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Effects of Excitatory Amino Acid Receptor Antagonists on Pulpal Blood Flow of the Rat Mandibular IncisorLaboratoire de Physiologie de la Manducation, Université Paris 7—Denis Diderot, 2 Place Jussieu, Bât. A, 2ème étage, 75005 Paris, France; Correspondence: *corresponding author, hofman{at}ccr.jussieu.fr
Pulpal blood flow (PBF) changes have been monitored by laser Doppler flowmetry on rat mandibular incisors. Electrical stimulation (10 sec, 50 µA, 2 ms, 20 Hz) of one incisor induced a blood flow decrease followed by a blood flow increase. The effect of intravenous administration of antagonists of ionotropic glutamate receptors (iGluRs) and antagonists of metabotropic glutamate receptors (mGluRs) was compared with that of those obtained from animals treated with the vehicle alone. No long-term effect on basal PBF was observed, except a remaining increase of 34.5% (p < 0.05, n = 5) for ketamine (10 mg/kg), an iGluR antagonist, and of 37% (p < 0.05, n = 5) for MCPG (7.5 mg/kg), an mGluR antagonist. In animals treated with iGluR antagonists, acute changes in PBF after stimulation were not significantly different from those observed with vehicle. In animals treated with mGluR antagonists, the blood flow decrease was significantly enhanced in amplitude and duration for MCPG (7.5 mg/kg), respectively, +73% and +92% (p < 0.05, n = 5). These results suggest that Group I mGluRs participate in the regulation of the immediate pulpal blood flow decrease induced by electrical stimulation of the lower incisor in the rat.
Key Words: rat • pulpal blood flow • laser Doppler flowmetry • glutamate receptors • sympathetic nerve fibers
Electrical stimulation of the dental pulp in the rat elicits a blood flow decrease related to the liberation of norepinephrine by autonomic nerve fibers acting mainly at adrenoreceptors and a blood flow increase involving the liberation of neuropeptides by primary sensory afferents (Raab et al., 1988). Among these neuropeptides, substance P (SP) and calcitonin gene-related peptide (CGRP) are liberated by capsaicin-sensitive small-diameter nerve fibers in many tissues, such as the skin or the dental pulp (Gazelius et al., 1987). However, neuropeptides are not the only neurotransmitters/neuromodulators that can induce vascular changes. Excitatory amino acids (EAAs), such as glutamate, can also elicit vascular changes. For example, glutamate, which exerts its biological action through ionotropic receptors (iGluRs) and metabotropic receptors (mGluRs), may induce cardiovascular changes in the awake rat through mGluR activation in the spinal cord (Li et al., 1999). Administration of EAA antagonists acting at the iGluR of the NMDA subtype produces variations in local cerebral blood flow in the rat (Sharkey et al., 1994). Glutamate contributes, via NMDA receptors, to the tonus of cerebral microvessels, and this effect is dependent on neuronal discharge activity and neuronal production of NO (Fergus and Lee, 1997). Besides these central effects, there is some evidence that glutamate may also exert a peripheral action on vascularization. Some arguments support this hypothesis. EAA receptors are present on the peripheral end of small-diameter primary sensory nerve fibers in the rat (Ault and Hildebrand, 1993). Glutamate has been identified by immunocytochemistry in dorsal root ganglion cells and trigeminal cell bodies (Kai-Kai and Howe, 1991) and, more specifically, in small-diameter cellular bodies of primary afferent neurons innervating the dental pulp (Azérad et al., 1992). Moreover, a study by Jackson and Hargreaves (1999) showed that in vitro superfusion of bovine dental pulp by agonists of glutamate receptors stimulated the release of immunoreactive CGRP. The aim of the present study was to investigate the possible role of EAAs in the regulation of the dental pulpal blood flow. Vascular changes induced by electrical stimulation of pulpal nerve fibers were monitored by laser Doppler flowmetry in response to administration of both iGluR and mGluR antagonists.
