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Photoperiodic variation in substance P induced plasma extravasation in the rat
Neuropharmacology Vol.28, No.8, pp.889~892, 1989 Printed in Great Britain
0028-3908,'89 $3.00+0.00 Maxwell Pergamon Macmillan plc
SJGibson,PVAndrews,andRDHelme NationalResearchInstitute of Gerontology and Geriatric Medicine Dt of Medicine, University of Mel.Mume,l%XmtRoyalHospital, Vie. 3052,Australia. Fop&r Rd. Parkville, fAccep.ted 23 Jwze 19891
was exsminedfollowing Photoperiodicvariation in plasma extravasation blisterbase on the rat exogenousperfusionof substanceP over a ~induced of plasmaextravasation hind footpad. The resultsshoweda general&&isq.pression during the dark or active phase of the light/darkcycle whichappearsto be independfsnt of the circsdianfluctuations in endogenouscorticosterone. These findingswise the need to considerphotoperiodic conditions as a possible sourceof variability in neurogenic inflametoryresponses. Nociceptivestirrpllation of cutaneousC fibres is known to result in a neurogenic inflalm&Ory response characterisedby an increasein vascular 1982). Weurogaicinflammation permeability and vasodilatation fLanbeck& GasLse occurs via an axon reflex and is presumedto be mediatedby the neuropaptide substanceP (SP) which is releasedfrcnncutaneousprimary afferentC fibres followingstimulation(White & Hehae 1985). It has been argued that local or flare of the axon reflex, providesan objective measureof vascdilatation, beingshownto correlate with subjective mciceptor activitywith flare reslxnses reportsof experimentally i.Mucedpain (Benjamin 1953). Several reports have demonstrated p&otopericdic variations in sensitivity to ncciceptivestirrUi in both experin-ental animslsfeg.Rosenfeld & Rice 19741and in ~~~~~al~i~icat~~t man (eg. Chapnan& Jones1944). ~c~~c the response of spinaldorsalhornunitsto noxiousthermalstirrurlation of cutaneous prinery afferentC fibresvariesoverthe light/dark cycle @k!nty1981). However, whether the axon reflexresponse acccepanying activation of cutaneous C fibresalso exhibits photoperiodic variation is presently unknown. We have recentlydevelopeda modelin the rat to investigate the neurogenic tPE)and vasodilatation, are xaeasured inflamaatory response. Plasmasrtravasation blister on the rat h&&paw in response to over the base of a ~induced fHelme, Sndrews& Watson19861. ClReaim mechanical,thexml, an3 dtwnicalstinnrli of the presentstudywas to seekphotoperiodic variation in PE duringeachphaseof levelspeak soonafter the light/darkcycle. As er&genousplashlcorticosterone the onset of the dark or activephase (O'Reilly, Coleran& mtrong 1981),and given that steroidsare known to inhibitPE and vascdilatation (Certel& Kaliner 1981),the concentration of thishomne was alsomXritOred duringthe experiment.
Male Sprague Dawley rats previouslyaccliimtized to a Y&r-12hrlight/dark cycle (lightson 0600-1800)and ranging in weight fram 240 to 340 graa~were r&-&znlyaSsignedto one of 3 grcqzs. %&groups were tested&ring the lightphase n=5, and 1530hrs n=6)whilea thirdgroupwas testedsoonafterthe (1100 hrs anaesthesia with sodiumpentobarbitone (60 onset of dark (1900ht-sn=4). Following ng/kg i.p.) a fenoral vein catheterwas inserta and a blisterinzluced on the glabrousskin of the hind footpadby applying a vacuumpressureof -40 kPa for 30 rains at 40°c, as described previously(Hehne et al, 19861. The blister epitheliumwas then removed arkdaperspexperfusion c32der withan inlet and outletportwas fixedoverthe blisterbaseand sealedwith silicongrease. Three perfusionperiods, each of 30 rainsduration,m&witha pm-stimulationperfusionof Ringer's solution. This was followedby a perfusionof l@+ SP, the Ec5o ,concentration for iming PE (Hebne& & 1986); and a post-stirsrlation with Ringer's solution. Perfusicui of all solutionswas deliveredat 4ml.hr via a peristalticpurap undera constant temperatureof 37% The parfusatewas collectedduringthe pre-sttiation and post-sttilationperiods with Ringer'ssolution, and at 10 mins intervals 889
890
Preliminary
Notes
during stinulation with SP. 733 obtain a quantitative measure of PE the protein content of each sample was assaysdaccordingto the method of Bradford (1976). Approximately M of blood was taken fran the femoralvein catheter just prior to blister induction, prior to the pm-stinnrlationperiod, and imneaiately following the post-sttilation period. samples were cooled to 4T, and the separated p1aslla was extracted and stored at -2O'C until assayed for corticosteroneusing a nrxdificationof the n&hod describedby Murphy (1967). All animals resained under anaesthesia and the prevailing photoperiodic conditions were rnaintain&during testing. A single 60 watt light providedthe only source of ill~nation during the dark phase and animalswere placed under an opaque cover cloth during ths blister and perfusionprocedures. At the completion of the experimentanimalswere killed by either cervicaldislocation or barbiturateoverdose.
