Central activation of the trigeminovascular pathway in the cat is inhibited by dihydroergotamine: A c-Fos and electrophysiological study

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Central activation of the trigeminovascular pathway in the cat is inhibited by dihydroergotamine: A c-Fos and electrophysiological study
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  Brain  (1996), 119, 249-256 Central activation of the trigeminovascular pathwayin the cat is inhibited by dihydroergotamine A c-Fos and electrophysiological study Karen L. Hoskin, 1  Holger Kaube 2  and Peter J. Goadsby 1 institute of Neurology, The National Hospital forNeurology and Neurosurgery, London, UK and the 2 Department of Neurology, Essen, GermanyCorrespondence to: Dr Peter  J Goadsby, Institute ofNeurology, Queen Square, London WC1N 3BG, UK Summary Recent studies have delineated a clear role for the trigeminalinnervation of pain-sensitive intracranial structures in thepathophysiology of migraine. The development of newcompounds for the treatment of  the  acute attack of migrainehas led to a greater understanding of serotonin 5-hydroxytryptamine; 5HT) receptor diversity. The ergotalkaloids have been used in the treatment of acute attacks ofmigraine for many years and parenteral administration ofdihydroergotamine DHE) can be a useful treatment strategy.In this study, the question of a possible central site ofaction of DHE is considered using both anatomical andphysiological approaches. The c-Fos method has been usedto map functional activation of central neurons in responseto stimulation of  the  superior sagittal sinus SSS) in the cat.  his  structure has been used  as  it  refers  pain to the ophthalmicdivision of the trigeminal nerve in humans, and in catsinduces changes in neuropeptides and cranial blood flowsimilar to those seen in migraine. In addition, the temporalaspects of the effect of DHE have been studied by makingextracellular recordings from cells in the most caudal aspectof the trigeminal nuclear complex. Stimulation of the SSSresults in Fos expression in the superfical laminae of thetrigeminal nucleus caudalis and in the dorsal horn of  C\  and C2.  This activation is blocked by a clinically relevant doseof  DHE Similarly, cells can be recorded in this region thatrespond to SSS stimulation. This linked cellular activity canbe inhibited by the same intravenous dose of DHE. Together,these studies show that DHE can inhibit activity in centraltrigeminal neurons. Since the sinus and its nerve supply aredirectly stimulated, the peripheral nerve/vessel innervationis bypassed and this inhibition cannot have happened at anyother site. These data imply that drugs acting at the centraltrigeminal neurons may have a role in the treatment of  acute attacks of migraine. Keywords: headache; migraine; acute treatment; trigeminal nucleus; ergotamineAbbreviations: DHE = dihydroergotamine; SSS = superior sagittal sinus; 5HT = 5-hydroxytryptamine = serotonin Introduction Recent advances in the understanding of the pathophysiologyof migraine have included more complete descriptions of thepathways that are involved in head pain. It is clear thatmigraine involves activation of the pain-sensitive innervationfrom the trigeminal nerve and that the second order neuronsreside in the most caudal part of the trigeminal nucleuscaudalis and in the dorsal horn of the first and second cervicalsegments of the spinal cord (Goadsby and Zagami, 1991;Kaube  et al.,  1993a). Drugs that are effective in migrainemay, therefore, have an action upon these neurons in additionto effects at more peripheral sites (Moskowitz, 1992).The therapy of migraine has, until recent times, focused©  Oxford University Press 1996 upon drugs used to treat acute attacks, that were thought tohave a vascular role, such as the ergots (Graham and  Wolff, 1966), or therapy for prevention of attacks, that has beenconsidered to interact with inhibitory serotonergic mecha-nisms, such as methysergide or pizotifen (Peroutka, 1988).The latter group includes drugs that may act upon 5HT 2 receptors (Hoyer  et al.,  1986). The synthesis of the novel5HT, D -like agonist sumatriptan (Humphrey  et al.,  1991) andits successful use in the treatment of the acute attack ofmigraine (Ferrari, 1991; Goadsby  et al.,  1991b) has againhighlighted serotonergic involvement in the syndrome.Dihydroergotamine is also widely used in clinical practice   b  y g u e  s  t   onA u g u s  t  2  ,2  0 1  6 h  t   t   p :  /   /   b r  a i  n . oxf   or  d  j   o ur n a