- Email: [email protected]
Tu1881 Chronic Stress and Pain: Evidence for Differential Effects on Visceral and Somatosensory Pain
AGA Abstracts
the IGVEs we recently identified in the colorectum. Greater than 90% of IGVEs and "simple" type varicose nerve endings in the CM layer were CGRP-positive (N=10). No intraganglionic laminar endings (IGLEs) were observed that resembled those reported for vagal afferent endings. The other types of spinal afferent endings consisted of "simple" fine varicose nerve endings that innervated internodal strands, the submucosa and mucosa, blood vessels and longitudinal muscle (N=10). Spinal afferent endings were rare in the esophagus, but in 2 animals consisted on "simple" type fine varicose endings that ramified within the skeletal muscle and/or myenteric ganglia. We present the first complete characterization of the different types of spinal afferent nerve endings that innervate the upper gastrointestinal (GI) tract of a mammal. The findings reveal a complex array of different types of primary afferent endings that innervate specific layers of the stomach. Some of the novel classes of nerve endings identified must underlie the transduction of noxious and/or innocuous stimuli from the upper GI-tract in vivo. Now that the morphology and neurochemistry of spinal afferent endings can be identified in this region of the GI-tract, future studies should be directed to understanding how pain signals originate in these sensory endings.
Tu1881 Chronic Stress and Pain: Evidence for Differential Effects on Visceral and Somatosensory Pain Gen Zheng, John M. Hayes, Shuangsong Hong, John W. Wiley Introduction: We reported previously that chronic water avoidance (WA) stress and corticosterone treatment in healthy rats resulted in visceral hyperalgesia and were associated with up-regulation of pro-nociceptive transient receptor potential vanilloid type 1 (TRPV1) and down-regulation of anti-nociceptive cannabinoid receptor 1 (CB1) in L6-S2 dorsal root ganglions (DRGs) innervating the colon. Recovery from visceral hyperalgesia takes ~30 days, whereas, somatosensory hyperalgesia recovers typically in 72 hr. The pathway involved in the earlier recovery from somatosensory hyperalgesia is unknown. Objectives: We examined the hypothesis that the earlier recovery from somatosensory hyperalgesia involves reversible changes in voltage-gated sodium ion channels Nav1.7 and Nav1.8 which are involved in somatosensory pain signaling. Methods: Male rats were subjected to WA stress 1 hr/day for 10 consecutive days. Visceromotor response (VMR) to colorectal distention (CRD) was measured at 24 hr and 72 hr after the 10-day stress phase, whereas somatosensory pain response was measured using the Hargreaves test (thermal hyperalgesia) and Von Frey test (mechanical allodynia). L4-L5 DRGs, which contribute to sciatic nerve somatosensory innervation to the lower extremities, were removed and processed for immunofluorescence analysis of Nav1.7, Nav 1.8 sodium channels, and double-labeled with the nociceptive neuronal marker, peripherin, Results: Hyperalgesia was observed in both visceral and somatosensory pain responses at 24 hr after completing the 10-day WA stress. Visceral hyperalgesia in response to CRD persisted at 72 hr after chronic stress and vanished after 6 weeks of recovery. In contrast, mechanical and thermal hyperalgesia observed in the hindpaw after chronic stress returned to baseline levels at 72 hr. No significant changes were observed in TRPV1 and CB1 protein levels in L4-L5 DRGs in stressed rats compared to the controls. In contrast, both Nav1.7/peripherin and Nav1.8/peripherin double-positive neurons were significantly increased in L4-L5 DRGs from stressed rats (41% ±2% and 75%±5%, respectively) compared to the controls (26%±2% and 61%%±6%, respectively) at 24 hr after chronic stress, and reverted to baseline levels at 72 hr after completion of the stress phase (p<0.05). Conclusion: These data indicate that chronic, intermittent stress has differential effects on visceral and somatosensory pain pathways. Visceral hyperalgesia involves enhancement in TRPV1 expression that recovers slowly over 30 days, whereas somatosensory hyperalgesia involves increased expression in Nav1.7 and Nav1.8 sodium channels that return to baseline levels 72 hr after chronic stress. These data suggest that chronic stress-induced visceral and lower extremity somatosensory hyperalgesia can be treated selectively and preferentially at different levels of the spinal cord.
