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Identification of the site of afferent nerve sensitivity to vascular perfusion in mesenteric vessels to the rat ileum
S1696
S1698
Lipopolysaccharide (LPS)-induced Changes in Mesenteric Afferent Sensitivity of Rat Jejunum In Vitro Btngxian Wang, Joerg Glatzle, Mano H. Mueller, Paul Enck, David Grundy
Role of Serotonin and Mast Cell Degrannulation in the Acute Jejunal Ischemia Evoked Afferent Excitation Wen Jiang, David Grundy
Background: Bacterial translocation across the intestinal mucosal bamer leads to a macrophage mediated inflammatory response, visceral hyperalgesia and ilens. (Eskandari et al. 1099. Am J Physiol. 277: G478-86; Coelho et al. 2000. Am J Physiol. 279: G781-90). The aim of the present study was to examine how mediators released into mesenteric lymph following LPS treatment influence intestinal afferent sensitivity and the role of local prostanoids in any sensitization. Method: Intestinal lymph was collected from awake male lymphhsmla rats (300-350g) during 12h of intestinal infusion with glucose-saline following treatment with either saline or LPS (5mg/kg, i.p.). Using a novel in vitro preparation of vascularly perlused recipient rat jejunum, extracellular muhninit afferent recordings were made from the paravascular mesenteric nerve bundle along with intraluminal pressure as an index of motor activity Control lymph (0.3ml), LPS lymph (0.3ml) and LPS (3mg/ml, 0.3ml) were perfused intra-arterially, separated by an interval of 15rain. In six experiments Naproxen iI0p.M) was added to the Kreb's buffer. Mesenteric afferent discharge (AD) following treatment was quantified as percentage increase above baseline (means -+ S.E.M.) and compared using paired and unpaired t-tests as appropriate. Results: AD increased significantly after LPSlymph compared to control lymph (123.1 _+ 27.2% vs 44.7 -+ 22.3%, n=8, P<0.05). Peak discharge occurred after approximately 100 seconds and remained elevated for 5-8 minutes. This increase was independent of motility which was inhibited by 4/8 LPS lymph samples and 2/8 samples of control lymph. LPS itself also stimulated mesenteric afferent discharge (117.8 _+ 20.9%) and markedly inhibited jejunal motility (8/8 experiments). The afferent respons~ to LPS lymph was attenuated by Naproxen (47.6 _+ 15.1%, n = 6, P<0.05) although that to LPS was maintained (142.9 _+ 32.3%, n = 6, P>0.05). Conclusions: Inflammatory mediators released from the gut into the lymph by LPS have a profound effect on afferent discharge and motility. These mediators influence afferent firing via a local cydooxygenase (COX) dependent mechanism. LPS also has a short-term effect on afferent firing and motility that is independent of prostanoids.
Introduction: A variety of mediators are released during intestinal inflammation, injury and ischemia to stimulate and sensitize intestinal afferents. However, the nature of these mediators and their mechanisms of action are poorly understood. The aim of the present study was to examine the involvement of mast cells in ischaemia-induced afferent hypersensitivity and in particular the role played by serotonin receptors. Methods: Extracellular recordings were made from mesenteric afferents supplying a 5 cm segment of jejunum in pentobarbitone sodium (60mg/kg, ip) anaesthetised rats. Brief ischemia (8 rain duration) was produced by clamping the mesenteric vessels supplying the loop, followed by 30rain of reperfusion after removing the clamp. Two episodes of ischemia/reperfusion were separated by systemic treatment with either vehicle, 2.Smg/kg metergolnie (a 5-HT1,2 receptor antagonist), 0.Smg/ kg ketanserine (5-HT2A receptor antagonist) 100wg/kg granisetron (5-HT3 receptor antagonist), or the mast cell stabilizer doxantrazole (10mg/kg). Data are presented as the mean + SEM before and after responses compared using Students paired t-tests. Results: Ischemia produced a reproducible biphasic marked increase in afferent discharges (AD). Doxantrozal markedly attenuated the afferent response to ischemia (mean AD before 1 5 0+- 13 vs. after treatment 79-+ 9 spikes/s, n = 4, P0.05) nor granisetron altered the afferent response to ischemia (mean AD before 115 -+2i vs. after 116 _+25, n = 6, P>O.05). Conclusions: Mast cells contribute to the afferent response to brief periods of focal intestinal ischemia. 5-HT appears to play a role in this afferent sensitivity but neither 5HT3 receptors on extrinsic sensory nerve endings nor 5-HT2A on intestinal smooth muscle cells are involved. The nature of the serotonergic influence on ischaemic sensitivity remains to be determined. The project was supported by the MRC.
