAs. The resultsdemonstrate regional and tissue-specific patterns of expression of neurotrophins in colonsfrom naive rats and differential changes in these patterns of mucosal and muscle expressionof neurotrophins in response to inflammation. In naive animals, NGF levels increased fromproximal to distal regions for mucosa but were equal in all regions for muscle. Inflammationcaused a time-dependent increase in NGF in mucosa of proximal (66%) and distal (76%)but not mid-colon by day 7. There was no significant change in NGF in muscle from anyregion or at any time point. In contrast, in naive animals, BDNF levels decreased fromproximal to distal regions for mucosa but increased from proximal to distal regions formuscle. In general, basal levels of BDNF were much lower than for the other neurotrophins(6 pg/mg for BDNF vs 540pg/mg for NGF and 410pg/mg for NT-3). Inflammation causeda time-dependent increase (2- to 9-fold) in BDNF in mucosa of all regions of colon by day7. In contrast, muscle from only the proximal region showed a time-dependent increase (9-fold) by day 7. The pattern for NT-3 was also distinct. In naïve animals, NT-3 levels increasedfrom proximal to distal regions for mucosa and decreased from proximal to distal regionsfor muscle. Inflammation caused a time-dependent increase (60%) in NT-3 in mucosal from
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