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Capsaicin-sensitive mechanisms are involved in cortical spreading depression-associated cerebral blood flow changes in rats
Neuroscience Letters 292 (2000) 17±20
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Capsaicin-sensitive mechanisms are involved in cortical spreading depression-associated cerebral blood ¯ow changes in rats F. Bari*, D. Paprika, G. JancsoÂ, F. Domoki Department of Physiology, Faculty of Medicine, University of Szeged, Szeged, H-6720, DoÂm teÂr 10, Hungary Received 30 June 2000; received in revised form 4 August 2000; accepted 4 August 2000
Abstract We tested the hypothesis that capsaicin-sensitive mechanisms play a role in the cortical spreading depression (CSD)related changes in cortical blood ¯ow (CBF). CBF was measured with laser Doppler ¯owmetry in anesthetized rats. The animals were treated with capsaicin before (48 h±2 weeks) or during the experiments. This agent is thought to stimulate small-diameter sensory nerve ®bers selectively and to deplete stored peptides. In the vehicle-treated group (n 8), the peak value of the CSD-associated hyperperfusion was 257 ^ 12% above the baseline (mean ^ SEM, P , 0:05). In the groups treated with 20 and 40 mg/kg or 20 mg/kg capsaicin, there were only small decreases in CBF. In the groups treated with 100 mg/kg capsaicin, the CSD-associated hyperemia was reduced at 48 h (158 ^ 7%, P , 0:05). However, at 96 h a transient hyperresponsiveness (390 ^ 38%, P , 0:05) was observed, which had disappeared by 2 weeks. These results indicate that the manipulation of sensory neuropeptide stores results in a biphasic effect on CSD-induced CBF responses. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Capsaicin; Cortical spreading depression; Laser Doppler; Rat; Trigeminal nerve
Cortical spreading depression (CSD), propagating at a rate of 2±3 mm/min across the cortical surface, is a transient perturbation of the cortical neuronal activity and cortical blood ¯ow (CBF). CSD, which involves neuronal depolarization, the release of neurotransmitters and changes in gene expression, has long been regarded as closely associated with migraine headache with aura [15]. Although several vasoactive substances of various origins are thought to be implicated in CSD-related transient changes in CBF, the mechanism has never been satisfactorily elucidated. Available experimental evidence suggests that, besides the known parenchymal metabolites such as K 1, adenosine, nitric oxide (NO) and glutamate, vasoactive peptides also in¯uence the cerebrovascular tone following CSD. Peptide-containing nerve ®bers have been identi®ed that are closely related to large blood vessels in the dura mater and pia mater [3]. A high proportion of these ®bers is sensory and arises mainly from the ophthalmic (nasociliary) division of the trigeminal nerve. Recent data indicate that chronic transection of the nasociliary nerve results in a signi®cant reduction of CBF during CSD [18]. The mode and speci®city of activation of the trigeminal system during * Corresponding author. Fax: 136-62-545842. E-mail address: [email protected] (F. Bari).
CSD are still debated [9,14]. Studies on the trigeminal system have revealed that neuropeptides, including calcitonin gene-related peptide (CGRP), are involved in various cerebrovascular functions. CGRP release contributes to cerebral vasodilation during hypotension and electrical stimulation of the trigeminal nerve [2]. CGRP has also been shown to mediate the capsaicin-induced relaxation of the cerebral blood vessels [11]. CSD associated as well as post-occlusive hyperemia and the elevation of cerebral blood ¯ow observed during seizures or after endotoxin exposure are, at least in part, mediated through CGRP receptors [2,4,20]. Capsaicin, a pungent algesic substance, has complex effects on a subpopulation of primary sensory neurons. It has been widely demonstrated that the systemic injection of capsaicin results in stimulation of these nerves, followed by depletion of their peptide content. Capsaicin may also induce neurodegeneration and a permanent loss of certain sensory functions [10]. The contribution of the trigeminal sensory system, with special emphasis on the capsaicin sensitivity of CBF regulation, has long been investigated [8,16]. The effects of acute and chronic capsaicin treatments on the trigeminal nerve function seem controversial and require further examination [17]. The present study was
0304-3940/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 0) 01 42 4- 5
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F. Bari et al. / Neuroscience Letters 292 (2000) 17±20
carried out to determine whether capsaicin-sensitive sensory nerve ®bers signi®cantly affect CSD-related CBF responses. All experimental procedures complied with the NIH guidelines and were approved by the Local Committee for Animal Care and Use. Adult male Wistar rats were used. The animals (n 54) were anesthetized with pentobarbital (45 mg/kg) and catheters were inserted into the right femoral artery and vein. The trachea was intubated and the animals breathed freely with a 70% air±30% oxygen mixture. Rectal temperature was kept near 378C with a heating pad. The animal's head was ®xed in a stereotaxic frame, the calvaria was exposed and four circular craniotomy holes (1±2 mm in diameter) were drilled under aqueous cooling. The holes on both sides were positioned about 2 mm laterally and 1 mm anteriorly, and 3 mm laterally and 3 mm posteriorly from the bregma. Care was taken to leave the dura mater intact. The cortical blood perfusion