- Email: [email protected]
1060 The Effect of Acute Tryptophan Depletion (ATD) On the Activity and Connectivity of An Emotional Arousal Network During Visceral Pain
depression. CONCLUSIONS: Our results show that depression reactivates inflammation in mice that have quiescent colitis and this vulnerability is reversed by a tricyclic antidepressant. These findings suggest that an episode of depression could influence the relapse IBD patient and that anti-depressants may play a role in attenuating both the depression and the severity of inflammation.
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Background and Aims: ATD temporarily reduces serotonin (5-HT) synthesis in the brain and has been to shown to produce disinhibition of central arousal circuits, and alterations in visceral perception and emotional memory in IBS patients (Kilkens et al. 2004). We hypothesized that ATD-induced reduction in 5-HT synthesis would alter activity in an emotional arousal network previously shown to be involved in central pain amplification. Methods: BOLD responses of 12 healthy women were assessed using fMRI (1.5 T) during 6 low and 6 high, individualized rectal balloon distensions (INF) and 12 non-INF or rest periods, following ATD by oral administration of a tryptophan-depleted drink, or placebo (PL). Multivariate spatiotemporal partial least squares (ST-PLS) was applied to test the interaction of treatment (ATD,PL) with a distributed pattern of brain activity discriminating the low and high INF conditions. Structural equation modeling (SEM) tested for group differences in the effective connectivity of the emotional arousal network. Results: ST-PLS revealed a network of brain responses that differentiated low and high INF and showed stronger engagement during ATD compared to PL. The network accounted for about 73% of the variance in the data analyzed, and permutation testing revealed significance at p<.01. This network included regions comprising the emotional arousal network (medial orbital frontal cortex (mOFC), rostral (rACC), and subgenual anterior cingulate cortices (sACC), and amygdala) as well as thalamus, insula, ventral tegmental area and periaqueductal grey. While most regions generally demonstrated sustained activity, amygdala and thalamic activity was only evident during the first 3 seconds (1st scan). Subjects receiving ATD showed the greatest engagement of the emotional arousal circuitry during high INF. Specifically, network analyses with SEM revealed significantly greater positive coupling between iACC →sACC and sACC → Amyg circuits (p values<.05) in ATD as compared to PL during the high INF. Conclusions: Together with our previous demonstration of ATD on visceral pain perception, these findings are consistent with the concept that acute lowering of 5-HT levels results in greater engagement of a central arousal network which is involved in central pain amplification. Support Contributed By NIH grants: K08 DK 071626 (JSL) NR04881, P50 DK64539 (EAM), R24 AT002681 (EAM), DK 64539 (EAM)
1058 Tryptophan Catabolism in Irritable Bowel Syndrome: Relationship to Cytokines, Severity and Psychiatric Co-Morbidity Peter Fitzgerald, Gerard Clarke, Paul Scully, John F. Cryan, Fergus Shanahan, Eamonn M. Quigley, Eugene Cassidy, Timothy G. Dinan Introduction: Irritable bowel syndrome (IBS) is a functional disorder which has been linked with abnormal serotonin functioning. It has a high psychiatric co-morbidity with depressive and anxiety disorders, conditions which result from a central serotonergic deficit. Tryptophan (TRP) forms the substrate for serotonin biosynthesis, but alternatively TRP can also be catabolised to kynurenine (KYN). Tryptophan catabolism along the kynurenine pathway is regulated by the enzyme indoleamine 2,3-dioxygenase (IDO), and therefore the ratio of kynurenine to tryptophan provides a measurement of IDO activity. The main inducer of IDO is the pro-inflammatory