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Mechanisms controlling lipopolysaccharide (LPS) induced changes in mesenteric afferent sensitivity in the rat small intestine
379) Tire"aims of this study were to compare the atferent neuronal responses to jejunal ramp and phasic distension in anesthetized Wistar and WKY rats, and to compare the effects of CRF upon this distention-induced activity Methods: Extracellnlar recordings of ielunal afferent nerve discharge were obtained trom pentobalbitone-anaesthetized male rats (330450g) Ranrp (2 mmHg/4 sets, up to 60mmHg) and phasic: distensions (10-50 mmHg 25 seconds were performed by intlation ofa 3cm balloon, usirg a barostat CRF was administered 5 minutes prior to a second set of distensions Data are mean • g E M and analyzed using an ANOVA with Bonfteroin corrections. Results: Ramp distensions evoked a biphasic activa lion of afferent uerve discMrge and phasic distensions evoked pressure dependent it• in altO:rent discharge, in both strains. The hrst (2d5mmHg) and second (20-60• phases of the alffrent response to ramp distension appeared as increases in atferent discharge above baseline of 3 • 1 o 22 .+_5 spikes/s and 28 :re5 to 60• 10 spike~s, n:spective]y in Wistars, and 0 • 1 to 10 _+5 spikes/s and 14 • cato 40 • 6 spikes/s, in WKY. Phasic distensions (10-50mmHg) evoked mean increases in discharge ove~the 25 second distension of i 34 +- 34 to 640 • 127 spikes/s, above baseline in Wistars and 100 • 21 to 406 • 68 spikes/s, above basehne in WRY'. Although the afterent responses to both ramp and phasic distensions appeared greater in Wistar rats compared to WKY, this dflf~:rence did not achieve statistical significance. Following administration ot CRF (10>g/kg, i.v), there was no change in the afferent respormes to ramp or phasic distension in Wistar rats. However, CRF evoked siginficantIy higher increases in afterent nerve discharge during phasic and ramp distension in WKY rats (15-60mmHg to* ranrps and 30-50mmHg for phasics P<0.05) Conclusions: These results indicate that sensory nerves innervating the GI tract in WKY rats are more sensitive to CRF than those of Wistar rats. It is possible that this penpherallyq)ased hypersensitivity may contnbute to the wsceral hyper,~lgesiaprevious~.ydemonstrated in these animals.
Peincillam ne and Zinc are both effective in treating WD, but Penicillamine side effects may be serious while Zinc is saint and can he adopted during pregnancy L~v-ertransplant remains the treatment of choice t0r end stage liver disease
W1419 Vagal Selective Eftects of Ruthenium Red on the Jejunal Afferent Fibre Response to Ischaemia David C F~llhner Wendy J Wiuchester~ Gareth A Hicks David Grundy Introduction grief periods of local ischaemia elicit a biphasic pattern of activation within }qunal af[erem ~brvs accompanied by a transient increase in firing rate [ollowing reperfusion 0iang & Grsndy, J Alrton Nerv Syst 2000 78:99-108) [nclvased levels of lactic acid have been reported fo/]owmg fbdominal ischaemia and contribute to f~,e activation of visceral atferenls (Sahl r l ong':mrst, Am j Pbysiol/992 262 H748-53). One possible mechanism by which protons may excite vrsr eral al~csents is the proton sensitive VR1 receptor (Caterina et al Nature, 1997 389:81&24) The aim of the present study was to determine the efkcts of the VR1 antagonists capsazepine and rntheninm red on the/qnnal afferent response to ischaemia / repeffusion Methods: Male Wistar mrs (300-400g) were anaesthetised with sodium pe~:tobarbitone and prepared for extmcellular recording o~mesenteric nerve bundles Two 10 • periods of iscbaemia were studied separated by a 30 • interval Prior to the second isdlaemia animals were treated with either cap~zepine (3mg/kg i v bdus; l m ~ j • ini\rsion started 20 min betore ischaemia), ruthenium red (3mg/kg iv. 5 • before ischaemia) nr saline (0.5ml&g i v 5 • beti~re ischaemia) Responses to ischaemia were also studied in chnmically