Subchronic in vivo effects of a high static magnetic field (9.4 T) in rats.

The potential adverse biologic effects of sub chronic (cumulatively 10 weeks) exposure to a high magnetic field (9.4 T) were evaluated in young adult male and female Fischer rats as well as in their progeny. Biologic end points in adult rats included changes in daily clinical observations; spatial memory tests; weekly heart rates, body weights, food and water consumption, and the feed efficiency ratio; terminal hematologic, blood biochemical and urinary parameter changes; gross pathologic findings; and major organ weights. In offspring, biologic end points included the gestation period, number of live births, number of pups, ratio of male to female pups/litter; postnatal time period of eye opening; birth and weekly body weights, behavioral changes, central nervous system responses, as well as hematologic, blood biochemistry, and urinary parameter changes; and gross pathologic findings. Findings from this study showed that there were no adverse biologic effects in male and female adult rats or their progeny that could be attributed to 10-week exposure to a 9.4-T static magnetic field.

Lactate turnover in rat glioma measured by in vivo nuclear magnetic resonance spectroscopy.

Elevated tissue lactate concentrations typically found in tumors can be measured by in vivo nuclear magnetic resonance (NMR) spectroscopy. In this study, lactate turnover in rat C6 glioma was determined from in vivo 1H NMR measurements of [3-13C]lactate buildup during steady-state hyperglycemia with [1-13C]glucose. With this tumor model, a narrow range of values was observed for the first-order rate constant that describes lactate efflux, k2 = 0.043 +/- 0.007 (n = 12) SD min-1. For individual animals, the standard error in k2 was small (< 18%), which indicated that the NMR data fit the kinetic model well. Lactate measurements before and after infusing [1-13C]glucose showed that the majority of the tumor lactate pool was metabolically active. Signals from 13C-labeled glutamate in tumors were at least 10-fold smaller than the [3-13C]lactate signal, whereas spectra of the contralateral hemispheres revealed the expected labeling of [4-13C]glutamate, as well as [2-13C] and [3-13C]glutamate, which indicates that label cycled through the tricarboxylic acid cycle in the brain tissue. Lack of significant 13C labeling of glutamate was consistent with low respiratory metabolism in this glioma. It is concluded that lactate in rat C6 glioma is actively turning over and that the kinetics of lactate efflux can be quantified noninvasively by 1H NMR detection of 13C label. This noninvasive NMR approach may offer a valuable tool to help evaluate tumor growth and metabolic responsiveness to therapies.

Relationships among lactate concentration, blood flow and histopathologic profiles in rat C6 glioma.

Increased capacity for glycolytic metabolism is a well-known characteristic of neoplastic cells. Because lactic acid is the end product of glycolysis, in vivo MRS measurements of tumor lactate concentration ([lac]t) may provide valuable information about tumor metabolism, which will aid the development of therapies and the clinical diagnosis and treatment of tumors. In the present study, several hemodynamic and histologic parameters were evaluated with respect to their influence on [lac]t. Pronounced differences in [lac]t in two distinct populations of tumors suggested a putative perfusion threshold. Above this threshold, [lac]t was independent of hemodynamic and histologic factors including tumor blood flow (measured using MRS and the method of D2O washout), extent of necrosis and inflammatory cell infiltrate. Thus, for most tumors, [lac]t was not determined by any one single factor such as hypoxia, venous clearance, glucose supply, extent of necrosis or degree of inflammatory cell infiltrate. Rather, [lac]t may be equilibrated, at least in part, by an interplay of forces involving hemodynamics and substrate supply. In general, the data are consistent with the hypothesis that elevated lactate in most tumors is related to the high glycolytic activity of adequately perfused, viable neoplastic cells.

Effects of a novel azaspirane (SK&F 105685) on arthritic lesions in the adjuvant Lewis rat: attenuation of the inflammatory process and preservation of skeletal integrity.