The protocol has been described elsewhere in detail (Boucher et al., 2001). Briefly, the experiment was conducted as follows:
General Preparation
Electrical Stimulation
Recording of PBF
Experimental Protocol and Administration of Drugs
Analysis of the Results
Effects of Electrical Stimulation of the Rat Mandibular Incisor Ninety minutes after the surgical preparation, the arterial blood pressure and the PBF of both incisors were stable. Bipolar electrical stimulation, without any injection of drug, induced an immediate blood flow decrease followed by a blood flow increase. The mean amplitude of blood flow decrease was -33.37 ± 4.18% (n = 18), compared with baseline PBF, and its duration was 34 ± 4 sec (n = 18). The mean amplitude of blood flow increase was +23.78 ± 2.34% (n = 18) compared with baseline PBF, and its duration was 954 ± 160 sec (n = 18). During electrical stimulation and consecutive changes of PBF, mean arterial blood pressure (MAP) remained stable (see Fig. 1A  ).
Removal of the dental pulp at the end of the experiment abolished the signal.
Effects of Injection of iGluR Antagonists
PBF changes after electrical stimulation of the rat mandibular incisor Related to the control group, which received vehicle i.v., none of the antagonists of iGluRs tested—MK801, ketamine, or GYKI 52466—modified immediate pulpal blood flow decrease and late blood flow increase responses. Differences among groups, for amplitude and for duration of the 2 vascular changes, were not statistically significant (see Fig. 2 ).
Effects of Injection of mGluR Antagonists Effects on basal parameters Intravenous injection of the drugs or their vehicle alone induced, a few seconds after the beginning of the injection, a decrease of PBF and MAP. At the end of the injection, values returned to baseline level. Only the injection of MCPG 7.5 mg/kg, after the initial decrease of PBF at the end of the injection, induced an increase of PBF; 15 min after MCPG (7.5 mg/kg) administration, a significant increase of PBF remained (0.70 ± 0.04 AU vs. control 0.51 ± 0.04 AU; ANOVA, F = 3.70, p = 0.02). The persisting increase of MAP was not statistically significant. For any of the drugs tested, whatever the dose, differential PBF between the 2 incisors registered on the same animal was equal to zero (see Table  and Fig. 1B).
PBF changes after electrical stimulation of the rat mandibular incisor
The same specific attention to stimulation and recording conditions was paid in this experiment as previously (Boucher et al., 2001). The bipolar stimulation of the dental pulp by electrodes applied directly to the crown of the incisor elicited the local immediate pulpal blood flow (PBF) decrease and late PBF increase observed by other authors (Kerezoudis et al., 1993; Raab et al., 1988) without changes in general blood pressure. This type of stimulation limits the risk of periodontal diffusion that can occur with monopolar stimulation (Hansson and Ekblom, 1987; Moody et al., 1989) and allows for the recruitment of the nerve fibers within the incisor dental pulp which are almost exclusively unmyelinated nerve fibers (Bishop, 1981), although some of them may be weakly myelinated outside of the pulp (Jiffry, 1981). Particular attention was paid to the isolation of the teeth, since the laser Doppler signal may be contaminated by the periodontal blood flow, as shown by Hartmann et al. (1996) and Vongsavan and Matthews (1996). The signal recorded during the experiment corresponded exclusively to the pulpal perfusion, since extirpating the dental pulp, at the end of the experiment, definitively abolished the signal. Separation of the 2 hemimandibles during the surgical procedure ensured that no cross-innervation influenced the contralateral incisor. The analysis of the present data focuses on the difference in the PBF of the 2 incisors, either stimulated or non-stimulated. This method allows us to cancel out any general variation of systemic origin that might occur during long sessions. Therefore, remaining variations result from local changes elicited by the electrical stimulation in the stimulated tooth. To evaluate the hypothesis that EAA receptors are involved in the physiological control of the rat's mandibular PBF, we administered different glutamate receptor antagonists during this study. Ionotropic glutamate receptors are divided into two classes: NMDA and non-NMDA (AMPA and kainate) (see Bigge, 1999, for review). In this study, iGluR antagonists administration transiently affected basal PBF, but no difference