vagotmnised animals Data are presented as tile mean • gEM change in a/fetvnt discharge (n = 3-5) and were compared betbre and atier treatnrent using a Student's paired t-tesl Results No significant diftkrence was observed in the increase in afterent discharge to iscbaemia / mperklsion }allowing capsazepine or saline. However, a signiticant reduction in the increased af[erem discharge during the' early phase response to ischaemia was obser~sed tol/owing rnthenium ted (53 _+ 9 3 Hz vs ~7 • 59 Hz; p<0.05), although no signihcant difference was observed in either the late phase response to ischaemia (113 ~: 242 llz vs 129 + 226 Hz: p>0 05) or reperfusion response (]4 • 7.8 Hz vs 9 :+- 6 0 Hz: p > 0 0 5 ) A similar reduction in the increase m aiffsent discharge during the early phase response to ischaemia with no change m the late phase or reperthsion response was observed in chronically vagotomised aninrals compared wrth control animals Conclusion: "fhe resuhs of this study demonstrate that ruthenium red attenuates the early phase af the jejunal allesent response o ischaemia, which is predominantly relayed in vagai atferents The mechanism nndedying his eft)ct is unknown, however, it does not appear to be media ed via tfie VRI receptor
WI42Z Mechanisms controlling Iipopolysaccharide (LPS) induced changes in mesenteric afferent sensitivity in the rat small intestine Chuan Yong Lm St., Wen Jiang, Mario Mueller, David Grundy, Martin Kreis Background: The precise mechanisms underlying endotoxin-induced hyperalgesia remain unknown. We aimed to study the mechanisms underlying the sensitizing action of LPS on intestinal aft;erent responses to mechanical and chemical stinrufi in the rat Methods: Extracellular recordings of jeiunal afferent nerve discharge were obtained in vivo hum pentobarbitone-anaesthetized male Wistar rats (300-400 g) 5-HT (1'5 ~g/kg, i v ) and ramp distension to 60 cmH20 were repeated at I 5 • intervals before and t01lowing LPS i v Data are mean + gEM and statistical ditterences were assessed using ANOVA tnllowed by dunnett analysis Results: LPS (6 mg/kg iv) stimulated a short-term, transient (<30 • augmentation of fire peak response to %HT and distension (both P<0 05) and a delayed hut maintained (>30 • increase in spontaneous afferent discharge (base] ne 154 • 13 imp/s; 547 § 56 imps/s 90-120 • alter LPS, p<005, n = 5) Pre-tmatment w~th naproxen (10mg/kg, iv) significantly attenuated both the short-term sensitization to 5-HI" and dislcnsion and the long-term inarease in baseline afl:erent firirg following kPS Protreatment with aminoguanidine (iNOS inhibitor, 15 mg/kg i.vd signiIicandy prolonged the period of afferent sensitizanon to distension and 5-HT without int]uencing the augmented baseline finng ram. Nikdipine (lmgz%g, iv) also prolonged the sensitization wrthout any change in the kPS-mediated increase in tiring, while omega-eonotoxin GV1Aatternkated the increase in aft}• discharge to LPS, without any change in sensitisation to mechanical and chemical stimuli. Conclusiona: ghe long term (>30 nrin) increase in altO'rentl]rmg folIowing systemic LPS involves release of prostanoids via neural rei]ex mechanism The short4erm (<30min) sensitization is prolonged by iNOS inhibition and by L- type cakium channd blockers suggesting that induced nimc oxide prnduction serves m down regulates LPSinduced atlmvnt hyperseusitix~ty Acknowledgement: This work was supported by DFG Kn 1816/3-3
