Immunocytochemical demonstration of heat shock protein 25 in the rat temporomandibular joint

The expression of heat shock protein 25 (Hsp 25) was investigated in the rat temporomandibular joint by immunocytochemistry combined with confocal and electron microscopy. Immunostaining with an antibody to Hsp25 was able to demonstrate various cellular elements in the synovial membrane of the joint. Intense immunoreaction for Hsp25 was recognized in certain cells comprising the synovial lining layer. Confocal microscopic observation revealed two characteristic profiles of the Hsp25-positive cells with cytoplasmic processes: one extended thick and long processes towards the articular cavity, and the other prejected horizontally slender processes which covered the synovial membrane. Under the electron microscope, the immunoreactive synovial lining cells were characterized by a well-developed rough endoplasmic reticulum and secretory granules, suggesting that they can be categorized as fibroblastic type B cells. The covering by the cytoplasmic extensions was confirmed by immuno-electron microscopic observations. This cytoplasmic covering presumably performs a barrier function and expedites the effective secretion/resorption of synovial fluids. Since it has been proposed that Hsp 25 is associated with an estrogen receptor, the immunopositive synovial lining cells were considered estrogen-target cells. Immunoreactivity for Hsp25 was also observed in the chondrocytes of the maturative and hypertrophic cell layers as well as in the cells of the articular disk. A suggestion was made that Hsp25 might be involved in the inhibition of apoptosis of those cells.     

Responses of odontoblasts to cavity preparation in rat molars as demonstrated by immunocytochemistry for heat shock protein (Hsp) 25

Responses of odontoblasts to cavity preparation in rat molars were investigated by immunocytochemistry for heat shock protein (Hsp) 25. In untreated control teeth, intense Hsp 25-immunoreactivity was found in the cell bodies of odontoblasts and their processes within the predentin. Confocal microscopy of Hsp 25-immunostained and rhodamine-labeled sections revealed that the immunoreactive odontoblasts were intensely labeled for phalloidin at the periphery of their cytoplasm and throughout their processes, but the reaction for phalloidin was limited within the inner half of the dentin. Cavity preparation caused an edematous reaction between the injured odontoblasts and predentin as well as a beaded swelling and successive destruction of the odontoblast processes. Immediately after cavity preparation, the odontoblasts beneath the edematous lesion showed an immunoreactivity for Hsp 25, which subsequently disappeared completely from the pulp-dentin border by 12 h after the operation. However, round cells without apparent cytoplasmic processes continued to be immunoreactive, suggesting the survival of a part of the odontoblasts against preparation stimuli. Numerous phalloidin-reactive but Hsp 25-immunonegative cells appeared along the pulp-dentin border and extended their processes deep into the exposed dentinal tubules, probably categorized in a lineage of immunocompetent cells. By postoperative 72 h, newly differentiated odontoblasts with Hsp 25-immunoreactivity were arranged at the pulp-dentin border. These findings indicate that the time course of changes in the expression of Hsp 25-immunoreactivity reflects the regeneration process of odontoblasts, and suggest that this protein is a useful marker substance for differentiated odontoblasts.     

Localization of CD44 and hyaluronan in the synovial membrane of the rat temporomandibular joint

Previous studies have pointed out a lack of adhesion structures in the synovial lining layer of the rat temporomandibular joint (TMJ) despite showing an epithelial arrangement. CD44, a major cell adhesion molecule, plays crucial roles as an anchor between cells and extracellular matrices by binding hyaluronan (HA) for the development of organs or the metastasis of tumors. The present study examined the localization of CD44 in the synovial membrane of the rat TMJ by immunocytochemistry for OX50, ED1, and Hsp25, which are markers for the rat CD44, macrophage-like type A, and fibroblast-like type B synoviocytes, respectively. Histochemistry for HA-binding protein (HABP) was also employed for the detection of HA. OX50 immunoreactions were found along the cell surface and, in particular, accumulated along the surface of the articular cavity. Observations by a double immunostaining and immunoelectron microscopy revealed that all the OX50-immunopositive cells were categorized as fibroblastic type B cells, which had many caveolae and a few vesicles reactive to intense OX50. However, the macrophage-like type A cells did not have any OX50 immunoreaction in the synovial lining layer. A strong HABP reaction was discernable in the extracellular matrix surrounding both OX50-positive and -negative cells in the synovial lining layers, exhibiting a meshwork distribution, but weak in its sublining layer. This localization pattern of CD44 and HABP might be involved in the formation of the epithelial arrangement of the synovial lining layer. Furthermore, OX50 immunonegativity in the type A cells suggests their low phagocytotic activity in the rat TMJ under normal conditions.     

