Correlation between the values of bone measurements using DXA, QCT and USD methods and the bone strength in calcanei in vitro.

In this study we used the calcanei from 32 female and 29 male cadavers, ages 58 to 100. The bone mineral density (BMD) and average bone density (ABD) were measured using dual energy X-ray absorptiometry (DXA) and quantitative computed tomography (QCT) respectively, while speed of sound (SOS), broadband ultrasound attenuation (BUA) and stiffness index (SI) were measured using ultrasound densitometry (USD). Thereafter, the bone strength was measured using a compressor to cause bone fracture, and evaluated in comparison with the values of the three measurement methods. The scatter diagrams of the values of the three different methods versus age displayed a negative linear regression in both sexes. Values for BMD and ABD were generally about 20% higher in males than in females, while SOS, BUA and SI were a few percents higher in males than in females. A significantly high correlation existed between BMD and ABD (r = 0.95), and a moderate correlation between BMD and either SOS, BUA or SI (r = 0.65; r = 0.39; r = 0.57, respectively). Thus, among the values measured using USD, SOS most closely corresponded to BMD of the calcanei. The bone strength of the calcanei indicated a moderate correlation with BMD, ABD and SOS (r = 0.38, P < 0.01; r = 0.43, P < 0.001; r = 0.45, P < 0.001, respectively). However, 42 calcanei fractured under pressures of less than 40 kgf, although the other 19 calcanei endured pressure of 40 kgf or more. Two calcanei with high BMD over 0.7 g/cm2 by DXA were very fragile, whereas a few with low BMD less than 0.4 g/cm2 were not very fragile. Similarly, high SOS, BUA and SI values by USD did not always correspond to high bone strength. Thus, some discrepancies among the bone strength and measurement values remained to be solved in the future.

[In vitro study of ultrasonic bone densitometry using dissected calcaneus].

Quantitative ultrasound (QUS) of the calcaneus is a widely utilized method for bone densitometry. However, the meaning of measured parameters, such as speed of sound (SOS) and broadband attenuation (BUA), is not well established. We performed in vitro measurement of dissected human calcaneus (n = 60; male 29, female 31; mean age 81 years) using two QUS machines and also measured bone densities using SXA, DXA and pQCT. Finally, we investigated breaking strength of the calcaneus and studied the correlation with QUS parameters and other assessed parameters. The two QUS measurements were in good agreement in most experiments. SOS correlated most closely with bone mineral densities assessed by pQCT and did not correlate with factors related to bone size, while BUA showed the highest correlation with BMC and association with parameters related with bone size such as bone area and bone width. With maximal breaking stress of the calcaneus, correlations were almost equal among QUS parameters and bone mineral density. We conclude that SOS is the parameter most closely associated with true bone mineral density (g/cm3), whereas BUA represents both bone mineral density and bone size, mimicing BMD assessed by DXA or SXA.

[Bone mineral density of the calcanei dissected from 30 cadavers--correlation of the values measured by the DXA, QCT and USD methods].

The bone mineral density (BMD) of calcanei dissected from 30 cadavers (14 males and 16 females; ages 61 to 100) was measured by dual-energy X-ray absorptiometry (DXA) and quantitative computed tomography (QCT), while the stiffness was measured by ultrasound bone densitometry (USD). There was high correlation between BMD by DXA and average bone density by QCT (R = 0.94), but poor correlation between BMD by DXA and stiffness by USD (R = 0.55). However, the correlation coefficient of stiffness to BMD was much higher in the female calcanei (R = 0.74) than in the male calcanei (R = 0.32). Analyzing the three elements, stiffness, speed of sound (SOS) and broad-band ultrasound attenuation (BUA), it was clear that SOS rather than stiffness correlated strongly to BMD by DXA and to bone density of trabeculated bones by QCT (R = 0.618; 0.699 respectively). The value of BUA shows more relationship to the width of the calcanei than the area of X-ray projection from the side (R = 0.561).

Cajal-Retzius neurons identified by GABA immunohistochemistry in layer I of the rat cerebral cortex.

