mu-Opioid receptor mRNA expression and immunohistochemical localization in the rat temporomandibular joint

This study was undertaken to examine the presence and distribution of the mu-opioid receptor (MOR) in the non-inflamed rat temporomandibular joint (TMJ) using non-radiographic in situ hybridization at the mRNA level and immunohistochemistry at the protein level. MOR mRNA and MOR-like immunoreactivity (MOR-LI) were found around the small blood vessels in the anterior part of the synovial membrane. The number of MOR mRNA signals in the anterior synovial membrane was significantly higher than that in the posterior part. Morphologically, MOR mRNA and MOR-LI were localized in amorphous materials considered to be nervous tissue, as well as some cell types considered to be macrophages, mast cells and endothelial cells. The present study showed the distribution of MOR in the rat TMJ synovial membrane and suggests that the opiate system plays an important role in endogenous analgesia in the TMJ.     

Capsaicin receptor expression in the rat temporomandibular joint

Experimentally, temporomandibular joint (TMJ) nerve units respond to capsaicin, which is used clinically to treat TMJ pain. However, the existence of capsaicin receptors in the TMJ has not previously been clearly demonstrated. Immunohistochemical analysis has revealed the presence of transient receptor potential vanilloid subtype 1 (TRPV1) expression in the nerves and synovial lining cells of the TMJ. TRPV1-immunoreactive nerves are distributed in the synovial membrane of the joint capsule and provide branches to the joint compartment. The disc periphery is supplied by TRPV1 nerves that are mostly associated with small arterioles, and occasional nerves penetrate to the synovial lining layer. Double immunofluorescence has shown that many TRPV1-immunoreactive nerves are labeled with neuropeptide calcitonin gene-related peptide, whereas few are labeled with IB4-lectin. The results provide evidence for the presence of TRPV1 in both nerves and synovial lining cells, which might thus be involved in the mechanism of nociception and inflammation in the TMJ. This study was supported by a grant-in-aid for Scientific Research (C), no. 15591938, from the Japanese Ministry of Education, Science, Sports, Culture, and Technology (to M.A.K.).     

Expression of mRNA encoding insulin‐like growth factors I and II by uterine tissues and placenta during pregnancy in the rat

The uterus and the placenta synthesize insulin‐like growth factors (IGFs) and insulin‐like binding proteins (IGFBPs). These growth factors are implicated in processes of proliferation and differentiation that occur in the uterus. To determine the patterns of expression of IGFs during rat pregnancy we used in situ hybridization with digoxigenin labeled probes on uterus from day 7 to day 16 of pregnancy. In early gestation days (7–8) both IGF mRNAs showed similar tissue distribution with relative abundance in the stroma and circular muscle layer. On days 11 and 12 expression for IGF‐I mRNA was found in the mesometrial decidua and metrial gland and in the ectoplacental cone while clear expression of IGF‐II mRNA could only be found in the latter. On days 13 and 14, expression for IGF‐I mRNA could be detected in the mesometrial decidua and metrial gland but no expression was observed for IGF‐II mRNA. A gradient of IGF‐I mRNA expression could be observed in the placenta on day 16, with the trophoblastic cells of the basal zone expressing the signal with stronger intensity than in the labyrinthine zone. For IGF‐II mRNA the highest expression was associated with the labyrinthine zone. Endovascular trophoblast was positive for both mRNAs. The spatial and temporal patterns of expression suggests a role for IGFs in the process of decidualization as well as in the establishment, growth and differentiation of the various trophoblast cells of the placenta. Mol. Reprod. Dev. 53:294–305, 1999. © 1999 Wiley‐Liss, Inc.     

Expression and localization of versican during postnatal development of rat temporomandibular joint disc

To analyze the growth-related changes in extracellular matrix components in temporomandibular joint (TMJ) discs, the expression and localization of the core protein of a large chondroitin sulphate proteoglycan, versican, in rat TMJ discs during postnatal development (2–32 weeks) were examined using Western blot analysis, real-time quantitative PCR and immunohistochemistry. Western blot analysis showed that rat TMJ discs predominantly expressed one isoform (V1) and the core protein sharply increased after birth, reached a peak at 8 weeks, and then gradually decreased up to 32 weeks. Real-time quantitative PCR with TaqMan probes indicated that mRNA expression of versican was highest at 2 weeks and gradually decreased with growth. An immunohistochemical study showed that staining for versican was weak and evenly distributed in TMJ discs at 2 weeks. Regional differences in staining for versican became prominent after 8 weeks; staining was intense in the anterior and posterior peripheral attachments, and weak in the central part of the discs. These results demonstrate that growth-related changes and regional differences exist in the expression of versican in the TMJ discs of growing rats, and these probably reflect the changes in the biomechanical environment caused by the development of orofacial functions.     