Analysis of variancewith a priori plannedcontrastsrevealeda significant change over the 5 sample periods in each of the 3 groups;F4 48= 18.27 (1100 F4 48= 4.83 (1900hrs), ~(0.65, suggestinga hrs), F4 48= 21.5 (1530 hrs), marked in&ease in PE during stinn0ationwith SP (see Fig. 1).
TIME
Fiaure
The effect of sp (lm) on the plasM extravasaticmresponseof 1. anima_l.s tested at three differenttimes during the light/darkcycle (1100 hrs, n = 5; 1530 hrs, n = 6; 1900 hrs, n = 4). The first column of each group represent the 30 min pm-stinnrlationcontrol period. The three hatched columns represent the tbme1Omins SPperfusionperiods, tithe final open column representsthe 30 min post-sttiation period. All values are expressedas mean + S.E.M. As can be seen in Fig. 1, there was no significantdifferencebetweenthe two light phase tested groups but, despitemaintainingthe sane pattern of response, aninaalstested in the dark phase exhibitedsignificantlylower levelsof PE during the pm-stirrmlationperiod,F1 12= 13.33,p(O.05, and the three 10 mins periods of sttilation with SP, F1'12= 5.55, Fl 12= ,5.22, 12= 4.48, pto.05. PE was also decreasedduring the post-stufUatlonper1 In anrma1.s tested during the dark but this trend failed to reach statisticalsignificance. ?.s shown in Fig. 2, the resting levels (pre blister) of plasnaa in this homone, with corticosterone reflect the known circadian rhm siqnficantly elevated levels during the late afternoon and early night, F2 12= 5.5855, pCO.05. Bowwer, there was no significant difference in steroid concentration between the 3 groups at either of the two sampleperiods was following blister induction. A significantchange in plasma corticosterone also observed across the 3 sample periods in each of the 3 groups,F2,24=38.22 rz,"s.;n ;fg24=33.95.(1530.hWI F2 24=22.94 .(1900hrs), p,O.O5. AS Can . 2 blister induction tesulted r.n a marked elevation of corticosteronewhich was followed by a subsequentfall on canpletionof the experiment,perhaps indicatinga generalisedeffect of prolongedanaesthesia.
