Glucocorticoid-induced osteopenia in the mouse as assessed by histomorphometry, microcomputed tomography, and biochemical markers.

Glucocorticoids are potent anti-inflammatory molecules used in the treatment of asthma, rheumatoid arthritis, inflammatory bowel disease, and other inflammatory and dermatological diseases, as well as in posttransplantation immunotherapy. Although glucocorticoids have been prescribed for many years, their potential side effects, when administered orally, can prevent their long-term use. The most serious side effect observed in the clinic is glucocorticoid-induced osteoporosis (GIOP). To develop a small animal model to characterize glucocorticoid-induced bone loss, we carried out a series of experiments using BALB/c mice given daily intraperitoneal doses of the synthetic glucocorticoid, dexamethasone. Following dexamethasone treatment, the mice became osteopenic, with highly significant decreases in bone formation rate and mineral apposition rate, as assessed by standard histomorphometry. Moreover, 3 week treatment with dexamethasone resulted in a decrease in trabecular thickness and trabecular number with an increase in surface-to-volume ratio of trabeculae in the distal femur, as measured using microcomputed tomography (micro-CT). The serum bone formation marker, osteocalcin, was dose-dependently decreased in all mice treated with dexamethasone and showed a parallel extent of regulation to the bone formation rate changes. In addition, serum levels of leptin, recently identified as playing a role in the regulation of bone mass, increased following dexamethasone treatment. BALB/c mice therefore represent a useful model system in which the detrimental effects of glucocorticoids on bone can be studied.

The application of the Landstuhl frame for air evacuation of patients with femur fractures.

The use of traction to transport patients with femur fractures is well accepted. This paper describes step-by-step the construction of a traction device suitable for use on military aircraft. This "Landstuhl frame" is easily constructed using materials readily available. It is quick and effective for the transportation of patients with lower extremity fractures.

Studies on the effects of adenosine A3 receptor stimulation on human eosinophils isolated from non-asthmatic or asthmatic donors.

OBJECTIVE: This study was designed to demonstrate the presence of adenosine A3 receptors on human peripheral blood eosinophils, and to investigate the effect of A3 receptor stimulation on eosinophil function. MATERIAL: Eosinophils from either non-asthmatic or asthmatic donors. METHODS: Eosinophils were isolated from peripheral venous blood by discontinuous gradient centrifugation and negative immunoselection. Receptor localisation was investigated by immunoblotting and by immunocytochemistry using a novel antibody specific for the human A3 receptor. Two pharmacological responses were studied: elevation of intracellular calcium in single eosinophils, measured by microfluorimetry, and hydrogen peroxide generation in cell suspensions. RESULTS: The expression of A3 receptors by eosinophils was confirmed using the selective antibody. Addition of the A3 receptor selective agonist, IB-MECA (100 nM), produced increases in intracellular calcium in less than 10% of the eosinophils isolated from non-asthmatic donors. These responses were only partially attenuated with the A3 receptor antagonist, I-ABOPX. IB-MECA (0.001-1000 nM) did not stimulate hydrogen peroxide (H2O2) generation, nor did it enhance fMLP- or C5a-stimulated generation of H2O2. In fact high concentrations of IB-MECA inhibited the generation of H2O2 (when stimulated by fMLP or C5a), an effect probably mediated by A2 receptors. Similar results were obtained using eosinophils from asthmatic donors. CONCLUSIONS: Stimulation of adenosine A3 receptors does not appear to be a prime mechanism for free radical generation by human peripheral blood eosinophils.

Tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta down-regulate intercellular adhesion molecule (ICAM)-2 expression on the endothelium.

