Ryoichi Tsukuda

In vivo stimulation of endosteal bone formation by basic fibroblast growth factor in rats.

Intravenous administration of human basic fibroblast growth factor (bFGF) for 2 weeks stimulated osteoblast proliferation and new bone formation in various skeletal bones in young and aged rats at dosage levels of 0.1 mg/kg/day and greater. Morphometry of the soft X-ray radiograms of cross sections of the tibia indicated about a 20% increase in the calcified bone area of the diaphysis at 0.1 mg/kg/day. The Ca and hydroxyproline contents showed statistically significant increases at this dosage. The new bone formation was found only on the endosteal side, and no periosteal bone formation was found. Similar systemic osteogenic potential was seen after intravenous administration of other growth factors of the FGF family, human acidic FGF and human heparin-binding secretory transforming protein-1. The above results suggest a potential therapeutic role for these growth factors in bone-loss diseases such as osteoporosis.

Increase of basic fibroblast growth factor immunoreactivity and its mRNA level in rat brain following transient forebrain ischemia

SummaryWe examined the time course of basic fibroblast growth factor (bFGF) immunoreactivity and its mRNA level mainly in the hippocampus after transient forebrain ischemia using immunohistochemistry, enzyme immunoassay (EIA), Western blot analysis and in situ hybridization. Neuronal death in the hippocampal CA1 subfield was observed 72 h after 20 min of ischemia. The number of bFGF-immunoreactive(IR) cells increased 48 h–5 days after ischemia in all hippocampal regions. At 10 and 30 days, the bFGF-IR cells in the CA1 subfield had further increased in numbers and altered their morphology, enlarging and turning into typical reactive astrocytes with the advancing neuronal death in that area. In contrast, the number of bFGF-IR cells in other hippocampal regions had decreased 30 days after ischemia. The EIA study showed a drastic increase in bFGF levels in the hippocampus 48 h after ischemia (150% of that in normal rat) which was followed by further increases. In Western blot analysis, three immunoreactive bands whose molecular weights correspond to 18, 22 and 24 kDa were observed in normal rat and ischemia increased all their immunoreactivities. In the in situ hybridization study of the hippocampus, bFGF mRNA positive cells were observed in the CA1 subfield in which many bFGF-IR cells existed after ischemia. These data demonstrate that transient forebrain ischemia leads to an early and strong induction of bFGF synthesis in astrocytes, suggesting that the role of bFGF is related to the function of the reactive astrocytes which appear following brain injury.

Effects of basic fibroblast growth factor (bFGF) on bone formation in growing rats

The effects of basic fibroblasts growth factor (bFGF) administered intravenously at dosages of 0.1 and 0.3 mg/kg per day for 7 days to growing rats are reported. Static and dynamic histomorphometry techniques were applied to the microradiographs and undecalcified ground sections of the proximal tibiae and tibial shafts. The bone histomorphometric analyses in the proximal tibia revealed that 0.1 mg/kg per day of bFGF increased longitudinal growth rate, cartilage cell production rate, and metaphyseal bone area. In the tibial shaft, the endocortical mineral apposition and bone formation rates, total bone area, total osteoid area, and medullary bone area were increased, but the periosteal mineral apposition and bone formation rates were depressed. Two weeks after the cessation of treatment, the increased osteoid bone on the endocortical surface and in the marrow cavity was completely calcified, and the total mineralized area in the tibial shaft was significantly increased. The rats given 0.3 mg of bFGF/kg per day showed retarded weight gain, defective calcification at the growth plate metaphyseal junction, and on the endocortical surface. The growth plate width was increased, and the longitudinal growth rate, cartilage cell production rate, endocortical labeled surface, and bone formation rate were decreased. Two weeks after the cessation of treatment, these changes were almost reversed, and the longitudinal growth rate and cartilage cell production rate were increased as rebound phenomena. These results suggest that a low dose (0.1 mg/kg per day) of bFGF stimulates endosteal and endochondral bone formation and depresses periosteal bone formation in growing rats.

Effect of ipriflavone on glucocorticoid-induced osteoporosis in rats.

Ipriflavone, 7-isopropoxy-3-phenyl-4H-1-benzopyran-4-one, was administered orally for 12 weeks to male rats with prednisolone-induced osteoporosis. Microdensitometric analysis of a roentgenograph of the femurs revealed that ipriflavone increased the density of the distal metaphysis dose-dependently and tended to increase the density of the diaphysis. It also inhibited dose-dependently the decrease in the mechanical strength of the tibia, breaking strain and breaking energy, and the fractional content of ash in femurs. These results indicate that ipriflavone markedly suppresses bone resorption at the metaphysis where the content of trabecular bone with a rapid turnover rate is high, and possibly inhibits bone reduction at the diaphysis.