Preliminary Notes
891
E
0
0 1100
1530
1900
TIME
Fiaure 2. l%e meanplasmacorticosterone concentration of animalstestedat three differenttimes during the light/dark cycle (1100hrs,n = 5; 1530 pre-blister hrs, n = 6; 1900 hi-s, n = 41. Plaza sampleswerecollected and following the completion of induction/J, post-blister inductiona as mean + S.E.M. theexperimentq . All valuesare expressed
The findingsof the present study clearly denxonstrate the presenceof p&otopericdic variationsin SP-induced PE. PE was significantly decreased when annirrals were testedduringthe darkor activephaseof the light/dark cycleand this occurredduring the pi-e-stian.rlation as well as the stinnrlation periods. to be a genera&& suppression of the PE respnse rather Hae, there apfzeers thsn a specificphotoperiodic variationin the effect of SP on vascular althoughthe possibilitythat a ccanbination of both factors permeability, contribute to the light/dark variation in PE cannotbe excluded. As corticosteroids are know to inhibitPE, vasodilatation, and erythaM (Certel& Kaliner1981)one obviousexplanation for the suppression in PE could be the presenceof elevated plasmacorticosterone levelssoonafterthe onsetof priorto dark. In supportof thisproposalthe levelsof plasmacorticosterone blister inductionare inverslyrelatedto the eventuallevelof PEA However, the lackof significant difference in plasm corticosterone betweenthe 3 groups followingblister inductioncontrasts nerkedlywiththe observedphotoperiodic variationsin PE duringthisperiod. Hence,it seemslikelythatcorticosterone does not play a majorrole in the generalised suppression of PE duringthe acute stages of SP-induced PE, and thatscmeothernechanism mediatesthe generalised candidates includea nocturnal reductionin PE during the night. Possible reductionin peripheral bloodflow,and an increase in syqxtheticactivity, as bothprocesses are knownto exerta generalised influence on PE (Engel1978). In conclusion,the present findingsemphasisethe needtoconsider photoperiodicconditions as a source of variabilityin the neurogenic inflamnatoxyresponse,althoughsuch variationsappear to result frcana generalisedsqzpressionof vascularpermeability ratherthana neuronspecific suchperipheral mechanisms contribute to effect. Finally,it is possible'that circadianvariationin patientreportsof pain,at leastin conditions whereC fibre activationis lsxxnto be involved, suchas rheumatoid arthritis(Levine, of neurogenic Hence, further investigation Ccderre & Easbaum 1988). inflann&ory responsesthroughoutthe light/dark cyclemay contribute towarda betterunderstanding of nocturnal merbation in pain. This work was supported by a projectgrantto R.D.Helmefromthe National Health and MedicalResearchCouncil(NH & MRC) of Australia and frcmI the Asthma Fourxlation of Victoria.
892
Preliminary Notes
betweeninjuryand pain in m skin.J. Benjamin,F.B. (1953) Relationship -1. Physiol.5: 740-745. methodfor the quantitation of Bradford,M.M. (1976) A rapid and sensitive microgramquantitiesof protein utilizingthe principleof protein-dye binding. Anal.Biochen.72: 248-260. Chaplwl,W.P. and Jones,C.M. (1944)Variations in cutaneous and visceralpain sensitivity in normalsubjects. J. Clin.Invest.23: 81-86. nervoussystanon capillary hsel, D.(1978) The influewe of the sympathetic permeability. Res. Eq& Med. 173:l-8. Helme, R.D., NZi?xws, P.V. and Watson, B.A. (1986) Neurcgenic inflation caused by wool fabric in the rat; Possiblemediationby substance P. Neurosci. L&t. 66: 333-337. of dorsalhorn unitsby (t981) Diurnal variationin excitation HW &, III the spinalisedcat suggest a circulatingopioid factor. Neurosci. 6: 1935-1942. P in perimeralsensoryprocesses.m R. Lembeck,F. snd Gamse,R., Substance Systen,Pitman, porter arxlM. O'conner teds) SubstancePintheNemus Lox&n, 1982pi. 35-54. Levine, J.D.,Coderre,T.J. and Basbwn, A.I.,The peripheral nervoussystenand the inflmtory process. In R. IXixer,G.F.Gebhrt and M.R. Bond teds), proceedings of the Vth World Congresson Pain Vol. 3, Elsevier, Amsterdam, 1988pp. 33-43. Iwrphy, B.E.P.(1967) Sanestudiesof the protein-binding of steroidsand their applicationto the routine micro and ultramicromeasuren-ent of various steroids in body fluidsby caqzetitive protein-binding radioassay. J. Clin. Fn&crin. 27: 973-990. of mastcellgranules Oertel, H. and Kaliner,M. (1981) The biologicactivity in the rat skin: effects of adrenocorticosteroid on the late phase inflammatory responsesinduced by mast cell granules.J. AllergyClin. -1. 68: 238-245. O'Reilly, H.M., Coleman, G.J. and Armstrong, S. (1981) meno= corticostercme alterationsand appetitivelearningin the rat.Pnysiol. Behav.27: 515-522. threshaL% Rosenfeld,J.P.and Rice,P.E. (1974) DiurnalrhythIwin nocicephive of rats.Physiol.Behav.23: 419-420. P frcnn peripheral nerve White, D.M. and Helme,R.D. (1985) Releaseof suhstarze terminalsfollowingsthlation of the sciaticnerve.BrainRes. 336:27.
