Seiichi Kawamata

Role of Osteoprotegerin in Arterial Calcification Development of New Animal Model

Objectives—Enhanced osteoclastogenesis, increased bone resorption, and osteoporosis have been reported in osteoprotegerin-deficient (OPG (−/−)) mice. OPG (−/−) mice available in Japan usually do not show vascular calcification. We have found that arterial calcification can be quickly induced by a simple procedure in OPG (−/−) mice. Methods and Results—Male OPG (−/−), OPG (+/−), and OPG (+/+) mice were fed a high phosphate diet from 6 to 10 weeks after birth, and then 1&agr;,25-dihydroxyvitamin D3 (calcitriol) was injected for 3 days. We found that severe calcification developed in the media of the aorta in OPG (−/−) mice. Under electron microscopy, calcium deposits were observed in the cytoplasm and extracellular matrix of vascular smooth muscle cells (VSMCs). Neither apoptosis of VSMCs nor infiltration of macrophages was observed. Alkaline phosphatase (ALP) activity of aortic tissue correlated with the calcified lesion area. Mouse aorta and bone extracts revealed an identical pattern by ALP electrophoresis. Conclusions—Our results demonstrated that OPG had anticalcification activity in the aorta, probably through the downregulation of ALP activity. Because the time course of arterial calcification after the injection of calcitriol is accurate and reproducible, this mouse model will be useful for further investigation of vascular calcification.

Regeneration of peripheral nerve after transplantation of CD133+ cells derived from human peripheral blood.

OBJECT Despite intensive efforts in the field of peripheral nerve injury and regeneration, it remains difficult to achieve full functional recovery in humans following extended peripheral nerve lesions. In this study, the authors examined the use of blood-derived CD133(+) cells in promoting the repair of peripheral nerve defects. METHODS The authors transplanted phosphate-buffered saline (control), mononuclear cells, or CD133(+) cells embedded in atelocollagen gel into a silicone tube that was used to bridge a 15-mm defect in the sciatic nerve of athymic rats (12 animals in each group). At 8 weeks postsurgery, molecular, histological, and functional evaluations were performed in regenerated tissues. RESULTS The authors found that sciatic nerves in which a defect had been made were structurally and functionally regenerated within 8 weeks after CD133(+) cell transplantation. From macroscopic evaluation, massive nervelike tissues were confirmed only in rats with CD133(+) cell transplantation compared with the other groups. Morphological regeneration in the samples after CD133(+) cell transplantation, as assessed using toluidine blue staining, was enhanced significantly in terms of the number of myelinated fibers, axon diameter, myelin thickness, and percentage of neural tissue. Compound muscle action potentials were observed only in CD133(+) cell-treated rats. Furthermore, it was demonstrated that the transplanted CD133(+) cells differentiated into Schwann cells by 8 weeks after transplantation. CONCLUSIONS The results show that CD133(+) cells have potential for enhancement of histological and functional recovery from peripheral nerve injury. This attractive cell source could be purified easily from peripheral blood and could be a feasible autologous candidate for peripheral nerve injuries in the clinical setting.

Magnetically labeled neural progenitor cells, which are localized by magnetic force, promote axon growth in organotypic cocultures.

Study Design. An in vitro, rat animal study was conducted. Objective. To assess the corticospinal axon growth potential in varying concentrations of neural progenitor cells (NPCs) and in magnetically localized labeled NPCs, quantitatively using our original organotypic coculture system. Summary of Background Data. Transplantation of NPCs for spinal cord injury has been anticipated as a possible future treatment. It is important not only to illuminate the mechanism of NPCs for spinal cord injury, but also to develop an effective cell delivery system for clinical use. In order to develop more effective, efficient, and minimally invasive cell delivery systems, we established a new system using magnetic targeting. Methods. Magnetically labeled NPCs were suspended with activated magnetic beads and individual NPCs, and were compared the characterization to nonlabeled NPCs in vitro. We transplanted varying concentrations of 102, 103, 104, 105, and 106 NPCs in 1 &mgr;L medium to coculture models. Then the 104 labeled NPCs were transplanted with or without magnet to the cocultures. Results. Magnetically labeled NPCs had similar potential in axon growth compared with nonlabeled NPCs, so there were few toxic effects of magnetically labeling NPCs. The differential potentials were not changed whether they were localized or scattered in vitro. Corticospinal axon growth was promoted in accordance with the transplanted NPC numbers around the organotypic coculture. Localized labeled NPCs with a magnet promoted axon growth much more than scattered labeled NPCs without a magnet, so magnetically localized labeled NPCs expressed higher potential in axon growth. Conclusion. Magnetically labeled NPCs, which were localized by magnetic force, could promote axon growth in this organotypic coculture system.

