Adequate dietary calcium mitigates osteopenia induced by chronic lead exposure in adult rats.

The purpose of the present study was to investigate bone changes in the adult rat exposed to low lead levels during intake of normal dietary calcium and to contrast these findings with data from our earlier studies performed with animals receiving low dietary calcium concurrent with lead exposure. The present study exposed adult rats to 100 ppm lead via drinking water for 12 weeks and assessed bone histology, 1,25-dihydroxyvitamin D, 25(OH)vitamin D and parathyroid hormone levels. No osteopenia was evident by quantitative bone histology, and circulating levels of 1,25-dihydroxyvitamin D, 25(OH) vitamin D and parathyroid hormone were normal. Bone ash findings documented incorporation of significant amounts of lead into bone mineral. These findings document absence of interference with vitamin D metabolism, absence of secondary hyperparathyroidism and absence of osteopenia following 12 weeks of low lead exposure in the adult rat maintained on normal calcium intake. Results stress the importance of adequate calcium intake in our elderly population who may be exposed to cumulative, low-level lead exposure.

Detection of apoptotic cells in vitro: a practical standardized experimental protocol using human fibroblasts for initial laboratory studies.

This technical report presents a practical experimental protocol using human fibroblasts that is useful for initiating in situ methods for detection of apoptotic cells in vitro. The protocol details the growth of cells in multichamber wells, and includes a negative control, two positive controls, and untreated fibroblasts to be evaluated for apoptosis localization. Technical tips on handling of solutions, cell culture designs, and separation of slides by treatment are valuable steps for the laboratory beginning such studies. The methods proposed are both time and cost effective.

Magnesium deficiency induces bone loss in the rat.

Disorders in which magnesium (Mg) depletion is common have an associated high incidence of osteoporosis. Mg depletion in humans results in hypocalcemia, low serum parathyroid hormone (PTH) and 1, 25(OH)2-vitamin D levels, as well as PTH and vitamin D resistance which may serve as mechanisms for the development of osteoporosis. In order to determine if isolated Mg depletion will result in bone loss, we have induced dietary Mg deficiency in the rat. Adult (290 g) female rats were given either a low-Mg diet (2 mg/100 g chow; n = 6) or a normal control Mg diet (63 mg/100 g chow; n = 6). Dietary calcium (Ca) was normal in both groups (592 mg/100 g chow). At 12 weeks, blood was obtained for serum Mg, Ca, PTH, 1,25(OH)2-vitamin D, and osteocalcin determinations. The rats were then euthanized and the femurs obtained for mineral analysis and histomorphometry. Serum Mg in the low-Mg group was less than control (0.4 +/- 0.2 vs. 1.9 +/- 0.2 mg/dl, p < 0.001; mean +/- SD) while serum Ca was higher (11. 7 +/- 0.5 vs. 9.3 +/- 0.4 mg/dl, p < 0.001). PTH was suppressed in the Mg-deficient group (36 +/- 16 vs. 109 +/- 30 pg/ml in controls, p < 0.002). Serum 1,25(OH)2-vitamin D was also suppressed in the Mg-deficient animals (7.1 +/- 4.8 vs. 28.5 +/- 8.2 pg/ml in controls, p < 0.002). Serum osteocalcin levels were not different (19.8 +/- 2.5 ng/ml in Mg-deficient rats vs. 15.3 +/- 3.4 ng/ml in controls). While the ash weight of Ca and phosphorus in the femur did not change, the ash weight of Mg fell (low-Mg group 0.55 +/- 0.01%, controls 0.65 +/- 0.02%, p < 0.001). Histomorphometry demonstrated reduction in bone mass; the trabecular bone volume in the femur of the low-Mg group was reduced from control (7.7 +/- 0.2 vs. 13.7 +/- 1.9%, p < 0.002). A surprising new observation was an increase in osteoclast (OC) bone resorption with Mg depletion. The number of OC per millimeter bone surface was 16.9 +/- 1.3 in the low-Mg group versus 7.8 +/- 1.5 in controls (p < 0.001). The percentage of bone surface occupied by OC was 38.3 +/- 3.7 in the low-Mg group versus 17.7 +/- 2.4 in controls (p < 0.001). This increased resorption occurred with an inappropriate non-altered bone-forming surface relative to control (% osteoid surface: low-Mg group 2.4 +/- 0.7 vs. controls 2.6 +/- 0.4; % osteoid volume: low-Mg group 0.25 +/- 0.09 vs. controls 0.38 +/- 0.06; number of osteoblasts per millimeter bone surface: low-Mg group 0.9 +/- 0.3 vs controls 1.3 +/- 0.3). No increase in bone-forming surface or osteoblast number despite an increase in OC-resorbing surface and OC number strongly suggests impaired activation of osteoblasts and an uncoupling of bone formation and bone resorption. Our data demonstrate that Mg depletion in the rat alters bone and mineral metabolism which results in bone loss.