Bilateral traumatic temporomandibular joint luxation in a rat

Bilateral temporomandibular joint (TMJ) luxation was diagnosed postmortem in a female, 6-mo-old CD rat (Rattus norvegicus) after probable head entrapment and subsequent disentanglement from a protective jacket. Clinical antemortem signs included inability to close her mouth, prehend food and drink water, anxiety, and linear skin erosions extending down the right and left commissures of the mouth. Radiography revealed rostral displacement of the mandible with concomitant malocclusion. The combination of clinical signs, acute nature of the presentation, and torn appearance of the protective jacket were strongly indicative of a traumatic etiology. To our knowledge, this is the first reported case of TMJ luxation in a rat.     

Collagen distribution in the mouse endometrium during decidualization

The distribution of collagen and reticular fibers was studied in the endometrium of virgin and pregnant mice. The collagen and reticular fibers were examined in picrosirius-stained sections observed in a polarizing microscope and in silver-impregnated sections. Picrosirius-stained sections of animals in estrus, diestrus and on the 2nd day of pregnancy had fine greenish fibers distributed irregularly in the endometrium and thicker red fibers concentrated near the myometrium. Argyrophyl fibers in virgin mice were scarce and irregularly distributed. On the 4th day of pregnancy very few fibers were observed in the endometrium. On the 5th, 6th, and 7th days of pregnancy long greenish fibers were found surrounding decidual cells. A network of argyrophyl fibers was observed in the silver-impregnated decidua. It is suggested that new fibers are produced by decidual cells.     

Collagen, collagenase and collagenolytic activity in rat Graafian follicles during follicular growth and ovulation

Follicles were dissected from the ovaries of immature rats at intervals after subcutaneous injection of 20 IU of pregnant mare's serum gonadotropin. A surge of luteinizing hormone was observed at 54 h and ovulation occurred at 64-66 h. The follicular volume between 36 and 48 h, then doubled again shortly before ovulation. The collagen content of the follicles increased 3-fold from 35 to 56 h, but decreased significantly (25%) from 61 to 66 h. Follicle homogenates, activated with trypsin or aminophenylmercuric acetate, digested Type I collagen at 28 degrees C to produce typical of a true collagenase. Collagenolytic activity assayed against endogenous collagen at 37 degrees C did not change significantly between 38 and 66 h.     

Developmental regulation of insulin-like growth factor II mRNA in different rat tissues

Insulin-like growth factor II (IGF-II) is present at high levels in fetal and early neonatal rat plasma, and decreases profoundly following birth. In the present study, the levels of IGF-II RNA in different rat tissues at different ages were determined by hybridization to a rat IGF-II cDNA probe. IGF-II RNA was present in 11 of 13 fetal or neonatal tissues examined: at higher levels in muscle, skin, lung, liver, intestine, and thymus; at lower levels in brain stem, heart, cerebral cortex, kidney, and hypothalamus; and undetectable in spleen and pancreas (although the latter RNA was partially degraded). In each tissue, Northern blot hybridization revealed the presence of six IGF-II RNAs: 6, 4, 3.8, 2.2, 1.7, and 1.2 kilobase pairs, consistent with results previously observed in the BRL-3A rat liver cell line and attributed to alternative RNA processing. Although differences in the relative abundance of these RNAs were observed in different tissues, the same size species occurred in all tissues with the 4-kilobase pair RNA the most abundant species. RNAs from the different tissues were examined at six developmental ages (days 16 and 21 of gestation; days 2, 11, 22, and 75 after birth) by hybridization to slot blots and Northern blots. In lung, thymus, kidney, and brain stem, IGF-II RNA was expressed at higher levels in the fetus than after birth, whereas in muscle, skin, liver, heart, and intestine, the high fetal levels of IGF-II RNA continued through day 11 or day 22 after birth. IGF-II RNA persisted into adulthood in cerebral cortex and hypothalamus. Although the significance of these tissue-specific differences in the developmental regulation of the expression of IGF-II RNA remains to be established, they exhibit intriguing temporal correlations with major maturational events in some tissues such as lung and muscle.     