Leukocyte recruitment is a crucial step in inflammation. Inflammatory stimuli upregulate the expression of some endothelial adhesion molecules, such as E-selectin or ICAM-1, but not of others such as ICAM-2. ICAM-2, a constitutively expressed endothelial ligand for beta2 integrins LFA-1 and Mac-1, is involved in leukocyte adhesion to resting endothelium and in transmigration in vitro, however its role in inflammation is unclear. We have studied the effect of TNF-alpha and IL-1beta on ICAM-2 expression on human umbilical vein endothelial cells (HUVECs). Prolonged treatment (24 h) of HUVECs with TNF-alpha (10 ng/ml) or IL-1beta (34 ng/ml) reduced ICAM-2 surface expression to 50% of control, while interferon (IFN)-gamma had no effect. The loss in ICAM-2 surface expression correlated with a reduction of ICAM-2 mRNA to approximately 40% of control after 24 h of cytokine treatment. The activity of an ICAM-2 promoter reporter plasmid transfected into HUVECs was down-regulated by TNF-alpha and IL-1beta to similar values. Thus inflammatory cytokines inhibit ICAM-2 transcription, despite the absence of known cytokine-responsive elements in the promoter. Immunocytochemistry on HUVEC monolayers showed that ICAM-2 expression, mainly at the cell junctions in resting cells, was markedly decreased by cytokine treatment. This data suggest that ICAM-2 expression on the endothelium may be regulated during inflammation.

Reduction by inhibitors of mono(ADP-ribosyl)transferase of chemotaxis in human neutrophil leucocytes by inhibition of the assembly of filamentous actin.

1. Chemotaxis of human neutrophils is mediated by numerous agents [e.g. N-formyl-methionyl-leucyl-phenylalanine (FMLP) and platelet activating factor (PAF)] whose receptors are coupled to phospholipase C. However, the subsequent transduction pathway mediating cell movement remains obscure. We now propose involvement of mono(ADP-ribosyl)transferase activity in receptor-dependent chemotaxis. 2. Human neutrophils were isolated from whole blood and measurements were made of FMLP or PAF-dependent actin polymerization and chemotaxis. The activity of cell surface Arg-specific mono(ADP-ribosyl)transferase was also measured. Each of these activities was inhibited by vitamin K3 and similar IC50 values obtained (4.67 +/- 1.46 microM, 2.0 +/- 0.1 microM and 4.7 +/- 0.1 microM respectively). 3. There were similar close correlations between inhibition of (a) enzyme activity and (b) actin polymerization or chemotaxis by other known inhibitors of mono(ADP-ribosyl)transferase, namely vitamin K1, novobiocin, nicotinamide and the efficient pseudosubstrate, diethylamino(benzylidineamino)guanidine (DEA-BAG). 4. Intracellular Ca2+ was measured by laser scanning confocal microscopy with two fluorescent dyes (Fluo-3 and Fura-Red). Exposure of human neutrophils to FMLP or PAF was followed by transient increases in intracellular Ca2+ concentration, but the inhibitors of mono(ADP-ribosyl)transferase listed above had no effect on the magnitude of the response. 5. A panel of selective inhibitors of protein kinase C, tyrosine kinase, protein kinases A and G or phosphatases 1 and 2A showed no consistent inhibition of FMLP-dependent polymerization of actin. 6. We conclude that eukaryotic Arg-specific mono(ADP-ribosyl)transferase activity may be implicated in the transduction pathway mediating chemotaxis of human neutrophils, with involvement in the assembly of actin-containing cytoskeletal microfilaments.

Retinal ganglion cell distribution and behaviour in procellariiform seabirds.

Retinal ganglion cell distribution in nine species of procellariiform seabirds was studied by Nissl staining of retinal whole-mounts and the construction of density contour maps. Most species showed a horizontal linear area of high cell density, but concentric distributions with dorsal and central cell concentrations were found in two species. These results are discussed in relation to the birds' foraging behaviour.

A new computer method for the measurement of the thickness of basement membranes.

A new computer method for determining basement membrane thickness is described. The method, tested with repeat measurements on model and real basement membrane, compares favourably with previous methods of BM thickness measurement.

Myopia: induced, normal and clinical.

A review is given of lid-suture myopia in monkeys, of the natural lower field myopia in the pigeon eye, and of myopias in chick eyes produced by visual occluders. The ametropias produced by ophthalmic lenses, and the pharmacology of experimental myopia, are reviewed. Human studies are reviewed which can be taken as models of the experimental myopias. Two theories of myopic growth are outlined and evaluated.

