Rat Temporomandibular Joint Research Articles

Influence of feeding a soft diet on proteoglycan expression in rat temporomandibular joint discs

ObjectivesExtracellular matrix components play a significant role in maintaining tissue integrity and pathological processes of the temporomandibular joint (TMJ). This study aimed to evaluate the influence of a soft diet on the mRNA expression of proteoglycans and glycosaminoglycans (GAGs) linked to proteoglycan core proteins in rat TMJ discs. MethodsThirty 4-week-old male Wistar rats were assigned to one of two groups: a control group fed a regular pellet diet and a soft diet group fed a powdered diet for 4 weeks. The mRNA expression levels of 12 proteoglycans in TMJ discs were evaluated using real-time polymerase chain reaction (PCR). In addition, histomorphometric and biochemical analyses were performed to evaluate the thickness and deoxyribonucleic acid (DNA), GAG, and water content of the TMJ discs. ResultsThe TMJ disc thickness in the anterior, intermediate, and posterior bands decreased significantly in the soft diet group. The GAG content decreased significantly in the soft-diet group, whereas no significant differences in DNA content or water content ratio were observed between the groups. Real-time PCR indicated that the expression levels of aggrecan, versican, biglycan, decorin, fibromodulin, lumican, and chondroadherin decreased in the soft diet group. The expression levels of all versican isoforms decreased in the soft diet group. ConclusionsThese results indicate that the biomechanical environment of the TMJ caused by a soft diet is closely related to the expression of proteoglycans in TMJ discs, which may eventually increase the fragility of the TMJ discs.

Changes of Trigeminal Ganglion Neurons Innervating the Temporomandibular Joint in Chronic Pain Rat Model.

Background: Phenotype alterations of nociceptive neurons have been shown to be a key step in the pathogenesis of many pain-related diseases. However, it is unclear if the characteristic changes of temporomandibular joint (TMJ) primary afferent neurons are related to the pathogenesis of temporomandibular joint osteoarthritis (TMJOA) chronic pain. This study aimed to determine the morphological and neurochemical changes in trigeminal ganglion (TG) neurons innervating the TMJ in TMJOA chronic pain rats. Materials and Methods: Monosodium iodoacetate (MIA)-induced TMJOA chronic pain rat model was established (n = 6), and saline was injected in rats of the control group (n = 6). TMJ primary afferent neurons were labeled with retrograde tracing (Dil). The spatial distribution and the expression of calcitonin gene-related peptide (CGRP), isolectin B4 (IB4), and neurofilament 200 (NF200) of TMJ primary afferent neurons in TG were investigated using immunofluorescence. Intracellular calcium signaling was recorded by calcium imaging (n = 20). Results: TMJ primary afferent neurons were located only in the V3 region of the TG from both saline- and MIA-injected rats. The number of TG neurons innervating the TMJ was increased in MIA-injected rats. Elevated number and intracellular calcium concentration of small- and medium-sized instead of large-sized Dil+ TG neurons were found in MIA-injected rats. The upregulated expression of CGRP and IB4, but not NF200, in TG neurons innervating the rat TMJs was accompanied by TMJOA chronic pain. Conclusion: This study suggests that sensitization of small- to medium-sized Dil+ TG neurons and CGRP- and IB4-positive Dil+ TG neurons might contribute to the development of TMJOA chronic pain in rats. This will provide valuable information for more efficient control of TMJOA chronic pain.

Glycyrrhizin regulates antioxidation through Nrf2 signaling pathway in rat temporomandibular joint osteoarthritis.

Regulation of redox homeostasis could reduce osteoarthritis severity and limit disease progression, while glycyrrhizin (GL) shows great antioxidant and anti-inflammatory capacity. The aim of this study was to investigate the role of GL on oxidative stress and the potential regulatory mechanism in rat temporomandibular joint (TMJ) chondrocytes under oxidative stress, and investigate the effect of GL in the rat temporomandibular joint osteoarthritis (TMJOA) model. Rat TMJ chondrocytes were cultured in oxidative stress with different doses of GL. The effect of glycyrrhizin on the nuclear factor-erythroid 2-related factor 2 (Nrf2) in oxidative stress was evaluated by western blot and immunofluorescence staining. A rat model of TMJOA was treated with GL. Micro-computed tomography, histological and immunohistochemical analysis were used to assess the pathological change of TMJOA. The expression of superoxide dismutase 1 (SOD1), heme oxygenase-1 (HO-1), and peroxiredoxin 6 (PRDX6) were decreased, and intracellular Nrf2 signaling pathway was activated in chondrocytes in oxidative stress. GL upregulates the expression of antioxidants, especially PRDX6, as well as increases Nrf2 expression and nuclear translocation in rat condylar chondrocytes. Administration of GL attenuates condylar bone destruction, cartilage degeneration, and synovitis in rats TMJOA. Meanwhile, GL alleviated oxidative stress and enhanced the antioxidant capacity of TMJOA cartilage. This study suggested that GL alleviates rat TMJOA by regulating oxidative stress in condylar cartilage.

