original article prolonged gum chewing evokes activation of the...

J Med Dent Sci 2010； 57： 35－43

Corresponding Author: Kiyoshi KamiyaFirst Author: Kiyoshi KamiyaE-mail: [email protected] Author: Hideho AritaTel: +81-3-3762-2335　Fax: +81-3-3762-8148E-mail: [email protected] September 28；Accepted November 13, 2009

Original Article

Kiyoshi Kamiyaa,b, Masaki Fumotob, Hiromi Kikuchib, Tamami Sekiyamab, Yuko Mohri-Ikuzawaa, Masahiro Uminoa and Hideho Aritab＊

a) Anesthesiology and Clinical Physiology, Department of Oral Health Sciences, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University. 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japanb) Department of Physiology, Toho University School of Medicine. 5-21-16 Omori-nishi, Ota-ku, Tokyo 143-8540, Japan

We have proposed a concept that prolonged rhythmic gum chewing causes a suppressed nociceptive flexion reflex via the serotonergic (5-HT) descending inhibitory pathway. However, the mechanism of activation of the 5-HT system by gum chewing remains undetermined. Several human and animal studies have reported that a direct connection exists between the prefrontal cortex (PFC) and 5-HT neurons in the dorsal raphe nucleus; therefore, we hypothesized that activation of the PFC region might be responsible for augmented 5-HT activity. To evaluate this hypothesis, oxygenated hemoglobin (oxyHb) and deoxygenated hemoglobin concentrations in the PFC were measured in the PFC during a 20-min time period of gum chewing using 24-channel near-infrared spectroscopy. A significant increase in oxyHb level was observed in the ventral part of PFC compared with the dorsal part of PFC. We confirmed the previous results in that the nociceptive flexion reflex was significantly suppressed and the 5-HT level in blood was significantly increased following prolonged gum chewing. These results support the hypothesis that activation of the ventral part of PFC during

Prolonged gum chewing evokes activation of the ventral part of prefrontal cortex and suppression of nociceptive responses: involvement of the serotonergic system

gum chewing evokes augmented activity of 5-HT neurons in the dorsal raphe nucleus, which in turn suppress nociceptive responses.

Key words: NIRS (near- infrared spectroscopy) ; Prefrontal cortex; Serotonin (5-HT); Analgesia

1. Introduction

We have proposed a concept that prolonged rhythmic gum chewing causes a suppressed nociceptive response1. Because we found augmented serotonin (5-HT) levels in the whole blood after prolonged gum chewing, we hypothesize that this chewing-induced analgesia may be derived from the activation of the serotonergic descending inhibitory pathway 2. The mechanism underlying activation of the 5-HT system during gum chewing has not been fully understood. To address this issue, we conducted the present study, in which we sought to evaluate the possibility that the prefrontal cortex (PFC) might activate the 5-HT system. The rationale for this idea is that there are several reports indicating direct axonal projections from the PFC to the 5-HT neurons in the raphe nuclei3,4,5, and that recent human studies have demonstrated that gum chewing induces activation of the PFC6,7,8. To evaluate this possibility, the present study was focused upon oxygenation changes in the PFC, namely, concentration changes in oxygenated hemoglobin (oxyHb) and deoxygenated hemoglobin (deoxyHb) in the PFC, using 24-channel near-infrared spectroscopy (NIRS).

36 J Med Dent SciK. Kamiya et al.

To confirm previous results 1 concerning the serotonergic descending inhibitory pathway, we repeated the same experiment in this study, evaluating the nociceptive flexion reflex (NFR) and 5-HT levels in whole blood before and after gum chewing for 20 min.

2. Materials and Methods

2.1. Subjects Ten subjects (aged 26-37 years, 5 men and 5 women) volunteered for the present study. All subjects were heal thy , and screened to exclude those with psychiatric, systemic neuromuscular diseases, history of head injury, or medications that might have affected regional cerebral blood f low, as wel l as 5-HT measurements. Oral and written informed consent was acquired from each subject. The study was approved by the Tokyo Medical and Dental University Ethics Committee and conducted in accordance with the Declaration of Helsinki. The subjects were informed that they were free to terminate the study at any time. No explanation was provided regarding the aim of the study.