Education-associated cortical glucose metabolism during sustained attention.

For many diseases and injuries of the central nervous system, transplantation of neural progenitor cells is being evaluated as a possible treatment option. Although local, intravenous and subarachnoid injections have been reported as administration methods of neural progenitor cells, each of these methods has limitations. More effective and minimally invasive cell delivery systems are necessary for transplanting neural progenitor cells. In this study, we have developed a technique to form magnetically labeled neural progenitor cells for a magnetic targeting system. We demonstrated that neural progenitor cells can couple with magnetic beads, and that the labeled neural progenitor cells preserve the characteristics of non-labeled neural progenitor cells, and that they can be localized by magnetic force in vitro. Labeled neural progenitor cells have the potential to be used in magnetic targeting systems in-vivo models.

Alteration of Knee Joint Connective Tissues during Contracture Formation in Spastic Rats after an Experimentally Induced Spinal Cord Injury

This study was made to elucidate the changes in the periarticular connective tissue that can underlie the contracture after spasticity development. Sixteen Wistar rats underwent a spinal cord injury and 16 rats were either sham- or nonoperated. The periarticular connective tissue of the knee joint was assessed with histological, histomorphometric, immunohistochemical, and biochemical analyses. Histological results showed a smaller synovial intima, a dense subintima and posterior joint capsule without fibrosis, and a disarranged posterior capsule in the spinal cord-injured knees with the flexion contracture. The synovial intima length was shortened only at the posterior capsule. Neither the distribution nor expression of type I and III collagen was affected. Contractures after spinal cord injuries are characterized by synovial intima adhesions. A dense and disarranged capsule may lead to joint stiffness. The alteration of periarticular connective tissues exhibits properties characteristic of the contracture after spasticity development.

Light- and electron-microscopic study of the endolymphatic sac of the tree frog, Hyla arborea japonica.

SummaryThe endolymphatic sac of the tree frog and its crystals were observed by light- and electron microscopy. Scanning electron microscopy revealed that the crystals have a faceted body and two pointed ends. Light- and transmission electron microscopy revealed that the endolymphatic sac is composed of many small chambers. In their lumina, numerous “ghosts” of crystals that resulted from decalcification were observed. The ghosts were demarcated by a linear dense material or embedded in a flocculent substance. The epithelium of the endolymphatic sac is simple squamous or cuboidal and peculiar cytoplasmic granules are found in most cells. The granules are surrounded by a limiting membrane and have varying electron density. Some granules contain a core and/or tubular structures. Vacuoles containing large ghosts are also found in the epithelial cells. These ghosts were quite similar to those in the lumen and sometimes coexist with cell debris. The fine structure of the endolymphatic sac and its crystals is discussed.

The fine structure of atypical ciliated cells in the human gastric epithelium

SummaryGastric mucosa obtained from the body and pyloric portions of the human stomach were observed by light and transmission electron microscopy. Ciliated cells were found in two of 18 subjects examined, one patient with gastric ulcer and the other one with gastric adenocarcinoma. The ciliated cells were found in epithelia at sites away from the main lesions. The tissues containing ciliated cells showed intestinal metaplasia combined with mild chronic gastritis in both cases. The epithelial layer facing the gastric lumen was composed of columnar cells with numerous uniform microvilli and goblet cells. This epithelium extended to the superficial parts of the tubules surrounded by the lamina propria. The deeper portions of the tubules were composed of mucous secretory, endocrine, and rarely ciliated cells. These ciliated cells were provided with numerous cilia the numbers of which varied considerably from cell to cell. This was in contrast to the primary cilium which is usually single. The central part of the apical cell membrane was sometimes concave in the area from where cilia tended to arise. It was also observed that numerous basal bodies as well as mucus-like granules were contained in the same cell. The axonemal pattern was different from that of ordinary cilia and showed 9 + 0 and 8 + 1 patterns. In longitudinal sections it was found that one peripheral doublet was displaced to the center of the axoneme as it left the basal body.