S1699 S1697
The Effect of Mg2+ and D-serine on the NMDA Responsiveness of Rat Inferior Splanchnic Afferents in vitro
Identification of the Site of Afferent Nerve Sensitivity to Vascular Perfusion in Mesenteric Vessels to the Rat Ileum Alan Brunsden, Simon Brookes, Samuel Jacob, Karnadev Bardhan, David Grundy
Yuhua Wang, Jen Yu Wei, James A. McRoberts, Emeran A. Mayer Background: In isolated cell preparations, activation of NMDA receptors requires the addition of a co-agonist (glycine or D-serine), and is enhanced by removal extracellular Mg2§ which inhibits channel opening in a voltage-dependent manner. However, early work with intact tissue or tissue slices failed to identify glycine as a co-agonist of NMDA receptors, presumably due to endogenous levels of glycine being able to saturate the high afflmty binding site ( 150 nM). Aims: Determine whether removal of Mg2+ and addition of a co-agonist are necessary to observe a direct effect of NMDA in our in vitro tissue preparation. Methods: An in vitro isolated colorectal-inferior splanchnic nerve preparation was employed as prevlonsly described (Gastroenterology 120:A328, 2000). The current report summarizes4 experiments. Results: In Mg2+-free perfusate, the response of polymodal inferior splanchnic aflerents (PISAs) to 40 p.g NMDA alone was not significantly different from that to NMDA + 5 v.g D-serine (P = 0.29, N = 7, paired t-test). Similarly, responses in the presence or absence of D-serine were not significantly different in Mg2+-containing buffers (P=0.33, n = 9 vs 8, unpaired t-test). There was no significant difference between responses to NMDA alone, or NMDA + D-serine, in the presence or absence of Mg2+ (P = 0.38, N = 9 vs 7 and p = 0.46, N = 8 vs 7, respectively, unpaired t-test). Conclusions: These findings are similar to those previously reported using brain tissue slices and suggest that: 1) endogenous glycine levels in the colon wall are high enough to saturate the glycine biding site on NMDA receptors, 2) ongoing, spontaneous firing of PISAs sufficiently depolarizes the neurons to remove voltage-dependent Mg2§ inhibition. Supported by NIH grants DK 58173 (EAM) and 1 PSO DK64539-01 (EAM)
Introduction: Axons of extrinsic primary afferents, running via the mesentery to the gut, are excited by stopping vascular perfnsion (flow-olD in rat ileum in vitro. This response persists after removal of the gut body (Brunsden et a1.,2002, AJP 283:G656). By applying nerve tracing techniques, in conjunction with separate functional recordings, we have examined the site of sensitivity to flow-off in the mesenteric fan. Method: Segments of rat ileum were pinned fiat, arterially perfused with modified Krebs solution, and perfnsion pressure monitored Extracellular afferent discharge was recorded from a paravascular nerve bundle. .~ons were anterograddy filled with biotinamide, applied at the recording site, and visualised with Texas Red fluorescence in wholemounts (methods: Zagorodnyuk & Brookes 2000, J Neurosci. 20:6249). Afferent responses to flow-off (5 rain) were recorded in the intact preparation and after cut 1 and cut 2 (see Figure). Results: When applied at site A, biotinamide hbelling revealed that axons typically followed the mesenteric artery, branching before site B,forming a loose network which projected down each arterial limb. No 'intimate' perivaseular contacts were seen above the level of Cut 2 (n = 6). Biotinamide application at site B revealed networks of biotinamide-labelled vancose axons, forming close perivascular contacts on terminal arteries and submucosal vessels. Afferent discharge (impulses in 5rain, mean -+ SEM, (n = 5)) was increased by flow-off in all intact preparations (2827 _+ 243). Flow-off responses were reduced by 81 _+ 3 % after cut 1 (to 513 _+ 98*)and by 94 -+ 0.5 % after cut 2 (to 188 _+ 23*, *p <005, Bonferroni multiple comparison after RM-ANOVA). Basal afl;erem discharge (13.0 _+ 3.2 imps ~) was reduced by 54 _+ 8 % after cut 1 (6.4 _+ 2.3 imps ~) and by 84 -+ 5.5 % after cut 2 (1.9 + 0.6* imps ~). Conclusions: Terminal arteries (and gut arterioles) are the major sites of aflemnt sensitivity to flow-off in the mesenteric [an. Since these resistance vessels are the principle sites for the regulation of gut blood flow, perivascular afterents are ideally located for a role in the local control of intestinal circulation. Supported by the Bardhan Research & Education Trust (BRET)