The primary structure of the rat insulin-like growth factor II gene region

Complete nucleotide sequences of the rat insulin-like growth factor II gene region including 5' 18 kilobases (kb) up to the insulin gene, all exonic and intronic, and 3' 6 kb sequences were determined. Among these sequences several repetitive stretches became evident besides integration of type II Alu and identifier sequences. They were: (1) twelve repetitions of about 100 base pair (bp) units; (2) duplication of 60 bp units; (3) triplication of a 24 bp unit; and (4) 41-fold expansion of 12-15 bp units.     

Viscoelastic properties of the central region of porcine temporomandibular joint disc in shear stress-relaxation

In this study, shear relaxation properties of the porcine temporomandibular joint (TMJ) disc are investigated. Previous studies have shown that, in fatigue failure and damage of cartilage and fibrocartilage, shear loads could be one of the biggest contributors to the failure. The aim of the present study is to develop an evaluation method to study shear properties of the disc and to do a mathematical characterization of it. For the experiments, twelve porcine discs were used. Each disc was dissected from the TMJ and, then, static strain control tests were carried out to obtain the shear relaxation modulus for the central region of the discs. From the results, it was found that the disc presents a viscoelastic behavior under shear loads. Relaxation modulus decreased with time. Shear relaxation was 10% of the instantaneous stress, which implies that the viscous properties of the disc cannot be neglected. The present results lead to a better understanding of the discs mechanical behavior under realistic TMJ working conditions.     

Phenotypic alterations of neuropeptide Y and calcitonin gene-related peptide-containing neurons innervating the rat temporomandibular joint during carrageenan-induced arthritis

The aim of this study was to identify immunoreactive neuropeptide Y (NPY) and calcitonin gene-related peptide (CGRP) neurons in the autonomic and sensory ganglia, specifically neurons that innervate the rat temporomandibular joint (TMJ). A possible variation between the percentages of these neurons in acute and chronic phases of carrageenan-induced arthritis was examined. Retrograde neuronal tracing was combined with indirect immunofluorescence to identify NPY-immunoreactive (NPY-IR) and CGRP- immunoreactive (CGRP-IR) neurons that send nerve fibers to the normal and arthritic temporomandibular joint. In normal joints, NPY-IR neurons constitute 78±3%, 77±6% and 10±4% of double-labeled nucleated neuronal profile originated from the superior cervical, stellate and otic ganglia, respectively. These percentages in the sympathetic ganglia were significantly decreased in acute (58±2% for superior cervical ganglion and 58±8% for stellate ganglion) and chronic (60±2% for superior cervical ganglion and 59±15% for stellate ganglion) phases of arthritis, while in the otic ganglion these percentages were significantly increased to 19±5% and 13±3%, respectively. In the trigeminal ganglion, CGRP-IR neurons innervating the joint significantly increased from 31±3% in normal animals to 54±2% and 49±3% in the acute and chronic phases of arthritis, respectively. It can be concluded that NPY neurons that send nerve fibers to the rat temporomandibular joint are located mainly in the superior cervical, stellate and otic ganglia. Acute and chronic phases of carrageenan-induced arthritis lead to an increase in the percentage of NPY-IR parasympathetic and CGRP-IR sensory neurons and to a decrease in the percentage of NPY-IR sympathetic neurons related to TMJ innervation.Key words: trigeminal ganglion, otic ganglion, superior cervical ganglion, arthritis, temporomandibular joint.     