was monitored with the needle probes of a laser Doppler ¯owmeter (Peri¯ux 4001, Perimed, Sweden) [1] through the posterior holes. CSD was induced by the injection of 0.5 ml 1 M KCl into the anterior craniotomy holes, using a micropipette connected to a Hamilton syringe. Cortical direct current (DC) potential was measured with Ag±AgCl electrodes connected to a high-impedance electrometer (Experimetria, UK). One recording electrode was positioned 1 mm laterally to the ¯ow probe; the other was inserted under the dorsal part of the scalp. The blood pressure, the laser Doppler signal and the DC potential were stored (sample interval 1 s) on a PC for detailed analysis. Group 1 animals were treated with the solvent of the capsaicin solution (1 ml, s.c.) 2±4 days before the experiments and served as controls (n 8). Six consecutive CSDs were evoked on both hemispheres. The time lags between the CSDs were 20 min. In Group 2 (n 10), the protocol was same as for the time-control studies, except that after the second CSD the animals were treated with capsaicin (20 mg/kg followed by 40 mg/kg, i.v., with a time lag of 30 min). Following a recovery period of 15 min after the capsaicin injections, CSDs were initiated. In Group 3 (n 8), a low dose of capsaicin (20 mg/kg, single dose, s.c.) was given. The CSD-related vascular responses were tested 48 or 96 h after capsaicin treatment (n 4 in both subgroups). Animals in Group 4 (n 16) were treated with 100 mg/kg capsaicin (single dose, s.c.), and three subgroups were formed (n 5, 5 and 6, respectively). CSD-related CBF changes were tested at 48, 96 h or 2 weeks after the capsaicin treatment. In Group 5 (n 6), the effect of high-dose capsaicin (total 300 mg/kg; in daily fractions of 20, 50, 100 and 130 mg/kg, s.c.) on the CSDrelated vascular responses was examined. In Group 6 (n 6), the hypothesis that stimulation of the trigeminal (Gasserian) ganglion (GG) results in a change in CSDrelated CBF responses was tested. The GG was stimulated with a concentric bipolar electrode (3.5 mm posteriorly from the bregma, 3 mm laterally and 9.5 mm deep). The electrical stimulation parameters were square wave pulses at 5 Hz, with a duration of 5 ms at 1 mA for 10 min. These
parameters have been shown to stimulate CGRP-containing perivascular trigeminal nerves [13]. Euthanasia was performed at the end of the experiment with an overdose of sodium pentobarbitone (250±300 mg/kg, i.v.). A stock solution of capsaicin (Biogal, Hungary) (2% (64 mmol/l)) dissolved in isotonic saline with the aid of ethanol (10%) and Tween 80 (10%) was further diluted with isotonic saline just prior to the experiments. Since CBF was measured in arbitrary units, the laser Doppler data were converted to the percentage change from the baseline level for statistical analysis. Baseline was de®ned as mean of CBF over a period of 60 s immediately before CSD was elicited. Data are expressed as means ^ SEM. All statistical analyses were done using the SigmaStat software package (version 1.0, Jandel Co, USA). Changes were analyzed by using repeated measures analysis of variance (ANOVA) (for Groups 1, 2 and 6), followed by pairwise comparisons with the Student± Newman±Keuls test where appropriate. One-way ANOVA on ranks was used with Dunn's post-hoc test to compare data of Groups, 3, 4 and 5. A value of P , 0:05 was considered to be statistically signi®cant. General physiological variables (arterial blood pressure (MABP), arterial blood gases and pH) were within the normal ranges and did not alter signi®cantly throughout the experiments. For example, in Group 1 values for MABP, pCO2 and pH were at the beginning and at the end of the experiments 130 ^ 3 vs. 116 ^ 10 mmHg, 38 ^ 3 vs. 41 ^ 4 mmHg, and 7.42 ^ 0.05 vs. 7.38 ^ 0.05, respectively. Application of KCl to the frontal cortical surface induced a de¯ection in DC potential and an elevation in CBF. In the control group, we analyzed six consecutive CSDs with a time lag of 20 min between each pair. The peak of the ipsilateral CSD±associated hyperperfusion was 258 ^ 12% above the baseline (n 48, P , 0:05). It is important to note that the average of the ®rst CSDs was 206 ^ 15% (n 8, P , 0:05), i.e. signi®cantly smaller that any of the following ones. However, there were no statistically significant differences in the amplitude between the 2nd and 6th hyperemic responses (261 ^ 30%, 277 ^ 35%, 242 ^ 35%, 241 ^ 17% and 229 ^ 21%, respectively). Acute administration of capsaicin had a transient, triphasic blood pressure response (lasting 3±5 min). There were no signi®cant differences in MABP when CSDs were initiated before or after 20 or 40 mg/kg capsaicin (113 ^ 4 and 110 ^ 5 mmHg vs. 112 ^ 4 and 106 ^ 6 mmHg, respectively). The i.v. administration of capsaicin resulted in a slight but not statistically signi®cant elevation of resting CBF (116 ^ 15% and 112 ^ 16% following 20 and 40 mg/kg, respectively). However, the capsaicin administration attenuated the CSD-related hyperemia (Fig. 1). In Groups 3±5 baseline MABP and CBF were unaffected by preceding capsaicin administration. In Group 3, capsaicin had no effect on CSD-induced hyperperfusion measured 48 h
F. Bari et al. / Neuroscience Letters 292 (2000) 17±20
Fig. 1. Effects of acute capsaicin treatment on CSD-related hyperemia. Bars represent peaks of the hyperemic response expressed as percentage change from the baseline. The effects were evaluated under control conditions (C1 and C2) and 15 min after systemic capsaicin applications (20 and 40 mg/kg, i.v.). Data are expressed as means ^ SEM. (n 10), *P , 0:05 as compared with the control.