By means of immunohistochemistry using anti-gamma-aminobutyric acid (GABA) antibodies, characteristic neurons of pyriform, bipolar or pleomorphic shapes, regarded as the Cajal-Retzius (CR) neurons, were clearly demonstrated in layer I of the rat cerebral cortex at the various experimental periods. On embryonic day 15, ovoid neurons only in the marginal zone indicated immunoreactivity for GABA. They gradually extended thick processes often in parallel with the pial surface and formed a dense GABA fiber network in immature layer I during the early postnatal periods. Some GABA neurons seemed to migrate into the underlying layers to settle as nonpyramidal cells. With the expansion of brain volume, GABA neurons relatively diminished and decreased in number. Nevertheless, a small number of GABA neurons did exist as essential CR neurons in layer I even in the adult rats aged 9 months.

Appearance of hematogenous cells in the white matter of myelin-deficient Jimpy mice.

Various cells from the monocyte-macrophage line, a few neutrophils and some lymphocytes were found in the non-myelinated white matter of Jimpy mice, though there was no vascular injury to the brain parenchyma. Some of the lymphocytes appearing in the white matter were in close contact with macrophages, and occasionally with young oligodendrocytes. Such lymphocytes were mainly of the small type with few cell organelles in a scanty cytoplasm, though a few were of the medium type with a moderate amount of cell organelles in an enriched cytoplasm. Macrophages in the non-myelinated white matter displayed a highly heterogeneous cytoplasm. In particular, "cytoplasmic compartments" in macrophages displayed specific structures differing from the inclusion bodies generally seen in phagocytic cells. The contents of such "cytoplasmic compartments" were rather similar to parts of the macrophage's own cytoplasm, though they were isolated from the cytoplasm by a membrane and gaps. Our findings would seem to support the previous presumption that inherited myelin deficiency in Jimpy mice is induced by a T-cell mediated autoimmune response.

Astrocytal changes in the white matter of Jimpy mice: immunohistochemistry using antisera to glial fibrillary acidic protein.

Immunohistochemistry using antisera to glial fibrillary acidic protein (GFAP) was performed to observe astrocytal changes in the white matter of myelin-deficient Jimpy mice. In wild-type controls, astrocytes stained with the GFAP immunoreaction appeared in the cerebellar medulla by postnatal day 3 and in the corpus callosum by postnatal day 6. The immunoreactivity gradually intensified with age to reach a peak of the GFAP level around postnatal day 12. With the progress of myelinogenesis, GFAP positive astrocytes decreased in number and became sparse in the white matter. On the other hand, the white matter of Jimpy mutant mice revealed intensified GFAP immunoreactivity, corresponding to a numerical increase in astrocytes and an enlargement of their cytoplasm. This condition continued from postnatal day 12 up to the end of life. Such astrocytes made up a dense framework with thickened processes in the non-myelinated white matter, where oligodendrocytes were few. Under the electron microscope, an accumulation of gliofilaments and glycogen particles was apparent in the enriched astrocytal cytoplasm. Immunostaining was observed not only on gliofilament bundles but also rough endoplasmic reticulum and cytoplasmic matrix. The present results demonstrate that astrocytal hypertrophy and hyperplasia occur in the entire white matter of Jimpy mice.

Macrophagic ameboid cells in the brain ventricles of the neonatal rat.

Scanning electron microscopy revealed numerous macrophagic ameboid cells on the ependymal surface of all brain ventricles in neonatal rats. Macrophagic ameboid cells aggregated in the sulcus medianus of the fossa rhomboidea, the recessus of the cerebral aqueduct and the recessus infundibuli, i.e. the ventromedial floor of the ventricular cavity covered mainly with non-ciliated ependyma. Macrophagic ameboid cells were numerous in the first few days after birth, often intermingling with extravasated erythrocytes. Subsequently, these cells decreased in number until 10 days after birth. Thus, it was rather difficult to find such ameboid cells in the brain ventricles of 21-day-old rats. Intravenous injection of primuline, a fluorescence dye used as a cytoplasmic marker in the previous study, enhanced the appearance of the ameboid cells and caused them to remain longer on the ventricular surface.

Distribution and fate of macrophagic ameboid cells in the rat brain.

