Effect and timing of parathyroid hormone analog administration for preventing medication-related osteonecrosis of the jaws in a murine model.

The aim of this study was to investigate the effect and timing of recombinant human parathyroid hormone analog (PTH) administration for preventing medication-related osteonecrosis of the jaws (MRONJ) using a murine model. After standardized MRONJ induction using zoledronic acid and dexamethasone injections, 48 female Sprague-Dawley rats were divided into four groups according to the timing of PTH administration before or after dental extraction. Rats were euthanized 3 weeks after dental extraction, followed by clinical and histologic analyses. No clinical improvements were observed in the preoperative and postoperative PTH groups, compared to controls (p = 0.638 and 0.496, respectively). However, on histological analysis, the number of empty lacunae reduced significantly, and the number of blood vessels increased in the preoperative PTH group (p = 0.004 and 0.002, respectively). The postoperative PTH group did not show significant differences for empty lacunae and blood vessels compared to controls (p = 0.075 and 0.194, respectively). The reduction in the empty lacunae and the increase in the blood vessels in the preoperative PTH group were significant compared to other groups, suggesting more viable bone tissue in this group. In perspective, preoperative PTH use may represent a better prophylactic regimen for preventing the occurrence of MRONJ after traumatic dental or surgical procedures, especially in patients with a history of long-term bisphosphonate administration or at high risk of developing MRONJ. However, the findings should be proven in further studies on other animals followed by clinical trials.

A Retrospective CBCT Study of the Relationship between Mandibular Symphysis Bone Density and Mandibular Growth Direction.

OBJECTIVE: The objective of this retrospective study was to investigate the relationship between mandibular symphysis bone density (BD) and mandibular growth direction in adolescent patients by facilitating the measurement of cortical and cancellous BDs at the mandibular symphysis using cone beam computed tomography (CBCT). STUDY DESIGN: 224 adolescent patients (98 males and 126 females) were categorized by sex, age, and mandibular growth direction. Cortical and cancellous BDs were measured along with a sagittal slice at multiple locations. RESULTS: Females exhibited higher cortical BD than males at menton (Me, P =0.002). Patients with a posterior growth direction exhibited a higher cortical BD than those with anterior and normal growth direction at Me (P <0.021, P <0.001, respectively), pogonion (Pog, P =0.037, P =0.037, respectively) and genion (Ge, P =0.007, P =0.008, respectively). Patients with a posterior growth direction exhibited a higher cortical BD than those with anterior growth direction at B point (P =0.009). CONCLUSIONS: Significant differences in BD were identified across anthropometric categories. These findings may be useful in determining mandibular growth direction in adolescents.

Effect of Hyaluronic Acid Filler Injection on the Interdental Papilla in a Mouse Model of Open Gingival Embrasure.

The black triangle resulting from interdental papilla (IDP) loss is associated with poor aesthetics and difficulty in pronunciation and food impaction. There is limited knowledge of gingival tissue inflammatory response to hyaluronic acid (HA) filler injection, a minimally invasive IDP reconstruction method. This study aimed to examine the morphological and histological changes in IDP and the inflammatory cytokine localization to the IDP post-HA filler injection using an open gingival embrasure (OGE) mouse model. Mice from the control, sham, and OGE groups were attached with reference, inactive, and activated wires for 5 days, respectively. The degree of IDP loss was determined based on the spring-papilla distance (SPD). Morphological and histological changes in the OGE group injected with phosphate-buffered saline (PBS) or HA fillers were examined on days 2 and 7 post-injection. Immunohistochemical analysis was performed to determine the localization patterns of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, myeloperoxidase (MPO), and Ki67. Five days post-wire attachment, the control and OGE groups exhibited a significantly higher SPD than the sham group (p < 0.0167). The SPD of the HA filler injection group was significantly lower than that of the PBS injection group on days 2, 4, and 7 post-injection (p < 0.05). The IDP of the OGE group was wide and flat. HA filler was stable in the connective tissue underlying the epithelial tissue even on day 7 post-injection. TNF-α, IL-1ß, IL-6, MPO, and Ki67 were highly localized to the connective tissue surrounding the filler on day 2, which decreased on day 7 post-injection. Thus, HA filler can safely and successfully reconstruct the IDP in cases of OGE.