Phenotypes of articular disc cells in the rat temporomandibular joint as demonstrated by immunohistochemistry for nestin and GFAP

The articular disc is a dense collagenous tissue containing disc cells that are phenotypically described as chondrocyte-like cells or fibrochondrocytes. Despite the possible existence of these phenotypes in systemic joints, little is known about the detailed classification of the articular disc cells in the temporomandibular joint. In this immunocytochemical study we examined the localization and distribution patterns of nestin and glial fibrillary acidic protein (GFAP) in the articular disc of the rat temporomandibular joint at postnatal day 1, and weeks 1, 2, 4 and 8, based on the status of tooth eruption and occlusion. Nestin and GFAP are intermediate filament proteins whose expression patterns are closely related to cell differentiation and cell migration. Both types of immunopositive cell greatly increased postnatally to a stable level after postnatal week 4, but they showed different distribution patterns and cell morphologies. Nestin-reactive disc cells, which were characterized by a meagre cytoplasm and thin cytoplasmic processes, were scattered in the articular disc, whereas GFAP-positive cells, characterized by broader processes, existed exclusively in the deeper area. In mature discs, the major proportion of articular disc cells exhibited GFAP immunoreactivity. Furthermore, a double-immunostaining demonstrated that the nestin-negative cells, consisting of GFAP-positive and -negative cells, exhibited immunoreactions for heat shock protein 25. These findings indicate that the articular disc cells comprise at least three types in the rat temporomandibular joint and suggest that their expressions closely relate to mechanical loading forces within the joint, including occlusal force, as observed through postnatal development.     

Expression patterns of heat shock protein 25 in carbon tetrachloride-induced rat liver injury

Heat shock protein 25 (Hsp25) is a molecular chaperone playing roles in cytoprotection. We investigated the distribution and localization of Hsp25 expression in CCl₄-induced rat hepatic lesions; liver samples were obtained from 3 h to 10 days after a single oral administration of CCl₄. Immunohistochemically, Hsp25-positive hepatocytes started to appear in the perivenular area at 6 h after CCl₄ administration. Their number and strength increased till day 1. Expression of Hsp25 mRNA significantly increased after 3 h and proceeded to increase with time till day 1. Apoptotic hepatocytes were detected around the perivenular area after 6 h. The area where Hsp25-positive hepatocytes were observed till day 1 corresponded to the area where apoptotic hepatocytes were seen. On days 2 and 3, degenerative and/or necrotic hepatocytes in the perivenular area were replaced by macrophages reacting to ED1 (for CD68) and ED2 (for CD163); Hsp25 expression was seen in hepatocytes around the perivenular area and there was a close relationship of reactive macrophages with Hsp25-positive hepatocytes, suggesting a potential role for Hsp25 in suppressing injury by inflammation. The mRNA expression of tumor necrosis factor-α, monocyte chemoattractant protein-1 and osteopontin, which can be produced by infiltrating macrophages, corresponded to that of Hsp25 from day 1 to day 3; these factors might be related to the induction of Hsp25 expression. The shift of the Hsp25 expression pattern in the liver lesion might have depended on microenvironmental conditions evoked by interactions between necrobiotic hepatocytes and infiltrating macrophages. Thus, Hsp25 expression analyses should be beneficial for evaluations of hepatotoxicants.     