Tu1879 Mechanosensitivity of Spinal Sensory Axons on Mesenteric Blood Vessels in the Guinea Pig Small Intestine Adam Humenick, Bao-Nan Chen, Lukasz Wiklendt, Philip G. Dinning, Nicholas J. Spencer, Marcello Costa, Simon J. Brookes Background: A major population of extrinsic afferent neurons innervating the gut have endings associated with blood vessels in the mesenteric membranes or in the gut wall. These medium-to-high threshold mechano-nociceptors are sometimes referred to as "mesenteric" or "serosal" afferents because of the apparent location of their endings. They are typically activated by large distensions, and by a variety of chemicals including capsaicin, bradykinin, prostaglandin and ischaemia. However, their responses to vascular perfusion pressure have not been studied in detail. Methods: Tubular preparations, 12-14mm long, of guinea pig ileum, with attached mesentery and blood vessels, were set up in vitro. Extramural arteries were cannulated and extracellular, single unit recordings were made from extrinsic afferent nerves innervating the vessels, with platinum wire electrodes in paraffin oil. Afferents were tested for their responses to increased intra-arterial pressure, probing with von Frey hairs, longitudinal distension of vessels and capsaicin or Krebs solution warmed to >42˚C. Results: Extrinsic afferents that were excited by either warmed Krebs solution or capsaicin, were also excited by either axial stretch of vessels and/or by raising intraluminal pressure from 5mm Hg (resting) to 60mmHg (within the physiological range). Vessel distension also also activated powerful waves of peristaltic contractions in the preparation after a short delay. The ends of the gut were open to the bath, so the effects of raised vascular pressure on motility were not due to leakage and distension of the gut lumen. The latency of these perfusion-induced contractions (but not their amplitude) was reduced by prior exposure to a desensitising exposure to capsaicin. Conclusion: Extrinsic mechano-nociceptive ("serosal and mesenteric") sensory neurons with endings associated with intramural and extramural arteries can be activated by physiological levels of intra-arterial pressure, as well as by traction on the vessels, suggesting that they respond to multiple types of mechanical distortion. Their activation contributes to initiation of propulsive motility by intravascular pressurisation but is not essential for it.
Tu1882 Distinct Structure and Mechanosensitive Function of Placodes- and Neural Crest-Derived Nociceptors in the Guinea Pig Esophagus Fei Ru, Alexander Sverstad, Robert Dusenka, Svetlana Grobarcikova, Peter Banovcin, Alzbeta Trancikova, Marian Kollarik Afferent nociceptive C-fibers in the esophagus are derived from two embryonic sources: spinal dorsal root ganglia (DRG) and vagal jugular fibers are derived from neural crest and vagal nodose fibers are derived from placodes. We have previously shown that these embryonic types differ in activation profile and neurotrphic regulation. Here we addressed the hypothesis that the neural crest- and placodes-derived C-fibers differ in mechanical responsiveness and the structure of their nerve terminals. Extracellular single unit recordings were made from the T2 DRG, nodose and jugular neurons projecting C-fibers into the esophagus in the isolated innervated guinea pig esophagus preparation. Nerve terminals in the esophagus were visualized following selective in vivo injection of adeno-associated virus (AAV) vectors encoding GFP into the nodose and T2 DRG ganglia and immunostaining for GFP in wholemount preparations. The majority of nodose C-fibers (15/17, 90%), but only a minority of DRG (94/124, 25%) and jugular (7/29, 24%) fibers were responsive to esophageal distention (p<0.05 vs. nodose). The longitudinal size of mechanosensitive receptive fields determined by focal compression was relatively small in nodose C-fibers (3±1mm, n=32), while DRG (10±1mm, n=139) and jugular (11±1mm, n=38) nerve fiber terminals were relatively large (P<0.05 vs. nodose). The