Following an intravenous injection of a fluorescent dye, primuline, into neonatal rats, the distribution of ameboid cells and their fate in the central nervous system were examined under incident fluorescence, light and electron microscopes. In the ameboid cells labeled with primuline, the ingested fluorogen spread throughout the cytoplasm, in which it appeared as a whitish yellow fluorescence. Such labeled cells displayed increased acid phosphatase (ACPase) activity. Combining primuline labeling with the ACPase reaction allowed for the identification of ameboid cells even after the transformation mentioned below. Ameboid cells were distributed throughout the white matter in neonatal rats, but were few in the gray matter. Particularly, they appeared as cell clusters in the corona radiata of the corpus callosum, the subependymal layer surrounding the ventricles, the fornical commissure, the internal and external capsules, the cerebellar peduncles and the medulla. Around 8 to 14 days after birth, the ameboid cells which had so far appeared spherical became gradually elongated with a few branched cytoplasmic processes, and scattered in the white matter. Thereafter, most primuline labeled cells transformed into cells with a dark nucleus and a thin cytoplasm including small phagosomes, i.e., into cells with features of microglia. However, a small number of ameboid cells underwent degeneration. The data indicate that the ameboid cells observed in the white matter during the postnatal period transform into microglia. Since other investigations suggest that the ameboid cells are derived from blood monocytes, microglia could initially come from monocytes.

Effects of neonatal administrations of 6-OHDA on brain development. III. Myelin degeneration in the mesencephalic tract of the trigeminal nerve.

In rats treated with 6-OHDA in early postnatal days, myelin degeneration was observed in large and medium-sized myelinated nerve fibers belonging to the mesencephalic tract of the trigeminal nerve. Splitting of myelin lamination at the intraperiodic line and irregular excess scrolls were frequent in these fibers, whereas the smaller sized fibers remained intact. Occasionally, naked axons with numerous neurofilaments and neurotubules appeared near the empty myelin scrolls, indicating autolysis of the once-formed myelin sheaths. Some sheaths showed decompaction through loose lamination around the axons. The perikarya of the mesencephalic neurons showed no visible damage, but dark terminals making axo-somatic synapses in the motor nucleus were regarded as degenerating features of the axon-collaterals of the mesencephalic neurons. Thus, the myelin abnormalities were partially attributed to a kind of Wallerian degeneration. A survey of the glial cells in the corpus callosum indicated no remarkable changes in the ratio of the various glial types and in the myelin structures in comparison with those of the controls, although insufficient maturation of oligodendrocytes resulted in a 5-day delay in the onset of myelinogenesis. These findings suggest that the myelin degeneration in the mesencephalic tract is a regionally specific alteration induced by 6-OHDA administrations, probably because of the abnormal accumulation of NA in this area.

Divergent projections of catecholamine neurons of the locus coeruleus as revealed by fluorescent retrograde double labeling technique.

Divergent projections of catecholamine (CA) neurons of the locus coeruleus have been studied by fluorescent retrograde double labeling in conjunction with monoamine histofluorescence technique. The present results indicate that the coerulo-cortical CA system is composed of two types of neurons. A predominant type possesses few divergent axons innervating a restricted region, while the other type projects widely to various areas of the cerebral cortex. The existence of divergent axonal projections of single CA neurons in the locus coeruleus to the cerebellum and the spinal cord, to the frontal cortex and the cerebellum, is also demonstrated.

Effect of neonatal administrations of 6-OHDA on the brain development. I. Alteration in the striatum.

Following repeated subcutaneous administrations of 6-OHDA into neonatal rats (a dose; 100 mu/g bw), the effect on the brain development was examined. The striatal dopamine fluorescence never disappeared completely even immediately after multiple administrations of 6-OHDA, however a slight reduction of the fluorescence lasted for a long time. Semithin sections stained with toluidine blue showed numerous dark necrotic neurons appearing at a 15-times higher ratio than that in controls. Such necrotic neurons always accompanied a few granular deposits around the wavy surface. In a certain area of the striatum (0.898 mm2), there were no clear changes in the cell population in neurons and glia, in the ratio of glia to neurons, and in the ratio of each cell type of glia to the total glia, but some neuronal cell loss was speculated to occur slowly. The ultrastructural alteration observed at 28, 35 and 42 postnatal days in 6-OHDA treated rats was characterized by an appearance of myelin-like membranous lamellar bodies in the boutons synapsing with neural soma. In some cases intact small synaptic vesicles were seen around the lamellar body which probably rose from cell membranes in the synaptic area. Most of the necrotic neurons corresponded to the post-synaptic side for the boutons including lamellar bodies. Necrotic neurons displayed a dense cytoplasmic matrix, an increase of polysomes and cisternal swelling in rough ER and in the Golgi apparatus. Membranous lamellar bodies were occasionally observed in the periphery of satellite glial cells, probably oligodendrocytes. In conclusion, a long lasting reduction of striatal dopamine caused by 6-OHDA might disturb the function around the synaptic area and the metabolic regulation in the post-synaptic striatal neuron, thus resulted in the appearance of rather chronic morphological alterations in those areas.

