Yoshito Yoshimine

Increased Expression of Cathepsins E and D in Neurons of the Aged Rat Brain and Their Colocalization with Lipofuscin and Carboxy‐Terminal Fragments of Alzheimer Amyloid Precursor Protein

Abstract: Age‐related changes in the expression and localization of two distinct intracellular aspartic proteinases, cathepsin E (CE) and cathepsin D (CD), were investigated in the rat cerebral cortex and the brainstem by immunocytochemical and quantitative methods using discriminative antibodies specific for each enzyme. Non‐lysosomal CE was barely detectable in these two brain tissues in the embryonic stages, whereas relatively high expression of lysosomal CD was observed in embryonic tissues. After birth, CE was increasingly expressed in these tissues with aging to attain maximal levels at 30 months of age. Western blot analyses revealed that CE existed predominantly as the mature enzyme at 2 and 17 months of age, whereas it was present as not only the mature enzyme but also the proenzyme at 30 months of age. On the other hand, CD was mainly present in the mature form throughout development, although its level in these tissues was also significantly increased with aging. The CE‐positive cortical and brainstem neurons of the aged rat corresponded well with cells emitting autofluorescence for lipopigments. By the double‐staining technique, most of the CE‐positive cortical and brainstem neurons of the aged rat were also positive for antibody to the carboxyl‐terminal fragments of amyloid precursor protein (APP634–695), intracellular accumulation of which is thought to be associated with age‐related changes in the endosome/lysosome system. It is important that electron microscopy revealed that CE in brainstem neurons of the aged rat colocalized with CD in the lipofuscin‐containing lysosomes. These results indicate that aging results in the increased expression and lysosomal localization of CE in cortical and brainstem neurons and changes in the endosomal/lysosomal proteolytic system, which may be related to lipofuscinogenesis and altered intracellular APP metabolism.

Visualization of Irrigant Flow and Cavitation Induced by Er:YAG Laser within a Root Canal Model

INTRODUCTION Laser-activated irrigation (LAI) has recently been introduced as an innovative method for root canal irrigation. However, there is limited information about the cleaning mechanism of an Er:YAG laser. In this study, we visualized the action of laser-induced bubbles and fluid flow in vitro to better understand the physical mechanisms underlying LAI. METHODS An Er:YAG laser was equipped with a novel cone-shaped tip with a lateral emission rate of approximately 80%. Laser light was emitted at a pulse energy of 30, 50, or 70 mJ (output energy: 11, 18, or 26 mJ) and a repetition rate of 1 or 20 pulses per second, without air or water spray. Fluid flow dynamics in a root canal model were observed by using glass-bead tracers under a high-speed camera. Moreover, laser-induced bubble patterns were visualized in both free water and the root canal model. RESULTS Tracers revealed high-speed motion of the fluid. A full cycle of expansion and implosion of vapor and secondary cavitation bubbles were clearly observed. In free water, the vapor bubble expanded for 220 microseconds, and its shape resembled that of an apple. In the root canal model, the vapor bubble expanded in a vertical direction along the canal wall, and bubble expansion continued for ≥700 microseconds. Furthermore, cavitation bubbles were created much more frequently in the canal model than in free water. CONCLUSIONS These results suggest that the cleaning mechanism of an Er:YAG laser within the root canal might depend on rapid fluid motion caused by expansion and implosion of laser-induced bubbles.

Bone-like nodules formed in vitro by rat periodontal ligament cells

The periodontal ligament has been shown to possess the ability to regenerate both new cementum and alveolar bone as well as a self-regenerative capacity; however, the source of cementoblasts and osteoblasts is not still clear. We investigated the development of bone-like tissue in vitro by periodontal ligament cells, in order to determine whether the periodontal ligament contains osteoprogenitor cells. Periodontal ligament cells were obtained from periodontal ligament tissue attached to the maxillary incisors of 6-week-old WKA rats by means of the explant technique. Cells at passage #3 were cultured for long term in α-minimum essential medium containing 10% fetal bovine serum, antibiotics, and 50 μg/ml ascorbic acid, and were then examined using phase-contrast microscopy, histochemistry, transmission electron microscopy, X-ray microanalysis, and electron diffraction. Nodules were formed in the cultures, and when 10 mM Na-β-glycerophosphate was added, these nodules became mineralized. The mineralized nodules were identified as bone-like elements in view of the presence of osteoblast-like and osteocyte-like cells, collagenous matrix, a mineral composed of hydroxyapatite, and intense alkaline phosphatase activity. The results show that the periodontal ligament contains osteoprogenitor cells, which differentiate into osteoblasts and produce bone-like tissue.

Specific immunocytochemical localization of cathepsin E at the ruffled border membrane of active osteoclasts

The immunocytochemical localization of cathepsin E, a non-lysosomal aspartic proteinase, was investigated in rat osteoclasts using the monospecific antibody to this protein. At the light-microscopic level, the preferential immunoreactivity for cathepsin E was found at high levels in active osteoclasts in the physiological bone modeling process. Neighboring osteoblastic cells were devoid of its immunoreactivity. At the electron-microscopic level, cathepsin E was exclusively confined to the apical plasma membrane at the ruffled border of active osteoclasts and the eroded bone surface. Cathepsin E was also concentrated in some endocytotic vacuoles of various sizes in the vicinity of the ruffled border membrane, some of which appeared to be secondary lysosomes containing the phagocytosed materials. These results strongly suggest that this enzyme is involved both in the extracellular degradation of the bone organic matrix and in the intracellular breakdown of the ingested substances in osteoclasts.