Human intervertebral disc cells from the annulus: three-dimensional culture in agarose or alginate and responsiveness to TGF-beta1.

Cell culture procedures were developed for use with surgical and normal control specimens of the annulus of the human intervertebral disc. Cells were established in monolayer explant culture and seeded into three-dimensional growth environments of alginate or agarose; under these growth conditions cells assumed a rounded phenotype and formed colonies. A novel method of layering suspensions of cells onto cell well inserts proved technically much easier than the microbead culture method. Immunohistochemistry was utilized to demonstrate in vitro production of the following extracellular matrix components: types I, II, III, and VI collagen, 4-S-chondroitin sulfate, and keratan sulfate. Young and old age- and gender-matched cells grown in the presence of TGF-beta1 showed significant enhancement of proliferation after 4 days of exposure to TGF-beta with a lessened mitogenic response present after 10 days. Molecular studies of proteoglycan gene expression showed that at 4 days young normal cells had increased biglycan, but not decorin, message levels. Decorin expression was unchanged at Day 4 and decreased or shut off by Day 10. Results support the use of three-dimensional culture systems for in vitro evaluation of human disc cell function and expand our understanding of the in vitro behavior of these cells.

Large specimen bone embedment and cement line staining.

Undecalcified embedment of large bone specimens is often challenging. A method is presented here that is suitable for methacrylate embedment of sections of canine vertebrae while retaining the ability to localize tartrate-resistant acid phosphatase and alkaline phosphatase activity. Specimens also retained tetracycline labelling, and sectioned preparations were readily stained with routine bone procedures. A modification of the Bodian silver stain, used for examining the nerves and spinal cord in these specimens, provided a useful stain for canaliculi and cement lines in trabecular and cortical bone. This stain is advantageous when both bone and nerve tissue are of interest, as in spinal fusion studies.

Characterization and phenotypic stability of human disc cells in vitro.

Successful in vitro studies of disc cells require interaction of the cell with a compatible microenvironment which favors expression of the disc cell phenotype. The objective of this study was to characterize cells grown by standardized methods of isolation and passage, and culture cells from the human annulus in three-dimensional culture. Cells from the annulus of 11 individuals were cultured in alginate or agarose for ten days, and extracellular matrix components were evaluated with immunohistochemistry and quantitative analysis of the percent of colonies producing Type I or II collagen, 4-sulfated chondroitin sulfate or keratan sulfate. Results show production of these four extracellular matrix products through multiple passages and support the phenotypic stability of disc cells in three-dimensional culture.

Olfactory sensory neurons are trophically dependent on the olfactory bulb for their prolonged survival.

In most neural systems, developing neurons are trophically dependent on contact with their synaptic target for their survival and for some features of their differentiation. However, in the olfactory system, it is unclear whether or not the survival and differentiation of olfactory sensory neurons depend on contact with the olfactory bulb (normally the sole synaptic target for these neurons). In order to address this issue, we examined neuronal life-span and differentiation in adult rats subjected to unilateral olfactory bulb ablation at least 1 month prior to use. Life-span of a newly generated cohort of olfactory neurons was determined by labeling them at their "birth" via the incorporation of 3H-thymidine. In the absence of the bulb, neurons are continually produced at a twofold greater rate. However, the epithelium on the ablated side is thinner, indicating that average neuronal life-span must be reduced in the targetless epithelium. Indeed, nearly 90% of the labeled neurons disappear from the bulbectomized side between 5 d and 2 weeks of neuronal age. Moreover, on electron microscopic examination, olfactory axons are degenerating in large numbers on the ablated side. Since labeled neurons migrate apically through the width of the epithelium during this same period, it appears that most, if not all, neurons on the ablated side have a life-span on the order of 2 weeks or less. In contrast, there is a more moderate degree of neuronal loss on the unoperated side of the same animals during the first 2 weeks after tracer injection, and that occurs while the neurons are concentrated in the deeper half of the epithelium, suggesting that there is a preexisting population of neurons in the control epithelium that does not die during this period. Likewise, degenerating axons are much less frequent on the unoperated side. We conclude that life-span is significantly shorter for olfactory neurons born in the targetless epithelium and that olfactory neurons are trophically dependent on the presence of the bulb for their prolonged survival. Neuronal differentiation in the absence of the bulb was assessed according to ultrastructural criteria and the pattern of protein expression using antisera to the growth associated protein GAP-43 and the olfactory marker protein. By both measures, most neurons in the epithelium on the bulbectomized side, but not all, are immature.(ABSTRACT TRUNCATED AT 400 WORDS)