Differences in loading of the temporomandibular joint during opening and closing of the jaw

Kinematics of the human masticatory system during opening and closing of the jaw have been reported widely. Evidence has been provided that the opening and closing movement of the jaw differ from one another. However, different approaches of movement registration yield divergent expectations with regard to a difference in loading of the temporomandibular joint between these movements. Because of these diverging expectations, it was hypothesized that joint loading is equal during opening and closing. This hypothesis was tested by predicting loading of the temporomandibular joint during an unloaded opening and closing movement of the jaw by means of a three-dimensional biomechanical model of the human masticatory system. Model predictions showed that the joint reaction forces were markedly higher during opening than during closing. The predicted opening trace of the centre of the mandibular condyle was located cranially of the closing trace, with a maximum difference between the traces of 0.45 mm. The hypothesis, postulating similarity of joint loading during unloaded opening and closing of the jaw, therefore, was rejected. Sensitivity analysis showed that the reported differences were not affected in a qualitative sense by muscular activation levels, the thickness of the cartilaginous layers within the temporomandibular joint or the gross morphology of the model. Our predictions indicate that the TMJ is loaded more heavily during unloaded jaw opening than during unloaded jaw closing.     

Testosterone and estrogen have opposing actions on inflammation-induced plasma extravasation in the rat temporomandibular joint

The present study was designed to test the hypothesis that estrogen exacerbates inflammation of the temporomandibular joint (TMJ). Evans blue dye was used to quantify plasma extravasation (PE) around the rat TMJ. In an initial set of experiments, TMJ PE was compared in na?ve intact male and female rats, as well as in both groups after complete Freund's adjuvant (CFA)-induced inflammation of the TMJ. In contrast to our hypothesis, TMJ PE was significantly greater in both na?ve and CFA-inflamed male rats than in females. To determine whether these differences were due to gonadal hormones, four additional groups of rats were studied: gonadectomized (Gx) males and females, Gx males with chronic testosterone (T) replacement, and Gx females with chronic estrogen (E) replacement. The sex difference in baseline TMJ PE appeared to reflect the actions of T. However, in the presence of TMJ inflammation, T augmented TMJ PE in males, while E attenuated TMJ PE in females. Changes in PE were also assessed in the contralateral TMJ. Results from this analysis indicated that there is a transient contralateral increase in TMJ PE in females but not males. Given that there is an inverse relationship between PE and joint damage, our results suggest that testosterone may mitigate, but estrogen may exacerbate, TMJ damage, particularly in the presence of overt inflammation. Importantly, our results may help explain both the higher prevalence and severity of temporomandibular disorder pain in females than males.     

A new 5'-non-coding region for human placental insulin-like growth factor II mRNA expression

A human placenta cDNA library was screened for insulin-like growth factor II (IGF-II). Four clones were selected, which exhibited an IGF-II cDNA coding sequence identical to those previously described for human adult liver IGF-II cDNA. Extensive sequence diversity was observed in the 5'-non-coding region, probably resulting from differential intron splicing.     

Pathological mechanism of chondrocytes and the surrounding environment during osteoarthritis of temporomandibular joint

Temporomandibular joint (TMJ) osteoarthritis is a common chronic degenerative disease of the TMJ. In order to explore its aetiology and pathological mechanism, many animal models and cell models have been constructed to simulate the pathological process of TMJ osteoarthritis. The main pathological features of TMJ osteoarthritis include chondrocyte death, extracellular matrix (ECM) degradation and subchondral bone remodelling. Chondrocyte apoptosis accelerates the destruction of cartilage. However, autophagy has a protective effect on condylar chondrocytes. Degradation of ECM not only changes the properties of cartilage but also affects the phenotype of chondrocytes. The loss of subchondral bone in the early stages of TMJ osteoarthritis plays an aetiological role in the onset of osteoarthritis. In recent years, increasing evidence has suggested that chondrocyte hypertrophy and endochondral angiogenesis promote TMJ osteoarthritis. Hypertrophic chondrocytes secrete many factors that promote cartilage degeneration. These chondrocytes can further differentiate into osteoblasts and osteocytes and accelerate cartilage ossification. Intrachondral angiogenesis and neoneurogenesis are considered to be important triggers of arthralgia in TMJ osteoarthritis. Many molecular signalling pathways in endochondral osteogenesis are responsible for TMJ osteoarthritis. These latest discoveries in TMJ osteoarthritis have further enhanced the understanding of this disease and contributed to the development of molecular therapies. This paper summarizes recent cognition on the pathogenesis of TMJ osteoarthritis, focusing on the role of chondrocyte hypertrophy degeneration and cartilage angiogenesis.