putative nerve terminals of nodose C-fibers (visualized by GFP expression) were located in the innermost circular muscle layer just beneath mucosa, structurally resembled intramuscular arrays described previously in the stomach, and were relatively small (1.3±0.8mm, n=26 from 3 guinea pigs). In contrast, the putative DRG nerve terminals were located in the myenteric layer as delimited areas of networks of branching fine fibers often forming intraganglionic structures and projecting into the mucosa. The DRG terminals were relatively large (ranging from 0.5 mm to 11 mm, n=15 from 5 guinea pig). We conclude that the placodes- and neural crest-derived esophageal nociceptors differ in mechanosensitive function and innervation area. The placodes-derived nodose C-fiber terminals are distention-responsive and are located in the innermost circular muscle. In contrast, the neural crest-derived fibers are mostly distention-unresponsive and innervate both myenteric layer and mucosa. Our data indicate that the information regarding noxious distension will be relayed to the CNS mostly via placodal nodose nociceptors, along with a minority of neural crest nociceptors. In contrast, the information regarding noxious substances in esophageal lumen will be relayed to the CNS via neural crest nociceptors. Supported by BioMed Martin (ITMS: 26220220187) co-funded by EU.
Tu1880 What Do Extrinsic Sensory Neurons "Tell" the Body About Gut Motility? Bao-Nan Chen, Adam Humenick, Lukasz Wiklendt, Nicholas J. Spencer, Philip G. Dinning, Marcello Costa, Simon J. Brookes Background. Extrinsic primary afferent neurons mediate reflexes and sensations from the gastrointestinal tract. These sensory neurons can be activated by mechanical and chemical stimuli (including inflammatory mediators). Much of our understanding of afferent mechanosensitivity is based on imposed mechanical stimuli, either in vivo or in vitro. Under physiological conditions, such stimuli often evoke enteric motor reflexes, which complicate analysis of mechanosensitivity. In the present study, we investigated the nature of sensory neurons activated during physiological motor activity using small tubular preparations of gut in vitro. Methods. In isolated segments of guinea-pig distal colon, mechanical stimuli of low intensity were delivered, and resulting motor patterns recorded in terms of both intraluminal pressure and intestinal wall movements. High resolution intraluminal fibre-optic manometry (1cm sensor spacing with a 1.3mm diameter catheter) was combined with spatiotemporal mapping and extracellular recording of sensory neuron firing from colonic nerves innervating 3-4 cm specimens. Intraluminal pressure could be increased, in the non-noxious range (4 15mmHg), triggering propagating peristaltic contractions that could empty the segment of fluid. Sensory firing could be related specifically to mechanical changes (pressure, diameter) in the sensory neuron's identified field of innervation, with fine spatial and temporal resolution. Results. Low levels of spontaneous firing increased dramatically during luminal distension with Krebs solution to 4-15 mmHg. Bursts of sensory firing correlated strongly with waves of contraction. Two distinct patterns of firing could be distinguished. Some low threshold afferents fired phasically, just prior to, or during muscle shortening. The other pattern was more prolonged bursts of firing associated with lengthening of the muscle after contractions. None of the afferents showed bursts of firing during the peaks of intraluminal pressure (ie: at periods of maximal shortening). Addition of 1uM nicardipine with 1uM hyoscine led to abolition of distension-evoked phasic contractile activity, leaving passive mechanical distension responses. Sensory firing was reduced, lacked bursts and showed slowly adapting firing. Conclusions: Afferent firing in the non noxious range is primarily associated with either the onset of contractions, or lengthening after contractions, but surprisingly, not with the peaks of intraluminal pressure.