l   s  . or  g /  D o wnl   o a  d  e  d f  r  om   250 K. L. Hoskin  et al.for the treatment of migraine (Goadsby, 1994) and particularlyin the management of persistent severe headache (Raskin,1986). In a similar way to sumatriptan it acts at the 5HT, D receptor (Peroutka, 1988) and its potential site of action istherefore of interest. It has been suggested that the action ofanti-migraine compounds may be at the peripheral trigemi-novascular synapse to block neurogenically mediated plasmaextravasation (Moskowitz and Cutrer, 1993). We haveproposed that, in addition, a peripheral action to it may bepossible for anti-migraine drugs to act within the CNS onthe central ramifications of the trigeminal system (Goadsby et al.,  1991a . There is good evidence  in vitro  that DHE binds with veryhigh affinity to both a-adrenoceptors and to 5HT ]D -likereceptors in the rat brain (Hamblin  et al.,  1987). The questionof whether peripherally administered DHE or ergotamineenters the CNS in humans  in vivo  is controversial. It hasbeen reported that intravenous DHE (Kanto  et al.,  1981),and orally or rectally (Hovdal  et al.,  1982) administeredergotamine cannot be detected in the CSF, although Ala-Hurula  et al.  (1979) could detect small quantities ofergotamine in the CSF after a 2 mg oral dose. Methodologicalconsiderations, such as assay detection limits, have beencited to explain the differences seen in these studies of theCSF (Eadie, 1983).We have shown that DHE binds to receptors in thetrigeminal nucleus caudalis and in the dorsal horn of the firstand second cervical segments of the spinal cord using both in vitro  and  ex vivo  methods in the cat (Goadsby andGundlach, 1991). Intravenous administration of DHE has notbeen studied in models of central trigeminal activation, yetit is a widely employed route in clinical practice. In thisstudy we have combined the functional/anatomical c-Fosmethod with extracellular electrophysiology to correlate moreprecisely the spatial relationships between the anatomicallocation of trigeminally activated cells and the possible sitesof action of DHE. Methods Eleven cats were anaesthetized initially with 1.5 halothaneand a-chloralose (60 mg/kg, intraperitoneally; Sigma,St Louis, USA) and prepared for physiological monitoring.The femoral artery and vein were cannulated in order tomeasure blood pressure and heart rate and provide access fordrug and fluid administration, respectively. Cardiovascularparameters and pupillary reaction to noxious pinching of theforepaw were used to determine the need for supplementaryanaesthesia. The animals were endotracheally intubated,ventilated with 40 oxygen and paralysed after thecompletion of the surgical procedures with repeated doses ofgallamine triethiodide (6 mg/kg intravenously; May andBaker, UK). Body temperature and end-expiratory CO? weremonitored and maintained within physiological limits. Theanimals were mounted in a stereotactic frame and a circularmidline craniotomy (2 cm in diameter) was performed foraccess to the SSS. The adjacent dura and falx were dissectedparallel to the sinus over 10-15 mm. To prevent dehydrationand for electrical insulation against the cortex, a paraffin bathwas built with a dam of dental acrylic around the craniotomyand in addition a small polyethylene sheet inserted under thevessel. Fluid (4 glucose with 0.18 saline or normal saline)was administered intravenously at a rate of 3-5 ml/kg/h,while gallamine (6 mg/kg intravenously) and a-chloralose(10-15 mg/kg intravenously) were administered every 2 h.Blood pressure and heart rate were stable and withinphysiological range for all animals throughout the wholeexperiment. Arterial blood gas parameters were monitoredintermittently as a guide to the end-expiratory CO2 output. Fos study Following completion of surgery, the animal was maintainedfor the following 24 h. After this resting phase, the SSS wassuspended over a pair of platinum hook electrodes. We haveshown that this would, of  itself,  not provoke Fos activation(Kaube  et al.,  1993a). The SSS was stimulated with a GrassS88 stimulator driving a stimulus isolation unit (SIU5A,Grass Instruments, Quincy, Mass., USA; 150V, 250 usduration) at a rate of 0.3/s for 1 h. After completion of theperiod of stimulation 1 h was allowed to elapse prior toperfusion. Perfusion Cats were perfused transcardially with 1—1.5  1  of 0.9 saline(containing 1000 IU of heparin and 0.5ml of 1 sodiumnitrite) this was followed by 2  1  of 4 paraformaldehyde inphosphate buffer (pH 7.4) and finally by 500-600 