s1700 Thalamic Stimulation Differentially Modifies Spinal Neuronal Responses to Colorectal Distension in Adult Rats Exposed to Neonatal Colon Pain Elie D. AI-Chaer, Young-Chang Park Adult chronic visceral pain induced by neonatal painful incidents is associated with central neuronal sensitization in the absence of identifiable pathology (A1-Chaer et al 2000). This central sensitization may be facilitated by positive feedback loops between spinal and supraspinal structures that alter the dynamics of descending modulatory pathways. In this study we examined the role of the ventral posterolateral (VPL) nucleus of the thalamus as a possible supraspinal modulator of visceral hypersensitivity. We used A1-Chaer's model of chronic abdominal pain in rats, to test the hypothesis that activation of the VPL nucleus can modulate visceral nociception at the lumbosacral spinal cord level. Experiments were carried out in 21 adult male Sprague-Dawley rats: 15 rats received colon irritation (CI) as neonates and 6 rats served as control. Single unit extracellular recordings were made of the responses to graded colorectal distension (CRD) of lumbosacral (L6-$2) dorsal horn neurons before and after thalamic electrical stimulation (10-50 wA, 100 Hz, 10s). The responses of 21 viscerosensinve neurons (Control, n = 6; CI, n = 15) to somatic stimuli and CRD were recorded. The effect of thalamic stimulation on the responses of dorsal horn neurons vaned in different rats. Inhibition of neuronal responses was observed in 6/6 control and 5/15 CI rats. However, facilitation of neuronal responses to CRD was seen in 8/15 CI rats. These effects were significant on the responses to CRD (e.g. Average response to CRD at 80 mmHg increased from 25.03 +/- 1.68 to 33.07 +A 2.55; p=0.007). The data were analyzed using a t-test (when normally distributed) or Mann-Whitney Rank Sum Test (when the normality test failed). At the end of the experiment, the brain tissue was collected for histological verification. The differential effect of thalamic stimulation in CI versus control rats may be caused by plastic changes in the wiring of the sensory systems caused by the neonatal injury and indicates that the VPL nucleus of the thalamus plays a critical role in modulating chronic visveral pain.
Sup.mesenterie
J
4t~y
A--* Mosenteric
-
~
gut
~
A-249
AGA Abstracts
S1698
Lipopolysaccharide (LPS)-induced Changes in Mesenteric Afferent Sensitivity of Rat Jejunum In Vitro Btngxian Wang, Joerg Glatzle, Mano H. Mueller, Paul Enck, David Grundy
Role of Serotonin and Mast Cell Degrannulation in the Acute Jejunal Ischemia Evoked Afferent Excitation Wen Jiang, David Grundy
Background: Bacterial translocation across the intestinal mucosal bamer leads to a macrophage mediated inflammatory response, visceral hyperalgesia and ilens. (Eskandari et al. 1099. Am J Physiol. 277: G478-86; Coelho et al. 2000. Am J Physiol. 279: G781-90). The aim of the present study was to examine how mediators released into mesenteric lymph following LPS treatment influence intestinal afferent sensitivity and the role of local prostanoids in any sensitization. Method: Intestinal lymph was collected from awake male lymphhsmla rats (300-350g) during 12h of intestinal infusion with glucose-saline following treatment with either saline or LPS (5mg/kg, i.p.). Using a novel in vitro preparation of vascularly perlused recipient rat jejunum, extracellular muhninit afferent recordings were made from the paravascular mesenteric nerve bundle along with intraluminal pressure as an index of motor activity Control lymph (0.3ml), LPS lymph (0.3ml) and LPS (3mg/ml, 0.3ml) were perfused intra-arterially, separated by an interval of 15rain. In six experiments Naproxen iI0p.M) was added to the Kreb's buffer. Mesenteric afferent discharge (AD) following treatment was quantified as percentage increase above baseline (means -+ S.E.M.) and compared using paired and unpaired t-tests as appropriate. Results: AD increased significantly after LPSlymph compared to control lymph (123.1 _+ 27.2% vs 44.7 -+ 22.3%, n=8, P<0.05). Peak discharge occurred after approximately 100 seconds and remained elevated for 5-8 minutes. This increase was independent of motility which was inhibited by 4/8 LPS lymph samples and 2/8 samples of control lymph. LPS itself also stimulated mesenteric afferent discharge (117.8 _+ 20.9%) and markedly inhibited jejunal motility (8/8 experiments). The afferent respons~ to LPS lymph was attenuated by Naproxen (47.6 _+ 15.1%, n = 6, P<0.05) although that to LPS was maintained (142.9 _+ 32.3%, n = 6, P>0.05). Conclusions: Inflammatory mediators released from the gut into the lymph by LPS have a profound effect on afferent discharge and motility. These mediators influence afferent firing via a local cydooxygenase (COX) dependent mechanism. LPS also has a short-term effect on afferent firing and motility that is independent of prostanoids.