A developmental study of the synovial membrane of the rat temporomandibular joint: changes in the three-dimensional configuration during postnatal development

The development of synovial membranes in the posterior synovial portion of the rat temporomandibular joint was studied and the three-dimensional structure of the posterior synovial portion reconstructed from sagittal semithin sections. Reconstructions showed that the synovial membrane expanded and that synovial folds increased in number and became complicated in shape with the growth of the joint. Using transmission-electron microscopy, it was observed that the synovial lining cells degenerated, that the synovial membrane split to make further synovial folds, and that the folded-end structures consisted of synovial lining cells that extended into the subsynovial connective tissue. It is suggested that in the development of the three-dimensional configuration of the synovial membrane, several processes proceed simultaneously to form the synovial folds: a splitting of the synovial membrane, infolding of the synovial membrane into the subsynovial connective tissue, and outgrowth of the synovial folds towards the synovial cavity.     

Topography and distribution of sympathetic nerve fibers in the rat temporomandibular joint: immunocytochemistry and ultrastructure

The distribution and fine structure of nerve fibers containing neuropeptide Y (NPY), tyrosine hydroxylase (TH), and vasoactive intestinal polypeptide (VIP) in the temporomandibular joint were investigated by both the avidin-biotin complex method and an indirect immunofluorescence technique. The innervation pattern of NPY- and TH-positive fibers differed from that of VIP-positive fibers. Specifically, the former was distributed in both the superficial and deep sublining layers, while the latter was mostly located in the deep sublining layer. NPY- and TH-immunoreactive fibers were largely confined to vascular elements; occasional fibers were observed in the synovial lining layer close to the joint cavity. More nerves with NPY and TH immunoreactivity were observed close to the upper joint compartment than near the lower compartment NPY and TH immunoreactivity was dramatically reduced in the TMJ of superior cervical ganglionectomized animals, indicating the sympathetic origin of these nerves. NPY immunoreactivity was found only in unmyelinated axons, which were located in the adventitia and adventitia-medial border of arteries or arterioles. Occasionally, axons were near the joint cavity, in areas free of vascular structures. These observations show that abundant sympathetic nerves supply the temporomandibular joint of the rat and provide a morphological basis for the involvement of different neuropeptides in vascular regulatory and modulatory functions in physiological and pathophysiological conditions.     

Relationship of heat shock protein 25 with reactive macrophages in thioacetamide-induced rat liver injury

Heat shock protein 25 (Hsp25), which has anti-inflammatory activity, was examined for the relationship of its expression to macrophage appearance in thioacetoamide (TAA)-induced rat acute hepatic lesions. TAA-induced lesions, consisting of hepatocyte coagulation necrosis and reactive macrophages, developed in the centrilobular areas. Macrophages immuno-reacting to ED1 (CD68; exudative macrophages) were mainly seen within the lesions, whereas macrophages reacting to ED2 (CD163; resident macrophages and Kupffer cells), which have abundant cytoplasm, appeared mainly in the periphery of the lesions. Hsp25-immunopositivity was seen in hepatocytes around the lesions in relation to ED1- and ED2-positive macrophages in and around the centrilobular lesions, respectively. Because macrophages appearing in early stages of hepatic lesions produce various pro-inflammatory factors, mRNA expressions of tumor necrosis factor-α (TNF-α), monocyte chemoattractant factor-1 (MCP-1) and osteopontin (OPN) were examined in relation to Hsp25 mRNA expression. Hsp25 mRNA expression generally was correlated with TNF-α, MCP-1 and OPN expressions, suggesting their direct or indirect association with Hsp25 expression. Thus, Hsp25 might have a cytoprotection function against macrophages appearing in hepatic lesions, and factors produced by macrophages in the very early stages of hepatic lesions may influence Hsp25 expression. Hsp25 expression should be useful as an index of anti-inflammatory action for evaluation of hepatotoxicants in vivo.     