OBJECTIVE: To determine the effects of SK&F 105685 (N,N-dimethyl-8,8-dipropyl-2-azaspiro[4.5] decane-2-propanamine dihydrochloride) on the arthritic lesions in the tibiotarsal joint of adjuvant arthritic (AA) rats. METHODS: Inhibition of hindpaw inflammation was measured by water displacement. The protective effects on joint integrity were determined by measuring radiographic and histological changes and by scanning electron microscopy. RESULTS: Compared to AA control rats, SK&F 105685 suppressed hindpaw edema 64% or 41-54% in AA rats receiving 30 or 20 mg/kg/day, respectively. Radiographic evaluation showed marked decreases in soft tissue swelling and in the severity of skeletal tissue loss at the tibiotarsal joint in both dose groups. Histologically SK&F 105685 markedly attenuated the extent and severity of the inflammatory lesion and preserved the basic integrity of bone and cartilaginous tissues, including the content and distribution of proteoglycans of the articular cartilages. Cellular changes included decreases in the inflammatory infiltrate and in the number of osteoclasts and chondroclasts. Whereas AA control rats exhibited marked to severe loss (41-70%) of skeletal tissue mass, the loss in rats given 30 mg/kg SK&F 105685 was mild (< 20%). Scanning electron microscopy of the talus revealed only slight erosion of the articular cartilage and general preservation of the underlying bone. In contrast, in AA controls, there was marked erosion of the talar articular cartilage and severe loss of subchondral bone. Spleen cells from SK&F 105685 treated rats had a reduced capacity to respond to concanavalin A and contained suppressor cell activity as measured in a coculture assay. CONCLUSION: Our studies show that SK&F 105685 has remarkable protective effects on the joints of AA rats and suggests that it may attenuate the overall inflammatory process and retard the degenerative loss of skeletal tissue in rheumatoid arthritis in humans.

Magnetic resonance microscopy in rat skeletal research.

Noninvasive evaluation of skeletal tissue, particularly certain sites that tend to be predisposed to disease, is critical in understanding the pathogenesis, progression, and successful treatment of various diseases like osteoporosis, rheumatoid arthritis, and osteoarthritis. Although several noninvasive techniques are currently available to evaluate skeletal tissues, they all have critical limitations. We report here a systematic study to compare the morphological changes (overall profile and tissue architecture) in the proximal tibiae and coccygeal vertebrae of a young growing rat and an older retired female breeder rat using 2- and 3-dimensional MR (magnetic resonance) microscopy and histology. We have obtained MR microimages of intact rat tibiae and vertebrae with resolution upto 24 24 250 microns and have found excellent correlations between MR microscopy results and histological assessment.

The role of prostaglandins in bone in vivo.

Prostaglandins of the E series, primarily E2 and E1, have the greatest activity in bone. Following discovery of their potent ability to stimulate bone resorption in vitro, clinical investigations have placed prostaglandins at sites of localized bone resorption associated with inflammatory or space occupying lesions in vivo. These studies have shown that prostaglandin production at such sites may be increased by cytokines such as interleukin-1 but the mechanisms by which prostaglandins stimulate bone resorption are not yet known. Observation of periosteal bone formation in patients given, pharmacological doses of prostaglandin has led to investigation of its bone forming activity. Young, growing rats have increased metaphyseal bone formation and this is accompanied by increased periosteal and endocortical bone formation in older animals. In the mature animals there is a generalized activation of remodelling with increased formation in the remodeling cycle. This is also seen in oophorectomized rats and results in repletion of the lost bone in this model of osteoporosis. In animal models of localized disuse osteopenia, prostaglandins are found to be elevated at the site of bone loss and prostaglandin inhibitors at least partially protect against the exaggerated resorption that occurs. This is also seen in models of orthodontic tooth movement, periodontitis and osteomyelitis. Prostaglandin synthesis inhibitors have been shown to delay healing of bone and this has led to limitations on their use clinically in some situations. Exogenously administered prostaglandins have been found to enhance periosteal callus formation, but healing is not uniformly enhanced. Prostaglandins have also been associated with hypercalcemia in certain animal tumors that model human hypercalcemia of malignancy but are probably most important in this condition as mediators in the localized resorption of bone at tumor sites. These in vivo studies have shown that prostaglandins are involved with increases in both bone formation and bone resorption. In vitro studies have shown that prostaglandins stimulate osteoblasts as well as osteoclastic bone resorption but understanding these effects under in vivo conditions will require further investigation.

An investigation of the pathological and physiological effects of intraosseous sodium bicarbonate in pigs.

Recent interest in the intraosseous (IO) route as an alternative venous access for drug and fluid administration has increased. This study examined the physiological and skeletal pathological effects of IO NaHCO3 in pigs. In the pathological studies, swine (8-10 kg) received NaHCO3 (1 mEq/kg) in one tibia and saline (1 ml/kg) in the other tibia via an 18-gauge spinal needle inserted into the anteromedial surface of the bone. The animals were then observed for one month, sacrificed, and the tibias were isolated, sectioned, and stained for pathological examinations. The physiological effects of IO NaHCO3 infusion were studied and compared with that of intravenous (IV) administration using a cardiac arrest model as previously described. The results demonstrated that NaHCO3 had no effect on the mean arterial blood pressure and plasma catecholamine levels, but increased arterial pH values within two minutes of administration. Similar effects were found with IV NaHCO3. Pathological data indicated signs of minimal local increase in skeletal turnover associated with IO NaHCO3 infusion. It is concluded that the IO route is a safe alternative venous access for NaHCO3 administration in swine.