Histological Alterations from Condyle Repositioning with Functional Appliances in Rats.

OBJECTIVE: This study was designed to assess the morphological and histological alterations of the condyle of rats undergoing forward mandibular repositioning via functional appliance. MATERIALS AND METHODS: Functional appliances were mounted onto the upper jaws of rats. Morphological analysis was conducted on micro-CT images of sacrificed animals. Histological changes in condyle were examined by immunohistochemistry using proliferating cell nuclear antigen (PCNA), matrix metalloproteases (MMPs), vascular endothelial growth factor (VEGF), tissue inhibitors of matrix metalloproteinases (TIMP-1), interleukin 1b (IL-1ß), Aggrecan and Type II collagen. Osteoclast activity was identified by tartrate-resistant acid phosphatase (TRAP) staining. RESULTS: Morphological analysis confirmed the forward positioning of the condyles of rats by the appliance, but the position gradually returned to normal on days 14 after treatment. An increase in PCNA positive cells was observed in the posterior region of the condyles on days 7, whereas PCNA positive cells decreased in the anterior region. Aggrecan and Type II collagen localization increased in the posterior region throughout the entire period, but decreased in the anterior region on days 14. In both regions, IL-1ß and VEGF localization was significantly increased for 14 days while MMPs localization was evident throughout the entire period. The TRAP positive cells were significantly elevated on days 3 and 7. CONCLUSIONS: These results suggest that the functional appliance therapy induces significant morphological and histological changes in the anterior and posterior regions of the condyle and subsequently causes adaptive cellular functions such as chondrocyte differentiation and cartilage matrix formation.

Cudraxanthone H Induces Growth Inhibition and Apoptosis in Oral Cancer Cells via NF-κB and PIN1 Pathways.

Cudraxanthone H (CH) is a natural compound isolated from a methanol extract of the root bark of Cudrania tricuspidata, a herbal plant also known as Moraceae. However, the effect of CH on human cancer cells has not been reported previously. The aim of this study was to investigate the anticancer effects and mechanism of action of CH on oral squamous cell carcinoma (OSCC) cells. CH exerted significant antiproliferative effects on OSCC cells in dose- and time-dependent manners. CH also induced apoptosis in OSCC cells, as evidenced by an increased percentage of cells in the sub-G1 phase of the cell cycle, annexin V-positive/propidium iodide-negative cells, and nuclear morphology. This antiproliferative effect of CH was associated with a marked reduction in the expression of cyclin D1 and cyclin E, with a concomitant induction of cyclin-dependent kinase inhibitor (CDKI) expression (p21 and p27). CH inhibited the phosphorylation and degradation of IκB-α and the nuclear translocation of NF-κB p65. Furthermore, CH treatment down-regulated PIN1 mRNA and protein expression in a dose-dependent manner. PIN1 overexpression by infection with adenovirus-PIN1 (Ad-PIN1) attenuated the CH-induced growth-inhibiting and apoptosis-inducing effects, blocked CH-enhanced CDKI expression and restored cyclin levels. In contrast, inhibiting PIN1 expression via juglone exerted the opposite effects. The present study is the first to demonstrate antiproliferative and apoptosis-inducing effects of CH, which exerts its effects by inhibiting NF-κB and PIN1. These data suggest that it might be a novel alternative chemotherapeutic agent for use in the treatment of oral cancer.

Effects of sodium tri- and hexametaphosphate on proliferation, differentiation, and angiogenic potential of human dental pulp cells.