W1420 Murine Nippostrongylus br~asiliensis (Nb) Infection Induces Changes in Intestinal Mucosal Mast Cells (IMMC), IgE Levels and Afferent Nerve Activity Anthony Kirkup, Christine Booth, Andrze i Stainsz Elaine Fraser, Gervais Tougas, David Grundy, Kirk Hiilsley Ronald H Stead Background: Microanatomi(al Mationships between mast cells and nerves have been shown r a variety ot species Using Nb inkction, the rdationship between IMMCs and nerves has been shown to be plastic in lats, but t is not known if them are hmctional changes with Nh intectlou The• IMMC numbers, IgE levels and the sensitBnty o{ extrinsic jejunal afterent nerves were assessed in Nb-inkcted mice Methods: Naive, sham-intected or Nbinterred Balb/c mice (n ::: 3-4) were sacrificed 1 2, 3, 6, 9 & ]2 weeks post-infection. IgE levels were measured in serum using ELISA, while segments of jejunum were tixed in acetic acid/alcohol embedded in paraffm, sectioned and stained with Alcian blue. AddtiotMly, animals at weeks 3, 6 and 12 weeks post-Nb were anaesthetized and att}rem responses recorded in response to balloon distension (to 60 mmHg), using standard techniques. Data are shown as mean § gEM Resnhs: [gE levels and mast ce/I counts charged afier intection as shown in the table Fhere was no sigmBcant diffFrence between the spontaneous discharge ofcomml and iniected animals (control - 34.4 • 5 (n = 14), infected = 4:3 + 4.6 (n= 14), p=0 22) Mean responses to bafloon distension were significantly greater in inlected animals iban sham/naive mice at all time points examined (grouped data control = 734 -4- 4 7 inffcted = 1296 • 103 imp/s, p<0 01) Analysis of variance (n=28) confirmed a significant • in nerve discharge frequency m response to intection (p=0.0004); whereas tune aBer inh*ction was not a significant factor (p = 0 6 3 ) Conclusions: These data confirm that there are changes iu the mast cell population and lee levels in response to Nb infection ill mice. Increased mesentenc atferent discharge in Nb-infected mice supports tire hypothesis of intlammation induced neural plasticity The availability ot a murine model fbr the-investigaion ofinflamnratinn-induced neural plasticity is a u~ihl tool in understanding the pathophysiulogy ot intestinal disea~s This study was supported by a generous grant from Janssen~ Week 19E(Pg/n~)
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W1423 Behavioral Abnormalities and Increased Local and Systemic Inflammatory Responses in Adult Mice Following Maternal Deprivation as Neonates Ashs*an K. Varghese, Elena F. Verdu, Psemysl Bercik, Waliul I. Khan, Patricia A. Blennertzassett, Henry Szechtman, Stephen M. Collins Backgrocmd/Aims We have prewously sho,aTt that stress induced by neonatal maternal deprivation increases the severity of subsequent experimental colitis ira adult mice The aim of this s}udy was to identify behavioral changes associated with the increased vulnerability to colitis in adult mice, and to investigate local and systemic markers ot inflammation tollowmg the induction of colitis Methods C57BU6 mouse pups we• separated from their mothers for 31gday at 1-21 days of age Maternally non-deprived litters served as controls. On day 23, nrale mice were identified and weaned. Behavmr studies and colitis studies were perR)nned at day 60 on separate sets of male mice. Behavior Tests: At day 60, mice stere expo~d to a rectang~llar open t:te]dand allowed to habituate for 10 minutes. At 10 minutes, a novel object was introduced into the field and mice were allowed to respond for a further 10 minutes Behavioral patterns of mice throughout the 2&minute trial were analyzed using sof~watv. Colitis induction: Colitis was reduced at day 60 using 6% dextrau sultate sodium (DSS) in drinking water tbr 5 days. hrflammation was assessed using colonic [L-I,8 and Serum Amyloid P (SAP) levels. Results Behavior Te~ts:A steady decline iu activity levels was seen {6r both deprived (MD) and non-deprived (ND) mice, as expected, during the first 10 minutes of the trial However, alter introduclinn of the novel o]~ject ND mice continued to show habituation of activity levels whereas MD mice showed impaired habituation. Deprived mice showed a 37% (p<0 05) and 44% (p<0.05) decrease in percent-decline of distance traveled and movement duration, respectively compared to non-deprived animals, lnflammatoU responses:No spontaneous inflammation was seen in any group prior to DSS administration. Foil• 6% DSS, colonic IL-113and systemic SAP leveIs increased by I22% (p<0.005) and 85% (p<0 005), respectively m deprived as compared to non-deprived trace. Couclusion Early maternal deprivation results m an impaired ability of • to cope with stress m adulthood. Maternal deprivation also leads to increased levels of colomc [L-1[-3and systemic gAP levels post-DSS colitis induction in adult mice. These fmdings indicate that stress expenenced in early life increases the walnerability of adult mice to stress-induced behar