2.2. Pain induction and measurement of nociceptive flexion reflex (NFR)

NFR was used in this study. Because NFR is known to be an objective and stable measurement for pain assessment, and is a type of withdrawal reflex9,10,11. NFR recording and stimulating electrodes were attached to the subject. All recording and stimulation electrode sites were cleaned and gently abraded. Reflex was induced by transcutaneous electrical stimulation of the sural nerve at the right ankle while the subject reclined in a comfortable armchair with the lower legs bent to an angle of less than 150 degrees to achieve a state of muscular relaxation. For the NFR recording, a pair of electrodes was placed over the right biceps femoris muscle, 10 cm superior to the popliteal fossa. Electrical stimulation consisted of a train of eight 1 ms square wave pulses at 250 Hz. The electrical pulse was delivered from a constant current isolator system (SS104J, Nihon Kohden, Japan) controlled by a timing controller (SEN3301, Nihon Kohden). The stimuli were delivered randomly every 5-15 seconds to avoid anticipation and habituation to the stimuli. EMG signals were amplified by a bioelectric amplifier (EEG-4217, Nihon Kohden) with a time constant of 0.03 s and a low-pass filter at 1 kHz (frequency range:0.5-120 Hz). As shown in Fig. 1, each NFR was assessed as the

area based on the shape of the response between 100 and 170 ms. Considering the onset latency (101–125 ms) of nociceptive response (R3) described by Skljarevski and Ramadan9, we determined the onset of the NFR as the minimal negative point which appeared between 101 and 125 ms. Note that the tactile component (R2) occurred between 40 and 70 ms. The end of the NFR was determined as the minimal negative point following a large positive wave of R3 component. The raw EMG signals including the NFR were digitized at a sampling rate of 5 kHz for a microcomputer-based analysis. The digitized EMG data were full wave rectified and 10 consecutive traces were averaged in the microcomputer system. Then, a shape of each NFR was determined as described above and the area of the NFR was calculated. The shaded area in Fig. 1 corresponded to the calculated NFR area. To determine the intensity of the stimulus, we assessed the relationship between NFR and visual analogue scale (VAS) prior to the experiment. As the intensity increased, the subject was asked to mark a 100 mm VAS with the subjective judgment of pain intensity for each stimulus. The endpoints of the VAS were labeled ‘no pain’ (left) and ‘extremely painful’ (right). The intensity of the stimulus was fixed at the level at which the subject obtained about 70 percent the tolerance threshold (the maximum intensity that they could bear) of VAS score. That level was maintained throughout the experiment. The NFR threshold was determined at both the beginning and the end of the experiment to check the conditions of electrodes and to exclude the influence of habituation.

2.3. HPLC analysis The blood sample was refrigerated as quickly as possible using the following procedures. Blood (5ml) was collected in heparin-sodium-salt-containing vacutainer tubes. For the whole blood analysis, we applied the method described in detail by Kremer et al.12. Half a milliliter of blood was suspended in 2.5 ml of water. Then, 30 μl of the internal standard and 10 μl of a 10% (weight per volume) solution of ascorbic acid in water were added to the suspended blood sample. The sample was then stored frozen at –20℃ until the assay. HPLC analysis was performed within 2 weeks after the experiment. Whole blood sample was thawed and 167 μl of methanol were added to 1 ml of the whole blood sample to remove proteins. Then, whole blood sample was centrifuged at 4670 × g for 10 min at 4℃. The 500 μl supernatant of the whole blood

37Activation of prefrontal cortex and analgesic effect

sample was suspended in 4.5 ml of mobile buffer. The mobile phase consisted of a phosphate buffer (Na+, 0.1 M) containing 50 mg/L EDTA· 2Na and an ion-pair (300 mg/L sodium-octyl-sulfate, Nacalai Tesque, Japan) and 20% methanol at pH 6.0. The whole blood sample was injected into the HPLC system. 5-HT levels were determined using an HPLC-ECD (EICOM 300, EICOM, Japan) system. The working electrode was a graphite carbon electrode set at a detector potential of +400 mV against an Ag/AgCl2 reference electrode. 5-HT was separated on a reversed phase column (EICOMPAK CA-5 ODS, EICOM). The flow rate was set at 0.22 ml/min and the analysis temperature was 35℃.