Effective technical protocol for producing a mono-iodoacetate-induced temporomandibular joint osteoarthritis in a rat model.

An animal model of osteoarthritis (OA) induced by monosodium iodoacetate (MIA) can be effectively adjusted based on the concentration of MIA to control the onset, progression, and severity of OA as required. The rat temporomandibular joint osteoarthritis (TMJOA) model using MIA is a useful tool for studying the effectiveness of disease-modifying OA drugs in TMJOA research. However, the intricate and complex anatomy of the rat TMJ often poses challenges in achieving consistent TMJOA induction during experiments. In the previous paper, a reference point was established by drawing parallel lines based on the line connecting the external ear and the zygomatic arch. However, this is not suitable for the anatomical characteristics of the rat. We used the zygomatic arch as a reference, which is a technical protocol that considers it. In our protocol, we designated a point approximately 1 mm away from the point where the zygomatic arch bends towards the ear as the injection site. To ensure precise injection of MIA and increase the likelihood of inducing OA, it is recommended to insert the needle at a 45° angle so that the needle tip contacts the joint projection. To confirm TMJOA induction, we identified changes in the condyle using in vivo micro-computed tomography (CT) in a rat model of MIA-induced OA and measured the degree of pain-related inflammation using head withdrawal threshold (HWT) measurements. Micro-CT scanning revealed typical OA-like lesions, including degenerative changes and subchondral bone remodeling induced by MIA in the TMJ. Pain, a major clinical feature of OA, showed an appropriate response corresponding to the structural changes shown in micro-CT scanning. In addition, the MIA concentration suitable for long-term observation of lesions was determined through ex vivo micro-CT imaging and HWT measurements. The 8 mg concentration exhibited a significant difference compared to others, confirming the sustained presence of lesions, particularly through changes in subchondral bone over an extended period. Consequently, we have successfully established a reliable rat TMJOA induction model and identified the MIA concentration suitable for long-term observation of subchondral bone research, which will greatly contribute to the study of TMJOA-an incurable disease lacking specific treatment options.

Soluble epoxide hydrolase inhibitor blockage microglial cell activation in subnucleus caudalis in a persistent model of arthritis

Rheumatoid arthritis (RA) is a chronic condition characterized by pain and infiltration of immune cells into the joint. Immune cells can be activated, producing inflammatory cytokines, leading to continuously degenerative and inflammatory reactions and the temporomandibular joint (TMJ) can be affected by RA. In this scenario, novel targets are needed to increase treatment efficacy with minimized side effects. The epoxy-eicosatrienoic acids (EETs), are endogenous signaling molecules, playing important roles in diminishing inflammation and pain but are promptly metabolized by soluble epoxide hydrolase (sEH), generating less-bioactive acids.Therefore, sEH inhibitors is an interest therapeutic target to enhance the beneficial effect of natural EETs. TPPU is a potent sEH inhibitor that is capable of dampening EETs hydrolysis. Thus, we aimed to assess the impact of pharmacological sEH inhibition on a persistent model of albumin-induced arthritis in the TMJ, in two scenarios: first, as post-treatment, in an installed arthritic condition, and second, the protective role, in preventing the development of an arthritic condition. In addition, we investigate the influence of sEH inhibition on microglia cell activation in the trigeminal subnucleus caudalis (TSC) and in vitro experiments. Finally, we examined the astrocyte phenotype. Oral administration of TPPU, acts in multiple pathways, in a protective and reparative post-treatment, ameliorating the preservation of the TMJ morphology, reducing the hypernociception, with an immunosuppressive action reducing neutrophil and lymphocytes and pro-inflammatory cytokines in the TMJ of rats. In TSC, TPPU reduces the cytokine storm and attenuates the microglia activated P2X7/Cathepsin S/Fractalkine pathway and reduces the astrocyte activation and glutamate levels. Collectively, our findings revealed that sEH inhibition mitigates hypersensitive nociception through the regulation of microglia activation and astrocyte modulation, demonstrating the potential use of sEH inhibitors as immunoresolvents in the treatment of autoimmune disorders.

