Occlusal Trauma Induces Neuroimmune Crosstalk for a Pain State.

Temporomandibular joint (TMJ) disorder caused by occlusal trauma is one of the most controversial topics in dentistry. Experimental traumatic occlusion (ETO) induced by metal crowns cemented to mandibular first molars in rats causes a long-lasting nociceptive response. This study aimed to elucidate whether ETO generates an increase in inflammatory mediators in the TMJ. In addition, the impact of ETO on trigeminal ganglia, neurotransmitter release, and satellite glial cell (SGC) activation was investigated. ELISA revealed enhanced inflammatory mediators, including TNF-α, IL-1ß, IL-6, CX3CL1, and ADAM-17 by Western blotting, in periarticular TMJ tissue after 28 d of ETO. In the trigeminal ganglia, ETO groups increased the release of the neurotransmitters substance P and glutamate. Overexpression of the AMPA receptor and upregulation of NMDA were observed in the 0.4- and 0.7-mm ETO groups, respectively, highlighting enhanced neuronal excitation. Increased IL-1ß and COX-2 mRNA levels in the 0.7-mm ETO group confirmed trigeminal ganglia SGC activation. Immunofluorescence and electrophoresis of SGC revealed increased pERK expression in the 0.7-mm ETO group. ERK phosphorylation was shown to be nociceptive specific, with its upregulation occurring in cases of chronic inflammatory pain. Increased PKA mRNA levels were observed in the 0.4-mm ETO group, while CREB mRNA levels were upregulated for both ETO groups. Electrophoresis showed overexpression of sodium channel Nav 1.7 in the 0.7-mm ETO group, while immunofluorescence revealed that Nav 1.7 is expressed in sensory trigeminal ganglia cells. The results of this study suggest that occlusal trauma induces neuroimmune crosstalk, with synthesis of proinflammatory/pronociceptive mediators, which increases neuronal activity in trigeminal ganglia via the activation of an inflammatory response cascade to develop a persistent neuroinflammatory state that leads to central sensitization.

Micro-computed tomography for evaluating alveolar bone resorption induced by hyperocclusion.

PURPOSE: Occlusal trauma, resulting in the destruction of alveolar bone, is a form of periodontal disease caused by excessive mechanical stress (MS) during hyperocclusion. Previously, we showed that CC chemokine ligand (CCL) 2/CCR2 receptor axis plays a crucial role in MS-dependent osteoclastogenesis. However, in the previous work, we were unable to precisely measure changes in alveolar bone profiles. In the present study, we sought to establish a precise method for evaluating alveolar bone resorption induced by hyperocclusion using micro-computed tomography. METHODS: Under anesthesia, a stainless steel wire was attached to the molars of 5-week-old C57/BL6 wild-type (WT) mice, CCL2-/- mice, and CCR2-/-mice to induce occlusal force overload. At days 0 and 7, hard tissue samples were harvested and analyzed by micro-computed tomography. RESULTS: In the WT mice, bone mineral density of the alveolar bone was significantly decreased at day 7 as compared with day 0, with marked alveolar bone resorption observed. Similarly, significant alveolar bone resorption was observed in the CCL2-/- and CCR2-/- mice at day 7 as compared with day 0. CONCLUSIONS: Micro-computed tomographic images can be used to measure changes in bone mineral density in a mouse model of hyperocclusion. This method may be useful for further investigating bone changes in other periodontal disease research fields.

Angiogenic mechanisms of human dental pulp and their relationship with substance P expression in response to occlusal trauma.

Angiogenesis is the formation of new blood vessels based on a pre-existing vasculature. It comprises two processes, sprouting of endothelial cells and the division of vessels due to abnormal growth of the microvasculature. It has been demonstrated that substance P (SP) can induce angiogenesis either by modulating endothelial cell growth (direct mechanism) or by attracting cells with angiogenic potential to the injury site (indirect mechanism). Therefore, the purpose of this article is to review the angiogenic mechanisms that regulate mineralized tissue formation in human dental pulp tissue and their relationship with SP expression as a defence response to stimuli such as the masticatory function and occlusal trauma. Articles included in this review were searched in PubMed, Scopus and ISI Web of Science databases, combining the following keywords: human dentine pulp, angiogenesis, angiogenic growth factors, neuropeptides, substance P, neurogenic inflammation, dentine matrix, dentinogenesis, occlusal trauma and dental occlusion. It is concluded that human dental pulp tissue responds to occlusal trauma and masticatory function with a neurogenic inflammatory phenomenon in which SP plays an important role in the direct and indirect mechanisms of angiogenesis by the action evoked via NK1 receptors at different cells, such as fibroblasts, endothelial and inflammatory cells, leading to new blood vessel formation which are needed to stimulate mineralized tissue formation as a defence mechanism.