Introduction: A variety of mediators are released during intestinal inflammation, injury and ischemia to stimulate and sensitize intestinal afferents. However, the nature of these mediators and their mechanisms of action are poorly understood. The aim of the present study was to examine the involvement of mast cells in ischaemia-induced afferent hypersensitivity and in particular the role played by serotonin receptors. Methods: Extracellular recordings were made from mesenteric afferents supplying a 5 cm segment of jejunum in pentobarbitone sodium (60mg/kg, ip) anaesthetised rats. Brief ischemia (8 rain duration) was produced by clamping the mesenteric vessels supplying the loop, followed by 30rain of reperfusion after removing the clamp. Two episodes of ischemia/reperfusion were separated by systemic treatment with either vehicle, 2.Smg/kg metergolnie (a 5-HT1,2 receptor antagonist), 0.Smg/ kg ketanserine (5-HT2A receptor antagonist) 100wg/kg granisetron (5-HT3 receptor antagonist), or the mast cell stabilizer doxantrazole (10mg/kg). Data are presented as the mean + SEM before and after responses compared using Students paired t-tests. Results: Ischemia produced a reproducible biphasic marked increase in afferent discharges (AD). Doxantrozal markedly attenuated the afferent response to ischemia (mean AD before 1 5 0+- 13 vs. after treatment 79-+ 9 spikes/s, n = 4, P
S1699 S1697
The Effect of Mg2+ and D-serine on the NMDA Responsiveness of Rat Inferior Splanchnic Afferents in vitro
Identification of the Site of Afferent Nerve Sensitivity to Vascular Perfusion in Mesenteric Vessels to the Rat Ileum Alan Brunsden, Simon Brookes, Samuel Jacob, Karnadev Bardhan, David Grundy
Yuhua Wang, Jen Yu Wei, James A. McRoberts, Emeran A. Mayer Background: In isolated cell preparations, activation of NMDA receptors requires the addition of a co-agonist (glycine or D-serine), and is enhanced by removal extracellular Mg2§ which inhibits channel opening in a voltage-dependent manner. However, early work with intact tissue or tissue slices failed to identify glycine as a co-agonist of NMDA receptors, presumably due to endogenous levels of glycine being able to saturate the high afflmty binding site ( 150 nM). Aims: Determine whether removal of Mg2+ and addition of a co-agonist are necessary to observe a direct effect of NMDA in our in vitro tissue preparation. Methods: An in vitro isolated colorectal-inferior splanchnic nerve preparation was employed as prevlonsly described (Gastroenterology 120:A328, 2000). The current report summarizes4 experiments. Results: In Mg2+-free perfusate, the response of polymodal inferior splanchnic aflerents (PISAs) to 40 p.g NMDA alone was not significantly different from that to NMDA + 5 v.g D-serine (P = 0.29, N = 7, paired t-test). Similarly, responses in the presence or absence of D-serine were not significantly different in Mg2+-containing buffers (P=0.33, n = 9 vs 8, unpaired t-test). There was no significant difference between responses to NMDA alone, or NMDA + D-serine, in the presence or absence of Mg2+ (P = 0.38, N = 9 vs 7 and p = 0.46, N = 8 vs 7, respectively, unpaired t-test). Conclusions: These findings are similar to those previously reported using brain tissue slices and suggest that: 1) endogenous glycine levels in the colon wall are high enough to saturate the glycine biding site on NMDA receptors, 2) ongoing, spontaneous firing of PISAs sufficiently depolarizes the neurons to remove voltage-dependent Mg2§ inhibition. Supported by NIH grants DK 58173 (EAM) and 1 PSO DK64539-01 (EAM)