AGA Abstracts
S-926
the IGVEs we recently identified in the colorectum. Greater than 90% of IGVEs and "simple" type varicose nerve endings in the CM layer were CGRP-positive (N=10). No intraganglionic laminar endings (IGLEs) were observed that resembled those reported for vagal afferent endings. The other types of spinal afferent endings consisted of "simple" fine varicose nerve endings that innervated internodal strands, the submucosa and mucosa, blood vessels and longitudinal muscle (N=10). Spinal afferent endings were rare in the esophagus, but in 2 animals consisted on "simple" type fine varicose endings that ramified within the skeletal muscle and/or myenteric ganglia. We present the first complete characterization of the different types of spinal afferent nerve endings that innervate the upper gastrointestinal (GI) tract of a mammal. The findings reveal a complex array of different types of primary afferent endings that innervate specific layers of the stomach. Some of the novel classes of nerve endings identified must underlie the transduction of noxious and/or innocuous stimuli from the upper GI-tract in vivo. Now that the morphology and neurochemistry of spinal afferent endings can be identified in this region of the GI-tract, future studies should be directed to understanding how pain signals originate in these sensory endings.
Tu1881 Chronic Stress and Pain: Evidence for Differential Effects on Visceral and Somatosensory Pain Gen Zheng, John M. Hayes, Shuangsong Hong, John W. Wiley Introduction: We reported previously that chronic water avoidance (WA) stress and corticosterone treatment in healthy rats resulted in visceral hyperalgesia and were associated with up-regulation of pro-nociceptive transient receptor potential vanilloid type 1 (TRPV1) and down-regulation of anti-nociceptive cannabinoid receptor 1 (CB1) in L6-S2 dorsal root ganglions (DRGs) innervating the colon. Recovery from visceral hyperalgesia takes ~30 days, whereas, somatosensory hyperalgesia recovers typically in 72 hr. The pathway involved in the earlier recovery from somatosensory hyperalgesia is unknown. Objectives: We examined the hypothesis that the earlier recovery from somatosensory hyperalgesia involves reversible changes in voltage-gated sodium ion channels Nav1.7 and Nav1.8 which are involved in somatosensory pain signaling. Methods: Male rats were subjected to WA stress 1 hr/day for 10 consecutive days. Visceromotor response (VMR) to colorectal distention (CRD) was measured at 24 hr and 72 hr after the 10-day stress phase, whereas somatosensory pain response was measured using the Hargreaves test (thermal hyperalgesia) and Von Frey test (mechanical allodynia). L4-L5 DRGs, which contribute to sciatic nerve somatosensory innervation to the lower extremities, were removed and processed for immunofluorescence analysis of Nav1.7, Nav 1.8 sodium channels, and double-labeled with the nociceptive neuronal marker, peripherin, Results: Hyperalgesia was observed in both visceral and somatosensory pain responses at 24 hr after completing the 10-day WA stress. Visceral hyperalgesia in response to CRD persisted at 72 hr after chronic stress and vanished after 6 weeks of recovery. In contrast, mechanical and thermal hyperalgesia observed in the hindpaw after chronic stress returned to baseline levels at 72 hr. No significant changes were observed in TRPV1 and CB1 protein levels in L4-L5 DRGs in stressed rats compared to the controls. In contrast, both Nav1.7/peripherin and Nav1.8/peripherin double-positive neurons were significantly increased in L4-L5 DRGs from stressed rats (41% ±2% and 75%±5%, respectively) compared to the controls (26%±2% and 61%%±6%, respectively) at 24 hr after chronic stress, and reverted to baseline levels at 72 hr after completion of the stress phase (p<0.05). Conclusion: These data indicate that chronic, intermittent stress has differential effects on visceral and somatosensory pain pathways. Visceral hyperalgesia involves enhancement in TRPV1 expression that recovers slowly over 30 days, whereas somatosensory hyperalgesia involves increased expression in Nav1.7 and Nav1.8 sodium channels that return to baseline levels 72 hr after chronic stress. These data suggest that chronic stress-induced visceral and lower extremity somatosensory hyperalgesia can be treated selectively and preferentially at different levels of the spinal cord.