following the treatment (226 ^ 15%, P . 0:05), whereas the responses were attenuated at 96 h (175 ^ 14%, P , 0:05). In Group 4, the hyperemic effect of CSD was reduced 48 h after capsaicin treatment. In contrast, the responses were highly elevated on day 4 after capsaicin treatment. Two weeks after the administration of 100 mg/kg capsaicin, the CSD-related CBF responses were similar to those observed at 48 h (Fig. 2). In Group 5, the CSD-related hyperemic responses were tested on day 4 following the end of treatment. We observed only a small decrease in the peak ¯ow during CSD hyperemia (237 ^ 21%, P . 0.05). In Group 6, stimulation of the GG resulted in small elevations in MABP (from 110 ^ 5 to 124 ^ 8 mmHg) and in CBF (by 31 ^ 7%, P , 0:05). During stimulation, CSDdependent hyperemia could also be observed (132 ^ 8%, n 6, P , 0:05). In the post-stimulation period, the hyperemic responses were suppressed (168 ^ 17% and 215 ^ 21%, after 20 and 40 min, respectively). The CSDrelated responses returned to normal within 1 h. The present study provides evidence that both the stimulation of capsaicin-sensitive nerve ®bers and the depletion
Fig. 2. Effect of chronic capsaicin administration on CSD-related hyperemia. Bars represent peaks of the hyperemic response expressed as percentage change from the baseline. The effects were evaluated 48, 96 h and 2 weeks after systemic capsaicin application (100 mg/kg, s.c.). The total numbers of hyperemic responses compared are 18, 20, 16 and 19 for the control group, for the 48, 96 h and 2 weeks subgroups, respectively. Data are expressed as means ^ SEM. *P , 0:05 as compared with the control, vehicle-treated group.
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of their peptide content modulate CSD-related cerebrovascular responses. These observations extend earlier ®ndings indicative of the modulator role of capsaicin-sensitive nerves in the regulation of CBF in various conditions [8,11,16]. Further, chemical depletion of sensory neuropeptides resulted in alterations of CSD-associated hyperemia similar to that observed following chronic transection of the ophthalmic division of the trigeminal nerve [18]. These ®ndings indicate the involvement of sensory nerves in the cerebrovascular responses associated with CSD. The trigeminal system has long been regarded as the principal pathway for the transmission of sensory signals from the surroundings of the cerebral vasculature. However, the chemical nature of the endogenous substance(s), which may stimulate of these nociceptive nerve ®bers is still unclear. During CSD, the concentrations of metabolites and ions in the extra- and intracellular space change dramatically [15]. It is assumed that elevated extracellular potassium concentration may cause depolarization of perivascular nerve ®bers and release neuropeptides including CGRP from the peripheral nerve terminals [12]. The mechanism of sensory activation during CSD is poorly explored. The ®nding that CSD causes an increase in c-fos expression in the trigeminal nucleus caudalis supports the theory that nociceptors have been activated [9]. Lambert and coworkers [14] have shown that a certain population of trigeminocervical sensory neurons remains insensitive when CSD is initiated. There is ample evidence that activation of trigeminal C-®bers results not only in the transmission of afferent impulses to the central nervous system, but also in the liberation of neuropeptides from stimulated nerve terminals. Electrical stimulation of C-nociceptors resulted in signi®cant vasodilation at very low frequencies or by single impulses, therefore it is proposed that vascular action could be activated without producing any sensory effects [7]. Hence, capsaicin-sensitive mechanisms have been shown to mediate axon re¯ex-like plasma extravasation in the dura mater [5]. An interesting ®nding of our study is the development of an apparent supersensitivity to the stimulus producing CSD and cortical hyperemia following capsaicin treatment. The mechanism of this increased hyperemic reaction is unclear. Systemic administration of capsaicin results in a long-lasting impairment or complete loss of the function of chemosensitive afferent ®bers, at least in part, due to degeneration of sensory neurons [10]. This involves the elimination of a population of peptidergic nerve ®bers which, by the release of CGRP, play a signi®cant role in cerebral vasodilation through an action on CGRP receptors [6]. In addition, recent ®ndings suggest the existence and vasodilator function of capsaicin-insensitive, CGRP containing trigeminal nerve ®bers [17]. We suggest that an interaction between the capsaicin-sensitive and capsaicin-insensitive nerves may explain the temporal changes in the magnitude of the CSD-related hyperemic responses. Hypersensitivity to transmitter substances of target tissues innervated by auto-
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F. Bari et al. / Neuroscience Letters 292 (2000) 17±20