Morphological changes in hind limb muscles elicited by adjuvant-induced arthritis of the rat knee

We investigated qualitative and quantitative changes in rat hind limb muscles caused by complete Freunds adjuvant (CFA)‐induced knee joint pain. One week after CFA injection, muscle atrophy was induced only on the CFA‐injected side. Wet weight of the rectus femoris (RF) and soleus (SOL) muscles were significantly decreased by 20% and 19%, respectively. The reduction in cross‐sectional areas by CFA was similar for fast and slow muscle fibers in the RF (10% vs 15%, respectively) and SOL muscles (16% vs 16%, respectively). At the light microscopic level, pathological changes were not found in the RF muscles on both sides, although the infiltration of mononuclear cells and muscle regeneration were found in the SOL muscles on CFA‐injected and contralateral control sides. On the other hand, electron microscopy revealed degenerative changes in the RF and SOL muscles on the CFA‐injected side. Interestingly, sarcomere hypercontraction, indicating overexercise, was observed to a limited extent in the SOL muscles on the control side. In conclusions, knee joint pain can trigger the rapid development of muscle atrophy with degenerative changes not only in thigh but also calf muscles. This indicates that early interventions to inhibit joint pain or inflammation may prevent muscle atrophy.

Analysis of Gene Expression in Experimental Pressure Ulcers in the Rat with Special Reference to Inflammatory Cytokines.

Pressure ulcers have been investigated in a few animal models, but the molecular mechanisms of pressure ulcers are not well understood. We hypothesized that pressure results in up-regulation of inflammatory cytokines and those cytokines contribute to the formation of pressure ulcers. We measured genome-wide changes in transcript levels after compression, and focused especially on inflammatory cytokines. The abdominal wall of rats was compressed at 100 mmHg for 4 hours by two magnets. Specimens were obtained 12 hours, 1, or 3 days after compression, and analyzed by light microscopy, microarray, Real-Time PCR, and ELISA. The skin and subcutaneous tissue in the compressed area were markedly thickened. The microarray showed that numerous genes were up-regulated after the compression. Up-regulated genes were involved in apoptosis, inflammation, oxidative stress, proteolysis, hypoxia, and so on. Real-Time PCR showed the up-regulation of granulocyte-macrophage colony stimulating factor (GM-CSF), interferon γ (IFN-γ), interleukin 1β (IL-1β), interleukin 1 receptor antagonist gene (IL1Ra), interleukin 6 (IL-6), interleukin 10 (IL-10), matrix metalloproteinase 3 (MMP-3), tissue inhibitor of metalloproteinase 1 (TIMP-1), and tumor necrosis factor α (TNF-α) at 12 hours, IFN-γ, IL-6, IL-10, MMP-3, and TIMP-1 at 1 day, and IFN-γ, IL-6, and MMP-3 at 3 days. Some genes from subcutaneous tissue were up-regulated temporarily, and others were kept at high levels of expression. ELISA data showed that the concentrations of IL-1β and IL-6 proteins were most notably increased following compression. Prolonged up-regulation of IL-1β, and IL-6 might enhance local inflammation, and continuous local inflammation may contribute to the pressure ulcer formation. In addition, GM-CSF, IFN-γ, MMP-3, and TIMP-1 were not reported previously in the wound healing process, and those genes may have a role in development of the pressure ulcers. Expression data from Real-Time PCR were generally in good agreement with those of the microarray. Our microarray data were useful for identifying genes involved in pressure ulcer formation. However, the expression levels of the genes didn’t necessarily correspond with protein production. As such, the functions of these cytokines need to be further investigated.

Development of arthrogenic joint contracture as a result of pathological changes in remobilized rat knees.

This study aimed to elucidate how rats recover from immobilization‐induced knee joint contracture. Rats’ right knees were immobilized by an external fixator at a flexion of 140° for 3 weeks. After removal of the fixator, the joints were allowed to move freely (remobilization) for 0, 1, 3, 7, or 14 days (n = 5 each). To distinguish myogenic and arthrogenic contractures, the passive extension range of motion was measured before and after myotomy of the knee flexors. Knee joints were histologically analyzed and the expression of genes encoding inflammatory or fibrosis‐related mediators, interleukin‐1β (1L‐1β), fibrosis‐related transforming growth factor‐β1 (TGF‐β1), and collagen type I (COL1A1) and III (COL3A1), were examined in the knee joint posterior capsules using real‐time PCR. Both myogenic and arthrogenic contractures were established within 3 weeks of immobilization. During remobilization, the myogenic contracture decreased over time. In contrast, the arthrogenic contracture developed further during the remobilization period. On day 1 of remobilization, inflammatory changes characterized by edema, inflammatory cell infiltration, and upregulation of IL‐1β gene started in the knee joint posterior capsule. In addition, collagen deposition accompanied by fibroblast proliferation, with upregulation of TGF‐β1, COL1A1, and COL3A1 genes, appeared in the joint capsule between days 7 and 14. These results suggest the progression of arthrogenic contracture following remobilization, which is characterized by fibrosis development, is possibly triggered by inflammation in the joint capsule. It is therefore necessary to focus on developing new treatment strategies for immobilization‐induced joint contracture.