Inflammatory and degenerative effects of induced osteoarthritis/rheumatoid arthritis models on temporomandibular joint of rats

ObjectiveThe present study compared three models of induction of osteoarthritis (OA) and rheumatoid arthritis (RA) in the temporomandibular joint (TMJ) of rats. DesignThe induction method was by injection of complete Freund’s adjuvant (CFA) + type II bovine collagen (CII). Twenty-four adult male rats were divided into four groups (n = 6): G1: Sham, 50 μL of 0.9% sodium chloride at the base of the tail and in each TMJ; G2: OA, 50 μL CFA+CII in each TMJ; G3: RA+OA, 100 μL of CFA+CII at the base of the tail and 50 μL CFA+CII in each TMJ; G4: RA, 100 μL of CFA+CII at the base of the tail. All injections were repeated 5 days later. Twenty-three days after the first injection, the animals were sacrificed and the TMJs were submitted to histomorphometric analysis and measurement of cytokines. The Kruskal-Wallis and Dunn tests were used (alpha=0.05). ResultsThe total thickness of the condylar cartilage increased in G2 in relation to the other groups, G3 and G4 reduced in relation to G1; and G2 and G4 reduced in relation to G2 and G3. The levels of IL-1β, IL-6 and TNF-α increased in the three induction models compared to G1. The level of IL-10 increased in G2 compared to the other groups and reduced in G3 and G4 compared to G1. ConclusionCFA+CII induced inflammation and degeneration compatible with RA (advanced chronic stage) when injected in the tail, and compatible with OA (acute stage or early disease) when injected only in the TMJ.

Piezo1 Affects Temporomandibular Joint Osteoarthritis by Influencing pSmad3.

Objective: The aim of this research was to study the expression of Piezo1 in a rat temporomandibular joint osteoarthritis animal model and to explore its mechanism for inducing inflammatory changes. Methods: A total of 24male SD rats aged approximately 8 weeks were randomly divided into three groups: the blank control group, complete Freund’s adjuvant group (CFA), and CFA + inhibitor (GsMTx4) group. After 3 weeks, the condylar heads of the rats were evaluated by micro-CT, HE, immunohistochemistry, safranin O staining, and other experimental techniques. Protein was extracted from the subchondral bone, and the changes in Piezo1, Smad3, and pSmad3 levels in each group were detected by Western blotting. p < 0.05 was considered to indicate statistical significance. Results: The degree of damage to the cartilage and subchondral bone in the Piezo1 inhibitor group was smaller than that in the CFA group. The expression level of Piezo1 in the CFA group was higher than that in the other groups, and the difference was statistically significant. The expression of pSmad3 in the CFA group was also higher than that in the other groups (p < 0.05). Conclusion: Piezo1 is expressed in the condylar cartilage and subchondral bone of rats, and the degree of condylar destruction can be improved by influencing the pSmad3 expression.

Chondrocyte apoptosis in temporomandibular joint osteoarthritis promotes bone resorption by enhancing chemotaxis of osteoclast precursors

This study aimed to explore the effect and mechanism of chondrocyte apoptosis on the chemotaxis of osteoclast precursors (OCPs) during bone destruction. The relationship between cartilage and bone destruction was verified with a rat temporomandibular joint osteoarthritis (TMJOA) model. The pan-caspase inhibitor Z-VAD-FMK (ZVAD) was applied to confirm the chemotactic effect of chondrocyte apoptosis on OCPs. Synthesis and release of the key chemokine CX3CL1 in apoptotic and non-apoptotic chondrocytes was assessed with IHC, IF, WB, and ELISA. The function of CX3CL1-CX3CR1 axis in the chemotaxis of OCPs was examined by CX3XR1 inhibitor AZD8797 (AZD) and si-CX3CL1. The regulatory effect of p38 MAPK on CX3CL1 release was verified by p38 inhibitor PH-797804. A temporal and spatial association between cartilage degradation and bone resorption was found in the TMJOA model. The caspase-dependent chondrocyte apoptosis promoted chemotaxis of OCPs, which can be restrained by ZVAD. CX3CL1 was significantly upregulated when chondrocytes underwent apoptosis, and it played a critical role in the recruitment of OCPs, blockage of CX3CL1-CX3CR1 axis resulted in less bone resorption in TMJOA. P38 MAPK was activated in apoptotic chondrocytes, and had a regulatory effect on the synthesis and release of CX3CL1. After inhibition of p38 by PH-797804, the chemotactic effect of apoptotic chondrocytes on OCPs was limited. This study indicates that apoptosis of chondrocytes in TMJOA enhances chemotaxis of OCPs toward osteoclast precursors through upregulation of the p38-CX3CL1 axis, thereby promoting the activation of local osteoclasts.

