RESEARCH REPORTS Clinical
P.F. Sowman1 and K.S. Türker1,2* 1Research
Centre for Human Movement Control, Discipline of Physiology, School of Molecular and Biomedical Science, University of Adelaide, SA 5005, Australia; and 2Department of Physiology, Faculty of Medicine & Centre for Brain Research, Ege University, Izmir 35100, Turkey; *corresponding author,
[email protected]
Periodontal-Masseteric Reflexes Decrease with Tooth Pre-load
J Dent Res 87(2):175-179, 2008
ABSTRACT The responses of incisal periodontal mechanoreceptors to increasing mechanical stimulation are known to follow a hyperbolicsaturating course. The implications of these properties for the reflexive control of bite-force have not been examined directly. In line with the abovementioned receptor characteristics, we hypothesized that the periodontal-masseteric reflex will reduce as a function of increasing incisal pre-load. In 10 participants, a central incisor was repeatedly tapped (0.4 N). We measured the modulation by pre-load (0.2-2.0 N) of the reflex frequency-response at between 3 and 20 Hz. The entrainment of the reflex increased with frequency up to 20 Hz and diminished with increasing pre-load. Importantly, the hyperbolic relationship shown here between the periodontalmasseteric reflex and tooth pre-load agreed with the load/response relationships predicted by single-receptor and tooth movement studies. This study demonstrated that periodontal mechanoreceptors are able to contribute to the ongoing control of only small bite-forces. KEY WORDS: periodontal mechanoreceptors, masseter, reflex, saturation.
INTRODUCTION
he saturating responses of periodontal mechanoreceptors to increasing Tanimal mechanical stimuli have been demonstrated directly in anesthetized preparations (Linden, 1990) and by microneurographic recordings in
humans [see, e.g., Trulsson and Johansson (1994)]. While these techniques have identified this property in single receptors, the reflex manifestation of this phenomenon within the masticatory apparatus has not been examined. Without such an examination, any inferences made in regard to the functional contributions of periodontal mechanoreceptors to the control of bite-force remain tenuous, since the receptor forms only part of the reflex circuit. The aim of this paper was to investigate the masseteric reflex response to periodontal mechanoreceptor stimulation under conditions of various tooth pre-loads. In line with work previously reported, we hypothesized that the reflex response would saturate with increasing pre-load of the tooth (Trulsson and Johansson, 1994). Should this hypothesis be confirmed, it would suggest that, functionally, periodontal mechanoreceptors are able to contribute to the control of masticatory forces only when minimal biteforces are used. In this study, we used a new method to examine this hypothesis. We applied a pulse train of identical stimuli at pre-determined rates to a central incisor and quantified entrainment of the reflex response at the frequency of stimulation. We hoped to identify the ideal frequency at which strong entrainment of periodontal mechanoreceptors occurs that could then be demonstrably blocked by local anesthesia. Following the results of previous frequency response studies of human reflexes (Cathers et al., 2006), our second hypothesis was that the entrainment of the periodontal-masseteric reflex would be strongest at a frequency that best matched the known mean firing rate of masseteric motoneurones, i.e., ~ 20 Hz.
METHODS Participants
Received February 14, 2007; Last revision September 25, 2007; Accepted October 18, 2007
Experiments were conducted with approval from the University of Adelaide human ethics committee and conformed to the Declaration of Helsinki. Ten participants (six females; age range, 18-28 yrs) were recruited for these experiments. All provided written informed consent. All participants had natural and healthy dentitions and were free of dental symptoms. Participants sat upright in a dental chair adjusted for height such that they could bite onto fixed metal bite plates (inter-incisal distance of 12 mm). The metal bite plates were coated with a dental impression material (3M Express™, St. Paul, MN, USA) molded to each person's teeth individually. The upper per-incisal part of this impression was cut away so that the right upper incisor was fully exposed. The thickness of the impression material between the molars ensured that the incisal edge was free from contact.
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J Dent Res 87(2) 2008 Electromyography (EMG) Recording Adhesive EMG electrodes (Duotrode ® , Myo-Tronics Inc., Seattle, WA, USA) were affixed to the skin over the left masseter muscle. Bipolar EMG was amplified (1000-10,000x), filtered (20-1000 Hz), and recorded at a sampling rate of 2000 Hz for 1 min for each stimulation condition, via a specially designed LabVIEW ® (National Instruments, Austin, TX, USA) data acquisition program (Brinkworth, 2004). Participants were presented with visual feedback as a percentage of maximal voluntary contraction (MVC) EMG on a dedicated monitor. EMG feedback signals were full-wave-rectified before being low-pass-filtered at 1 Hz. Participants were required to bite until a required target feedback level (10% MVC) was reached, and then maintain this level as accurately as possible for a period of 1 min.