nomic or motor nerves is well established following chronic denervation. It is conceivable that similar hypersensitivity of vascular peptide, i.e. CGRP receptors may develop following the elimination of capsaicin-sensitive perivascular afferent nerves. Previous ®ndings support this assumption by showing that an initial decrease in CGRPimmunoreactivity in the GG after capsaicin treatment was followed by an increase 48 h later [19]. Electrical stimulation of the GG resulted in characteristic changes of the CSD-related vascular responses. The observation that we were able to initiate CSD during stimulation is in accordance with the hypothesis that the trigeminal system is only one component in¯uencing the hyperemic responses. The decrease in CSD-related vasodilation could be explained by a diminished availability of CGRP or related peptides. Electrical stimulation of the GG with parameters similar to that used in the present study resulted in immunohystochemical alterations indicative of peptide release and decreased CGRP content [13] which may modify the vascular responses during CSD. It is worth mentioning that after acute transection of peripheral axons the local effector function of the sensory nerves remains intact for 1±2 days [7]. Several reports have shown altered cerebrovascular responses following cortical depolarization, which develops rapidly under hypoxic or ischemic conditions, in the course of CSD or after head trauma. Our ®ndings suggest that neurogenic release of vasoactive peptides may signi®cantly contribute to the modulation of altered cerebrovascular responses under various physiological and pathophysiological conditions. This study was supported by grants from OTKA (T026295) and ETT (50/2000). [1] Bari, F., Horvath, G. and Benedek, G., Dexmedetomidineinduced decrease in cerebral blood ¯ow is attenuated by verapamil in rats: a laser Doppler study, Can. J. Anaesth., 40 (1993) 748±754. [2] Brian Jr, J.E., Faraci, F.M. and Heistad, D.D., Recent insights into the regulation of cerebral circulation, Clin. Exp. Pharmacol. Physiol., 23 (1996) 449±457. [3] Busija, D.W., Nervous control of cerebral circulation, In T. Bennett and S.M. Gardiner (Eds.), Nervous Control of Blood Vessels, Overseas PA, Amsterdam, 1996, pp. 177±206. [4] Colonna, D.M., Meng, W., Deal, D.D. and Busija, D.W., Calcitonin gene-related peptide promotes cerebrovascular dilation during cortical spreading depression in rabbits, Am. J. Physiol., 266 (1994) H1095±H1102. [5] Delepine, L. and Aubineau, P., Plasma protein extravasation induced in the rat dura mater by stimulation of the parasympathetic sphenopalatine ganglion, Exp. Neurol., 147 (1997) 389±400.
[6] Edvinsson, L., Mulder, H., Goadsby, P.J. and Uddman, R., Calcitonin gene-related peptide and nitric oxide in the trigeminal ganglion: cerebral vasodilatation from trigeminal nerve stimulation involves mainly calcitonin generelated peptide, J. Auton. Nerv. Syst., 70 (1998) 15±22. [7] Holzer, P., Peptidergic sensory neurons in the control of vascular functions: mechanisms and signi®cance in the cutaneous and splanchnic vascular beds, Rev. Physiol. Biochem. Pharmacol., 121 (1992) 49±146. [8] Hong, K.W., Pyo, K.M., Lee, W.S., Yu, S.S. and Rhim, B.Y., Pharmacological evidence that calcitonin gene-related peptide is implicated in cerebral autoregulation, Am. J. Physiol., 266 (1994) H11±H16. [9] Ingvardsen, B.K., Laursen, H., Olsen, U.B. and Hansen, A.J., Possible mechanism of c-fos expression in trigeminal nucleus caudalis following cortical spreading depression, Pain, 72 (1997) 407±415. [10] JancsoÂ, G., Pathobiological reactions of C-®bre primary sensory neurones to peripheral nerve injury, Exp. Physiol., 77 (1992) 405±431. [11] Jansen, I., Alafaci, C., Uddman, R. and Edvinsson, L., Evidence that calcitonin gene-related peptide contributes to the capsaicin-induced relaxation of guinea pig cerebral arteries, Regul. Pept., 31 (1990) 167±178. [12] Kallner, G. and Franco-Cereceda, A., Ion channels involved in the release of calcitonin gene-related peptide by low pH, prostacyclin and capsaicin in the isolated guinea-pig heart, Eur. J. Pharmacol., 352 (1998) 223±228. [13] Knyihar-Csillik, E., Tajti, J., Samsam, M., Sary, G., Buzas, P. and Vecsei, L., Depletion of calcitonin gene-related peptide from the caudal trigeminal nucleus of the rat after electrical stimulation of the Gasserian ganglion, Exp. Brain Res., 118 (1998) 111±114. [14] Lambert, G.A., Michalicek, J., Storer, R.J. and Zagami, A.S., Effect of cortical spreading depression on activity of trigeminovascular sensory neurons, Cephalalgia, 19 (1999) 631± 638. [15] Lauritzen, M., Pathophysiology of the migraine aura. The spreading depression theory, Brain, 117 (1994) 199±210. [16] Moskowitz, M.A., Macfarlane, R., Tasdemiroglu, E., Wei, E.P. and Kontos, H.A., Neuroeffector functions of sensory nerve ®bers in the cerebral circulation after global cerebral ischemia, Arzneimittelforschung, 41 (1991) 315±318. [17] Peitl, B., Petho Í , G., PorszaÂsz, R., NeÂmeth, J. and SzolcsaÂnyi, J., Capsaicin-insensitive sensory-efferent meningeal vasodilatation evoked by electrical stimulation of trigeminal nerve ®bres in the rat, Br. J. Pharmacol., 127 (1999) 457±467. [18] Reuter, U., Weber, J.R., Gold, L., Arnold, G., Wolf, T., Dreier, J., Lindauer, U. and Dirnagl, U., Perivascular nerves contribute to cortical spreading depression-associated hyperemia in rats, Am. J. Physiol., 274 (1998) H1979±H1987. [19] Spears, R., Hutchins, B. and Hinton, R.J., Capsaicin application to the temporomandibular joint alters calcitonin generelated peptide levels in the trigeminal ganglion of the rat, J. Orofac. Pain, 12 (1998) 108±115. [20] Wahl, M., Schilling, L., Parsons, A.A. and Kaumann, A., Involvement of calcitonin gene-related peptide (CGRP) and nitric oxide (NO) in the pial artery dilatation elicited by cortical spreading depression, Brain Res., 637 (1994) 204±210.