was observed on electrically induced acute changes, either immediate PBF decrease or late PBF increase. Only ketamine administration induced a remaining increase of PBF (+34.5%, p < 0.05) 15 min after injection. The dosage of the drugs may be questioned. In vivo studies using MK801, which is a powerful NMDA antagonist acting at small concentrations (Wong et al., 1986), show a maximal effect, at 0.3 mg/kg i.v, on cerebral microcirculation, medullar reflexes, and neuronal activity in the rat CNS (Sharkey et al., 1994). Beyond 0.3 mg/kg, MK801 induces general manifestations such as ataxia and respiratory troubles, which may compromise the vital prognosis (Kelland et al., 1993). Ketamine, a non-competitive NMDA-antagonist, is a dissociating anaesthetic (Anis et al., 1983) which induces considerable systemic effects at 10 mg/kg i.v GYKI 52466, an antagonist of AMPA and kainate receptors, produces important physiological effects at 0.2 mg/kg (Donevan and Rogawski, 1993). In this study, the three iGluR antagonists were therefore administrated in the range of the maximal concentrations recommended. Thus, it can be reasonably assumed that the lack of effect of iGluR antagonists is not related to an underdosage. One explanation could be that these drugs did not find any target in the dental pulp. Glutamate present in the cell bodies of primary sensitive neurons innervating dental pulp (Azérad et al., 1992) either would not be released by peripheral endings or, if released, would not act on ionotropic receptors, at least in our experimental conditions. The lack of effect of iGluR antagonists observed in our study is difficult to reconcile with the observations of Jackson and Hargreaves (1999), who performed in vitro superfusion of bovine dental pulp by agonists of glutamate receptors. In their study, the administration of AMPA and kainate receptor agonists stimulated the release of immunoreactive CGRP (iCGRP) in a concentration-dependent manner. Pre-treatment and co-administration of CNQX, AMPA/kainate receptor antagonist, significantly reduced iCGRP release induced by these agonists. According to these results, ionotropic receptor antagonists, especially GYKI 52466, were expected to decrease the electrically induced blood flow increase, since CGRP is involved in the antidromic blood flow increase in oral tissues (Gazelius et al., 1987). However, species differences in tooth pulp innervation (rat vs. bovine) or differences in the protocol (in vivo vs. in vitro) may explain this discrepancy. Metabotropic glutamate receptors (mGluRs) are coupled with a variety of second-messenger systems via G proteins and are subdivided into 3 groups according to their amino acid sequence similarity, agonist pharmacology, and signal transduction mechanism coupling (see Conn and Pin, 1997, for review). MCPG is a non-selective antagonist of Group I and Group II metabotropic receptors; MPPG is an antagonist of Group II and Group III metabotropic receptors (Jane et al., 1995). Intravenous administration of these drugs transiently decreased PBF and MAP, which returned to basal values in 15 min. Only MCPG, 7.5 mg/kg, induced a significant persistent increase, +37%, of PBF (p < 0.05, n = 5), 15 min after administration. No differential effect was observed in comparisons of the stimulated and non-stimulated incisors. In this study, regarding the effects on the pulpal blood flow increase induced by electrical stimulation, MCPG and MPPG did not cause any modification of the changes. The low doses, 2.2 mg/kg for MCPG and 0.15 mg/kg for MPPG, were chosen closely related to the concentrations used in in vitro studies or when used i.c.v Higher doses, 7.5 mg/kg for MCPG and 2.18 mg/kg for MPPG, did not provoke any additional effect. It is therefore unlikely that the lack of effect on blood flow increase was the result of an underdosage. In contrast, both amplitude and duration of the immediate pulpal blood flow decrease were affected when a high dose of antagonist MCPG (7.5 mg/kg, i.v) was used. Pulpal blood flow decrease was significantly enhanced, +73% for amplitude and +92% for duration, compared with that in control animals (p < 0.05, n = 5). Relatively little is known about the peripheral processes in which these mGluRs may be involved in the regulation of peripheral vascularization. To the best of our knowledge, studies have focused only on the central role of mGluRs in cardiovascular regulation (D'Amico et al., 1996; Foley et al., 1999). One explanation for the enhancement of immediate PBF decrease may be