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c-fos Expression in Rat Brain Nuclei Following Incisor Tooth Movement
1 Department of Morphology, Stomatology and Physiology, Correspondence: * corresponding author, mjrocha{at}forp.usp.br
In the rat experimental model, molar tooth movement induced by Waldo’s method is known to cause a temporally and spatially defined pattern of brain neuronal activation. Since orthodontic correction usually involves the entire dental arch, we used a spring-activated appliance to extend the investigation to incisors, and we included brain regions related to antinociception. Adjustment of the non-activated appliance on incisors resulted in c-fos expression in the dorsal raphe, peri-aqueductal gray matter, and the locus coeruleus, in addition to trigeminal sensory subnuclei and the parabrachial nucleus, where neuronal activation has already been detected in previous studies on molar tooth movement. Appliance activation with a 70-g force resulted in a further increase in Fos-immunoreactive neurons in the trigeminal sensory subnucleus caudalis and in the dorsal raphe. This result suggests that there is a recruitment of neurons related to nociception and to antinociception when tooth movement is increased.
Key Words: Fos • antinociception • peri-aqueductal gray • dorsal raphe • locus coeruleus
Orthodontic tooth movement causes clinical sensations described as discomfort and pain. These sensations usually appear one day after the application of orthodontic force; they last for a few days and then disappear gradually (Ngan et al., 1989; Brown and Moerenhout, 1991). Previous studies correlated these clinical sensations with experimental findings on drastic changes in the distribution and formation of periodontal neural elements (Kato et al., 1996; Kobayashi et al., 1998). Tooth movement can be experimentally induced in rats by a procedure known as Waldo’s method, which consists of inserting a piece of elastic rubber between the upper molars (Waldo, 1953). Using this methodology, several investigators observed a temporally and spatially defined distribution of Fos-like immunoreactive neurons in the subnuclei of the trigeminal complex, in the parabrachial nucleus (PB), amygdala, paraventricular nucleus of the hypothalamus, and the paraventricular nucleus of the thalamus (Kato et al., 1994; Yamashiro et al., 1997, 1998; Aihara et al., 1999; Fujiyoshi et al., 2000; Hiroshima et al., 2001). These pioneering studies established a correlation between brain activation and clinical sensation of discomfort and pain resulting from tooth movement. Since orthodontic correction usually involves the entire dental arch and targets teeth other than the molars, these studies need to be extended. We investigated c-fos expression in response to incisor tooth movement induced by an orthodontic appliance previously activated (or not) with defined force, and compared activated pathways with the ones already reported in studies using Waldo’s method. Furthermore, we decided to extend the investigation to brain regions related to antinociception which were not previously studied in this context, such as the peri-aqueductal gray matter, dorsal raphe, and locus coeruleus.
Animals and Experimental Protocol Thirty male Wistar rats (each weighing from 250 to 300 g) were anesthetized by an intra-muscular injection of ketamine (100 mg/kg) and xylazine (14 mg/kg). We chose this anesthetic mixture because of its negligible effects on c-fos expression in the brainstem (Rocha and Herbert, 1997). The animals were divided into 2 experimental and 2 control groups. In the first experimental group (n = 10), an inactivated orthodontic appliance was fixed to the maxillary incisors of the rats and left there for 3 hrs. We chose this time because previous work with Waldo’s method showed that maximum c-fos expression in brain structures is observed within 2 to 4 hrs of activation (Kato et al., 1994; Hiroshima et al., 2001). In the second experimental group (n = 10), an orthodontic appliance activated with 70 g was fixed to the maxillary incisors of the rats, where it was left for 3 hrs. At the end of the experiment, the animals were re-anesthetized and transcardially perfused with 100 mL of phosphate-buffered saline (PBS 0.01 M, pH 7.4), followed by 400 mL of 4% paraformaldeyde in 0.1 M phosphate buffer. The brains were removed from the skulls, post-fixed in the same fixative for 4 hrs, placed in PBS containing 30% sucrose, and stored at 4°C until cryostat sectioning. Control groups consisted of anesthetized animals without orthodontic appliances (n = 5) or anesthetized animals where the activated appliance was placed on the incisors but was not cemented and was immediately removed (n = 5). All animal experiments were approved by the Ethics Committee on the Use of Experimental Animals of the University of São Paulo, Campus Ribeirão Preto.