ml of 30 sucrose solution in phosphate buffer. The brain and cervicalspinal cord were removed and stored in 50 sucrose withazide. Coronal sections (40 urn) of the caudal medulla andupper cervical spinal cord were cut on a freezing microtomeand every fifth section was collected for processing. Sectionswere cut from a block beginning at the level of the obexthrough to C 3  segment of the cervical cord. c-Fos procedure Free-floating sections were incubated at 4°C for 3-7 days ina commercially available rabbit, polyclonal antibody to Fosprotein ('Ab-2\ Oncogene Science Ltd, USA) in a  1:1000 dilution with 1 phosphate buffered horse serum, containing 0.1 bovine serum albumin and 0.2 Triton-XlOO. Fos-likeimmunoreactivity (hereafter simply called FosJ was visualizedusing standard avidin—biotin peroxidase immunohisto-chemical techniques. Following the primary incubation,sections were washed in 0.1 M phosphate buffer (pH 7.4)for 30 min and then incubated in a biotinylated goat anti-rabbit IgG (1:200 dilution) (Vector Labs, USA) for a minimumof 2 h at room temperature on a rotating table. Followingthe second incubation, the sections were washed again in   b  y g u e  s  t   onA u g u s  t  2  ,2  0 1  6 h  t   t   p :  /   /   b r  a i  n . oxf   or  d  j   o ur n a l   s  . or  g /  D o wnl   o a  d  e  d f  r  om   DHE inhibits trigeminal nucleus activity  2510.1 M phosphate buffer (pH 7.4) for 30 min. The sectionswere then incubated for 2.5 h in a  1:1000  dilution ofExtrAvidin-peroxidase (Sigma), sections were again washedin 0.1 M phosphate buffer (pH 7.4) for 30 min. The sectionswere then incubated in 20 ml of 0.1 M phosphate buffercontaining 0.05 diaminobenzidine (Sigma), 0.005 of 4 ammonium chJoride, 0.005 of 20 (+)-glucose and 0.02 of a  1 solution of nickel ammonium sulphate for 20 min.The sections were then placed in a fresh identical 20 mlsolution and 20 ul of glucose oxidase (Sigma) was added toinitiate the chromogenic reaction. The reaction was allowedto proceed until  Fos  positive nuclei could be clearly seenunder the microscope. The diaminobenzidine reaction productwas visible as a black precipitate due to the presence ofthe nickel ammonium sulphate. Following this reaction thesections were washed two or three times in 0.1 M phosphatebuffer (pH 7.4) and they were mounted on gelatinized slides.Fos-positive cells were distinguished by their black nickelenhanced nuclei from the background. Using the procedureadopted by Hammond  et  al (1992), cells were only consideredpositive if the black precipitate of the diaminobenzidinereaction within the cell nucleus was distinguishable from thebackground throughout a range of magnifications betweenX20 and X4 (Hammond  et al.,  1992). Fos-positive cellswere plotted onto sections of the caudal medulla and uppercervical spinal cord modified from the atlases of Berman(1968) and Rexed (1954). Control incubations in the presenceof the antigen were not carried out in this series ofexperiments. However, the omission of the primary antibodyin other experiments performed in this laboratory have failedto produce positive staining. Furthermore, preabsorption ofthe antibody with Fos protein has demonstrated its specificityin other studies.Distributions of cells were quantified for each individualanimal by taking 10 sections at random, from each of thelevels (SpV, Cl, C2 and C3) and plotting the label from asingle side onto one of the schematic sections describedabove. The plotting was performed by one person who,although they had knowledge of the experimental design,was not aware of the experimental group to which eachanimal belonged. Electrophysiology To activate trigeminal primary afferents, the SSS wasstimulated with a Grass S88 stimulator with stimulus isolationunit (150 V, 250 us, 0.3 Hz; SIU5A). Tungsten-in-glassmicroelectrodes (tip length/diameter: 50/15 urn, impedance:<200 k£2) were lowered into the dorsolateral spinal cord  4— 5 mm caudal to the mid-point of the C2-rootlets between 400and 800 urn below the surface with a hydraulic micro-positioner  Kopf,  Model 650, USA). Electrical responseswere amplified (NeuroLog, total system gain 20 000-30 000)and low-pass filtered (NeuroLog, high cut-off frequency 5.5kHz) to prevent aliasing. The signal from die amplifier waspassed to the analog input of an A/D converter (LabMaster,Ohio, USA) in an IBM-compatible microcomputer (80386/80387 based) for on-line analysis of single units responsesby custom written program (Microsoft C). Single unit activitywas analysed after digital online high