2.4. Cerebral Oxygenation We employed a 24 channel near-infrared spectroscopy (NIRS) system (OMM-3000, Shimadzu Corporation, Japan) to detect concentration changes in oxygenated hemoglobin (oxyHb), deoxygenated hemoglobin (deoxyHb), and totalHb using three types of near infrared light (wavelengths: 780, 805, and 830 nm). Each parameter was calculated according to the following equations13:　oxyHb = －1.49 × ∆A780 + 0.5970 × ∆A805 + 1.4847

× ∆A830

　deoxyHb = 1.845 × ∆A780 － 0.2394 × ∆A805 － 1.0947 × ∆A830

　totalHb = oxyHb + deoxyHb A780 , A805 , and A830 , represented detected optical absorbances at 780, 805, and 830 nm, respectively. These were calculated every 130 ms, and the cumulative sampling data in 1040 ms (130 ms ×8 points) for analysis purposes. Figure 2A shows the locations of optodes and channels. A total of 16 optodes, namely eight emitters and eight detectors, were placed on the subject’s frontal region, using an optode holder. The distance between the optodes was set to 3 cm. The optode of channel two was positioned at Fpz, according to the international 10-20 system used in electroencephalography. To confirm estimation of NIRS channel locations, we used a 1.5-T magnetic resonance imaging (MRI) scanner (Excelart™, Toshiba, Japan). An anatomical 3D T1-weighted MRI scan was performed with marking the optode location on the skull by vitamin D capsules. Based on MRI measurements in the present study and other previous reports14,15, these recorded areas may correspond to the bilateral superior, middle and inferior frontal area.

Figure 1 : Nociceptive flexion reflexes (NFR) recorded in biceps femoris muscle. The reflex was induced by transcutaneous electrical stimulation of the sural nerve at the right ankle. Electrical stimulation consisted of a train of 8 1 ms square wave pulses at 250 Hz. The three representative responses shown were obtained when the intensities of the electrical stimulation were 22 mA (top panel), 25 mA (middle panel), and 27 mA (bottom panel). Each NFR was assessed as the shaded area based on the shape of the response between 100 and 170 ms in this case. Note that a tactile reflex (known as R2) appeared at a latency of 72 ms following stimulation of the sural nerve (see bottom trace). For calculation of the NFR area, the NFR response (R3) was full wave rectified and integrated (see text for details).


2.5. Experimental protocols Each experiment began at 1:00 pm. Subjects were asked to chew gum voluntarily and rhythmically for 20 min at a comfortable speed (task experiment). To ensure intensity and rhythm of mastication, we monitored EMG of masseter muscle throughout gum chewing. The gum was specially prepared in the General Laboratory at Lotte Co. Ltd. (Saitama, Japan). The flavor was mint. The mean quantity was 1.0 ± 0.2g. Subjects were stimulated before, immediately after, and 30 min after cessation of gum chewing. To assess 5-HT neuronal activity, we sampled a blood before, immediately after, and 30 min after cessation of gum chewing. Blood samples took from a vein.

2.6. Data analysis Concentration changes in oxyHb were most sensitive to changes in regional cerebral blood flow, and provided the strongest correlation with BOLD signal among the three NIRS parameters16,17. Therefore, changes in oxyHb were considered the best indicator of brain activity. Left ventrolatelal (LVL), Ventromedial(VM), Right ventrolatelal (RVL), Left dorsolatelal (LDL), Dorsomedial (DM), and Right dorsolatelal (RDL) were used for statistical analysis, as shown in Fig. 2B. The 20 channels, except for #11, 12, 13, 14, were averaged into six regions, once raw data from each channel was converted into a z-score. A detailed description of the z-score is provided in the results section.