cytokine, interferon-gamma (IFN-γ). Aims: The primary aim of this study was to investigate tryptophan catabolism in IBS, and to relate such catabolism to immune activation and psychiatric co-morbidity. We hypothesised that there would be increased IFN-γ levels in IBS, and consequently, increased shunting of tryptophan along the kynurenine pathway at the expense of serotonin biosynthesis. Method: 42 female IBS subjects and 22 female healthy controls had plasma kynurenine and tryptophan concentrations measured using an HPLC method. Interferon-γ was assayed using an electro-chemiluminescence multiplex system imager. Co-morbid depressive and anxiety disorders were identified using a valid and reliable self-report tool, the Patient Health Questionnaire (PHQ), and the severity of IBS was assessed using a summary score of 4-point ordinal scales in accordance with a previously published method. Results: Of the 42 IBS subjects, 17 had severe IBS, 16 reported moderate symptoms and 9 reported mild IBS symptomatology. There was a significant positive correlation between IBS severity and Kyn/Trp ratio(r = 0.47, p=0.002). Those with severe IBS symptoms demonstrated increased tryptophan catabolism along the kynurenine pathway (i.e. increased Kyn/Trp ratio)compared to those with mildto-moderate IBS and healthy controls(p = 0.004); and those with severe IBS were over twice as likely to have depression or anxiety compared to those with less severe symptoms (RR = 2.2;95% C.I 1.2-3.9). No difference in IFN-γ levels was observed between the IBS patients and controls; however IFN-γ was positively correlated with Kyn:Trp ratio in the IBS group (r = 0.58, p = 0.005) and this was not seen in the controls (r = - 0.05, p = 0.8). Conclusion: Our results indicate an increased sensitivity to IFN-γ in the regulation of tryptophan catabolism in IBS. This may predispose to the increased rate of Kyn production observed in severe IBS cases and to the high co-morbidity with depressive and anxiety disorders in this group.
1061 Spinal NMDA NR-1 Expression Following Resolution of TNBS Colitis QiQi Zhou, Donald Price, Robert Caudle, G Nicholas Verne Background: N-methyl-D-aspartic acid (NMDA) receptors play an important role in the development of neural plasticity and hyperalgesia following inflammation or tissue injury. It is unclear, if changes in spinal-NMDA receptor expression occur following resolution of colitis. Objectives: To investigate the role of NMDA NR1receptors in the spinal cord (L4S1; T10-L1) of rats following resolution of TNBS colitis. We hypothesize that there will be altered expression of NMDA NR1 subunits in TNBS treated rats compared to controls. Methods: Male Sprague-Dawley rats (150g-250g) were treated with either intracolonic 20mg/ rat trinitrobenzene sulfonic acid (TNBS, Sigma Chemical Co.) in 50% ethanol (n=29) or an equivalent volume of saline (n=8). Sixteen weeks following induction of colitis, all rats underwent nociceptive visceral and somatic pain testing. (Zhou et al., 2007). Animals were sacrificed and their spinal cord (L4-S1; T10-L1) was retrieved from 3 groups of rats following nociceptive visceral and thermal pain testing. Group 1: saline control group (SC) (n=3); group 2: recovered rats (RC) from TNBS injection (n=3); group 3: hypersensitive rats (HC) from TNBS injection (n=3). Immunohistochemistry techniques were used to investigate spinal-NMDA receptor expression in the spinal cord (L4-S1; T10-L1) of all rats. Results: The NMDA NR1-N1,C1, and C2 expression were found in lamina I & II of spinal cord of T10-L1 and L4-S1 in HS rats but not SC and RC rats. Conclusions: Selective changes in the expression of NR1 splice variants of the NMDA receptor occur after TNBS induced colitis was healed in the rat. These results suggest a role of NMDA receptors NR1 splice variance in the development of neuronal plasticity and resulting visceral hyperalgesia/pain in the spinal cord.