pass filtering (cut-offfrequency 500 Hz) and passing a digital window discriminatorto create a post-stimulus histogram over 25 or 100 recordings(sweep length 50 ms) to identify linked responses. Baselinerecordings with 100 averages each were repeated at leastthree times to ensure that single unit responses to SSSstimulation were reproducible over time. Drugs Dihydroergotamine (Sandoz) was administered in a dose of15 ug/kg intravenously 30 min prior to stimulation for theFos studies. For the electrophysiological studies three controlpost-stimulus histograms were obtained before DHE wasadministered. Statistics Physiological data are reported as mean±SD, whereas Fosdata are reported as the median response with the interquartilerange. Although die Fos technique provides quantitation it islikely to result in ordinal rather than interval data (Siegel,1956) and therefore is better analysed by non-parametricmediods. To compare the two groups a Mann-Whitney  U test has been employed (Siegel, 1956). The electro-physiological data were compared using a Student's  t  test.All comparisons were considered significant at the P <  0.05 level. Results Eleven cats were studied in all with a mean weight of2.4±0.3 kg (mean±SD). The physiological data for theanimals reported are presented in the Table 1. Fos  studies Stimulation of die SSS results in expression of Fos-likeimmunoreactivity in restricted regions of the caudal medullaand high cervical spinal cord as has been previously reportedfrom our group (Kaube  et al.,  1993a). The data here extendthe number of animals that we have studied  n =  3) at a rateof stimulation of 0.3 Hz. The regions expressing  Fos  in thetrigeminal system are the ventrolateral portion of laminae Iand IIo of die trigeminaJ nucleus caudalis and Q dorsal horngrey matter and the medial aspect of laminae I and Ho ofthe C 2  dorsal horn grey matter. In addition to these cells Foswas also detected in the caudal portion of the medial nucleusof the solitary tract and in the nucleus retroambigualis at thelevel of die trigeminal nucleus caudalis and in laminae X indie high cervical spinal cord. The quantitative data for theseregions are presented in Table 2.   b  y g u e  s  t   onA u g u s  t  2  ,2  0 1  6 h  t   t   p :  /   /   b r  a i  n . oxf   or  d  j   o ur n a l   s  . or  g /  D o wnl   o a  d  e  d f  r  om   252 K. L Hoskin  et al. Table 1  Physiological data* c-Fos studies  n  = 6)Electrophysiological studiesWeight(kg)2.5±0.32.3±0.3Blood pressure(mmHg)110±8 101  ±7PH7.31 ±0.057.33±0.04 pCO 2 37±636±5 pO 2 211  ±47 221  ±23*Mean±SD for  n =  11 animals. Table  2  Fos expression following SSS stimulation is reduced after treatment with DHE* RegionCaudal medullaLaminae I/IIo cNTS f c, Laminae I/IIoLamina X c 2 Laminae I/IIoLamina XControlMedian763788259218stimulationRange50-9535-4872-10517-3971-9514-26DihydroergotamineMedian9* 4* 8*6* 7* 3* Range7-103-125-95-76-103-5•Median with interquartile range for  n  = 6 cats; ^cNTS, caudal extension of the medial nucleus of thesolitary tract; *P < 0.05. DHE Treatment of three animals with DHE resulted in markedchanges in Fos expression in the regions studied (Fig. 1).The number of Fos-positive cells in laminae I and Ho of thetrigeminal nucleus caudalis fell from the control level of 81(76-114) to 9 (7-10;  P <  0.05) following DHE (Fig. 2).Similarly, Fos expression in laminae I and IIo of the Q  P <  0.05) and C 2  P <  0.05) cervical spinal cord weremarkedly reduced by DHE administration (Table 2). Electrophysiology Cells were recorded at the C 2  level of the cervical spinalcord with a baseline probability of firing of 0.73±0.2 and alatency for the fastest component of the histogram of  8.3±1.1 ms  n =  5; Fig. 3). The probability of firing was reproducibleacross three consecutive averages each consisting of 100stimulations followed by 50 ms sweeps. The data for eachcontrol period and the post-treatment period are shown inFig. 