Changes in oxyHb concentrations were used as indicators for statistical analysis in the present study. Because raw NIRS data was originally reported as relative values, it was not possible to average the data across subjects or channels. Therefore, raw oxyHb data from each channel was converted into a z-score because z-scores can be averaged, regardless of the unit of measurement. The z-scores were calculated using mean the value and standard deviation of oxyHb before gum chewing. The mean value and standard deviation of oxyHb for 3 min before onset of gum chewing were converted to z-scores “0” and “1”, respectively, in each channel. Converted data were analyzed by using a one-way repeated measure ANOVA. If ANOVA results were significant, each treatment was compared with the basal level using Tukey’s post-hoc test. Furthermore, the difference between two PFC regions was analyzed, i.e., ventral and dorsal part of PFC. The ventral part of PFC regions included LVL, VM, and RVL, and the dorsal part of PFC regions included LDL, DM, and RDL. The difference of converted oxyHb concentrations between ventral part of PFC regions and dorsal part of PFC regions was analyzed by paired t-test. Changes in the mean NFR area and the 5-HT levels in the whole blood were analyzed by using a one-way repeated measure ANOVA. If ANOVA results were significant, each treatment was compared with the basal level using Dunnett’s post-hoc test. Effects were considered to be statistically significant when P values were less than 0.05. All data were

Figure 2 : (A)Locations of optodes and channels. This figure illustrates an overhead view of an optode holder. (B) Six PFC regions used for statistical analysis. 20 channels were averaged into 6 regions, termed LVL (left ventral

lateral region), VM (ventral medial region), RVL (right ventral lateral region), LDL (left dorsal lateral region), DM (dorsal medial region), and RDL (right dorsal lateral region), respectively.


expressed as the mean ± SE.

3. Results

3.1. Changes in NFR and 5-HT level Figure 3A shows changes in the mean NFR area before, immediately after, and 30 min after gum chewing, with data expressed as percentages of the basal level: 100% basal level represented the data obtained before gum chewing. Each mean NFR area was calculated from 30 successive NFR areas during the analysis period. Since 30 successive NFR stimuli were randomly delivered every 5-15 s, each analysis period was approximately 5 min. One-way ANOVA revealed significant changes in the mean NFR area after gum chewing (n=10, F2,27=16.22, P<0.05). A significant post hoc difference was calculated between before and immediately after gum chewing (P<0.05), and between before and 30 min after gum chewing (P<0.05). Figure 3B shows changes in 5-HT levels in the whole blood before, immediately after, and 30 min after gum chewing, with data expressed as percentages of the basal level: 100 % basal level indicated data obtained before gum chewing. One-way ANOVA revealed significant changes in 5-HT levels in the whole blood after gum chewing (n=10, F2,27=12.70, P<0.05). There was a significant post hoc difference between before and immediately after gum chewing (P<0.05), and between before and 30 min after gum chewing (P<0.05). 5-HT level in the whole blood observed immediately after of gum chewing increased by 7.7 ±1.1% from the basal level.

3.2. NIRS changes Figure 4 shows typical examples of changes in regional hemoglobin concentration in the PFC before, during, and after gum chewing. OxyHb and totalHb concentrations were slightly increased immediately after the onset of gum chewing in all PFC regions (Fig. 4). During 20 min of gum chewing, oxyHb and totalHb concentrations were increased in the ventral part of PFC regions (#1–10 in Fig. 4). By contrast, during the latter part of the 20-min gum chewing period, oxyHb and totalHb concentrations gradually decreased in the dorsal part of PFC regions (#15–24 in Fig. 4). Compared with the changes in oxyHb and totalHb concentrations, little or no changes in deoxyHb concentrations occurred during gum chewing.