Immunocytochemical localization of MAPKAPK-2 and Hsp25 in the rat temporomandibular joint

One series of our research has shown an intense expression of immunoreaction for heat shock protein 25 (Hsp25) in various cellular elements in the rat temporomandibular joint (TMJ). This protein is the major substrate of mitogen-activated protein kinase-activated protein kinase-2 (MAPKAPK-2), which mediates an intracellular stress-activated signaling pathway to stimulate cytosolic actin reorganization under various stresses. The present study was undertaken to examine the localization of MAPKAPK-2 in the rat TMJ by immunocytochemical techniques. Furthermore, confocal microscopy with double staining was employed to demonstrate the colocalization of MAPKAPK-2 and Hsp25. Immunocytochemistry for MAPKAPK-2 showed an intense immunoreaction in the cytoplasm of the synovial lining cells, the endothelial cells, and the fibroblasts in the synovial membrane of the rat TMJ. Double immunostaining under a confocal microscope succeeded in demonstrating the colocalization of MAPKAPK-2 and Hsp25 immunoreactions in the cytoplasm of fibroblastic type B synoviocytes in the TMJ. On the other hand, the macrophage-like type A-cells expressed MAPKAPK-2 immunoreactions but lacked Hsp25 immunoreactivity. The cells in the articular disk and the chondrocytes in the maturative and hypertrophic layer of the mandibular cartilage also showed intense immunoreactions for MAPKAPK-2 and Hsp25. In addition to cytoplasmic localization, MAPKAPK-2 immunoreactions were found in the nucleus of some synovial lining cells, cells in the articular disk, and chondrocytes. Current observations imply the presence of the phosphorylation of Hsp25 via activated MAPKAPK-2 in the cytoplasm. MAPKAPK-2 and Hsp25 possibly participate in the induction of cytoskeletal changes to the various cellular elements in rat TMJ under normal conditions.     

Immunohistochemical localization of heat shock protein 25 (HSP 25) during root formation of the rat molar

The present study investigated the immunohistochemical localization of heat shock protein 25 (HSP 25) of rat molar teeth during root formation. Most, probably all, cells of the epithelial rest of Malassez (ERM cells) had immunoreaction for laminin, a marker protein for basement membrane. During root formation, HSP 25 immunoreactivity was observed in odontoblasts, cells at the subodontoblastic layer, and those in close proximity to the acellular cementum. HSP 25-immunopositive cells at the subodontoblastic layer were present only at the apical region. Most HSP 25-immunoreactive cells in close proximity to the cementum lacked laminin immunoreactivity. However, at postnatal day 28 a small number of cells showed immunoreaction for both HSP 25 and laminin at the cervical and bifurcational regions. Under the electron microscope, most HSP 25-immunoreactive cells along the surface of the cementum were round and contained rich organelles such as mitochondria and rough endoplasmic reticulum. They lay between fiber bundles of the periodontal ligament. The localization and morphological features of these HSP 25-immunoreactive cells resemble those of cementoblasts. On the other hand, HSP 25-immunoreactive cells at the cervical region were oval and contained few cell organelles. They were closely apposed to each other, and separated from the surrounding tissues with basal lamina. These features were similar to those of mature ERM cells. In contrast, cells with microvillus-like processes and relatively rich mitochondria, which were similar to immature ERM cells, had no immunoreaction for HSP 25. These results suggest that HSP 25 may be involved in shape alterations of ERM cells, cementoblasts, and odontoblasts during differentiation.     

A hypothetical biological synovial fluid for treatment of temporomandibular joint disease

Temporomandibular Joint disorder (TMD) is a common disorder of mandibular motion system with distinct clinicopathological characteristics. TMD may cause to change in the components of synovial fluid, that affects the functions on lubrication and nutrition of cartilage. Boundary lubrication system contributing to the low friction of joint consists of three parts: lubricin, surface-active phospholipids and hyaluronan (HA). Diminishment of lubrication function is thereby implicated as an adverse contributing factor in degenerative joint diseases such as internal derangement, osteoarthrosis. Moreover, mesenchymal stem cells (MSCs) of synovial membrane can be obtained without irreversible damage, are easily expandable with limited senescence. We postulate that biological active components secreted from MSCs are separated and accumulated by gel permeation chromatography, and then we use the ultra-flirtation of serum and biologically active components to reconstruct the biological synovial fluid in order to rehabilitate the boundary lubrication system and the nutrition of cartilage. Further study investigating the components of biological synovial fluid provides with new treatment strategy for TMD.     