Upregulation of fibroblast growth factor-receptor messenger RNA expression in rat brain following transient forebrain ischemia

Recently, we demonstrated that transient forebrain ischemia in rats leads to an early and strong induction of basic fibroblast growth factor (bFGF) synthesis in astrocytes in the injured brain regions. In this study, in order to clarify the targets of such raised endogenous bFGF levels, the messenger RNA (mRNA) expression of its receptors (flg and bek) at in the hippocampus following transient forebrain ischemia induced by four-vessel occlusion for 20 min was investigated using an in situ hybridization technique. Transient forebrain ischemia induced an increase in the number of flg mRNA-positive cells from an early stage (24 h after ischemia) in the hippocampal CA1 subfield where delayed neuronal death occurred later (48–72 h after ischemia). This increase became more marked with the progression of neuronal death and was still evident in the same area 30 days later. The time course of the appearance and distribution pattern of flg mRNA-positive cells in the CA1 subfield were quite similar to those of bFGF mRNA-positive cells. On the other hand, in situ hybridization for bek mRNA showed only slight and transient (observed 72 h and 5 days after ischemia) increases in the number of mRNA-positive cells in the CA1 subfield following ischemia. The use of in situ hybridization and glial fibrillary acidic protein immunohistochemistry in combination demonstrated that the cells in the CA1 subfield that exhibited ischemia-induced flg or bek mRNA expression were astrocytes. These data indicate that transient forebrain ischemia induces upregulation of fibroblast growth factor-receptor expression, accompanied by increased bFGF expression in astrocytes, and suggest that the increased astrocytic bFGF levels in injured brain regions act on the astrocytes via autocrine systems and are involved in the development and maintenance of astrocytosis.

Expression of ICAM-1 on glomeruli is associated with progression of diabetic nephropathy in a genetically obese diabetic rat, Wistar fatty

We developed an animal model for non-insulin-dependent diabetes mellitus, a genetically obese rat strain, Wistar fatty. These rats show obesity-related features such as hyperinsulinemia and hyperlipemia, and only males develop diabetic features including hyperglycemia, glucoseuria and polyuria as they age. Histopathological study demonstrated a deposition of PAS-positive granules in the epithelial cells and a diffuse thickening of the mesangial area and moderate changes of the renal tubules. We found that ICAM-1 is expressed on the glomeruli of male Wistar fatty rats and the expression is associated with the development of nephropathy; it is weak at 5 weeks, becomes markedly strong at 15 weeks and progresses further at 29 weeks of age. We tried in vivo administration of monoclonal antibody, anti-ICAM-1 alone or together with anti-LFA-1 into male Wistar fatty rats during the period from 5 weeks to 17 weeks of age. The treatment, however, could not prevent the development of nephropathy. ICAM-1 expressed on the glomeruli of Wistar fatty rats seems not to play a key role in development of the nephropathy by mediating leukocyte infiltration. It will be a useful marker of the development of the disease.

Stimulatory effect of ipriflavone on formation of bone-like tissue in rat bone marrow stromal cell culture

SummaryThe effects of ipriflavone (IP) (10−5 M) on bone formation were studied in stromal cells from the femoral bone marrow of young adult rats cultured for 21 days in the presence of β-glycerophosphate and dexamethasone. Stereoscopic microscopy showed nodule formation after 14 days of culturing, and both the number and the size of the nodules increased with time. The alizarin-red-stained calcified area in the nodules in the IP group was nearly 4 times as large as that in the control after 21 days. Light and electron microscopy revealed the presence of many osteoblast-like cells with developed rough endoplasmic reticulum and Golgi apparatus in the nodules in the control group after 14 days, and a collagenous fibril network was seen among the cells. After 21 days, calcification of the dense collagenous fibril network and bone matrix-like tissue were observed in many nodules, resulting in the formation of bone-like tissue containing osteocyte-like cells. In the IP group, the collagenous fibril network area in the nodules was greater than that in the control after 14 days, and a further increase in both the dense collagenous fibril network area and calcified bone-like tissue area was observed after 21 days. These findings indicate that IP stimulates bone-like tissue formation in the rat bone marrow stromal cell culture, suggesting that the promotion of collagen production by osteoblasts is involved in the stimulation of bone-like tissue formation by IP.