Chondrocyte-derived exosomes promote cartilage calcification in temporomandibular joint osteoarthritis

BackgroundsAbnormal cartilage calcification is one of the pathological changes of temporomandibular joint (TMJ) osteoarthritis (OA). Recent studies have reported that exosomes can regulate the formation of abnormal calcified nodules in diseases including atherosclerosis and chronic kidney disease. However, the influences of chondrocyte-derived exosomes on abnormal cartilage calcification in TMJ OA are still unclear.MethodsTMJ OA was induced by unilateral anterior crossbite (UAC) for 4, 8, or 12 weeks in rats to observe abnormal calcification in TMJ condylar cartilage and exosome formation. Concomitantly, GW4869, the inhibitor of exosome formation, was locally injected to the TMJ of rats under stimulation of UAC, while the exosomes extracted from primary condylar chondrocytes stimulated with fluid flow shear stress (FFSS) were locally injected to rats TMJ.ResultsAbnormal calcification was enhanced in the degenerative cartilage of TMJ OA in UAC rats, and a large number of exosome-like structures with diameters of 50-150 nm were found in the calcified cartilage together with decreased expression of matrix Gla protein (MGP) and increased expression of CD63, tissue-nonspecific alkaline phosphatase (TNAP) and nucleotide pyrophosphatase/phosphodiesterase-1 (NPP1). After FFSS stimulation, the number of exosomes secreted by chondrocytes and the numbers of calcified nodules were increased in cultured cells, and the protein levels of MGP, TNAP, and NPP1 in exosomes were changed. Inhibition of exosome formation, TNAP, and NPP1 or supplementation with exogenous MGP effectively alleviated FFSS-induced chondrocyte calcification. Local injection of GW4869, the exosome inhibitor, alleviated TMJ OA-related cartilage degeneration and calcification in UAC rats. Local injection of exosomes obtained from chondrocytes stimulated by FFSS to the TMJs of normal rats induced cartilage degeneration and calcification similar to that in TMJ OA.ConclusionsAbnormal biomechanical loading leads to enhanced formation of chondrocyte-derived exosomes, in which promoters of calcification increased and inhibitors decreased, resulting in accelerating abnormal cartilage calcification in TMJ OA. The inhibition of degenerative chondrocyte-derived exosomes is expected to be a new way to prevent and treat TMJ OA.

Histone deacetylase inhibitors attenuated interleukin-1β-induced chondrogenesis inhibition in synovium-derived mesenchymal stem cells of the temporomandibular joint.

AimsIn the repair of condylar cartilage injury, synovium-derived mesenchymal stem cells (SMSCs) migrate to an injured site and differentiate into cartilage. This study aimed to confirm that histone deacetylase (HDAC) inhibitors, which alleviate arthritis, can improve chondrogenesis inhibited by IL-1β, and to explore its mechanism.MethodsSMSCs were isolated from synovium specimens of patients undergoing temporomandibular joint (TMJ) surgery. Chondrogenic differentiation potential of SMSCs was evaluated in vitro in the control, IL-1β stimulation, and IL-1β stimulation with HDAC inhibitors groups. The effect of HDAC inhibitors on the synovium and condylar cartilage in a rat TMJ arthritis model was evaluated.ResultsInterleukin (IL)-1β inhibited the chondrogenic differentiation potential of SMSCs, while the HDAC inhibitors, suberoylanilide hydroxamic acid (SAHA) and panobinostat (LBH589), attenuated inhibition of IL-1β-induced SMSC chondrogenesis. Additionally, SAHA attenuated the destruction of condylar cartilage in rat TMJ arthritis model. IL-6 (p < 0.001) and matrix metalloproteinase 13 (MMP13) (p = 0.006) were significantly upregulated after IL-1β stimulation, while SAHA and LBH589 attenuated IL-6 and MMP13 expression, which was upregulated by IL-1β in vitro. Silencing of IL-6 significantly downregulated MMP13 expression and attenuated IL-1β-induced chondrogenesis inhibition of SMSCs.ConclusionHDAC inhibitors SAHA and LBH589 attenuated chondrogenesis inhibition of SMSC induced by IL-1β in TMJ, and inhibition of IL-6/MMP13 pathway activation contributes to this biological progress. This study provides a theoretical basis for the application of HDAC inhibitors in the treatment of TMJ arthritis.Cite this article: Bone Joint Res 2022;11(1):40–48.