ANOVA p
W1421
Differential Sensitivity of Mechanosensitive Jejunal Mesenteric Afferent Nerves to Corticotrophin Releasing Factor in Wistar Kyoto and Wistar Rats Charlotte E, Booth, Wendy J Wm(hester, Mike Maline, Beverley Greenwood-Van meervald Gareth A Hicks Background: Stress or corticotropin releasing factor (CRF) enhance the viscemmotorresponse to cdorectal distension (CRD) in rats (Gun et al 1997, Neurogastroenterol Motil 9: 271) In additioo the high anxiety rat strain, Wistar Kyoto (WkW) display hypersensitivity" to CRD, which can he blocked by a CRF antagonist (Gunter et a! 2000, Physiol & Behav 69:
A-667
AGA Abstracts
Peincillam ne and Zinc are both effective in treating WD, but Penicillamine side effects may be serious while Zinc is saint and can he adopted during pregnancy L~v-ertransplant remains the treatment of choice t0r end stage liver disease
W1419 Vagal Selective Eftects of Ruthenium Red on the Jejunal Afferent Fibre Response to Ischaemia David C F~llhner Wendy J Wiuchester~ Gareth A Hicks David Grundy Introduction grief periods of local ischaemia elicit a biphasic pattern of activation within }qunal af[erem ~brvs accompanied by a transient increase in firing rate [ollowing reperfusion 0iang & Grsndy, J Alrton Nerv Syst 2000 78:99-108) [nclvased levels of lactic acid have been reported fo/]owmg fbdominal ischaemia and contribute to f~,e activation of visceral atferenls (Sahl r l ong':mrst, Am j Pbysiol/992 262 H748-53). One possible mechanism by which protons may excite vrsr eral al~csents is the proton sensitive VR1 receptor (Caterina et al Nature, 1997 389:81&24) The aim of the present study was to determine the efkcts of the VR1 antagonists capsazepine and rntheninm red on the/qnnal afferent response to ischaemia / repeffusion Methods: Male Wistar mrs (300-400g) were anaesthetised with sodium pe~:tobarbitone and prepared for extmcellular recording o~mesenteric nerve bundles Two 10 • periods of iscbaemia were studied separated by a 30 • interval Prior to the second isdlaemia animals were treated with either cap~zepine (3mg/kg i v bdus; l m ~ j • ini\rsion started 20 min betore ischaemia), ruthenium red (3mg/kg iv. 5 • before ischaemia) nr saline (0.5ml&g i v 5 • beti~re ischaemia) Responses to ischaemia were also studied in chnmically vagotmnised animals Data are presented as tile mean • gEM change in a/fetvnt discharge (n = 3-5) and were compared betbre and atier treatnrent using a Student's paired t-tesl Results No significant diftkrence was observed in the increase in afterent discharge to iscbaemia / mperklsion }allowing capsazepine or saline. However, a signiticant reduction in the increased af[erem discharge during the' early phase response to ischaemia was obser~sed tol/owing rnthenium ted (53 _+ 9 3 Hz vs ~7 • 59 Hz; p<0.05), although no signihcant difference was observed in either the late phase response to ischaemia (113 ~: 242 llz vs 129 + 226 Hz: p>0 05) or reperfusion response (]4 • 7.8 Hz vs 9 :+- 6 0 Hz: p > 0 0 5 ) A similar reduction in the increase m aiffsent discharge during the early phase response to ischaemia with no change m the late phase or reperthsion response was observed in chronically vagotomised aninrals compared wrth control animals Conclusion: "fhe resuhs of this study demonstrate that ruthenium red attenuates the early phase af the jejunal allesent response o ischaemia, which is predominantly relayed in vagai atferents The mechanism nndedying his eft)ct is unknown, however, it does not appear to be media ed via tfie VRI receptor