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Capsaicin-sensitive mechanisms are involved in cortical spreading depression-associated cerebral blood ¯ow changes in rats F. Bari*, D. Paprika, G. JancsoÂ, F. Domoki Department of Physiology, Faculty of Medicine, University of Szeged, Szeged, H-6720, DoÂm teÂr 10, Hungary Received 30 June 2000; received in revised form 4 August 2000; accepted 4 August 2000
Abstract We tested the hypothesis that capsaicin-sensitive mechanisms play a role in the cortical spreading depression (CSD)related changes in cortical blood ¯ow (CBF). CBF was measured with laser Doppler ¯owmetry in anesthetized rats. The animals were treated with capsaicin before (48 h±2 weeks) or during the experiments. This agent is thought to stimulate small-diameter sensory nerve ®bers selectively and to deplete stored peptides. In the vehicle-treated group (n 8), the peak value of the CSD-associated hyperperfusion was 257 ^ 12% above the baseline (mean ^ SEM, P , 0:05). In the groups treated with 20 and 40 mg/kg or 20 mg/kg capsaicin, there were only small decreases in CBF. In the groups treated with 100 mg/kg capsaicin, the CSD-associated hyperemia was reduced at 48 h (158 ^ 7%, P , 0:05). However, at 96 h a transient hyperresponsiveness (390 ^ 38%, P , 0:05) was observed, which had disappeared by 2 weeks. These results indicate that the manipulation of sensory neuropeptide stores results in a biphasic effect on CSD-induced CBF responses. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Capsaicin; Cortical spreading depression; Laser Doppler; Rat; Trigeminal nerve
Cortical spreading depression (CSD), propagating at a rate of 2±3 mm/min across the cortical surface, is a transient perturbation of the cortical neuronal activity and cortical blood ¯ow (CBF). CSD, which involves neuronal depolarization, the release of neurotransmitters and changes in gene expression, has long been regarded as closely associated with migraine headache with aura [15]. Although several vasoactive substances of various origins are thought to be implicated in CSD-related transient changes in CBF, the mechanism has never been satisfactorily elucidated. Available experimental evidence suggests that, besides the known parenchymal metabolites such as K 1, adenosine, nitric oxide (NO) and glutamate, vasoactive peptides also in¯uence the cerebrovascular tone following CSD. Peptide-containing nerve ®bers have been identi®ed that are closely related to large blood vessels in the dura mater and pia mater [3]. A high proportion of these ®bers is sensory and arises mainly from the ophthalmic (nasociliary) division of the trigeminal nerve. Recent data indicate that chronic transection of the nasociliary nerve results in a signi®cant reduction of CBF during CSD [18]. The mode and speci®city of activation of the trigeminal system during * Corresponding author. Fax: 136-62-545842. E-mail address: [email protected] (F. Bari).
CSD are still debated [9,14]. Studies on the trigeminal system have revealed that neuropeptides, including calcitonin gene-related peptide (CGRP), are involved in various cerebrovascular functions. CGRP release contributes to cerebral vasodilation during hypotension and electrical stimulation of the trigeminal nerve [2]. CGRP has also been shown to mediate the capsaicin-induced relaxation of the cerebral blood vessels [11]. CSD associated as well as post-occlusive hyperemia and the elevation of cerebral blood ¯ow observed during seizures or after endotoxin exposure are, at least in part, mediated through CGRP receptors [2,4,20]. Capsaicin, a pungent algesic substance, has complex effects on a subpopulation of primary sensory neurons. It has been widely demonstrated that the systemic injection of capsaicin results in stimulation of these nerves, followed by depletion of their peptide content. Capsaicin may also induce neurodegeneration and a permanent loss of certain sensory functions [10]. The contribution of the trigeminal sensory system, with special emphasis on the capsaicin sensitivity of CBF regulation, has long been investigated [8,16]. The effects of acute and chronic capsaicin treatments on the trigeminal nerve function seem controversial and require further examination [17]. The present study was
0304-3940/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 0) 01 42 4- 5
18
F. Bari et al. / Neuroscience Letters 292 (2000) 17±20
carried out to determine whether capsaicin-sensitive sensory nerve ®bers signi®cantly affect CSD-related CBF responses. All experimental procedures complied with the NIH guidelines and were approved by the Local Committee for Animal Care and Use. Adult male Wistar rats were used. The animals (n 54) were anesthetized with pentobarbital (45 mg/kg) and catheters were inserted into the right femoral artery and vein. The trachea was intubated and the animals breathed freely with a 70% air±30% oxygen mixture. Rectal temperature was kept near 378C with a heating pad. The animal's head was ®xed in a stereotaxic frame, the calvaria was exposed and four circular craniotomy holes (1±2 mm in diameter) were drilled under aqueous cooling. The holes on both sides were positioned about 2 mm laterally and 1 mm anteriorly, and 3 mm laterally and 3 mm posteriorly from the bregma. Care was taken to leave the dura mater intact. The cortical blood perfusion was monitored with the needle probes of a laser Doppler ¯owmeter (Peri¯ux 4001, Perimed, Sweden) [1] through the posterior