Enhancement of tumor induction in rats with Moloney murine sarcoma virus by a "new" method based on direct injection into fetuses.

Undiluted, fivefold-diluted, and 25-fold-diluted doses of a stock of Moloney murine sarcoma virus were injected directly, in a volume of 0.025 ml, into the backs of fetal Sprague-Dawley rats by laparotomy through the uterine wall at 18 days of gestation. During the first 8 weeks after birth the young responded to the virus with remarkably high but dose-dependent incidences of neoplasms. When a one-fifth dilution of the virus preparation was inoculated at fetal ages 16, 18, and 20 days, the incidences of lesions decreased with advancing fetal age. The tumors developed preferentially at the virus inoculation site and/or in the proximal parts of the extremeties; all were considered to be of mesenchymal derivation, i.e., malignant mesenchymoma, rhabdomyosarcoma, osteosarcoma, fibrosarcoma or fibromyxosarcoma, hemangiosarcoma, plasmacytoma, and a giant cell tumor. This injection procedure provided us with a valuable experimental tool for the rapid screening or testing of potential chemical carcinogens and other biologic studies.

Effect of ipriflavone on expression of markers characteristic of the osteoblast phenotype in rat bone marrow stromal cell culture.

The effects of ipriflavone on cellular proliferation and differentiation of osteoblasts were investigated using stromal cells isolated from the femoral bone marrow of young rats. To induce the formation of mineralized bone-like tissue in vitro, the cells were cultured in the presence of beta-glycerophosphate and dexamethasone. Ipriflavone was added when subculturing was started. After 14 days of culturing with ipriflavone (10(-7)-10(-5) M), increases in both the alkaline phosphatase activity and the hydroxyproline content per culture dish and a slight decrease in the saturated cell density were observed. Furthermore, continuous treatment with ipriflavone for 14-33 days resulted in an increase in the area of bone-like mineralized tissue accompanied by an increase in the secretion of osteocalcin. When culture medium lacking dexamethasone was used, rat bone marrow stromal cells neither differentiated into osteoblasts nor formed bone-like tissue, and under these conditions, ipriflavone had no effect on the proliferation or the phenotypic expression of the cells. These results suggest that ipriflavone directly stimulates markers of the osteoblast phenotype at a certain stage in bone formation without affecting undifferentiated cells that have not been committed to the osteogenic lineage.

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.

Increase in basic fibroblast growth factor-like immunoreactivity in rat brain after forebrain ischemia.

Using immunohistochemical techniques, a study was conducted to determine whether basic fibroblast growth factor (bFGF) is generated as one of the 'self-repair' responses in rat brain following transient forebrain ischemia. In normal brain, slight bFGF-like immunoreactivity was observed. However, in rats exposed to 20 min of forebrain ischemia, intense bFGF-like immunoreactivity was observed in the CA1 subfield of the hippocampus and the caudate putamen, and marked activity was evident in the temporal cortex, corpus callosum and the CA4 subfield of the hippocampus. Marked neuronal degeneration was also observed in these brain regions following forebrain ischemia. These results suggest that induction of bFGF-like immunoreactivity may be related to the healing which follows brain ischemia.

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.

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.

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.

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.

Evaluation of Perkin's applanation tonometer and the normal range of intraocular pressure in anesthetized rats.

To assess the reliability of noninvasive measurement of intraocular pressure (IOP) in rats, a Perkin's applanation tonometer was calibrated against direct manometry. The normal values of IOP in male Wistar rats were then detected. The mean tonometer readings against the transducer IOP produced regression formula: y = -0.198 + 1.071 x (r2 = 0.987). The mean IOP with standard deviation in rats was 17.7 +/- 3.5 mm Hg (95% and 99% confidence intervals: 17.2 and 18.1 mm Hg, 17.0 and 18.3 mm Hg for the lower and upper limits of the normal rat IOP, respectively). The IOP could be measured accurately using Perkin's applanation tonometer in anesthetized rats each weighing 300 g and over. Measurement of IOP using this tonometer was considered to be valuable allowing, repeated use in rats because of its small size, portability and noninvasiveness.

Relationship between caffeine-induced ocular hypertension and ultrastructure changes of non-pigmented ciliary epithelial cells in rats.