Radioautographic investigation of gliogenesis in the corpus callosum of young rats. I. Sequential changes in oligodendrocytes.

The corpus callosum of young rats was examined to clarify the behavior of the three subtypes of oligodendrocytes (the large organelle-rich "light oligodendrocytes," the smaller and more densely stained cells referred to as "medium oligodendrocytes," and the even smaller and denser "dark oligodendrocytes"). It was hoped to find out whether cells of the three subtypes undergo division and how they are related to one another. 3H-thymidine was given intraperitoneally as single or three shortly spaced injections to a first group of 19- to 20-day old rats weighing about 40 g, and to a second group of 25-day old rats weighing about 80 g. The animals were sacrificed at various time intervals from 2 hours to 35 days after 3H-thymidine administration. Pieces of corpus callosum were taken near the superior lateral angle of the lateral ventricles; and semithin sections were radioautographed and stained with toluidine blue. Two hours after 3H-thymidine injection, label is virtually absent from light, medium and dark oligodendrocytes, from microglia, and probably from astrocytes, but is present in about 10% of the immature glial cells, which include the poorly differentiated glioblasts and the partially differentiated oligodendroblasts and astroblasts. Hence, the cells undergoing DNA synthesis and mitosis in the corpus callosum are these three types of immature cells. During the week that follow the administration of 3H-thymidine, label appears in oligodendrocytes and astrocytes, which presumably have arisen from the initially labeled immature cells. The oligodendrocytes acquire label in a sequential manner: the light cells show label first and their labeling index reaches a peak at the seven-day interval; the medium oligodendrocytes become labeled next with a labeling peak toward the 14- and 21-day intervals and, finally, the dark oligodendrocytes with a peak around the 28-day interval. Analysis by the method of Zilversmit et al. ('42-'43) provides precise details on the sequence: immature cells presumed to be oligodendroblasts give rise to light oligodendrocytes which, after four to seven days, transform into medium oligodendrocytes which, after another 11 to 18 days, transform into dark oligodendrocytes. The dark cells may persist indefinitely or turn over at a very slow rate. It is concluded that oligodendrocytes arise from the last division of oligodendroblasts and develop in three main periods: a light stage lasting less than a week, a medium stage lasting about two weeks, and a very long lasting dark stage.

Radioautographic investigation of gliogenesis in the corpus callosum of young rats. II. Origin of microglial cells.

Microglial cells are absent from the corpus callosum of newborn rats. In the hope of finding out when and how microglial cells appear with age, 3H-thymidine was given intraperitoneally as single or three shortly spaced injections to 5-day-old rats weighing about 15 g; and these animals were sacrificed at various time intervals from 2 hours to 35 days later. Pieces of corpus callosum were taken near the superior lateral angle of the lateral ventricles; and semithin sections were radioautographed and stained with toluidine blue. The corpus callosum of 5-day-old rats is composed of loosely arranged unmyelinated fibers and scattered cells. Among these cells, microglia are rare; there are a few astrocytes, many immature glial cells, rare pericytes, and 6--7% of phagocytic "ameboid cells" consisting of a few monocytes and many macrophages. In the animals sacrificed two hours after 3H-thymidine administration, label is present only in immature cells and "ameboid cells." As time elapses and the fibers of corpus callosum become myelinated, oligodendrocytes and, later, microglial cells appear. At the age of 12 days, microglial cells are present in substantial number; and by 19 days, the number doubles to reach a plateau. Many of the new microglial cells are labeled, e.g., 78.1% in 12-day-old animals (7 days after 3H-thymidine administration). The labeled microglial cells must have come from the transformation of cells that acquired label early, that is, from the immature cells or the "ameboid cells." The height of the peaks of labeling--59.8% at nine days for immature cells and 77.8% at 12 days for "ameboid cells"--points to the latter as precursors of the highly labeled microglial cells. Furthermore, the "ameboid cells" disappear as microglial cells appear and there are transitional elements between these two cell types. Cell counts suggest that about a third of the "ameboid cells" transform into microglial cells, while the others degenerate and die. Thus, the microglial cells which appear in the corpus callosum during the first three weeks of life result from transformation of the "ameboid cells"--a group of macrophages showing various stages of transition from monocytes. As for the occasional microglial cell appearing after the third week or in the adult, they presumably come directly from monocytes. In either case, monocytes would be the initial precursors.