INTRODUCTION: This study aimed to investigate the effects of 2 inorganic polyphosphates (poly[P]) are linear polymers of orthophosphate (Pi) residues linked by energy-rich phosphoanhydride poly(P) compounds, sodium triphosphate (STP, Na5P3O10) and sodium hexametaphosphate (SHMP, Na15P13O40 âˆ¼ Na20P18O40) on the proliferation, odontoblastic differentiation, and angiogenic potential of human dental pulp cells (HDPCs). METHODS: Differentiation was measured by alkaline phosphatase activity, calcified nodule formation by alizarin red staining, and marker messenger RNA (mRNA) levels by reverse-transcription polymerase chain reaction. In vitro angiogenesis was quantified by migration, mRNA levels of angiogenic genes, and endothelial tube formation. RESULTS: STP and SHMP dose dependently increased the proliferation and ALP activity and enhanced mineralized nodule formation and odontoblast marker mRNAs of HDPCs. STP and SHMP resulted in the up-regulation of angiogenic genes in HDPCs. Endothelial cells treated with conditioned medium collected from STP- and SHMP-exposed HDPCs showed an increase in migration and capillary tube formation. Knockdown of the expression of the genes encoding of inorganic pyrophosphate by small interfering RNA attenuated the STP- and SHMP-induced odontogenic differentiation and angiogenic potential. CONCLUSIONS: This study showed that STP and SHMP promote the growth, differentiation, and angiogenic potential of HDPCs. These results suggest that STP and SHMP may be candidates for dental pulp tissue engineering and regenerative endodontics.

Comparison of the effects of human dental pulp stem cells and human bone marrow-derived mesenchymal stem cells on ischemic human astrocytes in vitro.

This study assesses the cytoprotective effects of human dental pulp stem cells (hDPSCs) and conditioned medium from hDPSCs (CM-hDPSCs) on ischemic human astrocytes (hAs) in vitro compared with human bone marrow-derived mesenchymal stem cells (hMSCs). Ischemia of hAs was induced by oxygen-glucose deprivation (OGD). CM-hDPSCs and hMSCs were collected after 48 hr of culture. Cell death was determined by 3-[4,5-dimethylthialzol-2-yl]-2,5-diphenyltetrazolium bromide and cellular ATP assays. The expression of glial fibrillary acidic protein (GFAP) and musashi-1 as markers of reactive astrogliosis was examined with immunochemical staining. mRNA expression and reactive oxygen species (ROS) were analyzed by RT-PCR and flow cytometry, respectively. OGD increased cytotoxicity in a time-dependent manner and decreased cellular ATP content concomitantly in hAs. Pretreatment and posttreatment with hDPSCs were associated with greater recovery from OGD-induced cytotoxicity in hAs compared with hMSCs. Similarly, CM-hDPSCs had a greater effect on OGD-induced cytotoxicity in a dose-dependent manner. Pre- and posttreatment with CM-hDPSCs or CM-hMSCs attenuated OGD-induced GFAP, nestin, and musashi-1 expression in hAs. Furthermore, treatment of cells with CM-hDPSCs and hMSCs blocked OGD-induced ROS production and interleukin-1ß upregulation. This study demonstrates for the first time that hDPSCs and CM-hDPSCs confer superior cytoprotection against cell death in an in vitro OGD model compared with hMSCs as shown by cell viability assay. Reactive gliosis, ROS production, and inflammatory mediators might contribute to this protective effect. Therefore, hDPSCs could represent an alternative source of cell therapy for ischemic stroke.

Melatonin promotes hepatic differentiation of human dental pulp stem cells: clinical implications for the prevention of liver fibrosis.