Macromolecular organization of collagen fibres in natural and tanned basement membrane.

The elastic constants and ultrastructure of natural and tanned basement membrane of the crystalline lens of the adult rat have been investigated. Sonicated and negatively stained specimens of both membranes show parallel filaments that have similar spacing of 3.5(+/- 0.1) nm and a different periodicity. In natural membrane the periodicity is 3.7(+/- 0.13) nm, whilst in tanned basement membrane the periodicity is 3.2(+/- 0.15) nm. The periodicity ratio of tanned membrane to natural membrane was 0.86 +/- 0.04, whilst the elongation ratio of tanned membrane compared with natural membrane was 0.88 +/- 0.05. In contrast to this, the thickness ratio of tanned to natural membrane was 1.098 +/- 0.045. Tanned basement membrane showed a shrinkage of 12% in length but an increase in thickness of about 10%. These data suggest, firstly, that the degree of extension of the superhelices of the filaments follows closely the degree of extension of the intact membrane and, secondly, that the coiled superhelices of tanned membrane have an angle of tilt of about 42 degrees compared with those of natural membrane, where the angle is about 50 degrees. The Young's modulus of elasticity and ultimate stress of tanned basement membrane are, respectively, eight times greater and one-third as great as natural membrane. The entropy change in basement membrane was calculated from the external work necessary to extend the tanned membrane, and was estimated to be -13.5(+/- 2.4) J K-1 mol-1. An estimate of the change in entropy from thermodynamic measurements made on a suspension of collagen tanned with glutaraldehyde was found to be -30.1(+/- 9.5) J K-1 mol-1. The two different estimates of the change in entropy of collagen following tanning suggest that in basement membrane only about 45% of the collagenous protein has an extensile helical structure.

Retinal magnification factor at the ora terminals: a structural study of human and animal eyes.

A geometrical method of calculating retinal magnification factor at the limits of the retinal field, adjacent to the ora terminalis, is described. The method is applied to the eyes of 11 mammalian and avian species, using new anatomical measurements and data from the literature. In the human and monkey eye, magnification at the far periphery is substantially smaller than at the posterior pole; in cat, rabbit, rat and mouse there is lesser reduction; in pigeon, tawny owl and starling magnification is closely similar at the far periphery and posterior pole.

The projection of the visual field upon the retina of the pigeon.

Co-ordinates in the visual field of the pigeon eye were located in the eye cup anatomically by marking the position of the trans-scleral image. The retinal horizon and vertical meridian at azimuths 0 deg (frontal field) and 90 deg (lateral field) were located. Approximately 18 deg of binocular field are available in frontal vision and the frontal meridian projects to temporal retina outside the red field. Spatial distributions of ganglion cells, displaced ganglion cells, and centrifugal terminals were located in the eye cup. Retinal magnification factors in the posterior pole and temporal periphery are close to 120 microns/deg.

A morphological analysis of experimental myopia in young chickens.

Devices that degrade vision were applied to the left eyes of 3-day old chicks. The dome device affected the entire visual field, and the arch device, only the lateral field. Control chicks wearing a circumorbital ring and untreated chicks were also examined. The dome device produced -15D and the arch device -4D of mean refractive error, while the ring and untreated chicks were emmetropic. Morphological measurements were made from macrophotographs of the intact and hemisected eyes fixed as for electron microscopy. The effects of the devices were analysed from the mean differences between the left (treated) and right (control) eyes. Nearly linear growth of the normal eye was found during the period in which measurements were taken (age 20-55 days). The ring device did not affect eye growth. The arch device significantly increased the dorsoventral equatorial diameter of the eye. The dome device had the greatest effect, and resulted in increases in both axial length and equatorial diameter during the treatment period. Dome eyes had a bulging cornea, increased anterior chamber depth, more open angle, and greater corneal diameter than controls. The axial length and equatorial diameter of the posterior segment also were increased. Two inflammatory responses of the eye were found, particularly in dome eyes; about 50% of treated eyes exhibited choroidal swelling, and vitreal clouding was found less frequently. The association between inflammation and excessive accommodation in producing the observed changes is discussed.