Histologic evaluation of tetracalcium phosphate-based cement as a direct pulp-capping agent

Histologic healing processes were observed at 1, 3, 7, and 10 days after application with either tetracalcium phosphate cement or calcium hydroxide cement to the exposed pulp of the rat maxillary incisors. In teeth applied with calcium hydroxide cement, necrotic tissue was present beneath the cement before new hard tissue formed. In contrast, tetracalcium phosphate cement elicited a dentine bridge formation with no evidence of either intervening tissue necrosis or marked inflammation. Furthermore on ultrastructural examination the newly formed hard tissue was in direct contact with the material. This study suggests that 4CP cement possesses a biocompatible property, which indicates its potential for use as a direct pulp-capping agent.

Biocompatibility of tetracalcium phosphate cement when used as a bone substitute

We evaluated the biocompatibility of tetracalcium phosphate (4CP) cement, made of 4CP powder and 40 wt% copolymer of polyacrylic acid/itaconic acid and 10 wt% citric acid solution. Light and electron microscopic characteristics were studied 3, 10 and 30 d after implantation. Neither inflammation nor foreign-body giant cell reaction was observed in the tissue adjacent to the implanted material. After 30 d, this material was surrounded with newly formed bone. Ultrastructural examination showed that osteogenesis occurred directly on the surface of the material. These findings suggest that this 4CP cement is biocompatible and possesses osteoconductive properties.

Cathepsin D deficiency induces oxidative damage in brain pericytes and impairs the blood-brain barrier

Recent evidence suggests that peripheral blood mononuclear cells (PBMCs) contribute to the pathogenesis of neuropathological changes in patients with neuronal ceroid lipofuscinosis (NCL) and lysosomal storage diseases. In order to examine the possible increase in the permeability of the blood-brain-barrier (BBB) and resultant infiltration of PBMCs due to cathepsin D (CatD) deficiency, a process underlying the onset of congenital NCL, we examined structural changes in brain vessels in CatD-/- mice. Consequently, the mean diameter of the brain vessels in the cerebral cortex on postnatal day 24 (P24) was significantly larger in CatD-/- mice than in wild-type mice. Furthermore, the mean number of brain pericytes in CatD-/- mice began to decline significantly on P16 and almost disappeared on P24, and oxidative DNA damage was first detected in brain pericytes on P12. Examinations with electron microscopy revealed that brain pericytes were laden with dense granular bodies, cytoplasmic vacuoles and lipid droplets. The infiltration of PBMCs characterized by segmented nucleus laden with dense granular bodies was also noted in the cerebral cortex of CatD-/- mice. When primary cultured microglia prepared from enhanced green fluorescent protein (GFP)-expressing transgenic rats were injected into the common carotid artery, GFP-positive microglia were detected in the brain parenchyma of CatD-/-, but not wild-type, mice. Moreover, pepstatin A, a specific aspartic protease inhibitor, induced mitochondria-derived reactive oxygen species (ROS) production in the isolated brain pericytes, which decreased the cell viability. These observations suggest that increased lysosomal storage due to CatD deficiency causes oxidative damage in brain pericytes, subsequently resulting in an increased vessel diameter, enhanced permeability of the BBB and the infiltration of PBMCs. Therefore, protecting brain pericytes against lysosomal storage-induced oxidative stress may represent an alternative treatment strategy for congenital NCL.

In Vitro Evaluation of the Cytocompatibility of a Glass-Ionomer Cement Sealer

The purpose of this study was to evaluate the cytocompatibility of two different types of root canal sealers in cell culture. Human periodontal ligament cells were cultured with set materials from an experimental glass-ionomer cement sealer (KT-308) and a commercially available zinc oxide-eugenol-based sealer (Canals) for 1, 3, and 7 days. Cytotoxic effects were evaluated from the morphological changes under a light microscope. Canals induced severe degenerative alteration of human periodontal ligament cells. In contrast, human periodontal ligament cells adjacent to KT-308 showed normal morphology and growth during the culture period. These results suggest that the glass-ionomer cement sealer, KT-308, is cytocompatible and has good potential as a root canal sealer.

Biocompatibility of an experimental glass-ionomer cement sealer in rat mandibular bone

OBJECTIVE The purpose of this study was to compare an experimental glass-ionomer cement sealer, KT-308, with a conventional zinc oxide-eugenol sealer, Canals, in terms of tissue compatibility and solubility. STUDY DESIGN Tissue reactions were examined under light and electron microscopes at 3 and 20 days after the implantation of either freshly mixed KT-308 sealer or Canals sealer into prepared cavities in rat mandibles. RESULTS At 3 days after implantation, no inflammatory reaction was seen around KT-308 sealer, which was in direct contact with the bone surface. In contrast, Canals sealer elicited an initially severe inflammation in the surrounding tissue. At 20 days, the majority of KT-308 sealer remained in the bone cavity. Canals sealer was largely absorbed and surrounded by fibrous tissue with many macrophages. An ultrastructural examination also revealed that no intervening tissue was present between the cut bone surface and the glass-ionomer cement sealer and that disintegrated zinc oxide-eugenol particles were engulfed by macrophages. CONCLUSIONS KT-308 sealer possesses better tissue compatibility and lower solubility compared with a conventional zinc oxide-eugenol sealer, suggesting its potential for use as a root canal sealant.