Melatonin's effect on hepatic differentiation of stem cells remains unclear. The aim of this study was to investigate the action of melatonin on hepatic differentiation as well as its related signaling pathways of human dental pulp stem cells (hDPSCs) and to examine the therapeutic effects of a combination of melatonin and hDPSC transplantation on carbon tetrachloride (CCl4 )-induced liver fibrosis in mice. In vitro hepatic differentiation was assessed by periodic acid-Schiff (PAS) staining and mRNA expression for hepatocyte markers. Liver fibrosis model was established by injecting 0.5 mL/kg CCl4 followed by treatment with melatonin (5 mg/kg, twice a week) and hDPSCs. In vivo therapeutic effects were evaluated by histopathology and by means of liver function tests including measurement of alanine transaminase (ALT), aspartate transaminase (AST), and ammonia levels. Melatonin promoted hepatic differentiation based on mRNA expression of differentiation markers and PAS-stained glycogen-laden cells. In addition, melatonin increased bone morphogenic protein (BMP)-2 expression and Smad1/5/8 phosphorylation, which was blocked by the BMP antagonist noggin. Furthermore, melatonin activated p38, extracellular signal-regulated kinase (ERK), and nuclear factor-κB (NF-κB) in hDPSCs. Melatonin-induced hepatic differentiation was attenuated by inhibitors of BMP, p38, ERK, and NF-κB. Compared to treatment of CCl4 -injured mice with either melatonin or hDPSC transplantation alone, the combination of melatonin and hDPSC significantly suppressed liver fibrosis and restored ALT, AST, and ammonia levels. For the first time, this study demonstrates that melatonin promotes hepatic differentiation of hDPSCs by modulating the BMP, p38, ERK, and NF-κB pathway. Combined treatment of grafted hDPSCs and melatonin could be a viable approach for the treatment of liver cirrhosis.

Effects of glutamine on proliferation, migration, and differentiation of human dental pulp cells.

INTRODUCTION: Although glutamine (Gln) is mitogenic in various cell types, little is known about its role in human dental pulp cells (HDPCs). This study investigated the effects of Gln on proliferation, migration, and odontoblastic differentiation of HDPCs and the underlying signal pathway mechanisms. METHODS: Growth and migration were assessed by cell counting and colorimetric cell migration kits. Differentiation was measured as alkaline phosphatase activity, calcified nodule formation by alizarin red staining, and marker mRNA expression by reverse transcriptase-polymerase chain reaction (RT-PCR). Chemokine expression was also evaluated by RT-PCR. Signal transduction pathways were examined by RT-PCR and Western blot analysis. RESULTS: Gln dose-dependently increased proliferation, migration, alkaline phosphatase activity, mineralized nodule formation, and odontoblast-marker mRNA of HDPCs. Gln also up-regulated expression of interleukin-6, interleukin-8, MCP-1, MIP-3α, CCL2, CCL20, and CXCL1. Gln increased BMP-2 and BMP-4 mRNA, phosphorylation of Smad 1/5/8, ß-catenin, and key proteins of the Wnt signaling pathway. Furthermore, Gln resulted in up-regulation of extracellular signal-regulated kinase, p38, and c-Jun N-terminal kinase. In addition, noggin, DKK1, inhibitors of p38, ERK, and JNK significantly attenuatted Gln-induced growth, migration, and odontoblastic differentiation. CONCLUSIONS: Collectively, this study demonstrated that Gln promoted growth, migration, and differentiation in HDPCs through the BMP-2, Wnt, and MAPK pathways, leading to improved pulp repair and regeneration.

Localization of ODAM, PCNA, and CK14 in regenerating junctional epithelium during orthodontic tooth movement in rats.

