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RESEARCH REPORT |
Laboratoire de Physiologie de la Manducation, Université Paris 7—Denis Diderot, 2 Place Jussieu, Bât. A, 2ème étage, 75005 Paris, France;
*corresponding author, hofman{at}ccr.jussieu.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: rat • pulpal blood flow • laser Doppler flowmetry • glutamate receptors • sympathetic nerve fibers
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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General Preparation
Forty-five male Sprague-Dawley rats (weighing from 300 to 400 g each), purchased from Harlan France Laboratory (Gannat, France), were anesthetized with pentobarbital (SANOFI SAINTE ANIMALE, Libourne, France; 50 mg/kg-body wt, i.m., followed by 20 mg/kg/body wt shots as required). Adequacy of the anesthesia was determined by the maintenance of a stable heart rate and blood pressure, with no fluctuations on the tail pinch test. The rats were allowed to breathe spontaneously through a canula placed in the trachea. Blood pressure was monitored through the femoral artery, and a catheter for the delivery of drugs was placed in the femoral vein. Animals were placed on a thermostatically controlled electric blanket. Mandibular incisors were exposed and separated. After the surgical preparation, a resting period of at least 1 hr was observed before the experimental procedures started. Rats showing a mean arterial pressure over 135 mm Hg (18 kPa) or under 80 mm Hg (10.3 kPa) were excluded from the study. The animal use protocol was reviewed and approved by the Institutional Animal Care and Use Committee at the University of Paris 7—Denis Diderot.
Electrical Stimulation
Electrical stimulation of one mandibular incisor (2 ms, 20 Hz, 50 µA) for 10 sec was achieved through bipolar electrodes (Ag/AgCl) fixed at the surface of the crown with a custom-made device consisting of Teflon screws supporting the electrodes inserted into a Teflon support wrapping the tooth. The layer of enamel under the electrodes was removed under microscope and copious saline irrigation. To facilitate the current flow to the pulp, a conductor gel (KCl) ensured the contact between the stimulating electrode and the tooth. A pulse generator (Master 8, AMPI, Jerusalem, Israel) triggered the A360R-C WPI constant current stimulator (World Precision Instruments, Sarasota, FL, USA).
Recording of PBF
PBF was monitored with a dual-channel laser Doppler flowmeter (Moor Instruments, Axminster, UK; 780-820 nm, 2.7 mW) on both mandibular incisors. The non-stimulated tooth was monitored as a control. Values were expressed as arbitrary units of perfusion (AU). Fiber-optic probes (type P2, Moor Instruments, Axminster, UK; fiber diameter, 200 µm, with 500 µm separation distance between incident and reflected flux fibers) were fixed at right angles to the surface of the tooth crown in the custom-made devices holding the stimulation electrodes. Calibration was carried out according to the manufacturer's specifications. The time constant was set at 0.5 sec and bandwidth at 4 kHz. Teeth were isolated by a thick black rubber dam so that gingival contamination would be avoided. As a control, the dental pulp was removed at the end of the experiment and the PBF recorded.
Experimental Protocol and Administration of Drugs
A control electrical stimulation was performed on one mandibular incisor of each animal. After an interval of 45 min so that PBF could return to baseline, administration of test drugs was performed i.v. (automatic injector-Precidor, Infors, Basel, Switzerland; velocity of injection, 50 µL/min; volume of injection, 200 µL). An electrical stimulation was then performed 15 min after EAA receptor antagonists' administration. The aim was to compare the PBF changes elicited by electrical stimulation before and after administration of drugs. Antagonists of iGluRs, dissolved in physiological saline, were (+)-MK-801 hydrogen maleate (RBI/Sigma, St. Louis, MO, USA; 0.3 mg/kg, n = 5), ketamine hydrochloride (RBI/Sigma, USA; 10 mg/kg, n = 5), and GYKI 52466 hydrochloride (RBI/Sigma, USA; 0.2 mg/kg, n = 5). Antagonists of mGluRs, first dissolved in 1M NaOH, were (S)-
-methyl-4-carboxyphenylglycine, MCPG (Tocris, Ellisville, MO, USA; 2.2 mg/kg, n = 5 and 7.5 mg/kg, n = 5), and (RS)--methyl-4-phosphonophenylglycine, MPPG (Tocris; 0.15 mg/kg, n = 5 and 2.18 mg/kg, n = 5). Two groups of control rats were injected i.v. with, respectively, 200 µL of physiological saline (n = 5, as controls for iGluR antagonists) or 200 µL of a mix of 1 M NaOH (105 µL/kg) and physiological saline (n = 5, as controls for mGluR antagonists).
Analysis of the Results
The analysis of the results focused on the effects of drugs on basal PBF and on PBF changes after bipolar electrical stimulation of the mandibular rat incisor. Results were expressed in terms of amplitude and duration. Reference level was constituted by basal PBF, stabilized before stimulation. After the data were collected, PBF from both the stimulated and control incisors was expressed as a percentage of the pre-stimulated levels. The basal flow of each incisor before stimulation was represented by the value 0. The two PBFs were then subtracted. The amplitude of the changes (peak variations) after electrical stimulation was determined by the minimal value of PBF, for the immediate blood flow decrease, and the maximal value for the late blood flow increase. The duration of the effect was determined by the period between the beginning of the response and the time when PBF returned to baseline. Reported changes were expressed as percentages (baseline to peak) and as means ± SEM. Differences were evaluated by one-way analysis of variance (ANOVA) with paired post-tests in case of significance (p < 0.05).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Effects of Injection of iGluR Antagonists
Effects on basal parameters
Intravenous injections of MK801, ketamine, and GYKI 52466 affected basal PBF and MAP differently. MK801 injection increased both PBF and MAP with a return to basal values after 5 min. Ketamine administration strongly decreased both PBF and MAP, with a return to baseline levels after about 8 min, followed by an increase of the 2 parameters. Fifteen minutes after initial administration, a residual increase of PBF (0.74 ± 0.03 AU vs. control 0.55 ± 0.04 AU; ANOVA, F = 3.36, p = 0.045) and MAP was still present, although not statistically significant for the latter. GYKI 52466 administration decreased PBF and MAP slightly. This decrease did not remain in effect 15 min after drug administration. Administration of vehicle did not modify any of the recorded parameters. Differential PBF recorded between the 2 incisors on the same animal was equal to zero with any of the drugs tested (see Table).
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and Fig. 1B).
PBF changes after electrical stimulation of the rat mandibular incisor
The statistical analysis indicated significant differences for both amplitude and duration of the immediate pulpal blood flow decrease for only MCPG 7.5 mg/kg compared with vehicle-treated animals (p < 0.05). Amplitude and duration were increased, respectively, -35.18 ± 2.06% vs. control -20.35 ± 3.58% (ANOVA, F = 3.81, p = 0.0249, Bonferroni p value < 0.05), and 47 ± 4 sec vs. control 25 ± 5 sec (ANOVA, F = 6.35, p = 0.0076, Bonferroni p value < 0.05). For pulpal blood flow increase, no statistical differences were found among the different groups (see Fig. 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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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.
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ACKNOWLEDGMENTS |
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Received February 1, 2001; Last revision December 14, 2001; Accepted January 23, 2002
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| Journal of Dental Research ® | Critical Reviews (1990-2004) |
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,