Electrophysiological optometry using Scheiner's principle in the pigeon eye.

A new electrophysiological optometer has been developed, based on Scheiner's principle. Using two light-emitting diodes driven in counterphase, and grating stimuli in Maxwellian view, the system has been used to refract the photoreceptor plane of the pigeon eye. When a grating is conjugate with the photoreceptors, the electroretinographic response (e.r.g.) is minimal, and the image is stationary. As defocus is introduced, so image shift occurs, and the e.r.g. rises on each side of the refractive minimum. Two experiments were devised to test whether a derived minimum point is primary, by using gratings of bar width 3, 5 and 7 units, and by using random checkerboard stimuli. Ray tracing was used to compute the lobe width of dioptric profiles for gratings of spatial frequency 1.47, 0.88 and 0.63 cycles/deg. The computed lobe widths agree well with experimentally determined e.r.g. profiles. Examination of the lateral visual field, on the horizon, shows that the pigeon eye is emmetropic, and well corrected for astigmatism.

Refractive sectors in the visual field of the pigeon eye.

Scheiner's principle has been used in electroretinographic optometry to refract the photoreceptor plane in different regions of the visual field of the pigeon eye. Along the horizon and in the upper visual field the eye is emmetropic, or nearly so. Below the horizon the eye becomes progressively more myopic at more negative elevations, refractive state falling to -5D at -90 deg. Lower field myopia is not an artifact of oblique astigmatism, nor of an aberration symmetrical about the optical axis. It is suggested that lower field myopia is a biological adaptation suited to keep the photoreceptors in the upper retina conjugate with the ground. Refractive state below the horizon can be fitted with a sine function by varying a parameter H (eye-ground height). The value of H agrees well with directly measured eye-ground height.

Cytoarchitectural fields and retinal termination: an axonal transport study of laminar organization in the avian optic tectum.

The cytoarchitecture in the retinoreceptive zone of the pigeon optic tectum has been studied in Nissl-stained sections taken in four planes. As suggested by a previous study, two cytoarchitectural fields are present. Reconstructed views of the tectum show that the fields are separated by a narrow transition zone approximating to the tectal representation of the retina's horizontal meridian. In field 1 (which is upper and rostral), sublayer IIb is wide, IIc wide and trilaminate, IId narrow and IIe continuous; in field 2, IIb and c are narrow, IId wide and IIe discontinuous. The distribution of retinal terminals was investigated by the anterograde axonal transport of [3H]proline or horseradish peroxidase from intravitreal injections. The depth distribution of grains or reaction product throughout the entire tectum was quantified by scanning with a microdensitometer. Both autoradiography and horseradish peroxidase transport show two patterns of lamination separated by a narrow transition zone and these two terminal fields correspond closely to the cytoarchitectural fields. In field 1 optic terminals are concentrated in sublayer IIb, superficial c, d, and to a lesser extent in f; in field 2 concentrations are present at the IIb/c boundary, across deep IIc and d, and a small concentration is found IIf. The patterns of retinal termination with depth in the tectum found by axonal transport are compatible with those found by electron microscopy, and are discussed in relation to the optic termination found by other techniques. Study of the time course of axonal transport shows that both radioactive material and horseradish peroxidase are fast transported to all the bands of optic terminals at about 150 mm/day. Horseradish peroxidase gradually accumulates in the retinoreceptive zone, filling clusters of terminals and horizontal processes. At 12 days, it has begun to disappear from the zone and a few diffusely filled profiles, that may be transcellularly labelled, are present. Electron microscope autoradiography of fast transported material shows clusters of grains over optic terminals and preterminals and a percentage density analysis confirms that these profiles are specifically labelled. The two tectal fields each contain the projection from specialized areas of the retina, suggesting functional specialization in the tectum for the processing of different kinds of visual information.