OBJECTIVE: To identify the regenerating junctional epithelium (JE) during orthodontic tooth movement in rats. MATERIALS AND METHODS: Closed-coil springs were used to create a 20 g mesial force to the maxillary first molars. On days 1, 3, 7, 10, and 14 after force application, histologic changes in JE were examined by immunohistochemistry using proliferating cell nuclear antigen (PCNA), odontogenic ameloblast-associated protein (ODAM), and cytokeratin 14 (CK14). RESULTS: On day 1, JE was destroyed and lost attachment to the tooth surface. Cell division activity was rarely observed in JE, and ODAM localization was weakly detected in damaged JE. By day 3, regenerating JE had not fully recovered. High cell proliferation activity and CK14 expression started to appear in most basal cells of JE. ODAM expression was reduced and appeared in a small area. By day 7, JE had almost recovered. Cell proliferation activity was still observed in several basal cells of JE, and ODAM expression was detected among JE cells. CK14 was hardly observed in JE except in the basal cells. By days 10 and 14, regenerated JE appeared. ODAM, PCNA, and CK14 expression was similar to that of the control. CONCLUSIONS: Damaged JE might recover rapidly during orthodontic tooth movement because basal cells of the remaining JE, which show higher proliferation activity, are involved in JE regeneration. Reduced ODAM expression during proliferation of JE cells may increase again after JE regeneration is complete. Therefore, ODAM may be associated with the normal function of JE.

The role of PIN1 on odontogenic and adipogenic differentiation in human dental pulp stem cells.

Recently, the involvement of PIN1, a peptidyl-prolyl cis/trans isomerase, has been reported in age-related bone homeostasis and adipogenesis. However, the role of PIN1 during odontogenic and adipogenic differentiation remains to be fully understood, particularly regarding human dental pulp stem cells (HDPSCs). Thus, in the present study, we have investigated the role of PIN1 in odontogenic and adipogenic differentiation of HDPSCs and signaling pathways possibly involved. PIN1 mRNA and protein level were upregulated in a time-dependent manner during adipogenic differentiation, increasing until 1 day of odontogenic induction and then steadily declined during odontogenic differentiation. Treatment of a known PIN1 inhibitor, juglone, significantly increased odontogenic differentiation as confirmed by alkaline phosphatase (ALP) activity, calcium deposition, and mRNAs induction of odontogenic markers [ALP, osteopontin (OPN), osteocalcin (OCN), dentin sialophosphoprotein (DSPP), and dentin matrix protein 1 (DMP-1)]. On the contrary, adipogenic differentiation was dramatically reduced upon juglone treatment, with concomitant downregulation of lipid droplet accumulation and adipogenic marker genes [peroxisome proliferation-activated receptor gamma (PPARγ), lipoprotein lipase (LPL), and adipocyte fatty acid-binding protein (AP2)]. In contrast to PIN1 inhibition, the overexpression of PIN1 via adenoviral infection (Ad-PIN1) in HDPSCs inhibited odontogenic differentiation but increased adipogenic differentiation, in which stem cell property markers such as stage-specific embryonic antigen-4 (SSEA-4) and STRO-1 were upregulated during odontogenic differentiation but downregulated in adiopogenic differentiation. Consistently, juglone-mediated inhibition of PIN1 augmented the osteogenic medium (OM)-induced activation of bone morphogenetic protein (BMP), Wnt/ß-catenin, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and nuclear factor-kappa B (NF-κB) pathway, which response was reversed by Ad-PIN1. Moreover, juglone blocked the adipogenic induction medium-induced activation of PPARγ, C/EBPα, C/EBPß, ERK, and NF-κB pathways, which was rescued by Ad-PIN1 infection. In summary, the present study shows for the first time that PIN1 acts as an important modulator of odontogenic and adipogenic differentiation of HDPSCs and may have clinical implications for regenerative dentistry.

Localization of osteopontin and osterix in periodontal tissue during orthodontic tooth movement in rats.