related to an effect on sympathetic endings, i.e., a block of mGluRs potentially present on post-ganglionic sympathetic fibers. Glutamate released from primary sensory neurons would then reduce the PBF decrease of sympathetic origin. In support of this hypothesis, and despite the lack of direct evidence of glutamate receptors present on post-ganglionic sympathetic nerve fibers, some studies indicate that EAA receptors are involved in sympathetic activity, at the pre-ganglionic level for mGluR (Nolan and Logan, 1998) or post-ganglionic for iGluR (Carlton et al., 1998; Coggeshall and Carlton, 1999). In addition, noradrenaline release is modulated by sympathetic iGluR activation (Cosentino et al., 1995). The enhancement of neurogenic PBF decrease observed in the present study could also be related to the action of neuropeptide Y (NPY), which is colocalized with noradrenaline in sympathetic nerve terminals originating from the superior cervical ganglion, around the pulpal vessels (Wakisaka and Akai, 1989). NPY potentiates the vasoconstrictor effects of NA when sympathetic nerves are stimulated with a high frequency (20 Hz) (Lundberg et al., 1986), as was the case in this study. It may be noted that some studies have reported interactions between NPY and metabotropic receptors at the central level. In the dentate gyrus of the rat, for example, activation of mGluR stimulates NPY mRNA and NPY-Y2 receptor expression (Schwarzer and Sperk, 1998). Another hypothesis arises from recently published studies by Walker et al. (2001) and Bhave et al. (2001), who recently demonstrated the presence of group I mGluRs on peripheral unmyelinated nerve fibers, presumably of sensory origin, and their involvement in peripheral inflammation. Activation of these receptors increases the sensitivity to noxious heat, while their antagonists prevent inflammation-induced pain and attenuate established inflammatory pain. Regarding the vascular changes occurring in inflammation, these results may fit with our own results. Glutamate liberated by primary afferent sensory neurons would then act at metabotropic autoreceptors to control the PBF. Finally, a direct action of mGluR antagonists on endothelial cells cannot be excluded, since Krizbai et al. (1998) have shown that EAA receptors are expressed in primary cultures of rat cerebral endothelial cells. Even so, in vivo and in vitro observations by Morley et al. (1998) suggest that both human and rat cerebral endothelial cells do not express functional glutamate receptors. Studies concerning the expression of functional glutamate receptors on peripheral endothelial cells would be necessary for the possibility of a direct action to be assessed. To date, the peripheral nerve-blood microvessel coupling has not been examined from this point of view. In conclusion, the effects of antagonists of ionotropic and metabotropic glutamate receptors on pulpal blood flow of the rat mandibular incisor, after bipolar electrical stimulation, suggest an involvement of group I metabotropic glutamate receptors in the regulation of the immediate electrically induced pulpal blood flow decrease. Further studies, such as RIA or immunochemistry, are necessary for assessment of the localization of metabotropic glutamate receptors in the dental pulp, and for clarification of the mechanisms of these effects.
This research was institutionally supported by the Université Paris 7—Denis Diderot, through the funds of the Laboratory of 'Physiologie de la Manducation'. The authors thank Drs. Morton Sobel and Chris Simons for the revision of the manuscript. Received for publication February 1, 2001. Revision received December 14, 2001. Accepted for publication January 23, 2002.
Journal of Dental Research, Vol. 81, No. 4,
253-258 (2002)
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ABSTRACT
INTRODUCTION
-methyl-4-carboxyphenylglycine, MCPG (Tocris, Ellisville, MO, USA; 2.2 mg/kg, n = 5 and 7.5 mg/kg, n = 5), and (RS)-
Control group, 
MK 801 group, 
Ketamine group, 
Gyki 52466 group. Control rats were injected i.v. with 200 µL of physiological saline (vehicle). Amplitude is expressed in percentage of variation of differential PBF from baseline level (difference between stimulated and control teeth). Duration is expressed in sec. Values are means ± SEM. Differences among experimental groups were evaluated by one-way ANOVA. For the immediate blood flow decrease and for the late blood flow increase, no statistical differences were found among groups.
Control group, 
MCPG 2.2 mg/kg group,