4. The variability between each of the control periodswas 12 in total. About 30 min after the intravenousadministration of DHE, the probability of firing began toreduce reaching a minimum at 45 min  (0.21  ±0.04). Thisrepresented a significant reduction from the control level(P < 0.01) with no change in the latency of the units thatremained. In no animal did the responses return to baselineafter 3 h observation. Discussion We have examined the activation of central trigeminal neuronsusing two approaches both of which have shown thatintravenous administration of DHE can inhibit trigeminalneurons. Fos activation provides a good overall picture ofthe neuronal activation during stimulation of a pain-sensitiveintracranial structure. The data show that there are robustincreases of Fos in trigeminal neurons after stimulation ofthe SSS which are markedly reduced by pretreatment withDHE. These data suggest that the effect involves a largeproportion of the caudal trigeminal neurons and is robust inthe sense that the data are mirrored by what is seen observingcell firing. The electrophysiological data supports the Fosdata in that cells studied had a marked reduction in probabilityof firing after DHE and demonstrated that this effect took-30 min to reach its maximum.These studies rely, in the first instance, on the validity ofthe model of SSS stimulation for their interpretation andsignificance. It is clear in humans that mechanical or electricalstimulation of the SSS  in vivo  is painful (Ray and  Wolff, 1940). Furthermore, accidental stimulation of the trigeminalganglion during thermocoagulation for trigeminal neuralgia,when anaesthesia is not adequate, is extremely painful andwell remembered by the few patients who experience thepain. Moreover, that pain from sagittal sinus stimulation isreferred to the ophthalmic division of the trigeminal nerveby patients. There is good evidence in migraine that there isactivation of the trigeminovascular system since theneuropeptide marker for that innervation, calcitonin gene-related peptide, is released during migraine (Goadsby  et al., 1990). In the cat, SSS stimulation leads to similar changesin neuropeptides (Zagami  et al.,  1990). The sinus model,therefore, acts as an excellent method by which to examineaspects of the trigeminovacular system that cannot be studiedin humans. The method of stimulation used in these studies   b  y g u e  s  t   onA u g u s  t  2  ,2  0 1  6 h  t   t   p :  /   /   b r  a i  n . oxf   or  d  j   o ur n a l   s  . or  g /  D o wnl   o a  d  e  d f  r  om   DHE inhibits trigeminal nucleus activity  253Electrical stimulated SSSElectrical stimulationpre-treated with DHEFig.  1  Individual Fos-positive cells (closed circles) from 10 40-|i.m sections of the caudal medulla (SpV), first (Q), second (C2) and third(C 3 ) cervical spinal cord segments of the cat. Data are presented for the control animals in which the superior sagittal sinus (SSS) wasstimulated and for animals also stimulated but treated with dihydroergotamine (DHE). The plots show a marked  reduction  n Fos-positivecells after treatment with intravenous dihydroergotamine. INT = intermediate grey matter; I/IIo = laminae I and no of the dorsal hom;cNTS = medial nucleus of the solitary tract; VH = ventral hom; X = lamina X; Gr = gracile nucleus; Cu = cuneate nucleus; LCN =lateral cervical nucleus; i = ophthalmic, ii = maxillary and iii = mandibular divisions of trigeminal nucleus.is very important. By stimulation of the sinus with a hookelectrode, the trigeminal nerves innervating the sinus aredirectly activated. This eliminates the nerve/vessel interfaceand in so doing the peripheral 5HTi D  receptor that mediatesplasma extravasation (Moskowitz and Cutrer, 1993). Theremaining site for the action of any drug, and in this studyDHE, must be within the trigeminal nucleus at the secondorder synapse. The only intervening structures are the axonsof passage and the cells bodies of the trigeminal ganglionboth of which are not known to have functional 5HT 1D receptors. The next available site for the drug is the trigeminalnucleus which is the structure examined by both the Fos andelectrical studies.Given that the model as it has been applied essentiallyexamines central trigeminal behaviour, at least for themeasurements that have been made, the data suggest thatDHE acts in the brain. There is good evidence  in vitro  thatDHE binds with very high affinity to both a-adrenoceptorsand to 5HT r like receptors in the rat brain (Hamblin  et al., 1987).  The question of whether peripherally administeredDHE or ergotamine enters the CNS in humans  in vivo  is notas clear. Neither intravenous DHE (Kanto  et al.,  1981)nor orally or rectally (Hovdal  et al.,  1982), administeredergotamine can be detected in the CSF although Ala-Humla et al.  (1979) could detect small quantities of ergotamine inthe CSF after a 2 mg oral dose. Methodological consider-   b  y g u e  s  t   onA u g u s  t  2  ,2  0 1  6 h  t   t   p :  /   /   b r  a i  n . oxf   or  d  j   o ur n a l   s  . or  g /  D o wnl   o a  d  e  d f  r  om 
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