Immunomodulating and articular protecting activities of a new anti-rheumatic drug, TAK-603

We investigated the pharmacological activities of a newly synthesized anti-rheumatic drug, TAK-603. (1) In vivo: In adjuvant arthritic (AA) rats, TAK-603 inhibited the hind paw swelling and the body weight loss. The minimum effective dose was 3.13 mg/kg/day (p.o.). Histological and radiographic studies showed that TAK-603 suppressed the development of synovial lesions and joint and bone destruction. TAK-603 was also effective in AA rats when administered for the first 7 days after the adjuvant injection. It suppressed type IV allergy (25 mg/kg/day, p.o.) but had no effect on type III allergy. It had little effect in acute inflammation, analgesic and antipyretic models. These data suggest that TAK-603 acts on the immune system, especially on cellular immunity. (2) In vitro: TAK-603 suppressed the mitogen-induced proliferation of mouse lymphocytes and the ConA-induced IFN-gamma and IL-2 production by rat lymphocytes at 10(-7) to 10(-5) M. It also significantly inhibited the IL-1 induced extracellular matrix reduction in rabbit chondrocytes. It had no effects on prostaglandin E2 (PGE2) production in rat peritoneal cells. These data show that TAK-603 has the ability to suppress the immune system and protect cartilage from destruction. TAK-603 is expected to be a promising drug for rheumatoid arthritis.

Dose-response relationships of cytotoxicity, PFC response and histology in the spleen in rats treated with alkylating agents.

Our previous study revealed the appropriate conditions for the plaque- forming cell (PFC) assay in rats. Using this assay in the present study, dose-response relationships of cytotoxicity, PFC response and histology in the spleen were evaluated in rats receiving alkylating agents. Rats were given a single intravenous administration of cyclophosphamide (CY) at a dose of 3, 10 or 30 mg/kg. Spleen weights and cellularity were decreased in the rats treated with 30 mg/kg. Suppressed PFC response wes observed in the rats receiving 10 mg/kg or more. In the rats treated with CY at 1, 3 or 10 mg/kg for 7 days, spleen weights and cellularity and PFC response were reduced at doses of 3 mg/kg or more. Treatment with the other alkylating agents, however, had a different consequence. Namely, in the rats treated with nitromin once or for 7 days, spleen weights and cellularity were decreased at a dose lower than that causing a reduction in the PFC response. In the rats treated with melphalan or chlorambucil, the weights and cellularity of the spleen tended to be decreased at a dose lower than that suppressing the PFC response. Histologically, in the case of CY, the marginal zone was narrow with cellular depletion in the rates receiving 3 mg/kg, whereas little change was seen in the periarteriolar lymphoid sheath (PALS). At a dose of 10 mg/kg, the marginal zone was markedly atrophied and slight atrophy of the PALS was seen. On the other hand, in the rats treated with nitromin, a dose-related decrease in the size of the spleen was seen without changes in the tissue architecture. Melphalan caused atrophy of both the marginal zone and the PALS at a dose suppressing PFC response. Regarding the red pulp, the extramedullary hematopoiesis disappeared with melphalan and nitromin, but not with CY. These results indicate that the decreases in weights and cellularity and histological changes in the spleen caused by the alkylating agents are detectable at the dose suppressing PFC response except for CY, which has a marked immunosuppressive action. Furthermore, the observed histological findings in the spleen were characteristic of each alkylating agent.

Suppressive effect of ipriflavone on bone depletion in the experimental diabetic rat: dose response of ipriflavone.

The dose dependent effect of ipriflavone (7-isopropoxy-isoflavone) on the femoral bone in streptozotocin-induced diabetic rats was studied by microdensitometric analysis. Diabetic rats showed severe hyperglycemia, glucosuria, hypoinsulinemia, associated with increased urinary calcium and hydroxyproline. Microdensitometric analysis revealed decreases in femoral length, bone width, and bone density. The dietary administration of ipriflavone (about 270 mg/kg/day) to the diabetic rats for 6 weeks prevented reduction of the cortical thickness index in the diaphysis and depletion of bone density in the distal metaphysis, and also reduced the inner diameter of the diaphysis; diabetic state was not improved. A simple correlation and linear regression analysis revealed that ipriflavone also significantly reduced the inner diameter in the diaphysis at a dose of 90 mg/kg/day, but not at one of 25 mg/kg/day. These results suggest that ipriflavone suppresses the depletion of the femoral bone through inhibition of bone resorption in a dose dependent fashion; its minimum effective dose is 90 mg/kg/day in experimental diabetes.