Upregulation of the Purinergic Receptor Subtype P2X3 in the Trigeminal Ganglion Is Involved in Orofacial Pain Induced by Occlusal Interference in Rats.

AIMS: To evaluate whether the purinergic receptor subtype P2X3 (P2X3R) in trigeminal ganglion (TG) neurons is involved in hyperalgesia of the temporomandibular joints (TMJs) and masseter muscles associated with placement of an occlusal interference. METHODS: Forty-five rats were randomized into five groups (ie, for days 1, 3, 7, 14, or 28; nine rats per group). Six rats from each group were chosen to receive the occlusal interference, and the remaining three rats were sham-treated controls. On days 1, 3, 7, 14, and 28 after placement of the occlusal interference, the mechanical pain threshold (MPT) to stimulation of the TMJs or masseter muscles was examined using von Frey filaments. Seven days after the occlusal interference placement, changes in MPT were tested after administration of the P2X3R antagonist A-317491 into the TMJs and masseter muscles (60 µg/site) in six rats. The expression of P2X3R in the TGs was investigated by immunohistochemistry and quantitative polymerase chain reaction (qPCR). Retrograde tracing was combined with immunofluorescence to identify TMJ and masseter muscle afferent neurons in the TGs of six premature rats. RESULTS: The TMJ and masseter muscle MPTs were decreased after placement of the occlusal interference, and the P2X3R antagonist reversed the mechanical hyperalgesia that was caused by the occlusal interference placement. The frequency of P2X3R-immunoreactive cells increased in small-sized neurons in the TG after occlusal interference. By contrast, there was no increase in medium-sized TG neurons. P2X3R mRNA increased on day 3. Retrograde tracing indicated that the TMJ and masseter muscle afferent neurons in the TG expressed P2X3R. CONCLUSION: Upregulated P2X3R expression in the TG may contribute to orofacial pain development induced by an occlusal interference. P2X3R may be a therapeutic target for chronic TMJ or masseter muscle pain.

Effect of experimentally induced occlusal trauma on substance p expression in human dental pulp and periodontal ligament.

INTRODUCTION: The purpose of this study was to quantify the effect of occlusal trauma experimentally induced with occlusal interferences on substance P (SP) expression in healthy human dental pulp and periodontal ligament. METHODS: Twenty-eight human dental pulp and periodontal ligament samples were obtained from healthy premolars in which extraction was indicated for orthodontic reasons. Before extraction, occlusal trauma was induced with experimental occlusal interferences in half of these premolars by placing a resin block over their occlusal surface and submitting patients to chew gum for 30 minutes. The remaining healthy premolars were extracted without occlusal trauma and served as a control group. All dental pulp and periodontal ligament samples were processed, and SP was measured by radioimmunoassay. RESULTS: There was 45% and 120% greater SP expression in dental pulp and periodontal ligament, respectively, of teeth with experimentally induced occlusal trauma. Paired t test showed statistically significant differences for both human dental pulp and periodontal ligament (P = .02 and P < .001, respectively) of teeth submitted to occlusal trauma when compared with control group values. CONCLUSIONS: SP expression in human dental pulp and periodontal ligament increases when teeth are submitted to occlusal trauma experimentally induced with occlusal interferences.

Hyperocclusion stimulates osteoclastogenesis via CCL2 expression.