Tu1879 Mechanosensitivity of Spinal Sensory Axons on Mesenteric Blood Vessels in the Guinea Pig Small Intestine Adam Humenick, Bao-Nan Chen, Lukasz Wiklendt, Philip G. Dinning, Nicholas J. Spencer, Marcello Costa, Simon J. Brookes Background: A major population of extrinsic afferent neurons innervating the gut have endings associated with blood vessels in the mesenteric membranes or in the gut wall. These medium-to-high threshold mechano-nociceptors are sometimes referred to as "mesenteric" or "serosal" afferents because of the apparent location of their endings. They are typically activated by large distensions, and by a variety of chemicals including capsaicin, bradykinin, prostaglandin and ischaemia. However, their responses to vascular perfusion pressure have not been studied in detail. Methods: Tubular preparations, 12-14mm long, of guinea pig ileum, with attached mesentery and blood vessels, were set up in vitro. Extramural arteries were cannulated and extracellular, single unit recordings were made from extrinsic afferent nerves innervating the vessels, with platinum wire electrodes in paraffin oil. Afferents were tested for their responses to increased intra-arterial pressure, probing with von Frey hairs, longitudinal distension of vessels and capsaicin or Krebs solution warmed to >42˚C. Results: Extrinsic afferents that were excited by either warmed Krebs solution or capsaicin, were also excited by either axial stretch of vessels and/or by raising intraluminal pressure from 5mm Hg (resting) to 60mmHg (within the physiological range). Vessel distension also also activated powerful waves of peristaltic contractions in the preparation after a short delay. The ends of the gut were open to the bath, so the effects of raised vascular pressure on motility were not due to leakage and distension of the gut lumen. The latency of these perfusion-induced contractions (but not their amplitude) was reduced by prior exposure to a desensitising exposure to capsaicin. Conclusion: Extrinsic mechano-nociceptive ("serosal and mesenteric") sensory neurons with endings associated with intramural and extramural arteries can be activated by physiological levels of intra-arterial pressure, as well as by traction on the vessels, suggesting that they respond to multiple types of mechanical distortion. Their activation contributes to initiation of propulsive motility by intravascular pressurisation but is not essential for it.
Tu1882 Distinct Structure and Mechanosensitive Function of Placodes- and Neural Crest-Derived Nociceptors in the Guinea Pig Esophagus Fei Ru, Alexander Sverstad, Robert Dusenka, Svetlana Grobarcikova, Peter Banovcin, Alzbeta Trancikova, Marian Kollarik Afferent nociceptive C-fibers in the esophagus are derived from two embryonic sources: spinal dorsal root ganglia (DRG) and vagal jugular fibers are derived from neural crest and vagal nodose fibers are derived from placodes. We have previously shown that these embryonic types differ in activation profile and neurotrphic regulation. Here we addressed the hypothesis that the neural crest- and placodes-derived C-fibers differ in mechanical responsiveness and the structure of their nerve terminals. Extracellular single unit recordings were made from the T2 DRG, nodose and jugular neurons projecting C-fibers into the esophagus in the isolated innervated guinea pig esophagus preparation. Nerve terminals in the esophagus were visualized following selective in vivo injection of adeno-associated virus (AAV) vectors encoding GFP into the nodose and T2 DRG ganglia and immunostaining for GFP in wholemount preparations. The majority of nodose C-fibers (15/17, 90%), but only a minority of DRG (94/124, 25%) and jugular (7/29, 24%) fibers were responsive to esophageal distention (p<0.05 vs. nodose). The longitudinal size of mechanosensitive receptive fields determined by focal compression was relatively small in nodose C-fibers (3±1mm, n=32), while DRG (10±1mm, n=139) and jugular (11±1mm, n=38) nerve fiber terminals were relatively large (P<0.05 vs. nodose). The putative