OBJECTIVE: To evaluate the localization of osteopontin (OPN) and osterix in periodontal tissue during experimental tooth movement with heavy force in rats. MATERIALS AND METHODS: Nickel-titanium closed-coil springs were used to create a 100 g mesial force to the maxillary first molars. On days 3, 7, 10, and 14 after force application, histological changes in periodontium were examined by immunohistochemistry using proliferating cell nuclear antigen (PCNA), OPN, and osterix. RESULTS: PCNA-positive cells were found close to the alveolar bone and cementum on both sides. OPN-positive cells were observed along the cementing line of the cementum and bone on both sides and also were visible along with newly formed fibers in the periodontal ligament on the tension side. Osterix-positive cells were strongly detected on the surface of the alveolar bone and cementum on both sides. CONCLUSIONS: During tooth movement, periodontal remodeling occurs on both sides. These results indicate that OPN and osterix may play an important role of differentiation and osteoblasts and cementoblasts matrix formation during periodontal tissue remodeling.

The effects of enamel matrix derivative on the proliferation and differentiation of human mesenchymal stem cells.

PURPOSE: This study was designed to investigate the effect of enamel derivative matrix (EMD) on the proliferation, mineralization, and differentiation of human mesenchymal stem cells (hMSCs). MATERIAL AND METHODS: For the proliferation assay, water-soluble tetrazolium salt-8 tests were carried out after culturing for 24 and 48 h. For the evaluation of mineralization, Alizarin red S (ARS) tests were performed after 21 days of culturing in an osteogenic medium. In order to investigate some of the bone-related proteins, namely type I collagen (Col I A2), bone sialoprotein (BSP), and bone gamma-carboxyglutamate (Gla) protein (BGLAP, osteocalcin), real-time polymerase chain reaction (RT-PCR) tests were carried out after 2, 3, and 4 weeks of culturing, respectively. RESULTS: The activity of proliferation and mineralization increased significantly depending on the concentration of EMD (P<0.05). In the control group, the expression of Col I A2 decreased, but EMD enhanced its expression over time and was correlated to the concentration. The amount of expression of BSP in this group increased over time, but EMD strikingly suppressed its expression in the fourth week. As well, the amount of expression of BGLAP increased as the culture duration lengthened in the control group. However, the expression of BGLAP was suppressed in the experimental group with EMD. CONCLUSION: Within the limits of this study, EMD enhanced the proliferation of hMSCs. After evaluation with ARS staining, EMD seemed to enhance mineralization, and the RT-PCR test revealed that EMD promoted early-stage osteoblast differentiation by enhancing Col I A2 expression, but exerted an inhibitory effect on the mineralization by lowering the gene expression of BSP and BGLAP. Mineralized nodules formed with EMD may be composed of substances other than normal bone. Because most of the organic matrix of bone is type I collagen, which acts as the mineralization site, bone or bone-like mineralized mass might have been formed in spite of the different components of the non-collagenous proteins.

Human dental pulp cell culture and cell transplantation with an alginate scaffold.

Many studies on tissue stem cells have been conducted in the field of regenerative medicine, and some studies have indicated that cultured dental pulp mesenchymal cells secrete dentin matrix. In the present study we used alginate as a scaffold to transplant subcultured human dental pulp cells subcutaneously into the backs of nude mice. We found that when beta-glycerophosphate was added to the culture medium, dentin sialophosphoprotein mRNA coding dentin sialoprotein (DSP) was expressed. An increase in alkaline phosphatase, which is an early marker for odontoblast differentiation, was also demonstrated. At 6 weeks after implantation the subcutaneous formation of radio-opaque calcified bodies was observed in situ. Immunohistochemical and fine structure studies identified expression of type I collagen, type III collagen, and DSP in the mineralizing transplants. Isolated odontoblast-like cells initiated dentin-like hard tissue formation and scattered autolyzing apoptotic cells were also observed in the transplants. The study showed that subcultured dental pulp cells actively differentiate into odontoblast-like cells and induce calcification in an alginate scaffold.

A morphological study on the classification of maxillary dental arches.