The purpose of this study was to morphologically assess a possible mechanism for caffeine-induced ocular hypertension. Taking into consideration the relationship between the secretion of aqueous humor and the ultrastructure of the ciliary body, the time course of the morphological features in the ciliary epithelium when caffeine was administered intravenously to male Wistar rats was investigated by electron-microscopy. These morphological findings were also compared with the changes in the intraocular pressure (IOP). A significant increase in IOP was noted 15 min and 1 hr after a single dosing of caffeine alone. This change disappeared in all animals within 2 hr after dosing. The IOP in the animals receiving caffeine and the beta-blocker befunolol, which lowers the IOP by inhibiting aqueous humor secretion, decreased significantly from 15 min after dosing, and this change persisted 2 hr after dosing. In electron-microscopy 15 min and/or 1 hr after dosing with caffeine, a slight dilatation in the lateral intercellular spaces near the basement membrane of the non-pigmented ciliary epithelium was observed and the interdigitations between the non-pigmented epithelial cells were intact. Reversal of these changes was observed 2 hr after dosing. On the other hand, the lateral intercellular spaces between the non-pigmented epithelial cells were markedly dilated and the interdigitations were disorganized following dosing with caffeine alone and in combination with befunolol. These results described here indicate that the intravenous administration of caffeine causes ocular hypertension and also changes in the non-pigmented ciliary epithelium, suggesting an enhancement of aqueous humor transportation. This paradigm in the rat is considered to be useful to further assess caffeine-induced ocular hypertension and for use as an animal model in glaucoma research associated with an aqueous humor secretion.

Systemic injection of FGF-2 stimulates endocortical bone modelling in SAMP6, a murine model of low turnover osteopenia.

The effects of systemically administered fibroblast growth factor-2 (FGF-2) at doses of 0.1 and 0.3 mg/kg/day for 7 days were investigated 5-week-old male SAMP6 mice, a model of low turnover osteopenia. The bone histomorphometry in the distal epiphyseal growth plate of the femur showed that 0.3 mg/kg/day of FGF-2 decreased the longitudinal growth rate and cartilage cell production rate and increased the growth plate width. Growth plate chondrocytes showed the features of defective endochondral ossification at the same dosage level. In the distal one third of the femur, the marrow trabecular area, endocortical mineral apposition rate and/or bone formation rate were increased in both the SAMP6 mice given 0.1 and 0.3 mg of FGF-2/kg/day. In this region, the endocortical osteoblasts were hypertrophied with some layers of overlying proliferated fibroblastic mesenchymal cells. The presence of small foci of bone formation within the layers of these mesenchymal cells indicates their osteogenic potential. On the other hand, the periosteal bone formation rate in the mid-shaft of the femur was depressed in the 0.3 mg/kg/day group. These results suggest that systemically administered FGF-2 may have the possibility to increase the peak bone mass in SAMP6 by stimulating the osteoprogenitor cells to proliferate and differentiate into osteoblasts and enhancing endocortical bone modelling. The higher dose of FGF-2, however, inhibited both endochondral and periosteal bone formation.

A characteristic of aspirin-induced hearing loss in auditory brainstem response of conscious rats.

The acute effects of aspirin on auditory functions were examined electrophysiologically in conscious rats with chronically implanted electrodes for auditory brainstem response (ABR) recording. A single intravenous injection of aspirin at a dose of 225 mg/kg caused a reduction in the amplitude of the ABR P1 wave evoked by a 2 kHz tone pip 1 and 24 hr after dosing at almost all sound intensity levels, while the P1 amplitude at 4 kHz was reduced mainly 1 hr after dosing, and the P1 amplitude at 8 kHz was not significantly affected at middle and high intensities even 1 hr after dosing. The audiogram obtained from the P1 amplitude showed a significant increase in the sound threshold 1 and 24 hr after dosing at 2 kHz, and 1 hr after dosing at 4 kHz, but not at 8 kHz. The peak latency of the P1 wave was also prolonged. Furthermore, reduction of the P2 and P4 wave amplitude and prolongation of the P1-P2 and P2-P4 interpeak latency were also observed at 2 kHz but not a 4 or 8 kHz. These results suggest that the rat auditory function for low frequency is vulnerable to the effects of aspirin. This paradigm, i.e., frequency selectivity, n rats may be useful to further assess the different outer hair cells along the cochlear duct and provide additional evidence for the mechanism(s) or site underlying aspirin ototoxicity.