Orthodontic Appliance
Fos Immunohistochemistry Coronal sections through the brainstem were cut at 40-µm thickness on a cryostat, and each third section was collected in PBS and processed for Fos immunohistochemistry. Free-floating sections were washed in PBS (0.01 M, pH 7.4) and incubated in PBS containing 3% hydrogen peroxide for 10 min to inactivate endogenous peroxidase activity. After several rinses in PBS for 30 min, the sections were placed in 5% normal goat serum (NGS, Vector, Burlingame, CA, USA) for 45 min and then incubated for 24–48 hrs at 4°C with polyclonal anti-c-fos serum generated in rabbits (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The serum was diluted (1:5000) in PBS containing 1% NGS, 1% bovine serum albumin, and 0.3% Triton X-100. After being rinsed in PBS, the sections were incubated for 1.5 hr at room temperature with biotinylated goat anti-rabbit IgG (1:200; Vector). Subsequently, they were washed in PBS and placed for 30 min in avidin-biotin peroxidase complex (ABC kit, Vectastain, Vector, Burlingame, CA, USA). Labeled neurons were visualized by a 10- to 15-minute incubation with 0.05% 3,3'-diaminobenzidine tetrachloride and 0.001% hydrogen peroxide. The sections were mounted on gelatin-coated slides, dehydrated through an ascending ethanol series, xylene-cleared, and coverslipped with Entellan. Since the antibody used in this study recognizes c-fos and Fos-related proteins, immunoreactive neurons are described as Fos-like immunoreactive (Fos-IR).
Analysis
To examine the effects of anesthesia, we evaluated c-fos expression in the nuclei 3 hrs after intramuscular injection of ketamine/xylazine mixture. No Fos-IR neurons appeared in the trigeminal spinal subnuclei [caudalis (SpVc), interpolaris (SpVi), and oralis (SpVo)], and only a few (mean ± SEM) were seen in the locus coeruleus (LC, 0.6 ± 0.3, n = 3), parabrachial nucleus (PB, 1.2 ± 0.5, n = 5), peri-aqueductal gray matter (PAG, 0.8 ± 0.4, n = 5), and dorsal raphe (DR, 0.6 ± 0.4, n = 5). This shows that ketamine/xylazine is a good anesthetic mixture in tooth movement experiments, because it had no marked effect on its own on c-fos expression in the nuclei cited above. Furthermore, sham rats that received the same anesthesia and were subject to all manipulations associated with appliance adjustment did not show Fos-IR neurons in the SpVi and SpVo, and only a few in the SpVc (0.2 ± 0.2, n = 5). Again, only a few Fos-IR neurons were seen in the LC (0.5 ± 0.2, n = 4), PB (1.2 ± 0.6, n = 5), PAG (0.8 ± 0.3, n =4), and DR (0.7 ± 0.4, n = 4). This demonstrates that appliance adjustment itself had no effect on c-fos expression.
In the experimental groups, however, the orthodontic appliance was able to promote c-fos expression in several nuclei, regardless of whether the appliance was activated or not. Since these nuclei exhibited almost identical Fos-IR labeling in both hemispheres, we chose the better-defined side for counting and photography. In none of the groups (experimental or controls) did we observe c-fos expression in the principal trigeminal and mesencephalic nuclei. In the SpVc, most of the immunoreactive neurons were localized in laminae I and II (Fig. 2
In the group where the appliance was fixed without prior activation, the largest number (mean ± SEM) of labeled neurons was detected in the DR (29.0 ± 4.0), followed by the SpVc (28.7 ± 2.6), PAG (25.0 ± 4.2), and PB (18.2 ± 3.6). Fewer labeled neurons were detected in the LC (10.3 ± 1.2) and SpVi (8.0 ± 1.2). When the appliance was previously activated with a force of 70 g, a further increase in Fos-IR neurons was detected in the DR (44.3 ± 3.5, P < 0.05) and in the SpVc (37.7 ± 3.1, P< 0.05). No statistical difference was observed in the PAG (28.1 ± 2.9), PB (27.8 ± 3.4), SpVi (9.5 ± 1.4), and LC (9.6 ± 1.6) (see Fig. 4  ).