Introduction: Axons of extrinsic primary afferents, running via the mesentery to the gut, are excited by stopping vascular perfnsion (flow-olD in rat ileum in vitro. This response persists after removal of the gut body (Brunsden et a1.,2002, AJP 283:G656). By applying nerve tracing techniques, in conjunction with separate functional recordings, we have examined the site of sensitivity to flow-off in the mesenteric fan. Method: Segments of rat ileum were pinned fiat, arterially perfused with modified Krebs solution, and perfnsion pressure monitored Extracellular afferent discharge was recorded from a paravascular nerve bundle. .~ons were anterograddy filled with biotinamide, applied at the recording site, and visualised with Texas Red fluorescence in wholemounts (methods: Zagorodnyuk & Brookes 2000, J Neurosci. 20:6249). Afferent responses to flow-off (5 rain) were recorded in the intact preparation and after cut 1 and cut 2 (see Figure). Results: When applied at site A, biotinamide hbelling revealed that axons typically followed the mesenteric artery, branching before site B,forming a loose network which projected down each arterial limb. No 'intimate' perivaseular contacts were seen above the level of Cut 2 (n = 6). Biotinamide application at site B revealed networks of biotinamide-labelled vancose axons, forming close perivascular contacts on terminal arteries and submucosal vessels. Afferent discharge (impulses in 5rain, mean -+ SEM, (n = 5)) was increased by flow-off in all intact preparations (2827 _+ 243). Flow-off responses were reduced by 81 _+ 3 % after cut 1 (to 513 _+ 98*)and by 94 -+ 0.5 % after cut 2 (to 188 _+ 23*, *p <005, Bonferroni multiple comparison after RM-ANOVA). Basal afl;erem discharge (13.0 _+ 3.2 imps ~) was reduced by 54 _+ 8 % after cut 1 (6.4 _+ 2.3 imps ~) and by 84 -+ 5.5 % after cut 2 (1.9 + 0.6* imps ~). Conclusions: Terminal arteries (and gut arterioles) are the major sites of aflemnt sensitivity to flow-off in the mesenteric [an. Since these resistance vessels are the principle sites for the regulation of gut blood flow, perivascular afterents are ideally located for a role in the local control of intestinal circulation. Supported by the Bardhan Research & Education Trust (BRET)
s1700 Thalamic Stimulation Differentially Modifies Spinal Neuronal Responses to Colorectal Distension in Adult Rats Exposed to Neonatal Colon Pain Elie D. AI-Chaer, Young-Chang Park Adult chronic visceral pain induced by neonatal painful incidents is associated with central neuronal sensitization in the absence of identifiable pathology (A1-Chaer et al 2000). This central sensitization may be facilitated by positive feedback loops between spinal and supraspinal structures that alter the dynamics of descending modulatory pathways. In this study we examined the role of the ventral posterolateral (VPL) nucleus of the thalamus as a possible supraspinal modulator of visceral hypersensitivity. We used A1-Chaer's model of chronic abdominal pain in rats, to test the hypothesis that activation of the VPL nucleus can modulate visceral nociception at the lumbosacral spinal cord level. Experiments were carried out in 21 adult male Sprague-Dawley rats: 15 rats received colon irritation (CI) as neonates and 6 rats served as control. Single unit extracellular recordings were made of the responses to graded colorectal distension (CRD) of lumbosacral (L6-$2) dorsal horn neurons before and after thalamic electrical stimulation (10-50 wA, 100 Hz, 10s). The responses of 21 viscerosensinve neurons (Control, n = 6; CI, n = 15) to somatic stimuli and CRD were recorded. The effect of thalamic stimulation on the responses of dorsal horn neurons vaned in different rats. Inhibition of neuronal responses was observed in 6/6 control and 5/15 CI rats. However, facilitation of neuronal responses to CRD was seen in 8/15 CI rats. These effects were significant on the responses to CRD (e.g. Average response to CRD at 80 mmHg increased from 25.03 +/- 1.68 to 33.07 +A 2.55; p=0.007). The data were analyzed using a t-test (when normally distributed) or Mann-Whitney Rank Sum Test (when the normality test failed). At the end of the experiment, the brain tissue was collected for histological verification. The differential effect of thalamic stimulation in CI versus control rats may be caused by plastic changes in the wiring of the sensory systems caused by the neonatal injury and indicates that the VPL nucleus of the thalamus plays a critical role in modulating chronic visveral pain.
Sup.mesenterie
J
4t~y
A--* Mosenteric
-
~
gut
~
A-249
AGA Abstracts