0028-3908,'89 $3.00+0.00 Maxwell Pergamon Macmillan plc
SJGibson,PVAndrews,andRDHelme NationalResearchInstitute of Gerontology and Geriatric Medicine Dt of Medicine, University of Mel.Mume,l%XmtRoyalHospital, Vie. 3052,Australia. Fop&r Rd. Parkville, fAccep.ted 23 Jwze 19891
was exsminedfollowing Photoperiodicvariation in plasma extravasation blisterbase on the rat exogenousperfusionof substanceP over a ~induced of plasmaextravasation hind footpad. The resultsshoweda general&&isq.pression during the dark or active phase of the light/darkcycle whichappearsto be independfsnt of the circsdianfluctuations in endogenouscorticosterone. These findingswise the need to considerphotoperiodic conditions as a possible sourceof variability in neurogenic inflametoryresponses. Nociceptivestirrpllation of cutaneousC fibres is known to result in a neurogenic inflalm&Ory response characterisedby an increasein vascular 1982). Weurogaicinflammation permeability and vasodilatation fLanbeck& GasLse occurs via an axon reflex and is presumedto be mediatedby the neuropaptide substanceP (SP) which is releasedfrcnncutaneousprimary afferentC fibres followingstimulation(White & Hehae 1985). It has been argued that local or flare of the axon reflex, providesan objective measureof vascdilatation, beingshownto correlate with subjective mciceptor activitywith flare reslxnses reportsof experimentally i.Mucedpain (Benjamin 1953). Several reports have demonstrated p&otopericdic variations in sensitivity to ncciceptivestirrUi in both experin-ental animslsfeg.Rosenfeld & Rice 19741and in ~~~~~al~i~icat~~t man (eg. Chapnan& Jones1944). ~c~~c the response of spinaldorsalhornunitsto noxiousthermalstirrurlation of cutaneous prinery afferentC fibresvariesoverthe light/dark cycle @k!nty1981). However, whether the axon reflexresponse acccepanying activation of cutaneous C fibresalso exhibits photoperiodic variation is presently unknown. We have recentlydevelopeda modelin the rat to investigate the neurogenic tPE)and vasodilatation, are xaeasured inflamaatory response. Plasmasrtravasation blister on the rat h&&paw in response to over the base of a ~induced fHelme, Sndrews& Watson19861. ClReaim mechanical,thexml, an3 dtwnicalstinnrli of the presentstudywas to seekphotoperiodic variation in PE duringeachphaseof levelspeak soonafter the light/darkcycle. As er&genousplashlcorticosterone the onset of the dark or activephase (O'Reilly, Coleran& mtrong 1981),and given that steroidsare known to inhibitPE and vascdilatation (Certel& Kaliner 1981),the concentration of thishomne was alsomXritOred duringthe experiment.
Male Sprague Dawley rats previouslyaccliimtized to a Y&r-12hrlight/dark cycle (lightson 0600-1800)and ranging in weight fram 240 to 340 graa~were r&-&znlyaSsignedto one of 3 grcqzs. %&groups were tested&ring the lightphase n=5, and 1530hrs n=6)whilea thirdgroupwas testedsoonafterthe (1100 hrs anaesthesia with sodiumpentobarbitone (60 onset of dark (1900ht-sn=4). Following ng/kg i.p.) a fenoral vein catheterwas inserta and a blisterinzluced on the glabrousskin of the hind footpadby applying a vacuumpressureof -40 kPa for 30 rains at 40°c, as described previously(Hehne et al, 19861. The blister epitheliumwas then removed arkdaperspexperfusion c32der withan inlet and outletportwas fixedoverthe blisterbaseand sealedwith silicongrease. Three perfusionperiods, each of 30 rainsduration,m&witha pm-stimulationperfusionof Ringer's solution. This was followedby a perfusionof l@+ SP, the Ec5o ,concentration for iming PE (Hebne& & 1986); and a post-stirsrlation with Ringer's solution. Perfusicui of all solutionswas deliveredat 4ml.hr via a peristalticpurap undera constant temperatureof 37% The parfusatewas collectedduringthe pre-sttiation and post-sttilationperiods with Ringer'ssolution, and at 10 mins intervals 889
890
Preliminary
Notes
during stinulation with SP. 733 obtain a quantitative measure of PE the protein content of each sample was assaysdaccordingto the method of Bradford (1976). Approximately M of blood was taken fran the femoralvein catheter just prior to blister induction, prior to the pm-stinnrlationperiod, and imneaiately following the post-sttilation period. samples were cooled to 4T, and the separated p1aslla was extracted and stored at -2O'C until assayed for corticosteroneusing a nrxdificationof the n&hod describedby Murphy (1967). All animals resained under anaesthesia and the prevailing photoperiodic conditions were rnaintain&during testing. A single 60 watt light providedthe only source of ill~nation during the dark phase and animalswere placed under an opaque cover cloth during ths blister and perfusionprocedures. At the completion of the experimentanimalswere killed by either cervicaldislocation or barbiturateoverdose.