Long-term changes in the dentoalveolar system of rats after experimental intra-abdominal hypertension

Aim. To establish the effect of experimental intra-abdominal hypertension (IAH) on the teeth-jaw system of rats in the long-term period based on the study of pro-inflammatory cytokines and morphological analysis of the elements of the temporomandibular joint (TMJ) and salivary glands.Materials and methods. The experiment was carried out on 60 newborn rats; IAH was modelled by injecting collagen into the abdominal cavity to a predetermined level of intra-abdominal pressure. The rats were evenly (n = 20) divided into 3 groups: 1st - control; 2nd and 3rd - with light and severe IAH, respectively. Serum levels of IL-18, MCP-1, NGAL were determined by multiplex analysis after 10 and 120 days, VEGF-C - after 10 days - by ELISA. The morphological examination of the TMJ and salivary glands was performed using a Leica DM2000 microscope after 120 days.Results. After 10 days and 120 days, blood serum levels of NGAL, IL-18 and MCP-1 were statistically significantly increased in groups 2 and 3 compared to the control; the concentration of MCP-1 increased in proportion to the severity of the IAH with the maximum values in group 3. After 10 days, the level of VEGF was significantly increased in group 2 compared to the control group (p &lt; 0.02). Inflammation of the TMJ was observed significantly more often in groups 2 and 3 than in the control group (p = 0.0002). In group 3, circulatory disorders and bone marrow degeneration of the TMJ, as well as inflammation, circulatory disorders and hyperplasia of the lymphoid tissue of the salivary glands were statistically significantly more often compared to groups 1 and 2.Conclusion. In the long term, experimental IAH showed an increase in the levels of markers of inflammation and hypoxia in the blood serum of rats; the severity of synovitis and sialadenitis grew with an increase in the level of intra-abdominal pressure; the maximum deviations in inflammation markers and morphological changes in the TMJ and salivary glands of rats were observed in the group with severe IAH.

Changes of the condylar cartilage and subchondral bone in the temporomandibular joints of rats under unilateral mastication and expression of Insulin-like Growth Factor-1

This study aimed to define changes in the rat condylar cartilage and subchondral bone using the unilateral mastication model. In this study, forty 4-week-old Wistar rats were randomly divided into experimental (n=20) and control group (n=20). In the experimental group, unilateral dental splints were placed on the occlusal surface of left maxillary molars. The rats were sacrificed at 1, 2, 3, and 4 weeks after placement of the splint. Micro-CT scanning and histological staining were performed to observe the changes in the mandibular condylar cartilage and subchondral bone. Levels of insulin-like growth factor-1 (IGF-1) were determined via immunohistochemistry to analyse the occurrence of osteogenic changes. Micro-CT scanning findings demonstrated the occurrence of asymmetric growth of condyle in the experimental group. The condylar cartilage and subchondral bone exhibited degradation on the chewing side of the experimental group and showed decreased bone mineral density, thinner cartilage thickness, and increased degree of degeneration and osteoclast activity. Compared with the control group, the expression of IGF-1 was remarkably higher on the non-chewing side. Long-term unilateral mastication can lead to the occurrence of degenerative changes in the condylar cartilage and subchondral bone during growth and development. IGF-1 may play a role in promoting the process of osteogenesis.

Photobiomodulation inhibits inflammation in the temporomandibular joint of rats.