Development of the human temporomandibular joint

A great deal of research has been published on the development of the human temporomandibular joint (TMJ). However, there is some discordance about its morphological timing. The most controversial aspects concern the moment of the initial organization of the condyle and the squamous part of the temporal bone, the articular disc and capsule and also the cavitation and onset of condylar chondrogenesis. Serial sections of 70 human specimens between weeks 7 and 17 of development were studied by optical microscopy (25 embryos and 45 fetuses). All specimens were obtained from collections of the Institute of Embryology of the Complutense University of Madrid and the Department of Morphological Sciences of the University of Granada. Three phases in the development of the TMJ were identified. The first is the blastematic stage (weeks 7–8 of development), which corresponds with the onset of the organization of the condyle and the articular disc and capsule. During week 8 intramembranous ossification of the temporal squamous bone begins. The second stage is the cavitation stage (weeks 9–11 of development), corresponding to the initial formation of the inferior joint cavity (week 9) and the start condylar chondrogenesis. Week 11 marks the initiation of organization of the superior joint cavity. And the third stage is the maturation stage (after week 12 of development). This work establishes three phases in TMJ development: 1) the blastematic stage (weeks 7–8 of development); 2) the cavitation stage (weeks 9–11 of development); and 3) the maturation stage (after week 12 of development). This study identifies the critical period of TMJ morphogenesis as occurring between weeks 7 and 11 of development. Anat Rec 255:20–33, 1999. © 1999 Wiley‐Liss, Inc.     

Expression of the heat shock protein 47 in gentamicin-treated rat kidneys

Heat-shock proteins (HSPs) are rapidly synthesized in cells in response to various cytotoxic agents. Although several stress proteins are actively involved in the gentamicin-induced renal damages, the possible role of HSP47 in this condition is not yet clear. In this study, the expression of HSP47 in the gentamicin nephrotoxicity was examined by immunohistochemistry. Twenty male Wistar rats were sacrificed at day 0, 3, 7, 14 and 28 after subcutaneous injection of gentamicin. Gentamicin treatment causes tubular necrosis at day 3, followed by tubular regenerative changes and interstitial fibrosis, which was most prominent at day 14. The renal structures returned to almost normal architectures at day 28. By immunohistochemistry, HSP47 was weakly expressed in most of the glomeruli and occasionally in interstitial cells in the control rat kidneys. In contrast, strong immunostaining for HSP47 was noted in the tubular epithelial cells and interstitial cells in gentamicin treated rat kidneys, and strongest staining was observed at day 7. The immunostaining for HSP47 then gradually decreased, and returned to the normal level at day 28. In the whole experimental period, staining pattern of HSP47 in the glomeruli was not changed. In addition, phenotypically altered tubulointerstitial cells including regenerative tubular epithelial cells (immuno-positive for vimentin) and interstitial cells (immuno-positive for α-smooth muscle actin) were found in gentamicin nephrotoxicity. Expression of type III collagen increased in the areas of interstitial fibrosis. By double immunostaining, the regenerated and phenotypically altered tubulointerstitial cells were found to express HSP47 in and around interstitial fibrosis. It is concluded that overexpression of HSP47 by phenotypically altered renal cells might play a significant role in the development of gentamicin nephrotoxicity.     

Possible role of heat shock protein (Hsp) 25 in the enamel organ during amelogenesis in the rat molar.