Mechanical Tooth Stimulation A 0.4-N pulse with a halfsinusoidal rising-phase profile of 8 msec (Fig. 1) was applied orthogonally in a train to the lower third of the upper left central incisor via a Perspex probe (Perspex, Southampton, UK). The frequency of this pulse train was varied to include 5 arbitrarily determined, discrete frequency steps (3, 5, 8, 10, and 20 Hz). All frequencies of stimulation were tested under 4 different tooth preloads (0.1, 0.2, 0.5, and 2.0 N) while the person was biting at 10% MVC. Stimulation frequency and pre-load conditions were randomized and interspersed with 30-second rest periods.
Local Anesthetic Figure 1. Left masseteric EMG responses to 10-Hz incisal stimulation from a single person. (A) Rectified averaged EMG (n = 60) in response to decreasing amounts of pre-load. Top trace (red) shows response at the lowest pre-load (0.1 N) when periodontium is anesthetized. Bottom trace (black) is the averaged stimulus force (n = 60) applied at the lowest pre-load (0.4 N superimposed on a 0.1-N pre-load). Pre-load was varied to include the 4 steps tested (0.1, 0.2, 0.5, and 2.0 N). (B) Stimulus to EMG coherence for the data shown in (A). Traces are color-matched to (A). The horizontal dotted line represents the 99% confidence interval for significant coherence. (C) Spike trigger averaged EMGs of the same data shown in (A) and (B). Traces are color-matched. Top trace in each frame is the rectified, averaged EMG (trigger at time = 0); bottom trace is the cumulative sum of that reflex. Vertical dotted color-matched lines in each frame illustrate the onset latency and offset latency for the inhibitory response elicited by the stimulus. In the top left corner, the reflex size (n = 60) is displayed. Reflex latencies and size calculated as per Brinkworth and Türker (2003).
Following the control period, participants had their upper and lower peri-incisal periodontium infiltrated with local anesthetic (4 mL, Xylocaine 2% ® , Astra Pharmaceuticals Pty. Ltd., North Ridge, NSW, Australia). Adequate anesthesia was achieved when the participant no longer had tactile or vibratory sensation in either labial or buccal aspects of the upper or
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lower incisor teeth, canine to canine (Türker and Jenkins, 2000; Brinkworth et al., 2003). All stimulation conditions were then repeated, with a different randomization, 5 min after completion of the anesthetization procedure (about 15 min after the initial dose of the local anesthetic).
Data Analysis We performed coherence analysis to determine the frequency domain correlation between the simultaneously recorded stimulation-force and EMG signals. The application of coherence analysis to EMG data has been described in detail (Halliday et al., 1995) and has been implemented here with a custom-designed LabVIEW®-based computer program. For statistical analysis of the average response across the sample, the coherence values at the frequency of stimulation were converted to Z-scores to normalize their distribution, then analyzed with a three-way [pre-load, frequency, and condition (control or local anesthetic)] repeatedmeasures ANOVA. Where significant interactions occurred, we performed Tukey's post hoc pairwise comparisons to identify the location of significant differences. Data are presented as mean ± SEM, and significance was set at the 5% level. Statistical analysis was performed with SPSS 13.0© for Windows (SPSS, Chicago, IL, USA) and curve-fitting with SigmaPlot 9.0© (Aspire Software International, Ashburn, VA, USA). For those frequency conditions where, within an individual, there was significant coherence for all pre-loads, the reflex gain (output power/input power at the frequency of interest) was also calculated. We normalized the gain response for each person by dividing each of the reflex gains, within a frequency condition, by the maximum gain recorded for that frequency condition. For illustrative purposes, we performed a conventional spiketrigger averaging analysis (Brinkworth and Türker, 2003) on a representative dataset to demonstrate the shape of the reflex response clearly (Fig. 1).
RESULTS Periodontal-Masseteric Reflexes The reflexes elicited by mechanical stimulation of the tooth were primarily characterized by an inhibition in the EMG at a latency of ~ 20 msec (Fig. 1C). At the lowest pre-load, where the largest responses were observed, the inhibitory response was sometimes followed by a period of increased EMG activity (Fig. 1C, bottom frame). Pure excitation was not observed.