1059 Differences in Localization of TRPV1 Channels and Contractile Effect of Capsaicin in Mouse Isolated Lower Gastrointestinal Tract: Higher Abundance and Sensitivity of TRPV1 Channels in Rectum and Distal Colon Than in Transverse and Proximal Colon Kenjiro Matsumoto, Kimihito Tashima, Syunji Horie BACKGROUND & AIMS: Transient receptor potential vanilloid subtype 1 (TRPV1) is a nonselective cation channels expressed in primary sensory neurons, and is activated by low pH, heat and capsaicin, a constituent of chili peppers. In lower gastrointestinal tract, TRPV1 channels are involved in visceral pain. TRPV1 channels are reported to be increased in colonic nerve fibers of patients with inflammatory bowel disease and irritable bowel syndrome. However, the distribution and the roles of afferent neurons expressing TRPV1 in motor function of lower gastrointestinal tract remain to be fully elucidated. In the present study, we investigated the differences in immunohistochemical distribution of TRPV1 channels and contractile effect of capsaicin on smooth muscle in lower gastrointestinal tract. METHODS: The rectum and distal, transverse and proximal colon were surgically isolated from male, ddY mice. The longitudinal change in smooth muscle tone was isotonically measured using Magnus apparatus. The expression of mRNA of TRPV1 was examined by RT-PCR. Extensive TRPV1-immunoreactivity was detected by using immunohistochemical staining with fluorescein-conjugated tyramide amplification. RESULTS: RT-PCR analysis revealed that mRNA expression of TRPV1 channels in all part of the lower gastrointestinal tract. The localization of TRPV1 was studied in the lower gastrointestinal tract by an immunohistochemical technique. Numerous TRPV1-immunoactive nerve fibers were found in submucosal layer, smooth muscle layer and myenteric nerve plexus. The large numbers of TRPV1-immunoreactive axons were observed in rectum and distal colon. In contrast, the TRPV1-immunoreactive axons were sparsely distributed in transverse and proximal colon. The density of TRPV1-immunoreactive axons was much lower in transverse and proximal colon than in rectum and distal colon. In the Manus experiment with rectum and distal colon, capsaicin induced transient contraction followed by long-lasting contraction. Meanwhile, in transverse and proximal colon, only transient contraction was observed. The reactivity of smooth muscle in rectum and distal colon was more sensitive than those in transverse and proximal colon. Capsaicin-induced contraction was significantly inhibited by tachykinin receptor antagonists, especially the tachykinin NK2 antagonist GR159897. CONCLUSIONS: The present results suggest that TRPV1-expressing sensory nerves facilitate lower gastrointestinal motility, especially through the release of neurokinin A. Differences in the distribution of TRPV1 and contractile effect of capsaicin, suggest the part-dependent role of TRPV1expressing sensory nerves from proximal colon to rectum.
1062 RET Activation Drives Neuronal Phenotype in CNS-Derived Neural Stem Cells Maria-Adelaide Micci, Kristen M. Kahrig, Pankaj J. Pasricha Introduction Neural stem cell (NSC) transplantation is a promising technique for the treatment of disorders of the peripheral (PNS) and central nervous system (CNS). It has been shown that CNS-derived NSC can successfully engraft in the gut of a mouse model of gastroparesis and differentiate into nNOS expressing neurons and partially rescue gastric function. However, much needs to be learnt about the mechanisms responsible for differentiation and survival of NSC in the gut and whether these processes require classical factors such as GDNF and its cognate receptor, RET. Methods CNS-NSC were transfected with a constitutively active RET mutant gene (RET-PTC2) and were either (a) cultured in standard differentiating conditions (NB27 medium) or (b) co-cultured with a fresh preparation of mouse longitudinal muscle-myenteric plexus (LMMP) placed inside transwells (no-contact). Subsequently, cell proliferation and phenotypic fate were assesses using a variety of techniques. Results Activation of the RET pathway in transfected CNS-NSC was confirmed by the phosporylation of RET as well as of ERK and Akt. This was associated with a significant increase in proliferation in CNS-NSC as compared to CNS-NSC transfected with the control vector (abs @ 490 nm were 0.097+/-0.004 in RET-PTC2 transfected CNS-NSC vs 0.078+/0.003 in ctrl cells; N=3 P<0.05). The expression of the neuronal markers βIII-tubulin and PGP9.5 was also significantly increased in CNS-NSC transfected with RET-PTC2 (1.20+/0.05 and 1.38+/- 0.11 folds respectively by Western blotting, N=3 P<0.05) along with a decrease in the glial marker GFAP (0.67+/-0.10 folds vs ctrl by Western blotting, N=3 P<0.05). Further, the expression of nNOS and peripherin were also increased (1.20 and 1.30 folds respectively by qRT-PCR). When co-cultured with mouse LMMP, CNS-NSC
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AGA Abstracts
AGA Abstracts
The Effect of Acute Tryptophan Depletion (ATD) On the Activity and Connectivity of An Emotional Arousal Network During Visceral Pain Jennifer S. Labus, Emeran A. Mayer, Tessa Kilkens, Elisabeth A. Evers, Robert-Jan Brummer, Walter H. Backes, Michiel A. van Nieuwenhoven