To evaluate the morphology of dental arches, 62 (male: 36, female: 26) paired casts having normal dentitions and occlusion were selected from 396 (age: 18 to 26 years old; male: 257, female: 139) sets of dental study models. The maxillary dentitions were preliminarily classified as square, round-square, round and round V-shaped arches based on the conventional morphological descriptions. Midpoints of the incisor edge (I1(R), I1(L), I2(R), & I2(L), summits of the cuspids (CR & CL), buccal cusps of the premolars (P1(R), P1(L), P2(R), & P2L), mesial buccal cusps of the first and second molars (M1(R), M1(L), M2(R), & M2(L)), and the midpoint (A) of line I1(R)-I1(L) were designated as reference points. From A, let a vertical line intersected line M2(R)-M2(L) at reference point B. The line A-B intersected C(R)-C(L) at reference point E. We evaluated 1) the protrusion of the cuspids by (1) angle I2(R)-C(R)-P1(R) (angle R) + angle I2(L)-C(L)-P1(L) (angle L); 2) the curvature of the anterior teeth by (2) A-B/C(R)-C(L), (3) 180 degrees - angle C(R)-A-C(L), and (4) A-E/C(R)-C(L); 3) the length to width ratio of the dental arch by (5) A-B/M2(R)-M2(L); 4) the degree of roundness of the maxillary arch by estimation of (6) (rtheta5 - rtheta4)R + (rtheta5 - rtheta4)L; and 5) an item (7) for the differentiation of type I and type II round-square arches by relating the bilateral contour and position of break line P1-P2-M1-M2 (i) to line P1-M2 (ii). The data of items (1), (2), (3), (4), (5), and (6) were further standardized and summarized into three essential principal components: 1) the curvature of the anterior teeth, 2) the curvilinear contour of the dental arch, and 3) the length-to-width ratio of the dental arch. The results indicated that: 1) 60% of the maxillary dentitions were round-square arches which showed no prominent principal component; 2) square maxillary arches distinctly showed a small (1) angle R + angle L; 3) round arches were characteristic by small (6) (rtheta5 - rtheta4)R + (rtheta5 - rtheta4)L values; and 4) round V-shaped arches had large (1), (3) and (4) values.

Differential rostral projections of caudal brainstem neurons receiving trigeminal input after masseter inflammation.

To understand the functional significance of orofacial injury-induced neuronal activation, this study examined the rostral projection of caudal brainstem neurons that were activated by masseteric inflammation. Rats were injected with a retrograde tracer, Fluorogold, into the nucleus submedius of the thalamus (Sm), parabrachial nucleus (PB), lateral hypothalamus (LH), or medial ventroposterior thalamic nucleus (VPM) 7 days before injection of an inflammatory agent, complete Freund's adjuvant (CFA), into the masseter muscle. Rats were perfused at 2 hours after inflammation, and brainstem tissues were processed for Fos-Fluorogold double immunocytochemistry. Although there was no difference in Fos expression among the four groups (n=4 per site), the rostral projection of Fos-positive neurons showed dramatic differences. In the ventral portion of the trigeminal subnuclei interpolaris/caudalis (Vi/Vc) transition zone, the percentage of Fos-positive neurons projecting to the Sm (39.7%) was significantly higher than that projecting to the LH (5.4%) or VPM (5.6%; P<.001). The anesthesia alone also induced Fos expression in ventral Vi/Vc neurons, but these neurons did not project to Sm. In the caudal laminated Vc and dorsal Vi/Vc, the PB was the major site of rostral projection of Fos-positive neurons. In the caudal ventrolateral medulla and nucleus tractus solitarius, Fos-positive neurons projected to the Sm, PB, and LH. Most VPM-projecting neurons examined did not show Fos-like immunoreactivity after masseter inflammation. These findings emphasize the importance of the trigeminal Vi/Vc transition zone in response to orofacial deep tissue injury. Furthermore, the results differentiate the ventral and dorsal portions of the Vi/Vc transition zone, in that the Sm received projection mainly from activated neurons in the ventral Vi/Vc. The activation of Vi/Vc neurons and associated ascending pathways may facilitate somatoautonomic and somatovisceral integration and descending pain modulation after orofacial deep tissue injury.