WI42Z Mechanisms controlling Iipopolysaccharide (LPS) induced changes in mesenteric afferent sensitivity in the rat small intestine Chuan Yong Lm St., Wen Jiang, Mario Mueller, David Grundy, Martin Kreis Background: The precise mechanisms underlying endotoxin-induced hyperalgesia remain unknown. We aimed to study the mechanisms underlying the sensitizing action of LPS on intestinal aft;erent responses to mechanical and chemical stinrufi in the rat Methods: Extracellular recordings of jeiunal afferent nerve discharge were obtained in vivo hum pentobarbitone-anaesthetized male Wistar rats (300-400 g) 5-HT (1'5 ~g/kg, i v ) and ramp distension to 60 cmH20 were repeated at I 5 • intervals before and t01lowing LPS i v Data are mean + gEM and statistical ditterences were assessed using ANOVA tnllowed by dunnett analysis Results: LPS (6 mg/kg iv) stimulated a short-term, transient (<30 • augmentation of fire peak response to %HT and distension (both P<0 05) and a delayed hut maintained (>30 • increase in spontaneous afferent discharge (base] ne 154 • 13 imp/s; 547 § 56 imps/s 90-120 • alter LPS, p<005, n = 5) Pre-tmatment w~th naproxen (10mg/kg, iv) significantly attenuated both the short-term sensitization to 5-HI" and dislcnsion and the long-term inarease in baseline afl:erent firirg following kPS Protreatment with aminoguanidine (iNOS inhibitor, 15 mg/kg i.vd signiIicandy prolonged the period of afferent sensitizanon to distension and 5-HT without int]uencing the augmented baseline finng ram. Nikdipine (lmgz%g, iv) also prolonged the sensitization wrthout any change in the kPS-mediated increase in tiring, while omega-eonotoxin GV1Aatternkated the increase in aft}• discharge to LPS, without any change in sensitisation to mechanical and chemical stimuli. Conclusiona: ghe long term (>30 nrin) increase in altO'rentl]rmg folIowing systemic LPS involves release of prostanoids via neural rei]ex mechanism The short4erm (<30min) sensitization is prolonged by iNOS inhibition and by L- type cakium channd blockers suggesting that induced nimc oxide prnduction serves m down regulates LPSinduced atlmvnt hyperseusitix~ty Acknowledgement: This work was supported by DFG Kn 1816/3-3
W1420 Murine Nippostrongylus br~asiliensis (Nb) Infection Induces Changes in Intestinal Mucosal Mast Cells (IMMC), IgE Levels and Afferent Nerve Activity Anthony Kirkup, Christine Booth, Andrze i Stainsz Elaine Fraser, Gervais Tougas, David Grundy, Kirk Hiilsley Ronald H Stead Background: Microanatomi(al Mationships between mast cells and nerves have been shown r a variety ot species Using Nb inkction, the rdationship between IMMCs and nerves has been shown to be plastic in lats, but t is not known if them are hmctional changes with Nh intectlou The• IMMC numbers, IgE levels and the sensitBnty o{ extrinsic jejunal afterent nerves were assessed in Nb-inkcted mice Methods: Naive, sham-intected or Nbinterred Balb/c mice (n ::: 3-4) were sacrificed 1 2, 3, 6, 9 & ]2 weeks post-infection. IgE levels were measured in serum using ELISA, while segments of jejunum were tixed in acetic acid/alcohol embedded in paraffm, sectioned and stained with Alcian blue. AddtiotMly, animals at weeks 3, 6 and 12 weeks post-Nb were anaesthetized and att}rem responses recorded in response to balloon distension (to 60 mmHg), using standard techniques. Data are shown as mean § gEM Resnhs: [gE levels and mast ce/I counts charged afier intection as shown in the table Fhere was no sigmBcant diffFrence between the spontaneous discharge ofcomml and iniected animals (control - 34.4 • 5 (n = 14), infected = 4:3 + 4.6 (n= 14), p=0 22) Mean responses to bafloon distension were significantly greater in inlected animals iban sham/naive mice at all time points examined (grouped data control = 734 -4- 4 7 inffcted = 1296 • 103 imp/s, p<0 01) Analysis of variance (n=28) confirmed a significant • in nerve discharge frequency m response to