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Background and Aims: ATD temporarily reduces serotonin (5-HT) synthesis in the brain and has been to shown to produce disinhibition of central arousal circuits, and alterations in visceral perception and emotional memory in IBS patients (Kilkens et al. 2004). We hypothesized that ATD-induced reduction in 5-HT synthesis would alter activity in an emotional arousal network previously shown to be involved in central pain amplification. Methods: BOLD responses of 12 healthy women were assessed using fMRI (1.5 T) during 6 low and 6 high, individualized rectal balloon distensions (INF) and 12 non-INF or rest periods, following ATD by oral administration of a tryptophan-depleted drink, or placebo (PL). Multivariate spatiotemporal partial least squares (ST-PLS) was applied to test the interaction of treatment (ATD,PL) with a distributed pattern of brain activity discriminating the low and high INF conditions. Structural equation modeling (SEM) tested for group differences in the effective connectivity of the emotional arousal network. Results: ST-PLS revealed a network of brain responses that differentiated low and high INF and showed stronger engagement during ATD compared to PL. The network accounted for about 73% of the variance in the data analyzed, and permutation testing revealed significance at p<.01. This network included regions comprising the emotional arousal network (medial orbital frontal cortex (mOFC), rostral (rACC), and subgenual anterior cingulate cortices (sACC), and amygdala) as well as thalamus, insula, ventral tegmental area and periaqueductal grey. While most regions generally demonstrated sustained activity, amygdala and thalamic activity was only evident during the first 3 seconds (1st scan). Subjects receiving ATD showed the greatest engagement of the emotional arousal circuitry during high INF. Specifically, network analyses with SEM revealed significantly greater positive coupling between iACC →sACC and sACC → Amyg circuits (p values<.05) in ATD as compared to PL during the high INF. Conclusions: Together with our previous demonstration of ATD on visceral pain perception, these findings are consistent with the concept that acute lowering of 5-HT levels results in greater engagement of a central arousal network which is involved in central pain amplification. Support Contributed By NIH grants: K08 DK 071626 (JSL) NR04881, P50 DK64539 (EAM), R24 AT002681 (EAM), DK 64539 (EAM)
1058 Tryptophan Catabolism in Irritable Bowel Syndrome: Relationship to Cytokines, Severity and Psychiatric Co-Morbidity Peter Fitzgerald, Gerard Clarke, Paul Scully, John F. Cryan, Fergus Shanahan, Eamonn M. Quigley, Eugene Cassidy, Timothy G. Dinan Introduction: Irritable bowel syndrome (IBS) is a functional disorder which has been linked with abnormal serotonin functioning. It has a high psychiatric co-morbidity with depressive and anxiety disorders, conditions which result from a central serotonergic deficit. Tryptophan (TRP) forms the substrate for serotonin biosynthesis, but alternatively TRP can also be catabolised to kynurenine (KYN). Tryptophan catabolism along the kynurenine pathway is regulated by the enzyme indoleamine 2,3-dioxygenase (IDO), and therefore the ratio of kynurenine to tryptophan provides a measurement of IDO activity. The main inducer of IDO is the pro-inflammatory cytokine, interferon-gamma (IFN-γ). Aims: The primary aim of this study was to investigate tryptophan catabolism in IBS, and to relate such catabolism to immune activation and psychiatric co-morbidity. We hypothesised that there would be increased IFN-γ levels in IBS, and consequently, increased shunting of tryptophan along the kynurenine pathway at the expense of serotonin biosynthesis. Method: 42 female IBS subjects and 22 female healthy controls had plasma kynurenine and tryptophan concentrations measured using an HPLC method. Interferon-γ was assayed using an electro-chemiluminescence multiplex system imager. Co-morbid depressive and anxiety disorders were identified using a valid and reliable self-report tool, the Patient Health Questionnaire (PHQ), and the severity of IBS was assessed using a summary score of 4-point ordinal scales in accordance with a previously published method. Results: Of the 42 IBS subjects, 17 had severe IBS, 16 reported moderate symptoms and 9 reported mild IBS symptomatology. There was a significant positive correlation between IBS severity and Kyn/Trp ratio(r = 0.47, p=0.002). Those with severe IBS symptoms demonstrated increased tryptophan catabolism along the kynurenine pathway (i.e. increased Kyn/Trp ratio)compared to those with mildto-moderate IBS and healthy controls(p = 0.004); and those with severe IBS were over twice as likely to have depression or anxiety compared to those with less severe symptoms (RR = 2.2;95% C.I 1.2-3.9). No difference in IFN-γ levels was observed between the IBS patients and controls; however IFN-γ was positively correlated with Kyn:Trp ratio in the IBS group (r = 0.58, p = 0.005) and this was not seen in the controls (r = - 0.05, p = 0.8). Conclusion: Our results indicate an increased sensitivity to IFN-γ in the regulation of tryptophan catabolism in IBS. This may predispose to the increased rate of Kyn production observed in severe IBS cases and to the high co-morbidity with depressive and anxiety disorders in this group.