Photobiomodulation (PBM) has been applied as a non-invasive technique for treating temporomandibular joint symptoms, especially on painful condition's relief, however the anti-inflammatory mechanism underlying the effect of PBM remains uncertain. This study aims to evaluate the mechanisms of action of PBM (808nm) in a carrageenan-induced inflammation on temporomandibular joint (TMJ) of rats. In this study male Wistar rats were pre-treated with irradiation of a low-power diode laser for 15s on TMJ (infra-red 808nm, 100mW, 50J/cm2 and 1.5J) 15min prior an injection in the temporomandibular joint of carrageenan (100μg/TMJ). 1h after the TMJ treatments, the rats were terminally anesthetized for joint cavity wash and periarticular tissues collect. Samples analysis demonstrated that PBM inhibit leukocytes chemotaxis in the TMJ and significantly reduces amounts of TNF-α, IL-1β and CINC-1. In addition, Western blotting analysis demonstrated that PBM significantly decreased the protein levels of P2X3 and P2X7 receptors in the periarticular tissues. On the other hand, PBM was able to increase protein level of IL-10 (anti-inflammatory cytokine). In summary, it is possible to suggest that PBM inhibit inflammatory chemotaxis, modulation the balance of the pro- and anti-inflammatory characteristics of inflammatory cells.

The effect of association of aspirin and omega 3 in rat temporomandibular joint with induced arthritis

This study aimed to evaluate the effects of omega-3 (ω3) polyunsaturated fatty acids, in association with aspirin (AA), on the morphology of cytokine release in the temporomandibular joint (TMJ) of rats induced with rheumatoid arthritis (IR) by injecting 100 μL of complete Freund’s adjuvant with bovine type II collagen at the tail base. Thirty-two adult male rats were divided into treatment groups: Sham, treated with 0.9% sodium chloride (NaCl) p.o.; IR-control, treated with 0.9% NaCl p.o.; IR-ω3 treated with ω3 PUFAS (85 mg/kg/day p.o.); and IR-ω3 + AA treated with ω3 (85 mg/kg/day p.o.) + AA (20 mg/kg/day i.p.). After maintained treatment for seven days, the animals were euthanized. Bilateral TMJs from each rat were removed and one was subjected to histological immunoassays and enzyme-linked immunosorbent assays to assess interleukin (IL)-1β, tumor necrosis factor-α, and IL-10 levels. Data analysis was performed using the Kruskal–Wallis and Dunn tests. In the IR-ω3 and IR-ω3 + AA groups, the TMJ was greater than in the IR-control group (P < 0.0001). The addition of AA did not improve the effects of ω3 (P = 0.0698). Similarly, the addition of AA conferred no additional effects on the cytokine levels (P > 0.05); however, it increased the proteoglycan density, compared with ω3 alone. We found that ω3 exhibited anti-inflammatory activity in arthritic rats, and the addition of AA increased proteoglycan density, but did not affect cytokine expression.

Melatonin Abates TMJOA Chronic Pain by MT2R in Trigeminal Ganglion Neurons

Temporomandibular joint osteoarthritis (TMJOA) is one of the most common diseases causing chronic pain in the oral and maxillofacial region. So far, there are few ways to relieve the pain of TMJOA. Melatonin (MT) has a good analgesic effect in many diseases, including fibromyalgia, neuropathic pain, chronic headache, and burn pain, with very low acute toxicity and side effects. This study was to investigate the role and mechanism of MT in TMJOA chronic pain. In rats TMJOA chronic pain occurred at day 14 after an intra–temporomandibular joint injection of monosodium iodoacetate, which we previously reported. The enzyme-linked immunosorbent assay results showed that MT levels were higher in the synovial fluid from patients and rats with TMJOA as compared with those from control. Fluorescent retrograde tracing (Dil) identified that upregulation of MT type 2 receptor (MT2R) in trigeminal ganglion (TG) neurons innervating rat temporomandibular joints was accompanied by TMJOA chronic pain. Nociceptive behavior as assessed by von Frey and the Rat Grimace Scale demonstrated that exogenous administration of MT relieved chronic pain in TMJOA rats, whereas blocking MT2R with 4P-PDOT reversed the analgesic effect of MT. Immunofluorescence analysis also confirmed that MT inhibited CGRP and IB4 expression of TG neurons, and this inhibition was reversed by administering the MT2R antagonist in TMJOA rats. By using Fluo-3 AM-based calcium imaging in vitro, MT elicited calcium transients in Dil+ TG neurons, which were significantly abolished by 4P-PDOT. Collectively, this study suggested that MT relieves the TMJOA chronic pain of rats through downregulation of sensitized CGRP+ and IB4+ neurons in TG via MT2R. This will be helpful for health care professionals utilizing MT as an option against TMJOA chronic pain.