The present study shows that incisor movement induced by a three-hour application of an orthodontic appliance resulted in neuronal activation in several brain structures. A substantial number of Fos-IR neurons could be seen bilaterally in the spinal trigeminal caudalis, parabrachial nucleus, peri-aqueductal gray matter, and dorsal raphe. Few labeled neurons were seen in the trigeminal interpolaris and locus coeruleus, and none at all in the principal and mesencephalic nuclei or in the trigeminal oralis. The observation that neurons already expressed c-fos when the appliance was fixed but not activated indicates that it is not only an irritation of intra-oral structures by the spring located at the palate that may activate sensory neurons but, rather, that the simple adjustment of the orthodontic bands can already induce some tooth movement by a mechanical action of the orthodontic bands on periodontal ligaments. Accordingly, activation of the appliance with 70 g of force induced a further increase in the number of Fos-IR neurons in the spinal trigeminal caudalis and dorsal raphe. The spinal trigeminal caudalis (SpVc) is one of the important relay nuclei for processing oro-facial sensory information, and previous studies have shown that experimental tooth movement can induce c-fos expression in this nucleus. The labeled neurons were mainly located in the superficial laminae (laminae I-II), in the dorso-medial and -ventral edges of the nucleus, predominantly near the obex, and also in the transitional zone to the interpolaris (Kato et al 1994; Yamashiro et al., 1998; Aihara et al., 1999; Fujiyoshi et al., 2000). Furthermore, pre-treatment with morphine significantly reduced the induction of c-fos in this layer, and naloxone reversed this effect (Aihara et al., 1999). These superficial laminae of the SpVc are known to contain nociceptive-specific neurons (Dawson et al., 1980; Hu and Sessle, 1984). Therefore, our findings suggest that experimental incisor tooth movement caused by the appliance evoked nociception that was increased when orthodontic force was applied. Experimental molar tooth movement has been shown to induce c-fos expression in the lateral PB (Yamashiro et al., 1997; Hiroshima et al., 2001). This brainstem nucleus is innervated by layer I of SpVc (Jasmin et al., 1997) and is activated by trigeminal mediated sensations, particularly nociception (Bester et al., 1997). The reduction of this expression by morphine treatment which could be reversed by pre-treatment with naloxone suggests that this neuronal activation is, at least partly, due to noxious stimulation (Hiroshima et al., 2001). Moreover, since the PB is a relay area for the central autonomic/emotional control circuitry, it is possible that cardiovascular changes that may occur in response to tooth movement can also contribute to the c-fos expression that we observed in this nucleus. However, force application and consequent further increase in tooth movement were not able to increase PB c-fos expression, as they did in the SpVc and dorsal raphe (DR). The descending pathways modulating pain are composed of several neural connections that form a circuitry (Millan, 2002). The DR is an important nucleus in pain modulation and participates in a central antinociception circuitry together with the PAG and LC (Li et al., 1993; Wang and Nakai, 1994; Stamford, 1995). This latter nucleus is a source of noradrenergic input to the SpVc (Fritschy and Grzanna, 1990; Simpson et al., 1997). Since the ventro-lateral part of the DR also projects to the SpVc and LC, its neurons could affect pain transmission in the caudalis directly, as well as indirectly via the LC pathway (Klatt et al., 1988; Li et al., 1993; TerHorst et al., 2001). The PAG can also send descending inhibitory fibers directly to the trigeminal sensory complex in the rat (Morgan et al., 1997). Some of these fibers have serotonin as mediator, and nociception induced by experimental tooth movement has recently been shown to activate the bulbospinal serotonergic pathway (Yamashiro et al., 2001). So far, only two studies, in addition to ours, report findings on c-fos expression in the SpVi following experimental tooth movement. Yamashiro et al.(1997) reported finding a few labeled neurons following a 24-hour molar tooth movement experiment. In contrast, Kato et al.(1994) did not detect immunoreaction in this structure. This subnucleus, which is adjacent to the caudalis, receives a heavy input from pulpal afferents (Marfurt and Turner, 1984). It is possible that incisor tooth movement stimulated afferent dental pulp fibers, and this could account for the slightly elevated number of labeled neurons observed in our experiments. We did not see any labeled neurons in the SpVo, neither in the mesencephalic nucleus nor in the principal trigeminal. This is in accordance with results from investigators using Waldo’s method (Kato et al., 1994; Aihara et al., 1999). Yet, c-fos expression was seen to be up-regulated in the SpVo only 12 hrs after the onset of experimental tooth movement, suggesting that a cumulative effect of primary nociceptor activation might be necessary for c-fos induction (Fujiyoshi et al., 2000). In conclusion, our results suggest that sensory information derived from incisor tooth movement activates neurons related to antinociception, in addition to neurons already reported to be activated following molar tooth movement. Furthermore, we found an apparent recruitment of neurons in the trigeminal caudalis and dorsal raphe when tooth movement was increased.
We thank Nadir M. Fernandes for technical assistance, Christie R.A. Leite Panissi and Leda Menescal de Oliveira for critically reading the manuscript, and Klaus Hartfelder for language corrections. Luis Antonio Salata provided access to the image analysis system. The project received financial support from CNPq and FAPESP. Received for publication March 19, 2003. Revision received September 25, 2003. Accepted for publication September 29, 2003.
Journal of Dental Research, Vol. 83, No. 1,
50-54 (2004) This article has been cited by other articles:
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