holes. CSD was induced by the injection of 0.5 ml 1 M KCl into the anterior craniotomy holes, using a micropipette connected to a Hamilton syringe. Cortical direct current (DC) potential was measured with Ag±AgCl electrodes connected to a high-impedance electrometer (Experimetria, UK). One recording electrode was positioned 1 mm laterally to the ¯ow probe; the other was inserted under the dorsal part of the scalp. The blood pressure, the laser Doppler signal and the DC potential were stored (sample interval 1 s) on a PC for detailed analysis. Group 1 animals were treated with the solvent of the capsaicin solution (1 ml, s.c.) 2±4 days before the experiments and served as controls (n 8). Six consecutive CSDs were evoked on both hemispheres. The time lags between the CSDs were 20 min. In Group 2 (n 10), the protocol was same as for the time-control studies, except that after the second CSD the animals were treated with capsaicin (20 mg/kg followed by 40 mg/kg, i.v., with a time lag of 30 min). Following a recovery period of 15 min after the capsaicin injections, CSDs were initiated. In Group 3 (n 8), a low dose of capsaicin (20 mg/kg, single dose, s.c.) was given. The CSD-related vascular responses were tested 48 or 96 h after capsaicin treatment (n 4 in both subgroups). Animals in Group 4 (n 16) were treated with 100 mg/kg capsaicin (single dose, s.c.), and three subgroups were formed (n 5, 5 and 6, respectively). CSD-related CBF changes were tested at 48, 96 h or 2 weeks after the capsaicin treatment. In Group 5 (n 6), the effect of high-dose capsaicin (total 300 mg/kg; in daily fractions of 20, 50, 100 and 130 mg/kg, s.c.) on the CSDrelated vascular responses was examined. In Group 6 (n 6), the hypothesis that stimulation of the trigeminal (Gasserian) ganglion (GG) results in a change in CSDrelated CBF responses was tested. The GG was stimulated with a concentric bipolar electrode (3.5 mm posteriorly from the bregma, 3 mm laterally and 9.5 mm deep). The electrical stimulation parameters were square wave pulses at 5 Hz, with a duration of 5 ms at 1 mA for 10 min. These
parameters have been shown to stimulate CGRP-containing perivascular trigeminal nerves [13]. Euthanasia was performed at the end of the experiment with an overdose of sodium pentobarbitone (250±300 mg/kg, i.v.). A stock solution of capsaicin (Biogal, Hungary) (2% (64 mmol/l)) dissolved in isotonic saline with the aid of ethanol (10%) and Tween 80 (10%) was further diluted with isotonic saline just prior to the experiments. Since CBF was measured in arbitrary units, the laser Doppler data were converted to the percentage change from the baseline level for statistical analysis. Baseline was de®ned as mean of CBF over a period of 60 s immediately before CSD was elicited. Data are expressed as means ^ SEM. All statistical analyses were done using the SigmaStat software package (version 1.0, Jandel Co, USA). Changes were analyzed by using repeated measures analysis of variance (ANOVA) (for Groups 1, 2 and 6), followed by pairwise comparisons with the Student± Newman±Keuls test where appropriate. One-way ANOVA on ranks was used with Dunn's post-hoc test to compare data of Groups, 3, 4 and 5. A value of P , 0:05 was considered to be statistically signi®cant. General physiological variables (arterial blood pressure (MABP), arterial blood gases and pH) were within the normal ranges and did not alter signi®cantly throughout the experiments. For example, in Group 1 values for MABP, pCO2 and pH were at the beginning and at the end of the experiments 130 ^ 3 vs. 116 ^ 10 mmHg, 38 ^ 3 vs. 41 ^ 4 mmHg, and 7.42 ^ 0.05 vs. 7.38 ^ 0.05, respectively. Application of KCl to the frontal cortical surface induced a de¯ection in DC potential and an elevation in CBF. In the control group, we analyzed six consecutive CSDs with a time lag of 20 min between each pair. The peak of the ipsilateral CSD±associated hyperperfusion was 258 ^ 12% above the baseline (n 48, P , 0:05). It is important to note that the average of the ®rst CSDs was 206 ^ 15% (n 8, P , 0:05), i.e. signi®cantly smaller that any of the following ones. However, there were no statistically significant differences in the amplitude between the 2nd and 6th hyperemic responses (261 ^ 30%, 277 ^ 35%, 242 ^ 35%, 241 ^ 17% and 229 ^ 21%, respectively). Acute administration of capsaicin had a transient, triphasic blood pressure response (lasting 3±5 min). There were no signi®cant differences in MABP when CSDs were initiated before or after 20 or 40 mg/kg capsaicin (113 ^ 4 and 110 ^ 5 mmHg vs. 112 ^ 4 and 106 ^ 6 mmHg, respectively). The i.v. administration of capsaicin resulted in a slight but not statistically signi®cant elevation of resting CBF (116 ^ 15% and 112 ^ 16% following 20 and 40 mg/kg, respectively). However, the capsaicin administration attenuated the CSD-related hyperemia (Fig. 1). In Groups 3±5 baseline MABP and CBF were unaffected by preceding capsaicin administration. In Group 3, capsaicin had no effect on CSD-induced hyperperfusion measured 48 h
F. Bari et al. / Neuroscience Letters 292 (2000) 17±20
Fig. 1. Effects of acute capsaicin treatment on CSD-related hyperemia. Bars represent peaks of the hyperemic response expressed as percentage change from the baseline. The effects were evaluated under control conditions (C1 and C2) and 15 min after systemic capsaicin applications (20 and 40 mg/kg, i.v.). Data are expressed as means ^ SEM. (n 10), *P , 0:05 as compared with the control.