Excessive mechanical stress (MS) during hyperocclusion is known to result in disappearance of the alveolar hard line, enlargement of the periodontal ligament (PDL) space, and destruction of alveolar bone, leading to occlusal traumatism. We hypothesized that MS induces expression of osteoclastogenesis-associated chemokines in PDL tissue, resulting in chemotaxis and osteoclastogenesis during occlusal traumatism. We examined the effect of MS on relationships between chemokine expression and osteoclastogenesis using in vivo and in vitro hyperocclusion models. In an in vitro model, intermittent stretching-induced MS was shown to up-regulate the expression of CC chemokine ligand (CCL)2, CCL3, and CCL5 in PDL cells. The expression levels of CCL2 in PDL tissues, its receptor CCR2 in pre-osteoclasts, and tartrate-resistant acid-phosphatase-positive cells in alveolar bone were significantly up-regulated 4-7 days after excessive MS during hyperocclusion in in vivo rodent models. Hyperocclusion predominantly induced CCL2 expression in PDL tissues and promoted chemotaxis and osteoclastogenesis, leading to MS-dependent alveolar bone destruction during occlusal traumatism.

The effect of experimental occlusal interferences on nerve growth factor levels in periodontal tissues.

OBJECTIVE: To test the hypothesis that experimental occlusal interferences increase the nerve growth factor (NGF) levels in periodontal tissues and cause an up-regulation of preprotachykinin-A (PPTA) mRNA. BACKGROUND: NGF is related to hyperalgesia and inflammation. PPTA mRNA, a primer of substance P, is a possible factor in the aetiology of pain. METHODS: Experimental interferences were created by placing inlays in the right maxillary molars of 15 dogs. The right side molars formed the experimental group. The left side molars served as controls. Three dogs with cavities prepared without changing the occlusion formed a sham group. The dogs in the first group were sacrificed, 3 at each time, after 3, 7, 14, 30, and 60 days. The sham group was sacrificed after 14 days. The levels of NGF in periodontal tissues and PPTA mRNA in the trigeminal ganglions were detected by ELISA and TR-PCR. Comparisons were made with paired t-tests and a multivariate MANOVA test. RESULTS: On all measurement days, there were higher levels of NGF mRNA, PPTA mRNA, and NGF on the experimental than on the control side in 14 of 15 comparisons and in the sham group. NGF production in periodontium was time-dependent. No differences in NGF protein levels were observed between the control and the sham groups. CONCLUSION: The results which need confirmation in further tests are of clinical interest. They indicate that occlusal experimental interferences may be an etiologic factor in oral facial pain by increasing mRNA and NGF protein levels in the periodontal tissues.

Hyperalgesia in response to traumatic occlusion and GFAP expression in rat parabrachial [correction of parabranchial] nucleus: modulation with fluorocitrate.

We have examined, by immunocytochemical methods and nociceptive behavior assessment in rats, whether astrocytes in the parabrachial nucleus (PBN) are involved in the regulation of traumatic occlusion. The expression of glial fibrillary acidic protein (GFAP) in PBN of ipsilateral and contralateral sides was up-regulated 4 h after occlusal changes in molars, reached peak levels at 24 h, and was then gradually down-regulated. PBN astrocytes activated by traumatic occlusion were found to have enlarged cell bodies and thickened processes within 8 h. An inhibitor of glia metabolism (FCA, fluorocitrate) reduced astrocyte activation and significantly attenuated the development of pain hypersensitivity in this model. The results suggested that the GFAP-immunoreactive astrocytes in PBN within the bridge of Varolius were activated by traumatic occlusion, and that they were involved in the transmission and modulation of nociceptive information in the central nervous system. However, although astrocytes in PBN are thus probably involved in causing post-occlusal hyperalgesia, we have not been able to exclude that astrocytes at other locations also contribute to this effect.

Biting suppresses stress-induced expression of corticotropin-releasing factor (CRF) in the rat hypothalamus.

Corticotropin-releasing factor (CRF) expressed in the hypothalamus plays an important role in mediating behavioral responses to stressors. Restraining the body of an animal has been shown to activate and induce an enhanced expression of CRF in paraventricular neurons of the rat hypothalamus. Since aggressive biting behavior is known to suppress stress-induced noradrenaline secretion in the central nervous system and the formation of gastric ulcers, we investigated the effect of biting on restraint-induced CRF expression in the rat hypothalamus. The number of CRF-expressing neurons in the paraventricular nucleus increased significantly after short time restraint (30 or 60 min) followed by a 180-minute post-restraint period. Biting of a wooden stick during the restraint stress significantly suppressed the restraint-induced enhancement of CRF expression in the paraventricular nucleus. These observations suggest a possible anti-stress effect of biting and an important role of para-functional masticatory activity in coping with stressful events.