Effects of orally administered prostaglandin E-2 on cortical bone turnover in adult dogs: a histomorphometric study.

The effects of Prostaglandin E-2 (PGE-2) on cortical bone turnover in ribs and femurs of 32 intact adult dogs were evaluated following 3 months treatment. Static and dynamic histomorphometric skeletal changes were characterized using terminal in vivo tetracycline double labeling. PGE-2 caused a dose dependent increase in the formation of subperiosteal fibrous-lamellar new bone in femurs, and an increase in bone remodeling within the (original) cortical compacta of both femurs and ribs. Increased cortical remodeling resulted in a new steady state, but only in ribs. Increased Haversian remodeling in ribs and femurs was characterized by increases in the activation frequency, the number of bone resorbing and forming foci, the percent of osteons with single labels, and the radial closure and bone formation rates, with no effect on appositional rate. While the mean ratios of the number of resorption to formation foci (R/F) were unremarkable in femurs of treated versus control males, the R/F ratios in treated females were approximately 50% lower than matched controls. In treated males, both femoral osteon resorption and formation times were 50% shorter than matched controls. In treated females, femoral osteon resorption time was 2-4-fold shorter than the decrease in osteon formation time. Calcium and phosphorus levels were normal in all treated dogs. Serum alkaline phosphatase levels were increased approximately two-fold in high dose (10.0 mg/kg) dogs and correlated well with the histologic findings of increased skeletal turnover and bone formation.

The effects of 1,25-dihydroxycholecalciferol, parathyroid hormone, and thyroxine on trabecular bone remodeling in adult dogs. A histomorphometric study.

The effects of 1,25-dihydroxycholecalciferol (1,25-(OH)2D3), parathyroid hormone (PTH), and L-thyroxine (T4) on trabecular bone remodeling were evaluated by histomorphometric methods in adult female beagle dogs. Intravenous 1,25-(OH)2D3 (1.25 micrograms/day in equally divided doses) was administered intermittently for 6 days and withdrawn 14 days for three complete cycles. PTH was administered intravenously (2.5 U/kg/day) in divided doses 6 hours apart for 60 days. Thyroxine was given orally (1.0 mg/kg/day) in divided doses for a similar interval. Static and dynamic changes were evaluated using tetracycline and DCAF (2,4 BIS) N, N', Di (carboxymethyl) (amino methyl fluorescein) in vivo double labeling of bone from the iliac crest taken before treatment and after 60 days. The intermittent administration of 1,25-(OH)2D3 stimulated the bone resorption rate and depressed the formation rate. 1,25-(OH)2D3 increased trabecular resorption surfaces; osteoid surface, volume, and thickness; mineralization lag time; and osteoblast number but decreased the bone volume. Multiple small daily doses of PTH resulted in an overall negative balance in trabecular bone. This was associated with an increased trabecular surface-to-volume ratio, bone resorption and formation rates, active forming surfaces, osteoid volume and surface, life span of bone forming and resorbing sites, and the number of osteoclast nuclei. Thyroxine appeared to increase bone mass by enhancing the switch-over from the resorptive to the formative phase of remodeling. Coupling between osteoid apposition and mineralization was increased by recruiting more forming sites and prolonging their life span. Thyroxine increased bone resorption and formation rates, trabecular bone volume and balance, number of osteoclast nuclei, and life span of bone forming sites. The osteoid seam thickness and mineralization lag time were decreased. The present study demonstrated that 1,25-(OH)2D3, PTH, and thyroxine at the dose and schedule used, markedly altered stimulators of remodeling in trabecular bone of adult dogs.

Histomorphometric evaluation of the effects of intermittent 1,25-dihydroxycholecalciferol administration on cortical bone remodeling in adult dogs.