Figure 5 shows the grand average for the time course of converted oxyHb concentrations. For this analysis, the PFC regions were converted into six combined regions, namely, LVL, VM, RVL, LDL, DM, and RDL. It was noted that there were small increases in converted oxyHb concentrations immediately upon onset of gum chewing in all six regions. Thereafter, converted oxyHb concentrations in the ventral part of PFC regions (LVL, VM, and RVL regions) further increased during gum chewing. Higher oxyHb concentrations were observed for at least several minutes after the cessation of gum chewing. However, converted oxyHb concentrations in the dorsal part of PFC regions (LDL, DM, and RDL regions) exhibited no further increase during the latter part of the 20-min gum chewing period. Based on this difference in t ime course of converted oxyHb concentrations among PFC regions, we averaged the converted oxyHb concentrations during a 2-min time period before cessation of gum chewing (21–23 min).

F igure 6 shows the mean converted oxyHb concentrations in six combined PFC regions. One-way ANOVA revealed significant differences in converted oxyHb concentrations among the six PFC regions during gum chewing (n =10, F 5,54 =3.29, P<0.05). Significant differences between the RVL and DM (P<0.05), as well as between the RVL and RDL (P<0.05), were observed. In addition, we evaluated regional differences in converted oxyHb concentrations among the three ventral part of PFC regions (LVL, VM, and RVL) and among the three dorsal part of PFC regions (LDL, DM, and RDL). There were no significant differences in converted oxyHb concentrations among the three ventral (F2,27 =0.81, P =0.45) and dorsal (F2,27 =0.64, P =0.54) part of PFC regions. Because converted oxyHb concentrations in the three ventral part of PFC regions were greater than the corresponding levels in the three dorsal part of PFC regions, the averaged converted oxyHb concentrations in the three ventral part of PFC regions were compared with those in the dorsal part of PFC regions. Figure 7 shows the statistical comparison of the averaged converted oxyHb concentrations between ventral and dorsal part of PFC regions. The averaged converted oxyHb concentration in the ventral part of PFC region (3.79 ± 1.04, n =10) was significantly greater (P<0.05) than that in the dorsal part of PFC region (-0.08 ± 1.00).


Figure 3 : (A) Changes in the mean NFR area before, immediately after, and 30 min after gum chewing (GC) (n=10), with data expressed as percentage of the basal level. Each mean NFR area was calculated from 30 successive NFR areas during an analysis period. Since 30 successive NFR stimuli were randomly delivered every 5-15 s, each analysis period was approximately 5 min.

(B) Changes in 5-HT levels in the whole blood before, immediately after, and 30 min after cessation of gum chewing (GC), with data expressed as percentages of basal level. Vertical lines show mean ± SE (n=10). ＊ P<0.05 as compared with the before GC. See text for detail.

Figure 4 : Typical examples of changes in regional hemoglobin concentration in the PFC before, during, and after gum chewing. 1-10 squares indicate the data recorded from the ventral PFC regions and 15-24 squares from the dorsal PFC regions. Black lines indicate oxyHb concentrations, light black lines indicate deoxyHb concentrations, and gray lines indicate totalHb concentrations. The vertical dotted lines at 3 and 23 min represent the starting and the endpoints of gum chewing, respectively.


Figure 5 : The grand averages for the time course of oxyHb concentration in the PFC. The black line indicates mean change in oxyHb, while the gray lines indicate standard error. The black vertical dotted lines at 3 and 23 minutes represent the starting and the endpoints of gum chewing, respectively.

Figure 6 : Mean converted oxyHb concentrations in 6 combined PFC regions during gum chewing. Data are expressed as mean ± SE (n=10). ＊ P<0.05. See text for details.

Figure 7 : Statistical comparison of the averaged converted oxyHb concentrations between ventral and dorsal PFC regions . Data are expressed as mean ± SE (n=10). ＊ P<0.05. See text for details.