Analysis of variancewith a priori plannedcontrastsrevealeda significant change over the 5 sample periods in each of the 3 groups;F4 48= 18.27 (1100 F4 48= 4.83 (1900hrs), ~(0.65, suggestinga hrs), F4 48= 21.5 (1530 hrs), marked in&ease in PE during stinn0ationwith SP (see Fig. 1).
TIME
Fiaure
The effect of sp (lm) on the plasM extravasaticmresponseof 1. anima_l.s tested at three differenttimes during the light/darkcycle (1100 hrs, n = 5; 1530 hrs, n = 6; 1900 hrs, n = 4). The first column of each group represent the 30 min pm-stinnrlationcontrol period. The three hatched columns represent the tbme1Omins SPperfusionperiods, tithe final open column representsthe 30 min post-sttiation period. All values are expressedas mean + S.E.M. As can be seen in Fig. 1, there was no significantdifferencebetweenthe two light phase tested groups but, despitemaintainingthe sane pattern of response, aninaalstested in the dark phase exhibitedsignificantlylower levelsof PE during the pm-stirrmlationperiod,F1 12= 13.33,p(O.05, and the three 10 mins periods of sttilation with SP, F1'12= 5.55, Fl 12= ,5.22, 12= 4.48, pto.05. PE was also decreasedduring the post-stufUatlonper1 In anrma1.s tested during the dark but this trend failed to reach statisticalsignificance. ?.s shown in Fig. 2, the resting levels (pre blister) of plasnaa in this homone, with corticosterone reflect the known circadian rhm siqnficantly elevated levels during the late afternoon and early night, F2 12= 5.5855, pCO.05. Bowwer, there was no significant difference in steroid concentration between the 3 groups at either of the two sampleperiods was following blister induction. A significantchange in plasma corticosterone also observed across the 3 sample periods in each of the 3 groups,F2,24=38.22 rz,"s.;n ;fg24=33.95.(1530.hWI F2 24=22.94 .(1900hrs), p,O.O5. AS Can . 2 blister induction tesulted r.n a marked elevation of corticosteronewhich was followed by a subsequentfall on canpletionof the experiment,perhaps indicatinga generalisedeffect of prolongedanaesthesia.