[A study of calcitonin gene-related peptide-immunoreactive nerve fibers of rat molar pulp during traumatic occlusion and after removal].

OBJECTIVE: To study the effect of traumatic occlusion on CGRP-immunoreactive (CGRP-IR) nerve fibres in rat molar pulp and observe the recovery of CGRP-IR nerve fibres after removal of traumatic occlusion. METHODS: To observe immunohistochemically the change of CGRP-IR nerve fibres in molar pulp during traumatic occlusion and after removal. RESULTS: The increase of number, density and morphology of CGRP-IR nerve fibres in traumatic occlusion group was more than in control group, however, the changes of CGRP-IR nerve fibres in removal of traumatic occlusion group were less than in control group. CONCLUSIONS: The changes of CGRP-IR nerve fibres in number, morphology, and density are induced by traumatic occlusion in rat molar pulp, however, the nerve fibres recover to normal by removal of traumatic occlusion.

Occlusal hypofunction causes changes of proteoglycan content in the rat periodontal ligament.

The biological functions of proteoglycans and glycosaminoglycans are closely associated with mechanical stress on the tissue. In order to reveal the relationship between proteoglycans in the periodontal ligament and mechanical stress such as occlusal stimuli, occlusal hypofunction of rat unilateral mandibular molars was induced by extraction of the opposing first, second and third maxillary molars. Immunohistochemical analyses were performed using antibodies for chondroitin sulfate, decorin, biglycan, heparan sulfate and keratan sulfate, and hyaluronic acid-binding protein. Chondroitin sulfate, observed more strongly in the cervical side than in the apical side of the periodontal ligament of the unextracted sides of mandible, and uniformly present in the extracellular matrix of the periodontal ligament, decreased significantly from 1 wk post-extraction of the antagonists, with a decrease in thickness and disarrangement in fibrous components. Decorin core protein, uniformly present in the periodontal ligament of the unextracted sides, decreased as early on as 2 d post-extraction. Heparan sulfate, mainly localized on the cell surface of vascular endothelial cells and osteoclastic cells as well as in the extracellular matrix of the unextracted sides, decreased significantly in association with the decreased number of blood vessels and osteoclastic cells as early on as 2 d post-extraction. Biglycan, keratan sulfate and hyaluronic acid, uniformly distributed in the periodontal ligament of the unextracted sides, showed little change after the extraction. These results demonstrate that occlusal hypofunction causes tissue remodeling of the periodontal ligament, with a significant decrease of chondroitin sulfate, decorin and heparan sulfate.

Occlusal disharmonies modulate central catecholaminergic activity in the rat.

Occlusal disharmonies have classically been thought to be involved in the etiopathogenesis of bruxism, as have, more recently, alterations in central neurotransmission, particularly dopaminergic neurotransmission. However, the connection between these two factors has still not been established. In this study, we assessed the effects of diverse occlusal disharmonies, maintained for either 1 day or 14 days, on neurochemical indices of dopaminergic and noradrenergic activity in the striatum, frontal cortex, and hypothalamus of the rat. The in vivo activity of tyrosine hydroxylase, determined as the accumulation of 3,4-dihydroxyphenylalanine (DOPA), 30 min after the administration of 3-hydroxybenzylhydrazine, a DOPA decarboxylase inhibitor, and dopamine and noradrenaline contents were quantified by high-performance liquid chromatography with electrochemical detection. The wearing of an acrylic cap on both lower incisors for 1 day induced a significant increase in DOPA accumulation in the regions analyzed, with parallel increases in dopamine levels in the hypothalamus and dopamine and noradrenaline in the frontal cortex. After the cap was maintained for 14 days, DOPA accumulation tended to return to control values, except in the left striatum, thereby causing an imbalance between hemispheres. In contrast, 1 or 14 days after the lower left and the upper right incisors were cut, less pronounced changes in catecholaminergic neurotransmission were found in the brain areas studied. Moreover, the cutting of one lower incisor did not modify either DOPA accumulation or dopamine and noradrenaline contents in the striatum or hypothalamus. These results provide experimental evidence of a modulation of central catecholaminergic neurotransmission by occlusal disharmonies, being dependent on the nature of the incisal alteration and on the time during which it was maintained.