4. Discussion

The present study revealed that gum chewing for 20 min evoked a significant increase in oxyHb concentration in the ventral part of prefrontal cortex (PFC), and significant suppression of the nociceptive flexion reflex (NFR), accompanied by a significant increase in serotonin (5-HT) level in whole blood. We have previously demonstrated that prolonged rhythmic gum chewing suppresses nociceptive responses via the 5-HT descending inhibitory pathway1. Those previous results were confirmed by the results of the present study. NFR was significantly suppressed following 20 min of gum chewing, and the analgesic effects were accompanied by significant increases in 5-HT level in whole blood. To assess activation of the 5-HT system in humans in this study we measured 5-HT levels in whole blood. This method is based on the hypothesis that activation of the 5-HT system can be evaluated on the basis of an increase in 5-HT level in whole blood. This hypothesis was recently proven by our in vivo animal experiment18, demonstrating that augmented 5-HT within the brain can cross the blood-brain barrier into the bloodstream through 5-HT transporters in rats. It is thus reasonable to speculate that the increase in 5-HT levels in whole blood after gum chewing may be derived from augmented 5-HT levels in the brain, which are induced by increased 5-HT system activation in the brain. We would like to emphasize the fact that we measured 5-HT levels in whole blood, and not in platelet poor plasma containing 5-HT temporally. 5-HT released from the brain into the blood is considered to be quickly taken up into platelets. To assess 5-HT release from the brain, it is necessary to measure 5-HT levels in both the plasma and platelets, that is, whole blood. Because we found augmented 5-HT levels in whole blood, it is likely that prolonged gum chewing induced augmented activity of the 5-HT system in the brain, which was accompanied by suppression of the NFR. The main purpose of this study was to evaluate our hypothesis that the PFC might be one of the possible sources augmenting the 5-HT system in the brain. The rationale for this hypothesis was based on the following anatomical and functional observations. Steinbusch3 revealed two major excitatory inputs in rats : glutamatergic inputs from the PFC and noradrenergic inputs from the locus coeruleus. Regarding the former input from the PFC, Hajós et al.5 showed electrophysiological and neuroanatomical

evidence indicating a direct axonal projection from the PFC to the raphe nuclei in rat. In addition, Celada et al.4 revealed that electrical stimulation of the PFC evoked increased 5-HT neuronal activity of the dorsal raphe nucleus. Based on these observations, we proposed the hypothesis that the PFC would be a possible source augmenting the 5-HT system. The present study revealed that gum chewing for 20 min evoked a significantly increase in oxyHb concentrations in the ventral part of PFC regions. Because there was no significant change in oxyHb concentrations in the dorsal part of PFC regions, it is reasonable to speculate that the activation of the ventral part of PFC was produced by local oxygen demands within the PFC, not by changes in systemic circulation. Regarding activation of the PFC during gum chewing, there have been several previous reports6,7,8. Onozuka et al.6 reported that gum chewing for 4 min induced activation of the right dorsal PFC. Takahashi et al. 8 demonstrated dorsolateral PFC activation by gum chewing for 2 min. Note that these studies showed activation of the dorsal PFC, not the ventral PFC; activation of the dorsal PFC was elicited by a relatively shorter period of gum chewing for 2-4 min. By contrast, Takada et al.7 showed that gum chewing for 14 min evoked activation of the ventral and dorsolateral PFC. We measured oxyHb concentrations in the PFC during prolonged gum chewing for 20 min, because significant NFR suppression started 5–7 min after the onset of gum chewing1. For this reason, prolonged gum chewing for 20 min was used in the present study, with continuous measurements of oxyHb levels during this period. As a result, a significant increase in oxyHb concentrations was found in the ventral part of PFC regions, although t h e r e w a s o n l y a s m a l l i n c r e a s e i n o x y H b concentrations in the dorsal part of PFC regions. These results suggest that prolonged gum chewing would be necessary to induce activation of the ventral part of PFC region. A recent report by Gonçalves 19 revealed the existence of robust PFC projections to the dorsal raphe nucleus, mainly derived from the ventral PFC in rats. This result further supports the current hypothesis that activation of ventral part of PFC by prolonged gum chewing would induce activation of the 5-HT system. In conclusion, the present study revealed that prolonged gum chewing evoked a significant increase in oxyHb concentration in the ventral part of PFC, which causes augmented activity of the 5-HT system and suppression of nociceptive responses.


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