intection (p=0.0004); whereas tune aBer inh*ction was not a significant factor (p = 0 6 3 ) Conclusions: These data confirm that there are changes iu the mast cell population and lee levels in response to Nb infection ill mice. Increased mesentenc atferent discharge in Nb-infected mice supports tire hypothesis of intlammation induced neural plasticity The availability ot a murine model fbr the-investigaion ofinflamnratinn-induced neural plasticity is a u~ihl tool in understanding the pathophysiulogy ot intestinal disea~s This study was supported by a generous grant from Janssen~ Week 19E(Pg/n~)
Mastr villi
/
0 0.2 0.1
1 0.4 ' 0.2
2 198 Z2
3 40 ~ 7.9
6 11.9 4.7
9 61 , 2.2
6.0
20.8
342,
19,1
10,9
122
?.S,
1.8
5.5
2.2
0.9
3.8
2.0
r
12 12.2 4.2
W1423 Behavioral Abnormalities and Increased Local and Systemic Inflammatory Responses in Adult Mice Following Maternal Deprivation as Neonates Ashs*an K. Varghese, Elena F. Verdu, Psemysl Bercik, Waliul I. Khan, Patricia A. Blennertzassett, Henry Szechtman, Stephen M. Collins Backgrocmd/Aims We have prewously sho,aTt that stress induced by neonatal maternal deprivation increases the severity of subsequent experimental colitis ira adult mice The aim of this s}udy was to identify behavioral changes associated with the increased vulnerability to colitis in adult mice, and to investigate local and systemic markers ot inflammation tollowmg the induction of colitis Methods C57BU6 mouse pups we• separated from their mothers for 31gday at 1-21 days of age Maternally non-deprived litters served as controls. On day 23, nrale mice were identified and weaned. Behavmr studies and colitis studies were perR)nned at day 60 on separate sets of male mice. Behavior Tests: At day 60, mice stere expo~d to a rectang~llar open t:te]dand allowed to habituate for 10 minutes. At 10 minutes, a novel object was introduced into the field and mice were allowed to respond for a further 10 minutes Behavioral patterns of mice throughout the 2&minute trial were analyzed using sof~watv. Colitis induction: Colitis was reduced at day 60 using 6% dextrau sultate sodium (DSS) in drinking water tbr 5 days. hrflammation was assessed using colonic [L-I,8 and Serum Amyloid P (SAP) levels. Results Behavior Te~ts:A steady decline iu activity levels was seen {6r both deprived (MD) and non-deprived (ND) mice, as expected, during the first 10 minutes of the trial However, alter introduclinn of the novel o]~ject ND mice continued to show habituation of activity levels whereas MD mice showed impaired habituation. Deprived mice showed a 37% (p<0 05) and 44% (p<0.05) decrease in percent-decline of distance traveled and movement duration, respectively compared to non-deprived animals, lnflammatoU responses:No spontaneous inflammation was seen in any group prior to DSS administration. Foil• 6% DSS, colonic IL-113and systemic SAP leveIs increased by I22% (p<0.005) and 85% (p<0 005), respectively m deprived as compared to non-deprived trace. Couclusion Early maternal deprivation results m an impaired ability of • to cope with stress m adulthood. Maternal deprivation also leads to increased levels of colomc [L-1[-3and systemic gAP levels post-DSS colitis induction in adult mice. These fmdings indicate that stress expenenced in early life increases the walnerability of adult mice to stress-induced behar
ANOVA p
W1421
Differential Sensitivity of Mechanosensitive Jejunal Mesenteric Afferent Nerves to Corticotrophin Releasing Factor in Wistar Kyoto and Wistar Rats Charlotte E, Booth, Wendy J Wm(hester, Mike Maline, Beverley Greenwood-Van meervald Gareth A Hicks Background: Stress or corticotropin releasing factor (CRF) enhance the viscemmotorresponse to cdorectal distension (CRD) in rats (Gun et al 1997, Neurogastroenterol Motil 9: 271) In additioo the high anxiety rat strain, Wistar Kyoto (WkW) display hypersensitivity" to CRD, which can he blocked by a CRF antagonist (Gunter et a! 2000, Physiol & Behav 69:
A-667
AGA Abstracts