Optimally dosed nanoceria attenuates osteoarthritic degeneration of joint cartilage and subchondral bone

• Nanoceria injection to inflamed TMJ rescued the degeneration cartilage and subchondyle bone in vivo . • Anti-apoptotic and anti-inflammatory actions of nanoceria were proven to be the major molecular mechanisms. • Nanoceria is considered a nanotherapeutic candidate for the local treatment of degenerative joint diseases. Osteoarthritis entails excessive ROS generation, severe inflammatory responses, and anatomical destruction of cartilage and subchondral bone. Nanoceria holds antioxidant activity, exerting therapeutic efficacy under high ROS conditions. Here we investigate if the nanoceria properly administered to an osteoarthritic joint would be effective in protecting the cartilage and bone against osteoarthritic degeneration. Three-days post-induction of osteoarthritis in a rat temporomandibular joint, nanoceria (~20 nm) at varying doses (100 ~ 2000 µg/mL) were locally administered once, and tissue samples were analyzed at 10 days post-treatment. The nanoceria-administered groups could preserve the cartilage and subchondral anatomical structure of joint; particularly at 500 µg/mL, the preservation of quantitated hypertrophic layer and cartilage proteoglycan was as high as ~ 80% ( vs . ~ 30% in an osteoarthritic group), albeit higher doses gradually shielded the efficacy (~50–60%). The degeneration of subchondral bone was thus significantly prevented by the nanoceria treatment; ~70–80% of intact control with 500 µg/mL vs. ~ 50% in the osteoarthritic group. Moreover, the nanoceria treatment profoundly reduced the degenerative symptoms including cellular apoptosis and the secretion of catabolic proteins (COX2/PGE2) and pro-inflammatory cytokines (IL-1β/TNFα) while boosting regenerative events such as the polarization of M2 macrophages (CD206/163) and the production of anti-inflammatory cytokines (IL-10) and chondrogenic glycoproteins. The excessive ROS generated in the osteoarthritis joint was observed to be effectively scavenged by the nanoceria. Such therapeutic effects of nanoceria witnessed in vivo were further demonstrated in parallel with the in vitro experiments particularly involving the osteoarthritis-mimicking chondrocytes/macrophages co-culture models, where the anti-apoptotic and immunomodulatory roles of nanoceria were revealed to be major molecular mechanisms. The present findings may place the nanoceria a promising nanotherapeutic candidate for the treatment of degenerative joint diseases.

The role of adenosine A1 receptor in the peripheral tramadol's effect in the temporomandibular joint of rats

Peripheral tramadol's delivery in the temporomandibular joint (TMJ) leads to significant analgesic outcomes and inflammatory process's resolvent actions. Mechanistically, these properties are apart from the opioid system. Nevertheless, the molecular mechanisms behind these effects are still unclear. Therefore, the present study investigated the hypothesis that adenosine A1 receptors are involved in the tramadol-induced analgesic and anti-inflammatory effects in the TMJ. Animals were pretreated with an intra-TMJ injection of DPCPX (antagonist of A1 receptor) or tramadol and subsequent nociceptive challenge with an intra-TMJ injection of 1.5% formalin. For over 45 min, the nociceptive behavior was quantitated, and by the end of this assessment, the animals were euthanized, and the periarticular tissue was collected. Lastly, an in vitro assay of BMDM (Bone Marrow-Derived Macrophages) was performed to investigate tramadol activity in macrophages. The intra-TMJ injection of tramadol ameliorates formalin-induced hypernociception along with inhibiting leukocyte migration. The tramadol's peripheral anti-inflammatory effect was mediated by the adenosine A1 receptor and was associated with increased protein expression of α2a-adrenoceptor in the periarticular tissues (p < 0.05: ANOVA, Tukey’s test). Also, tramadol inhibits formalin-induced leukocyte migration and protein expression of P2X7 receptors in the periarticular tissue (p < 0.05); however, DPCPX did not alter this effect (p > 0.05). Moreover, DPCPX significantly reduced the protein expression of the M2 macrophage marker, MRC1. In BMDM, tramadol significantly reduces inflammatory cytokines release, and DPCPX abrogated this effect (p < 0.05). We identify tramadol's peripheral effect is mediated by adenosine A1 receptor, possibly expressed in macrophages in the TMJ tissue. We also determined an important discovery related to the activation of A1R/α2a receptors in the tramadol action.