1061 Spinal NMDA NR-1 Expression Following Resolution of TNBS Colitis QiQi Zhou, Donald Price, Robert Caudle, G Nicholas Verne Background: N-methyl-D-aspartic acid (NMDA) receptors play an important role in the development of neural plasticity and hyperalgesia following inflammation or tissue injury. It is unclear, if changes in spinal-NMDA receptor expression occur following resolution of colitis. Objectives: To investigate the role of NMDA NR1receptors in the spinal cord (L4S1; T10-L1) of rats following resolution of TNBS colitis. We hypothesize that there will be altered expression of NMDA NR1 subunits in TNBS treated rats compared to controls. Methods: Male Sprague-Dawley rats (150g-250g) were treated with either intracolonic 20mg/ rat trinitrobenzene sulfonic acid (TNBS, Sigma Chemical Co.) in 50% ethanol (n=29) or an equivalent volume of saline (n=8). Sixteen weeks following induction of colitis, all rats underwent nociceptive visceral and somatic pain testing. (Zhou et al., 2007). Animals were sacrificed and their spinal cord (L4-S1; T10-L1) was retrieved from 3 groups of rats following nociceptive visceral and thermal pain testing. Group 1: saline control group (SC) (n=3); group 2: recovered rats (RC) from TNBS injection (n=3); group 3: hypersensitive rats (HC) from TNBS injection (n=3). Immunohistochemistry techniques were used to investigate spinal-NMDA receptor expression in the spinal cord (L4-S1; T10-L1) of all rats. Results: The NMDA NR1-N1,C1, and C2 expression were found in lamina I & II of spinal cord of T10-L1 and L4-S1 in HS rats but not SC and RC rats. Conclusions: Selective changes in the expression of NR1 splice variants of the NMDA receptor occur after TNBS induced colitis was healed in the rat. These results suggest a role of NMDA receptors NR1 splice variance in the development of neuronal plasticity and resulting visceral hyperalgesia/pain in the spinal cord.