The postnatal expression of heat shock protein (Hsp) 25 during the amelogenesis of rat molars was investigated by immunocytochemistry and confocal microscopy. The localization pattern of Hsp 25-immunoreactivity in the inner enamel epithelium and ameloblast cell layer of the rat molars was almost identical to that in the rat incisors which we have previously reported: an intense Hsp25-immunoreactivity, which first appeared in the preameloblasts, was recognized in secretory ameloblasts and ruffle-ended ameloblasts with stage-specific immunointensity. Confocal microscopy with Hsp 25-antibody and rhodamine-labeled phalloidin clearly demonstrated the co-localization of Hsp 25 and actin filaments in the ameloblast layer, supporting our hypothesis that this molecule might serve to reinforce the ameloblast layer during enamel formation as well as the formation and maintenance of the ruffled border in ruffle-ended ameloblasts. Interestingly, the enamel free area cells, which essentially lack the ability for enamel formation, showed the Hsp 25-immunoreactivity during 4-11 days when they developed a ruffled border, but decreased in that immunoreactivity after postnatal 15 days following apoptosis. Since Hsp 25 has been shown to be a specific inhibitor of apoptosis, the enamel-free area cells contribute to determine the outline of dentin at the cusped area. These data support our previous hypothesis on the diverse functions of Hsp 25 in amelogenesis.     

Expression of caveolin-1 in the rat temporomandibular joint

This immunocytochemical study revealed the expression of caveolin-1, a major protein of caveolae, in the rat temporomandibular joint. In the synovial lining layer, immunoreactive products for caveolin-1 were detected on the cell membrane of the fibroblast-like type B cells, as confirmed by immunocytochemistry for heat shock protein 25. The cells in the articular disk, the articular layer, and zone of proliferation of the mandibular condyle also showed intense immunoreactions for caveolin-1.     

Development of the human temporomandibular joint.

A great deal of research has been published on the development of the human temporomandibularjoint (TMJ). However, there is some discordance about its morphological timing. The most controversial aspects concern the moment of the initial organization of the condyle and the squamous part of the temporal bone, the articular disc and capsule and also the cavitation and onset of condylar chondrogenesis. Serial sections of 70 human specimens between weeks 7 and 17 of development were studied by optical microscopy (25 embryos and 45 fetuses). All specimens were obtained from collections of the Institute of Embryology of the Complutense University of Madrid and the Department of Morphological Sciences of the University of Granada. Three phases in the development of the TMJ were identified. The first is the blastematic stage (weeks 7-8 of development), which corresponds with the onset of the organization of the condyle and the articular disc and capsule. During week 8 intramembranous ossification of the temporal squamous bone begins. The second stage is the cavitation stage (weeks 9-11 of development), corresponding to the initial formation of the inferior joint cavity (week 9) and the start condylar chondrogenesis. Week 11 marks the initiation of organization of the superior joint cavity. And the third stage is the maturation stage (after week 12 of development). This work establishes three phases in TMJ development: 1) the blastematic stage (weeks 7-8 of development); 2) the cavitation stage (weeks 9-11 of development); and 3) the maturation stage (after week 12 of development). This study identifies the critical period of TMJ morphogenesis as occurring between weeks 7 and 11 of development.     

Radioprotective effect of heat shock protein 25 on submandibular glands of rats

Irradiation (IR) is a fundamental treatment modality for head and neck malignancies. However, a significant drawback of IR treatment is irreversible damage of salivary gland in the IR field. In the present study, we investigated whether heat shock protein (HSP) 25 could be used as a radioprotective molecule for radiation-induced salivary gland damage in rats. HSP25 as well as inducible HSP70 (HSP70i) that were delivered to the salivary gland via an adenoviral vector significantly ameliorated radiation-induced salivary fluid loss. Radiation-induced apoptosis, caspase-3 activation, and poly(ADP-ribose) polymerase cleavage in acinar cells, granular convoluted cells, and intercalated ductal cells were also inhibited by HSP25 or HSP70i transfer. The alteration of salivary contents, including amylase, protein, Ca+, Cl-, and Na+, was also attenuated by HSP25 transfer. Histological analysis revealed almost no radiation-induced damage in salivary gland when HSP25 was transferred. Aquaporin 5 expression in salivary gland was inhibited by radiation; and HSP25 transfer to salivary gland prevented this alteration. The protective effect of HSP70i on radiation-induced salivary gland damage was less or delayed than that of HSP25. These results indicate that HSP25 is a good candidate molecule to protect salivary gland from the toxicity of IR.