Frequency selectivity on aspirin-induced hearing loss in rats with auditory stimulus-induced conditioned suppression.

The conditioned suppression technique was employed to examine the acute effects of aspirin on auditory function in rats. Lever pressing behavior for water reinforcement was suppressed in the presence of an auditory stimulus that had been previously paired with electric shocks. A single intravenous injection of aspirin at a dose of 225 mg/kg caused an erroneous lever pressing response in the broad sound intensities of 2 kHz tone stimulus during the conditioned stimulus period. A statistically significant increase in the threshold for 2 kHz was found 1 to 72 hr after dosing but not for 4, 8 and 10 kHz. These results suggest that the hearing for low sound frequency in rats is vulnerable to the effects of aspirin. This paradigm in rats may be useful to further assess the different outer hair cells along the cochlear duct and provide an additional evidence for the aspirin ototoxicity research.

Aqueous humor dynamics in beagle dogs with caffeine-induced ocular hypertension.

In order to investigate the possible mechanisms for caffeine-induced ocular hypertension, the intraocular pressure (IOP) and the outflow through the trabecular meshwork were measured in beagle dog eyes after dosing with intravenous caffeine (30 mg/kg) alone or in combination with the topical beta-blocker befunolol [applied as 100 microliters of a 1% (w/v) solution] which inhibits aqueous humor formation in the ciliary body. Intravenous injections of caffeine significantly increased the IOP at 0.25 and 1 hr after a single dose. The ocular hypertension recovered within 2 hr following dosing. Over time, there were no differences in the outflow between the caffeine and control groups. The instillation of befunolol lowered outflow and produced ocular hypotension. The levels of the IOP and outflow in dogs treated with caffeine and befunolol in combination were almost the same as those in dogs treated with befunolol alone. Single-dose and combination-dose studies demonstrate that intravenous caffeine increases the IOP in normal beagle dogs possibly by increasing aqueous humor formation and not by the inhibition of aqueous humor drainage through the trabecular meshwork.

Immunopathology of rhino mouse, an autosomal recessive mutant with murine lupus-like disease.

Detection of high incidence of antinuclear antibodies (ANA) was reported in young homozygous rhino mice employing formalinized chicken erythrocyte nuclei as substrate for indirect immunoflourescence (IF) assay. The titers of ANA heightened with increasing age, and attained to 1:1024 by the time mice reached 5 months of age. The occurrence of ANA was associated with development of splenic and hepatic fibrosis, glomerulonephritis and abnormalities of lymphoreticular tissue. The granular deposits of IgG and C3 detected by direct IF were initially found at the basement membrane of dermal-epidermal junction of rhino mice aged 2.5 months. These deposits distributed progressively in the fibrotic areas of spleen and liver, and renal glomerular tufts at 5 months of age. Dense deposits revealed by electron microscopy were found in the regions where IF of IgG and C3 was observed. Acid buffer eluates from liver and kidney contained IgG reactive with nuclear antigens. Importance of homozygous rhino gene was discussed in relation to development of autoimmune disorders of these mice.

Histomorphometric study of the parathyroid glands of the spontaneously hypercholesterolemic (SHC) rats with chronic renal failure.

Light and electron microscopic morphometry was performed on the parathyroid (PT) glands of the SHC rats with naturally occurring chronic renal failure. Macroscopically, the PT glands were distinctively hypertrophied in 24-week-old male SHC rats when compared with the corresponding controls, Sprague Dawley (SD) rats. Light microscopic morphometry on consecutive sections of the PT glands showed that the volume was about 3 times greater in the SHC rats, whereas there was no difference in the size of the chief cells. Mitoses were often found in the PT glands of the SHC rats. The total number of mitoses was about 8.5 times greater in the SHC rats, and was closely related to the volume of the PT glands. Ultrastructural morphometry of the chief cells revealed an increase in the cell surface area by the interdigitation of the plasma membrane and increases in the volume of mitochondria and Golgi complex. Secretory granules sometimes existed close to the cell surface in the SHC rats, but not in the SD rats. These results suggest that the PT glands of the SHC rats are hyperplastic mainly because of the proliferation of the chief cells. Concurrently, an increase in volume of the cell organelles suggests enhanced secretion activity in the chief cells.