following the treatment (226 ^ 15%, P . 0:05), whereas the responses were attenuated at 96 h (175 ^ 14%, P , 0:05). In Group 4, the hyperemic effect of CSD was reduced 48 h after capsaicin treatment. In contrast, the responses were highly elevated on day 4 after capsaicin treatment. Two weeks after the administration of 100 mg/kg capsaicin, the CSD-related CBF responses were similar to those observed at 48 h (Fig. 2). In Group 5, the CSD-related hyperemic responses were tested on day 4 following the end of treatment. We observed only a small decrease in the peak ¯ow during CSD hyperemia (237 ^ 21%, P . 0.05). In Group 6, stimulation of the GG resulted in small elevations in MABP (from 110 ^ 5 to 124 ^ 8 mmHg) and in CBF (by 31 ^ 7%, P , 0:05). During stimulation, CSDdependent hyperemia could also be observed (132 ^ 8%, n 6, P , 0:05). In the post-stimulation period, the hyperemic responses were suppressed (168 ^ 17% and 215 ^ 21%, after 20 and 40 min, respectively). The CSDrelated responses returned to normal within 1 h. The present study provides evidence that both the stimulation of capsaicin-sensitive nerve ®bers and the depletion
Fig. 2. Effect of chronic capsaicin administration on CSD-related hyperemia. Bars represent peaks of the hyperemic response expressed as percentage change from the baseline. The effects were evaluated 48, 96 h and 2 weeks after systemic capsaicin application (100 mg/kg, s.c.). The total numbers of hyperemic responses compared are 18, 20, 16 and 19 for the control group, for the 48, 96 h and 2 weeks subgroups, respectively. Data are expressed as means ^ SEM. *P , 0:05 as compared with the control, vehicle-treated group.
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of their peptide content modulate CSD-related cerebrovascular responses. These observations extend earlier ®ndings indicative of the modulator role of capsaicin-sensitive nerves in the regulation of CBF in various conditions [8,11,16]. Further, chemical depletion of sensory neuropeptides resulted in alterations of CSD-associated hyperemia similar to that observed following chronic transection of the ophthalmic division of the trigeminal nerve [18]. These ®ndings indicate the involvement of sensory nerves in the cerebrovascular responses associated with CSD. The trigeminal system has long been regarded as the principal pathway for the transmission of sensory signals from the surroundings of the cerebral vasculature. However, the chemical nature of the endogenous substance(s), which may stimulate of these nociceptive nerve ®bers is still unclear. During CSD, the concentrations of metabolites and ions in the extra- and intracellular space change dramatically [15]. It is assumed that elevated extracellular potassium concentration may cause depolarization of perivascular nerve ®bers and release neuropeptides including CGRP from the peripheral nerve terminals [12]. The mechanism of sensory activation during CSD is poorly explored. The ®nding that CSD causes an increase in c-fos expression in the trigeminal nucleus caudalis supports the theory that nociceptors have been activated [9]. Lambert and coworkers [14] have shown that a certain population of trigeminocervical sensory neurons remains insensitive when CSD is initiated. There is ample evidence that activation of trigeminal C-®bers results not only in the transmission of afferent impulses to the central nervous system, but also in the liberation of neuropeptides from stimulated nerve terminals. Electrical stimulation of C-nociceptors resulted in signi®cant vasodilation at very low frequencies or by single impulses, therefore it is proposed that vascular action could be activated without producing any sensory effects [7]. Hence, capsaicin-sensitive mechanisms have been shown to mediate axon re¯ex-like plasma extravasation in the dura mater [5]. An interesting ®nding of our study is the development of an apparent supersensitivity to the stimulus producing CSD and cortical hyperemia following capsaicin treatment. The mechanism of this increased hyperemic reaction is unclear. Systemic administration of capsaicin results in a long-lasting impairment or complete loss of the function of chemosensitive afferent ®bers, at least in part, due to degeneration of sensory neurons [10]. This involves the elimination of a population of peptidergic nerve ®bers which, by the release of CGRP, play a signi®cant role in cerebral vasodilation through an action on CGRP receptors [6]. In addition, recent ®ndings suggest the existence and vasodilator function of capsaicin-insensitive, CGRP containing trigeminal nerve ®bers [17]. We suggest that an interaction between the capsaicin-sensitive and capsaicin-insensitive nerves may explain the temporal changes in the magnitude of the CSD-related hyperemic responses. Hypersensitivity to transmitter substances of target tissues innervated by auto-
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F. Bari et al. / Neuroscience Letters 292 (2000) 17±20
nomic or motor nerves is well established following chronic denervation. It is conceivable that similar hypersensitivity of vascular peptide, i.e. CGRP receptors may develop following the elimination of capsaicin-sensitive perivascular afferent nerves. Previous ®ndings support this assumption by showing that an initial decrease in CGRPimmunoreactivity in the GG after capsaicin treatment was followed by an increase 48 h later [19]. Electrical stimulation of the GG resulted in characteristic changes of the CSD-related vascular responses. The observation that we were able to initiate CSD during stimulation is in accordance with the hypothesis that the trigeminal system is only one component in¯uencing the hyperemic responses. The decrease in CSD-related vasodilation could be explained by a diminished availability of CGRP or related peptides. Electrical stimulation of the GG with parameters similar to that used in the present study resulted in immunohystochemical alterations indicative of peptide release and decreased CGRP content [13] which may modify the vascular responses during CSD. It is worth mentioning that after acute transection of peripheral axons the local effector function of the sensory nerves remains intact for 1±2 days [7]. Several reports have shown altered cerebrovascular responses following cortical depolarization, which develops rapidly under hypoxic or ischemic conditions, in the course of CSD or after head trauma. Our ®ndings suggest that neurogenic release of vasoactive peptides may signi®cantly contribute to the modulation of altered cerebrovascular responses under various physiological and pathophysiological conditions. This study was supported by grants from OTKA (T026295) and ETT (50/2000). [1] Bari, F., Horvath, G. and Benedek, G., Dexmedetomidineinduced decrease in cerebral blood ¯ow is attenuated by verapamil in rats: a laser Doppler study, Can. J. Anaesth., 40 (1993) 748±754. [2] Brian Jr, J.E., Faraci, F.M. and Heistad, D.D., Recent insights into the regulation of cerebral circulation, Clin. Exp. Pharmacol. Physiol., 23 (1996) 449±457. [3] Busija, D.W., Nervous control of cerebral circulation, In T. Bennett and S.M. Gardiner (Eds.), Nervous Control of Blood Vessels, Overseas PA, Amsterdam, 1996, pp. 177±206. [4] Colonna, D.M., Meng, W., Deal, D.D. and Busija, D.W., Calcitonin gene-related peptide promotes cerebrovascular dilation during cortical spreading depression in rabbits, Am. J. Physiol., 266 (1994) H1095±H1102. [5] Delepine, L. and Aubineau, P., Plasma protein extravasation induced in the rat dura mater by stimulation of the parasympathetic sphenopalatine ganglion, Exp. Neurol., 147 (1997) 389±400.