Responses of periodontal nerve terminals to experimentally induced occlusal trauma in rat molars: an immunohistochemical study using PGP 9.5 antibody.

The response of periodontal nerves to experimentally induced occlusal trauma in rat molars was assessed by immunohistochemistry for protein gene product 9.5 (PGP 9.5) at light and electron microscopic levels, and by computerized image analysis. The occlusal surface on the left upper first molar of 8-wk-old male Wistar rats was raised approximately 1 mm under ether anaesthesia. The rats were perfusion-fixed on d 1, 2, 3, 4, and 7 after bite-raising and then decalcified for 2-3 wk. Frozen sagittal cryostat sections were stained by the avidin-biotin complex method. By the second day after bite-raising many Ruffini endings were swollen and their outline unclear at the light microscopic level. Transmission electron microscopy disclosed PGP 9.5 reaction products within Ruffini endings that had unusually long cytoplasmic projections extending through enlarged slits of the Schwann sheaths and also diffuse extracellular PGP 9.5-immunoreactivity near the Ruffini endings. From d 2 to 4, thin nerve fibres on the pressure side of the periodontal ligament were orientated irregularly and had a prominent beaded appearance. An increase in beaded nerve terminals occurred at d 2-4 post elevation, and decreased later. These results suggest that occlusal trauma indices specific changes in the distribution and shape of nerve terminals in the periodontal ligament.

Effect of traumatic occlusion on CGRP and SP immunoreactive nerve fibre morphology in rat molar pulp and periodontium.

Traumatic occlusion provides a trauma that affects the whole tooth and its supporting tissues. To study the effect of this trauma on CGRP and SP immunoreactive nerve morphology in pulp and periodontium, traumatic occlusion was induced in 2-months-old rats. The occlusal surface of the first maxillary molar in 30 rats were unilaterally raised 1 mm with a composite material. At different observation periods up to 30 days, the rats were transcardiacally perfused, the jaws demineralized, sectioned and processed for immunohistochemistry with the avidin-biotin-peroxidase method. Changes in nerve morphology, distribution and density in first and second molars and their supporting tissues were analyzed and compared in experimental (n = 30) and control rats (n = 14). Already after 5 days with traumatic occlusion, 22% of the experimental teeth had increased density of CGRP and SP immunoreactive nerves locally in gingiva, the periodontal ligament and the pulp, while in 15%, axonal proliferation and changed nerve morphology were found in the whole pulp (severe reaction). During a 20-day period, the pulpal nerve reactions progressed and included the whole pulp in 46% of the experimental teeth. The periodontal nerve responses were still localized only to the cervical and apical regions, and they remained local in these areas throughout the experimental periods. After 20 days the number of teeth with severe nerve changes seemed to decrease. The study shows that an unilateral change in occlusion of the first molar initiate nerve responses in the total molar dentition. In this experimental model the pulpal axons containing CGRP and SP reacted more serious to occlusal trauma than the nerves in the periodontium. The results indicate that the nerve changes in some cases might be transient.

[Carbohydrate-energy metabolism of periodontal bony tissue in the artificial modelling of traumatic occlusion]. / Uglevodno-énergeticheskii obmen kostnoi tkani parodonta pri iskusstvennom modelirovanii travmaticheskoi okkliuzii.

Experiments with 40 dogs have demonstrated that overexercise of the maxillodental system for 30 days results in reduction of the bioenergy potential of the periodontal bone tissue of the overloaded teeth. When the traumatic overload was eliminated for the next 30 days, the functional parameters gradually recovered, but the biochemical characteristics of the regenerated bone did not fully normalize.

The uptake of horseradish peroxidase in monkey temporomandibular joint synovium after occlusal alteration.

We conducted an electron microscopic study on the alterations in the temporomandibular joint (TMJ) synovium of the monkey after it was subjected to unilateral bite-raising, by means of a maxillary occlusal splint. The uptake of intravenously injected horseradish peroxidase (HRP) by type A cells was demonstrated in the disturbed TMJ. The uptake of HRP decreased three to six wk after occlusal alteration, and was the same as in normal type A synovial cells after 16 wk.