Effect of Local Anesthetic on Entrainment The application of local anesthetic significantly reduced the coherent response at the frequency of stimulation at all frequencies. The ANOVA showed a significant main effect of condition. During control, the average coherence Z-score was 0.097 ± 0.019; during local anesthetic, it was 0.023 ± 0.003, F(1,9) = 14.15 (p < 0.01, N = 200).
Effect of Frequency of Stimulation on Entrainment Prior to local anesthetic, there was an increase in stimulus to EMG coherence with increasing frequency of stimulation. The effect of frequency was statistically significant, F(1.9,17.4) = 11.286, (p < 0.01, N = 80), the average Z-score increasing from 0.023 ± 0.005 at 3 Hz up to 0.103 ± 0.018 at 20 Hz. There was a significant interaction between frequency and condition F (2.0,17.6) = 8.0, (p < 0.01, N = 40), since the increase in coherence Z-score seen with increasing frequency during
Figure 2. Significant interactions identified by the repeated-measures ANOVA performed on the coherence Z-scores across all participants (n = 10). (A) Averages (± SEM) for the condition (control or local anesthetic) by pre-load interaction (n = 50). (B) Averages (± SEM) for the condition by frequency interaction (n = 40). Pairwise comparisons performed with Tukey's HSD.
control was not evident after the application of local anesthetic (Fig. 2).
Effect of Pre-load on Entrainment The main effect of pre-load was significant: F(3,27) = 4.17, (p < 0.05, N = 100), as the pre-load increased, the Z-score decreased from an average of 0.077 ± 0.016 at a pre-load of 0.1 N down to 0.044 ± 0.005 at a pre-load of 2 N. Pre-load also interacted significantly with condition, since the decline in coherence Zscore seen with increasing pre-load during control was not evident after the application of local anesthetic (Fig. 2).
Reflex Gain Due to the low coherence estimates in the low stimulation frequency and high pre-load conditions, the calculation of reflex gain was generally inappropriate. However, at the highest frequency of periodontal mechanoreceptor stimulation, i.e., 20 Hz, there was statistically significant coherence between the stimulus and masseteric EMG at all of the pre-load levels in six persons. For these six persons, reflex gain was calculated. The relationship between pre-load and reflex gain (Fig. 3) followed that seen between coherence and pre-load.
DISCUSSION In this study, we mechanically stimulated periodontal mechanoreceptors under various conditions of incisal pre-load, to examine the reflex consequences of periodontal mechanoreceptor saturation. We used a stimulus profile (60 Hz rising-edge/8 msec rise-time) known to be adequate for periodontal mechanoreceptor activation (Linden and Millar, 1988), expected to elicit primarily inhibition of masseteric EMG and not suitable for an excitatory response alone (Türker et al., 1997). Accordingly, a short latency inhibition was the primarily observed response. With the lowest pre-loads (and largest reflex responses), an 'excitation' following the inhibition was also observed. This 'excitation' is now known to be an artefact of surface EMG recording methods, created by the
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Figure 3. Effects of pre-load on the entrainment of left masseteric EMG to 20-Hz stimulation of an incisor. The data displayed in this Fig. come from the six participants who had significant coherence values at all pre-loads when 20-Hz stimulation was applied (n = 6). (A) Average normalized gain response ± SEM (red) and average coherence Z-scores plotted against pre-load. The strength of correlation is expressed alongside the respective plot. Hyperbolic curves were fitted. (B) Coherence data presented in (A). (black) Compared with (i) the tooth movement/force relationship presented in Fig. 3 in Parfitt (1960); and (ii) the periodontal mechanoreceptor steady-state response/force relationship presented in Fig. 1b in Trulsson and Johansson (1994). Datapoints coinciding with the pre-load forces used in the current study were extracted directly from the Figs. presented in Parfitt (1960) and Trulsson and Johansson (1994). Y-axes for the current data have been inverted to illustrate the similarity to the previous data.