[6] Edvinsson, L., Mulder, H., Goadsby, P.J. and Uddman, R., Calcitonin gene-related peptide and nitric oxide in the trigeminal ganglion: cerebral vasodilatation from trigeminal nerve stimulation involves mainly calcitonin generelated peptide, J. Auton. Nerv. Syst., 70 (1998) 15±22. [7] Holzer, P., Peptidergic sensory neurons in the control of vascular functions: mechanisms and signi®cance in the cutaneous and splanchnic vascular beds, Rev. Physiol. Biochem. Pharmacol., 121 (1992) 49±146. [8] Hong, K.W., Pyo, K.M., Lee, W.S., Yu, S.S. and Rhim, B.Y., Pharmacological evidence that calcitonin gene-related peptide is implicated in cerebral autoregulation, Am. J. Physiol., 266 (1994) H11±H16. [9] Ingvardsen, B.K., Laursen, H., Olsen, U.B. and Hansen, A.J., Possible mechanism of c-fos expression in trigeminal nucleus caudalis following cortical spreading depression, Pain, 72 (1997) 407±415. [10] JancsoÂ, G., Pathobiological reactions of C-®bre primary sensory neurones to peripheral nerve injury, Exp. Physiol., 77 (1992) 405±431. [11] Jansen, I., Alafaci, C., Uddman, R. and Edvinsson, L., Evidence that calcitonin gene-related peptide contributes to the capsaicin-induced relaxation of guinea pig cerebral arteries, Regul. Pept., 31 (1990) 167±178. [12] Kallner, G. and Franco-Cereceda, A., Ion channels involved in the release of calcitonin gene-related peptide by low pH, prostacyclin and capsaicin in the isolated guinea-pig heart, Eur. J. Pharmacol., 352 (1998) 223±228. [13] Knyihar-Csillik, E., Tajti, J., Samsam, M., Sary, G., Buzas, P. and Vecsei, L., Depletion of calcitonin gene-related peptide from the caudal trigeminal nucleus of the rat after electrical stimulation of the Gasserian ganglion, Exp. Brain Res., 118 (1998) 111±114. [14] Lambert, G.A., Michalicek, J., Storer, R.J. and Zagami, A.S., Effect of cortical spreading depression on activity of trigeminovascular sensory neurons, Cephalalgia, 19 (1999) 631± 638. [15] Lauritzen, M., Pathophysiology of the migraine aura. The spreading depression theory, Brain, 117 (1994) 199±210. [16] Moskowitz, M.A., Macfarlane, R., Tasdemiroglu, E., Wei, E.P. and Kontos, H.A., Neuroeffector functions of sensory nerve ®bers in the cerebral circulation after global cerebral ischemia, Arzneimittelforschung, 41 (1991) 315±318. [17] Peitl, B., Petho Í , G., PorszaÂsz, R., NeÂmeth, J. and SzolcsaÂnyi, J., Capsaicin-insensitive sensory-efferent meningeal vasodilatation evoked by electrical stimulation of trigeminal nerve ®bres in the rat, Br. J. Pharmacol., 127 (1999) 457±467. [18] Reuter, U., Weber, J.R., Gold, L., Arnold, G., Wolf, T., Dreier, J., Lindauer, U. and Dirnagl, U., Perivascular nerves contribute to cortical spreading depression-associated hyperemia in rats, Am. J. Physiol., 274 (1998) H1979±H1987. [19] Spears, R., Hutchins, B. and Hinton, R.J., Capsaicin application to the temporomandibular joint alters calcitonin generelated peptide levels in the trigeminal ganglion of the rat, J. Orofac. Pain, 12 (1998) 108±115. [20] Wahl, M., Schilling, L., Parsons, A.A. and Kaumann, A., Involvement of calcitonin gene-related peptide (CGRP) and nitric oxide (NO) in the pial artery dilatation elicited by cortical spreading depression, Brain Res., 637 (1994) 204±210.