Pharmacological modulation of immediate and late airway response and leukocyte infiltration in the guinea pig.

We established an experimental model of late asthmatic response (LAR) using conscious guinea pigs actively sensitized by antigen aerosol inhalation. In actively sensitized guinea pigs, antigen challenge by aerosol inhalation caused an immediate increase in specific airway resistance (SRaw) (immediate airway response; IAR) followed by a LAR which occurred 4 to 8 hr after antigen challenge. SRaw in the challenged animals was still increased 23 hr after antigen challenge. Examination of bronchoalveolar lavage (BAL) fluid and histology of the lungs revealed increases in eosinophils and neutrophils during LAR. The beta-2 agonist salbutamol inhibited only IAR and not LAR. Dexamethasone inhibited LAR but not IAR. A low dose of theophylline had little effect on both IAR and LAR. A novel thromboxane A2 (TXA2) receptor antagonist, AA-2414, orally administered before antigen challenge dose-dependently inhibited both IAR and LAR, and oral administration of AA-2414 after the IAR inhibited LAR. Also, thromboxane synthetase inhibitors, CV-4151 and OKY-046, reduced both IAR and LAR. Salbutamol significantly reduced the increase in neutrophils in BAL fluid, and dexamethasone significantly reduced the increase in eosinophils and neutrophils in BAL fluid. Theophylline also reduced the increase in eosinophils in BAL fluid. However, AA-2414 did not inhibit the accumulation of these inflammatory cells in BAL fluid or the airway tissues. These results suggest that asthmatic responses in guinea pigs are similar to those in asthmatic subjects and that TXA2 plays an important role in both IAR and LAR but not in inflammatory cell infiltration in this model of allergic asthma.

Histopathologic observations on the senescence-accelerated mice (SAM) reared under specific pathogen free conditions.

Five to 12 months old senescence-accelerated mice reared under specific pathogen free conditions (SPF SAM) were subjected to histopathologic examination. Senescence-prone SAM-P/1/Ta, P/3/Ta, and P/8/Ta exhibited marked depletion in thymic cortical lymphocytes which were associated with lymph follicle formation in the medulla, and marked infiltration of lymphocytes in the submaxillary gland, kidney, and other peripheral tissues. Morphometrical analysis of the adrenal gland revealed cortical atrophy and medullary hyperplasia in male SAM-P/8/Ta at 5 months of age. Other characteristic changes were marked vacuolation of the renal proximal tubules in male P/3/Ta and P/8/Ta mice, and degeneration of vascular wall in the testis of P/3/Ta mice. The pathogenesis of these changes appeared to be related to the adrenal lesions. Systemic amyloid deposition, one of the characteristic features of SAM housed in conventional facilities was not observed under the SPF condition. Acceleration of senescence in SPF SAM may arise from disorders of the thymus and adrenal.

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

The 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 beta-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.

[Effects of ipriflavone (TC-80, an anti-osteoporotic drug) on acute and chronic pain].

The effects of TC-80 on acute and chronic pain in rats and mice were examined. Single oral administration of TC-80 at 50-300 mg/kg was not analgesic in the phenylquinone writhing, acetic acid writhing, and hot plate tests in mice or the tail flick test in rats. Three-weeks administration of TC-80 in a dose of 100 mg/kg/day, p.o., to rats had no analgesic action in the acetic acid writhing and tail flick tests. When TC-80 was given orally in a dose of 100 mg/kg/day for 3 weeks to rats with adjuvant arthritic chronic pain, analgesic effects were observed 2 weeks after the start of administration in males and in ovariectomized estrone-supplemented females; the effect seen in the females was statistically significant. Changes in the bones of the hind paws were examined radiologically, and synovitis, periosteal new bone formation and bone destruction were examined histopathologically in the females. These variables were improved by treatment with TC-80 for 3 weeks. It is concluded that TC-80 has no analgesic effect, but may inhibit chronic pain by anti-osteoporotic action on bone disease.