1059 Differences in Localization of TRPV1 Channels and Contractile Effect of Capsaicin in Mouse Isolated Lower Gastrointestinal Tract: Higher Abundance and Sensitivity of TRPV1 Channels in Rectum and Distal Colon Than in Transverse and Proximal Colon Kenjiro Matsumoto, Kimihito Tashima, Syunji Horie BACKGROUND & AIMS: Transient receptor potential vanilloid subtype 1 (TRPV1) is a nonselective cation channels expressed in primary sensory neurons, and is activated by low pH, heat and capsaicin, a constituent of chili peppers. In lower gastrointestinal tract, TRPV1 channels are involved in visceral pain. TRPV1 channels are reported to be increased in colonic nerve fibers of patients with inflammatory bowel disease and irritable bowel syndrome. However, the distribution and the roles of afferent neurons expressing TRPV1 in motor function of lower gastrointestinal tract remain to be fully elucidated. In the present study, we investigated the differences in immunohistochemical distribution of TRPV1 channels and contractile effect of capsaicin on smooth muscle in lower gastrointestinal tract. METHODS: The rectum and distal, transverse and proximal colon were surgically isolated from male, ddY mice. The longitudinal change in smooth muscle tone was isotonically measured using Magnus apparatus. The expression of mRNA of TRPV1 was examined by RT-PCR. Extensive TRPV1-immunoreactivity was detected by using immunohistochemical staining with fluorescein-conjugated tyramide amplification. RESULTS: RT-PCR analysis revealed that mRNA expression of TRPV1 channels in all part of the lower gastrointestinal tract. The localization of TRPV1 was studied in the lower gastrointestinal tract by an immunohistochemical technique. Numerous TRPV1-immunoactive nerve fibers were found in submucosal layer, smooth muscle layer and myenteric nerve plexus. The large numbers of TRPV1-immunoreactive axons were observed in rectum and distal colon. In contrast, the TRPV1-immunoreactive axons were sparsely distributed in transverse and proximal colon. The density of TRPV1-immunoreactive axons was much lower in transverse and proximal colon than in rectum and distal colon. In the Manus experiment with rectum and distal colon, capsaicin induced transient contraction followed by long-lasting contraction. Meanwhile, in transverse and proximal colon, only transient contraction was observed. The reactivity of smooth muscle in rectum and distal colon was more sensitive than those in transverse and proximal colon. Capsaicin-induced contraction was significantly inhibited by tachykinin receptor antagonists, especially the tachykinin NK2 antagonist GR159897. CONCLUSIONS: The present results suggest that TRPV1-expressing sensory nerves facilitate lower gastrointestinal motility, especially through the release of neurokinin A. Differences in the distribution of TRPV1 and contractile effect of capsaicin, suggest the part-dependent role of TRPV1expressing sensory nerves from proximal colon to rectum.
1062 RET Activation Drives Neuronal Phenotype in CNS-Derived Neural Stem Cells Maria-Adelaide Micci, Kristen M. Kahrig, Pankaj J. Pasricha Introduction Neural stem cell (NSC) transplantation is a promising technique for the treatment of disorders of the peripheral (PNS) and central nervous system (CNS). It has been shown that CNS-derived NSC can successfully engraft in the gut of a mouse model of gastroparesis and differentiate into nNOS expressing neurons and partially rescue gastric function. However, much needs to be learnt about the mechanisms responsible for differentiation and survival of NSC in the gut and whether these processes require classical factors such as GDNF and its cognate receptor, RET. Methods CNS-NSC were transfected with a constitutively active RET mutant gene (RET-PTC2) and were either (a) cultured in standard differentiating conditions (NB27 medium) or (b) co-cultured with a fresh preparation of mouse longitudinal muscle-myenteric plexus (LMMP) placed inside transwells (no-contact). Subsequently, cell proliferation and phenotypic fate were assesses using a variety of techniques. Results Activation of the RET pathway in transfected CNS-NSC was confirmed by the phosporylation of RET as well as of ERK and Akt. This was associated with a significant increase in proliferation in CNS-NSC as compared to CNS-NSC transfected with the control vector (abs @ 490 nm were 0.097+/-0.004 in RET-PTC2 transfected CNS-NSC vs 0.078+/0.003 in ctrl cells; N=3 P<0.05). The expression of the neuronal markers βIII-tubulin and PGP9.5 was also significantly increased in CNS-NSC transfected with RET-PTC2 (1.20+/0.05 and 1.38+/- 0.11 folds respectively by Western blotting, N=3 P<0.05) along with a decrease in the glial marker GFAP (0.67+/-0.10 folds vs ctrl by Western blotting, N=3 P<0.05). Further, the expression of nNOS and peripherin were also increased (1.20 and 1.30 folds respectively by qRT-PCR). When co-cultured with mouse LMMP, CNS-NSC
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AGA Abstracts
AGA Abstracts
The Effect of Acute Tryptophan Depletion (ATD) On the Activity and Connectivity of An Emotional Arousal Network During Visceral Pain Jennifer S. Labus, Emeran A. Mayer, Tessa Kilkens, Elisabeth A. Evers, Robert-Jan Brummer, Walter H. Backes, Michiel A. van Nieuwenhoven