nerve terminals of nodose C-fibers (visualized by GFP expression) were located in the innermost circular muscle layer just beneath mucosa, structurally resembled intramuscular arrays described previously in the stomach, and were relatively small (1.3±0.8mm, n=26 from 3 guinea pigs). In contrast, the putative DRG nerve terminals were located in the myenteric layer as delimited areas of networks of branching fine fibers often forming intraganglionic structures and projecting into the mucosa. The DRG terminals were relatively large (ranging from 0.5 mm to 11 mm, n=15 from 5 guinea pig). We conclude that the placodes- and neural crest-derived esophageal nociceptors differ in mechanosensitive function and innervation area. The placodes-derived nodose C-fiber terminals are distention-responsive and are located in the innermost circular muscle. In contrast, the neural crest-derived fibers are mostly distention-unresponsive and innervate both myenteric layer and mucosa. Our data indicate that the information regarding noxious distension will be relayed to the CNS mostly via placodal nodose nociceptors, along with a minority of neural crest nociceptors. In contrast, the information regarding noxious substances in esophageal lumen will be relayed to the CNS via neural crest nociceptors. Supported by BioMed Martin (ITMS: 26220220187) co-funded by EU.
Tu1880 What Do Extrinsic Sensory Neurons "Tell" the Body About Gut Motility? Bao-Nan Chen, Adam Humenick, Lukasz Wiklendt, Nicholas J. Spencer, Philip G. Dinning, Marcello Costa, Simon J. Brookes Background. Extrinsic primary afferent neurons mediate reflexes and sensations from the gastrointestinal tract. These sensory neurons can be activated by mechanical and chemical stimuli (including inflammatory mediators). Much of our understanding of afferent mechanosensitivity is based on imposed mechanical stimuli, either in vivo or in vitro. Under physiological conditions, such stimuli often evoke enteric motor reflexes, which complicate analysis of mechanosensitivity. In the present study, we investigated the nature of sensory neurons activated during physiological motor activity using small tubular preparations of gut in vitro. Methods. In isolated segments of guinea-pig distal colon, mechanical stimuli of low intensity were delivered, and resulting motor patterns recorded in terms of both intraluminal pressure and intestinal wall movements. High resolution intraluminal fibre-optic manometry (1cm sensor spacing with a 1.3mm diameter catheter) was combined with spatiotemporal mapping and extracellular recording of sensory neuron firing from colonic nerves innervating 3-4 cm specimens. Intraluminal pressure could be increased, in the non-noxious range (4 15mmHg), triggering propagating peristaltic contractions that could empty the segment of fluid. Sensory firing could be related specifically to mechanical changes (pressure, diameter) in the sensory neuron's identified field of innervation, with fine spatial and temporal resolution. Results. Low levels of spontaneous firing increased dramatically during luminal distension with Krebs solution to 4-15 mmHg. Bursts of sensory firing correlated strongly with waves of contraction. Two distinct patterns of firing could be distinguished. Some low threshold afferents fired phasically, just prior to, or during muscle shortening. The other pattern was more prolonged bursts of firing associated with lengthening of the muscle after contractions. None of the afferents showed bursts of firing during the peaks of intraluminal pressure (ie: at periods of maximal shortening). Addition of 1uM nicardipine with 1uM hyoscine led to abolition of distension-evoked phasic contractile activity, leaving passive mechanical distension responses. Sensory firing was reduced, lacked bursts and showed slowly adapting firing. Conclusions: Afferent firing in the non noxious range is primarily associated with either the onset of contractions, or lengthening after contractions, but surprisingly, not with the peaks of intraluminal pressure.
AGA Abstracts
S-926