Fast and accurate protocol for histology and immunohistochemistry reactions in temporomandibular joint of rats

ObjectivePropose a standard, fast and accurate protocol for the processing of the temporomandibular joint (TMJ) of adults’ rats for histology and immunohistochemistry reactions. DesignWistar male rats were perfused with paraformaldehyde (4 %). The heads were fixed in formaldehyde 10 % solution for 48 h. After that, the heads were sectioned in a sagittal plane and fixed for plus 48 h. Decalcification was performed using 20 % formic acid for 96 h and delimitation of TMJ area was done. Detailed methodology to a standard extraction and processing of TMJ to histological sections is described. Different buffers, equipment, temperature and time were tested to optimize immunostaining. Morphological preservation and antigenicity were evaluated by hematoxylin and eosin staining and immunohistochemistry reaction. ResultsThe current findings demonstrated that TMJ fixed in 10 % formaldehyde and decalcified in 20 % formic acid optimized decalcification processing time with preservation of cell morphology. Antigen retrieval with citrate buffer in pressure cooker (2 min at 100 °C and 5 min at room temperature) demonstrated the best protocol to preservation of the structures of TMJ. ConclusionsThis work demonstrates in detail a methodology of a fast and accurate TMJ processing for histology and immunohistochemistry reactions that guarantee tissue integrity and quality of staining.

Extracellular vesicles from M1-polarized macrophages promote inflammation in the temporomandibular joint via miR-1246 activation of the Wnt/β-catenin pathway.

Macrophage-mediated regulation of chondrocytes plays an important role in promoting temporomandibular joint (TMJ) inflammation. We investigated whether extracellular vesicles (EVs) derived from M1 macrophages (M1-EVs) have a proinflammatory effect on TMJ inflammation and what the associated mechanisms are. In vitro, purified THP-1 cell-derived M1-EVs were applied to human TMJ condylar chondrocytes, and in vivo M1-EVs derived from bone marrow-derived macrophages (BMDMs) were injected into rat TMJs. The levels of IL-6, IL-8, IL-1β, and matrix metalloproteinase were then evaluated and found to be upregulated in the chondrocytes and rat TMJs. MicroRNA sequencing analysis was performed to identify differential expression of miRNAs, including miR-1246. High expression of miR-1246 in M1-EVs from synovial fluid of patients with TMJ osteoarthritis and synovitis was verified by RT-PCR. We then identified miR-1246 targets GSK3β and Axin2 and found that miR-1246 inhibits GSK3β and Axin2 expression, causing activation of the Wnt/β-catenin pathway and inflammation in condylar chondrocytes. Our study found that M1-EVs promote inflammation by transfer of miR-1246 to condylar chondrocytes, thus providing new insight into one mechanism that can promote TMJ inflammation.

Temporomandibular inflammation regulates the matrix metalloproteinases MMP-2 and MMP-9 in limbic structures.

Temporomandibular disorder (TMD) is characterized by acute or chronic orofacial pain, which can be associated with inflammatory processes in the temporomandibular joint (TMJ) and emotional disorders. Peripheral and central sensitization in painful orofacial processes is common, and it can be triggered by peripheral inflammatory challenge with consequent neuroinflammation phenomena. Such neuroinflammation comes from inflammatory products from supportive cells, blood-brain barrier, and extracellular matrix. Here, we evaluated the possible recruitment of limbic structures for modified matrix metalloproteinases (MMPs) expression and activity during temporomandibular inflammation-induced orofacial persistent pain. The inflammatory process in TMJs of rats was induced by Freund's Complete Adjuvant (CFA) administration. The activity and expression of MMPs-2 and 9 were assessed by in situ zymography and conventional zymography, respectively. A glial colocalization with the MMPs was performed using immunofluorescence. The results evidenced both short- and long-term alterations on MMP-2 and -9 expression in the limbic structures following CFA-induced temporomandibular inflammation. The gelatinolytic activity was increased in the central amygdala, hippocampus, hypothalamus, ventrolateral periaqueductal gray (vlPAG), superior colliculus, and inferior colliculus. Finally, an increase of colocalization of MMP-2/GFAP and MMP-9/GFAP in CFA-induced inflammation groups was observed when compared with saline groups in the central amygdala and vlPAG. It is possible to suggest that glial activation is partly responsible for the production of gelatinases in the persistent orofacial pain, and it is involved in the initiation and maintenance of this process, indicating that inhibition of MMPs might be pursued as a potential new therapeutic target for TMD.