clustering of motor unit spikes that have been delayed by the inhibitory stimulus (Brinkworth et al., 2003; Türker and Powers, 2005). The frequency response of this reflex was examined between 3 and 20 Hz. Previous studies with similar methods of stimulation used for the investigation of other reflexes have utilized sinusoidal stimulation profiles (Joyce et al., 1974; Cathers et al., 2006). However, when, with sinusoidal stimulation, the frequency is increased, the slope of the rising-edge also increases. Given the known rate-dependence of periodontal mechanoreceptor response to mechanical stimulation (van Steenberghe and de Vries, 1978; Linden and Millar, 1988), the coherent response to sinusoidal stimulation would be expected to follow a predictable positive correlation with stimulus frequency. In this scenario, it would be impossible to delineate between frequency-response relationships that were due to ratedependency or reflex loop resonances. For this reason, we chose to use a pulse train, whereby the rate of the stimulus was constant and the frequency of its application could be varied
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accordingly. There were two novel findings in this study: This study showed that the reflex response in the masseter, elicited by mechanical stimulation of a tooth, is negatively correlated with the amount of pre-load applied to that tooth. This finding is significant, in that it shows that periodontal mechanoreceptors in humans are unlikely to contribute to force fluctuations during mastication, where bite-forces are much higher than the level at which the reflex saturates (Gibbs et al., 1981). Previous studies have shown a tendency for the periodontal mechanoreceptor response to saturate at forces of stimulation above 2 N (Trulsson and Johansson, 1994). Others have contended that the location of periodontal mechanoreceptors means that when a force is applied to a tooth, it causes a stretching of the periodontal ligament and depolarization of what is a Ruffini-like stretch receptor (Linden, 1990). Increasing the force applied causes an incremental diminution of available tooth movement (Parfitt, 1960; Wills et al., 1978; Moxham et al., 1995), and hence a saturating level of stimulation is reached. We have demonstrated that the tooth movement measured by Parfitt (1960) is hyperbolically proportional to the force applied to the tooth. Therefore, the 'adequate stimulus' for investigating the linear response characteristics of periodontal mechanoreceptors is a controlled displacement, rather than a controlled force (Beith et al., 2006). The results of this study support that hypothesis. The relationship demonstrated here, between both reflex gain and stimulus/EMG coherence and pre-load, follows a hyperbolic relationship that is almost identical to the toothmovement/force relationship shown previously (Parfitt, 1960). Significantly, this relationship mimics that demonstrated previously (Trulsson and Johansson, 1994), which describes the way in which the steady-state firing rates of 80% of typeidentified periodontal mechanoreceptors respond to increasing force application via a tooth. Our second novel finding was the frequency range over which a periodontal mechanoreceptor reflex response can occur where maximal coherence was observed, as hypothesized at the carrier frequency of jaw muscle motor units (~ 20 Hz). Given the unique nature of the stimulation protocol used in this study, we can be sure that the negative correlation seen here between the stimulus interval and the degree of coupling that occurred between the stimulus and the masseteric EMG was not due to the rate sensitivity of the periodontal mechanoreceptors. Rather, we ascribe this effect to a carrier-dependence phenomenon, whereby the entrainment of motoneurones to a periodic signal became strongest when the input signal approached the mean motoneuronal firing rate (Cathers et al., 2006). Applying this theory to the masticatory muscles, which have generally higher firing rates than those seen in limb muscles, one would expect to see a linear increase in entrainment up to and above 20 Hz (van Boxtel and Schomaker, 1983), as we observed in this study. The only departure from this relationship that could be observed was the upward inflection that occurred at 8 Hz. This was a notable result, in that it corresponded to the periodontal mechanoreceptor loop time observed in previous studies (Sowman and Türker, 2005). This observation strengthens the hypothesis that an 8-Hz mandibular physiological tremor may be partly due to resonance in the periodontal mechanoreceptor reflex loop (Sowman et al., 2006, 2007).
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Correlation methods, like the coherence function used as the primary measure here, give no direct estimate of the size of the reflex. However, by using the gain function, obtainable when spectral methods are used, one can estimate this. For these data, the coherence results suggest that, for those frequencies of stimulation below 20 Hz, there is a significant proportion of uncorrelated activity existent in the EMG record at the frequency of stimulation that is not due to the driving effect of the stimulus. Under such conditions, the estimation of a gain value is likely to provide spurious information regarding reflex size. Therefore, we can comment only on the degree to which the EMG response is correlated with the stimulus, a measure that incorporates both amplitude covariance and phase-locking. Nevertheless, this information gives a very good indication as to the degree of modulation of coupling strength between receptor and muscle that occurs when the tooth is pre-loaded. Furthermore, we have established that, by using a stimulation frequency that matches the known average firing rate of active masseter motor units (van Boxtel and Schomaker, 1983), i.e., 20 Hz, the entrainment of EMG to the stimulus is strong enough to establish a meaningful gain estimate, even at high pre-loads. The use of very low pre-loads would allow this technique to be used for reflex gain calculations at lower frequencies also. This study showed that the action of periodontal mechanoreceptors can reflexively alter bite-force during biting tasks only where the force exerted is small. This was evidenced by the sharp decline in evocable reflex activity when the tooth was pre-loaded above 1 N.
ACKNOWLEDGMENT This research was supported by the NH&MRC of Australia.
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