Preliminary Notes
891
E
0
0 1100
1530
1900
TIME
Fiaure 2. l%e meanplasmacorticosterone concentration of animalstestedat three differenttimes during the light/dark cycle (1100hrs,n = 5; 1530 pre-blister hrs, n = 6; 1900 hi-s, n = 41. Plaza sampleswerecollected and following the completion of induction/J, post-blister inductiona as mean + S.E.M. theexperimentq . All valuesare expressed
The findingsof the present study clearly denxonstrate the presenceof p&otopericdic variationsin SP-induced PE. PE was significantly decreased when annirrals were testedduringthe darkor activephaseof the light/dark cycleand this occurredduring the pi-e-stian.rlation as well as the stinnrlation periods. to be a genera&& suppression of the PE respnse rather Hae, there apfzeers thsn a specificphotoperiodic variationin the effect of SP on vascular althoughthe possibilitythat a ccanbination of both factors permeability, contribute to the light/dark variation in PE cannotbe excluded. As corticosteroids are know to inhibitPE, vasodilatation, and erythaM (Certel& Kaliner1981)one obviousexplanation for the suppression in PE could be the presenceof elevated plasmacorticosterone levelssoonafterthe onsetof priorto dark. In supportof thisproposalthe levelsof plasmacorticosterone blister inductionare inverslyrelatedto the eventuallevelof PEA However, the lackof significant difference in plasm corticosterone betweenthe 3 groups followingblister inductioncontrasts nerkedlywiththe observedphotoperiodic variationsin PE duringthisperiod. Hence,it seemslikelythatcorticosterone does not play a majorrole in the generalised suppression of PE duringthe acute stages of SP-induced PE, and thatscmeothernechanism mediatesthe generalised candidates includea nocturnal reductionin PE during the night. Possible reductionin peripheral bloodflow,and an increase in syqxtheticactivity, as bothprocesses are knownto exerta generalised influence on PE (Engel1978). In conclusion,the present findingsemphasisethe needtoconsider photoperiodicconditions as a source of variabilityin the neurogenic inflamnatoxyresponse,althoughsuch variationsappear to result frcana generalisedsqzpressionof vascularpermeability ratherthana neuronspecific suchperipheral mechanisms contribute to effect. Finally,it is possible'that circadianvariationin patientreportsof pain,at leastin conditions whereC fibre activationis lsxxnto be involved, suchas rheumatoid arthritis(Levine, of neurogenic Hence, further investigation Ccderre & Easbaum 1988). inflann&ory responsesthroughoutthe light/dark cyclemay contribute towarda betterunderstanding of nocturnal merbation in pain. This work was supported by a projectgrantto R.D.Helmefromthe National Health and MedicalResearchCouncil(NH & MRC) of Australia and frcmI the Asthma Fourxlation of Victoria.
892
Preliminary Notes
betweeninjuryand pain in m skin.J. Benjamin,F.B. (1953) Relationship -1. Physiol.5: 740-745. methodfor the quantitation of Bradford,M.M. (1976) A rapid and sensitive microgramquantitiesof protein utilizingthe principleof protein-dye binding. Anal.Biochen.72: 248-260. Chaplwl,W.P. and Jones,C.M. (1944)Variations in cutaneous and visceralpain sensitivity in normalsubjects. J. Clin.Invest.23: 81-86. nervoussystanon capillary hsel, D.(1978) The influewe of the sympathetic permeability. Res. Eq& Med. 173:l-8. Helme, R.D., NZi?xws, P.V. and Watson, B.A. (1986) Neurcgenic inflation caused by wool fabric in the rat; Possiblemediationby substance P. Neurosci. L&t. 66: 333-337. of dorsalhorn unitsby (t981) Diurnal variationin excitation HW &, III the spinalisedcat suggest a circulatingopioid factor. Neurosci. 6: 1935-1942. P in perimeralsensoryprocesses.m R. Lembeck,F. snd Gamse,R., Substance Systen,Pitman, porter arxlM. O'conner teds) SubstancePintheNemus Lox&n, 1982pi. 35-54. Levine, J.D.,Coderre,T.J. and Basbwn, A.I.,The peripheral nervoussystenand the inflmtory process. In R. IXixer,G.F.Gebhrt and M.R. Bond teds), proceedings of the Vth World Congresson Pain Vol. 3, Elsevier, Amsterdam, 1988pp. 33-43. Iwrphy, B.E.P.(1967) Sanestudiesof the protein-binding of steroidsand their applicationto the routine micro and ultramicromeasuren-ent of various steroids in body fluidsby caqzetitive protein-binding radioassay. J. Clin. Fn&crin. 27: 973-990. of mastcellgranules Oertel, H. and Kaliner,M. (1981) The biologicactivity in the rat skin: effects of adrenocorticosteroid on the late phase inflammatory responsesinduced by mast cell granules.J. AllergyClin. -1. 68: 238-245. O'Reilly, H.M., Coleman, G.J. and Armstrong, S. (1981) meno= corticostercme alterationsand appetitivelearningin the rat.Pnysiol. Behav.27: 515-522. threshaL% Rosenfeld,J.P.and Rice,P.E. (1974) DiurnalrhythIwin nocicephive of rats.Physiol.Behav.23: 419-420. P frcnn peripheral nerve White, D.M. and Helme,R.D. (1985) Releaseof suhstarze terminalsfollowingsthlation of the sciaticnerve.BrainRes. 336:27.