The effects of intermittent low doses (1.25 mug daily, administered intravenously for 6 days and withdrawn for 14 days for 3 complete cycles) of 1,25-dihydroxycholecalciferol (1,25-[OH](2)D(3)) on cortical bone were determined and compared in ribs with steady state and regionally accelerated remodeling in adult intact female dogs. The bone changes were analyzed by dynamic bone histomorphometric methods, using tetracycline and DCAF (2,4 BIS) N, N' di (carboxymethyl) (amino methyl fluorescein) in vivo double labeling of bones before treatment and after 60 days of intermittent 1,25-(OH)(2)D(3) administration. Serum calcium and phosphorus levels increased during 1,25-(OH)(2)D(3) administration. Urinary hydroxyproline excretion increased during the first interval of 1,25-(OH)(2)D(3) administration but was not changed significantly during the last two intervals. In normal cortical bone (11th rib) following the administration of 1,25-(OH)(2)D(3) there was a marked decrease in the activation frequency, bone formation rate, osteoid seam thickness, seam circumference, and mean appositional rate. Although recruitment of new remodeling sites was decreased after 1,25-(OH)(2)D(3), previously existing remodeling units continued to completion. These effects resulted in a preponderance of mature osteons in normal cortical bone. The morphometric changes in cortical bone (9th rib) exposed to both 1,25-(OH)(2)D(3) and periosteal elevation were characterized by a marked increase in both the activation frequency and bone formation rate and associated with a decrease in the osteon formation time. Other morphometric parameters that were increased included radial closure rate, numbers of osteoid seams and resorption cavities, ratio of bone resorbing to forming sites, percentage labeled and circumference of osteoid seams, and total and cortical bone areas. The combined effect of periosteal elevation and 1,25-(OH)(2)D(3) were markedly different from those observed with 1,25-(OH)(2)D(3) alone. These findings suggest that the rapid bone turnover induced by tissue injury will mask or alter the effects of hormones on bone remodeling when studied over a relatively short period of time.

Histomorphometric evaluation of the effects of low dose parathyroid hormone administration on cortical bone remodeling in adult dogs.

The effects of low dose levels of parathyroid hormone (PTH) on cortical bone remodeling of adult dogs were evaluated under conditions of normal turnover and accelerated remodeling produced by periosteal elevation. Six female dogs were administered PTH (2.5 units per kg. per day) intravenously in equally divided doses 6 hours apart for 60 days. The level and dose schedule of PTH used in this study resulted in a significant elevation in blood calcium above baseline values only at experimental day 5, no change in serum phosphorus and magnesium, and a transient increase in urinary hydroxyproline excretion on day 10. Accelerated cortical bone remodeling was induced by surgical elevation of periosteum of the 9th rib prior to PTH administration. Static and dynamic bone changes were analyzed using histomorphometric methods following tetracycline and DCAF ((2,4 bis) N, N'-di-(Carboxymethyl) aminomethyl fluorescein) in vivo double labeling. The results of this study suggested that the response of cortical bone to PTH was dependent upon the existing remodeling rate. PTH increased the radial closure rate and decreased the osteon formation time in unaltered cortical bone. These findings suggested that the initial anabolic effect of PTH may be due to increased bone formation at the osteoblastic level in existing Haversian units. In ribs with periosteal elevation, PTH decreased the circumference and thickness of osteoid seams, and there was a trend toward prolonged osteon formation time. The number of bone resorption sites was greater than the number of formation sites. PTH administration did not significantly change activation frequency or remodeling in cortical bone of adult dogs under conditions of normal turnover or accelerated remodeling. The accelerated remodeling produced by periosteal elevation appeared to mask certain responses to cortical bone to low dose levels of PTH.

Effects of thyroxine on cortical bone remodeling in adult dogs: a histomorphometric study.

The effects of thyroxine on cortical bone remodeling were studied under conditions of normal and accelerated remodeling induced by surgical elevation of the periosteum. Six adult female beagle dogs were administered orally 1.0 mg of L-thyroxine per kg body weight daily for 60 days. Static and dynamic changes were evaluated using tetracycline and DCAF (2,4-bis-N,N'-dicarboxymethyl aminomethyl fluorescein) in vivo double labeling of rib specimens taken before treatment and after 60 days. Thyroxine administered in moderate pharmacologic doses increased the activation frequency, number of bone-forming and -resorbing sites, and the osteoid seam circumference in unaltered bone. Thyroxine, by activating skeletal remodeling, increased bone turnover and both formation and resorption at the tissue level. In ribs with periosteal elevation and accelerated remodeling, thyroxine increased the activation frequency, number of bone resorption and formation sites, and ratio of bone resorptive-to-formative surfaces. In addition, thyroxine under these conditions resulted in an increase in the osteoid seam circumference, radial closure rate, and bone formation rate at the tissue level but decreased the osteon formation time. Under conditions of accelerated remodeling, thyroxine increased osteoblastic and resorptive activity to a greater degree than in unaltered bone and resulted in bone changes similar to that described in human beings with thyrotoxicosis. The increased serum calcium and phosphorus levels and urinary hydroxyproline excretion at several intervals during thyroxine administration were consistent with the morphometric evidence of increased bone turnover and resorption. These findings suggest that thyroxine is a potent activator of skeletal remodeling under conditions of both normal and accelerated remodeling.