Immunohistochemical localization of phosphatidylcholine in rat mandibular condylar surface and lower joint cavity by cryotechniques.

The immunolocalization of phospholipids has not yet been clearly demonstrated in temporomandibular joints (TMJs). We have examined the distribution of one of phospholipids, phosphatidyl-choline (PC), in the rat mandibular condylar surface and lower joint cavity. Some fresh resected TMJs with their disks attached were immediately plunged into isopentane-propane cryogen (-193 degrees C). Cryostat sections were cut, mounted on NH3+-coated slides, and fixed with paraformaldehyde (PF). Cryosections were first immunostained with anti-mouse PC antibody (JE-1). Subsequently, they were labeled with immunogold particles following silver enhancing for light microscopic analyses. Some cryosections were subjected to double immunofluoresecence labeling with anti-fibronectin antibody or hyaluronic acid-binding protein in combination with the anti-PC antibody. As an immunocontrol, other cryosections were pretreated with phospholipase A2 before such immunofluorescence labeling. We have confirmed the presence of PC in the lower joint cavity of rat TMJs as well as on the mandibular condylar surface layer, which was colocalized with hyaluronic acid and fibronectin respectively. However, by treatment with phospholipase A2, such immunolabeling for PC was clearly decreased, showing that the PC is a component in the rat in vivo TMJ. These findings suggest that PC, hyaluronic acid and fibronectin may interact each other in the TMJ articular surface areas to play a functional role for lubrication in TMJ.

Immunohistochemical study of the upper surface layer in rat mandibular condylar cartilage.

Both hyaluronic acid and fibronectin localizations were examined in the upper surface layer of rat mandibular condylar cartilages by immunohistochemical techniques. Their delicate structure was successfully preserved by preparation procedures of joint condyles with disks. Paraformaldehyde-fixed cartilaginous tissues were cut in a cryostat, and cryosections were analyzed using streptavidin-peroxidase and indirect immunofluorescence methods. Another immunogold method with conventional preparation procedures and a quick-freezing method was performed for their ultrastructural analyses. Both hyaluronic acid-binding protein and anti-fibronectin antibody were used to localize hyaluronic acid and fibronectin in the mandibular condylar cartilage, respectively. Some cryosections were pre-treated with hyaluronidase and chondroitinase before such labeling. The upper surface layer was composed of double laminar structures. One bordered with the cartilage matriceal surface, which was positive for fibronectin. The hyaluronic acid was localized over the fibronectin layer. Therefore, the hyaluronic acid in vivo was bound with fibronectin in the cartilaginous matrix, performing lubrication for the mandibular joint movement.

Ultrastructural study of upper surface layer in rat mandibular condylar cartilage by quick-freezing method.

The purpose of the present study is to clarify native ultrastructures of upper surface layers of the rat mandibular condylar cartilage in vivo by a quick-freezing method. The mandibular cartilaginous tissues were removed with their articular discs attached without opening the lower joint cavity. The specimens were processed for light microscopy, transmission or scanning electron microscopy. Deep-etching replica membranes were also prepared after the routine quick-freezing method. The upper surface layer was well preserved by the quick-freezing method. The cartilaginous tissues, which were fixed without opening their articular discs, appeared to keep better morphology than those after opening them. The upper surface layer was thicker than the corresponding layer as reported before. It consisted of atypical extracellular matrices with lots of apparently amorphous components, which were distributed over typical collagen fibrils, by conventional electron microscopy. As revealed with the replica membranes, it also consisted of variously sized filaments and tiny granular components localized on the typical collagen fibrils. A pair of stereo-replica electron micrographs three-dimensionally showed compact filaments within the upper surface layer. The quick-freezing method was useful for keeping native ultrastructures of the fragile upper surface layer in the mandibular condylar cartilage, which may be functionally important to facilitate smooth movement of the temporomandibular joint.

Effects of anoxia on serum immunoglobulin and albumin leakage through blood-brain barrier in mouse cerebellum as revealed by cryotechniques.

The purpose of this study was to examine time-dependent topographical changes of leaking proteins from blood vessels in the mouse cerebellum to assess the effect of normal blood circulation on the blood-brain barrier (BBB). The distribution of leaking serum proteins was immunohistochemically examined by various cryotechniques including our "in vivo cryotechnique". The cryofixed cerebellar tissues were processed for the freeze-substitution method, and finally embedded in the common paraffin wax. Serial de-paraffinized sections were immunostained by anti-mouse immunoglobulin-G (IgG) or albumin antibody. By combination of the "in vivo cryotechnique", in which normal blood flow into the cerebellum was always kept in vivo, with the freeze-substitution method, serum IgG and albumin were clearly localized inside of cerebellar blood vessels. To examine abnormal leakage of blood vessels as a model of anoxia, some cerebellar tissues were partially removed from brains in the mouse skull and quickly frozen in the isopentane-propane within a minute. In such resected cerebellar tissues, serum IgG and albumin were diffusely immunostained in large areas around the blood capillaries, probably because of easy leakage of the serum components through the immediately changed BBB. To the contrary, no serum protein could be identified outside blood capillaries under living conditions of the anesthetized mice. The present combination method, both "in vivo cryotechnique" and freeze-substitution, for immunohistochemistry enabled us to examine the in vivo localization of serum components in mouse brains due to alteration of the BBB.