Experimental myopia in chicks: ocular refraction by electroretinography.

Application of devices that degrade the retinal image has been reported to produce enlargement of the ocular globe in young domestic chicks. Two such device types (domes and arches) were applied to 3-day-old chicks. The domes affected the entire visual field whereas the arches affected only the lateral field. A third group wore a thin circumorbital ring to control for possible mechanical impediments to growth. Untreated control chicks comprised a fourth group. At ages ranging from 3 to 7 wk, the chicks were refracted in their lateral visual fields with a Maxwellian view optometer based on Scheiner's principle, which yields an objective assessment of the refractive state of the photoreceptor image plane. One to seven measurements were taken from each of 48 urethane-anesthetized chicks. These indicated that the mean refractive states of the untreated eyes and the ring eyes were -0.20 D and -0.19 D, respectively, which did not differ significantly from emmetropia. In contrast, the mean refractive states of the arch eyes and the dome eyes were -4.11 D and -14.88 D, respectively, which differed significantly from emmetropia and from each other. The results indicate that early retinal image degradation can result in the relatively rapid development of a substantial myopia in these experimental animals.

Ultrastructural appearances of a lens with marked polychromatic lustre: evidence for diffraction as a cause.

A human lens showing polychromatic lustre associated with cortical opacities (Christmas tree cataract) has been examined by slit-lamp photography, thin-section light microscopy, and electron microscopy. Anterior epithelial cells were fibroblast-like, and an area of breakdown of cortical lens fibres was observed in the anterior lens, containing feathery fibres, whorls, and process bodies. Parallel sided stacks of fused cell membranes were found beneath the watery area at the same depth as the polychromatic lustre seen in the slit-lamp. The dimensions of these membrane plates are consistent with the diffraction of light by a parallel thin film to give coloured points of light. Deeper areas of small granules between the lens fibres are associated with the main trunk-like opacity and its branches seen with the slit-lamp.

Sensory physiology, anatomy and immunohistochemistry of Rohon-Beard neurones in embryos of Xenopus laevis.

Rohon-Beard neurones show substance P-like immunoactivity in their somas and in their centrally projecting axons. Peripherally, the morphology of their free nerve endings within the trunk skin has been shown using horseradish peroxidase staining. The excitation of Rohon-Beard neurones by natural and electrical stimulation of the skin has been examined using intracellular micro-electrodes in the late embryo of Xenopus laevis. Rohon-Beard cells are sensitive to transient, local indentation of the trunk skin, responding with one or a few impulses. They adapt rapidly to repeated stimulation. They can also be excited by a brief current pulse to the skin. They are not sensitive to slow indentation of the skin, nor are they excited by epithelial action potentials. The responses to skin stimulation are not abolished by a Ringer solution containing 12 mM-Mg2+ and only 0.5 mM-Ca2+. Intracellularly evoked action potentials in single Rohon-Beard cells are sometimes sufficient to evoke sustained episodes of fictive swimming. The results indicate that Rohon-Beard cells are responsible for detecting light touch stimuli to the embryo's body and for initiating swimming in response to this stimulus.

Cell populations of the ganglion cell layer: displaced amacrine and matching amacrine cells in the pigeon retina.

The proportion and size distribution of ganglion and non-ganglion cells in the ganglion cell layer of different areas of the pigeon retina was examined in whole-mounts of the retina by retrograde axonal transport of horseradish peroxidase (HRP) from large brain injections. A maximum of 98% of cells were labelled in the red field and a maximum of 77% in the peripheral yellow field. Unlabelled cell bodies were 30% smaller than labelled ganglion cells and had a mean diameter of 6.2 microns and a size range of 4 to 9 microns. The morphology of cells in the ganglion cell layer was examined by Golgi staining of retinal whole-mounts. Small glia, displaced amacrine and ganglion cells were found. Displaced amacrine cell bodies were about 30% smaller than ganglion cells and their size distribution was similar to the unlabelled cells in HRP preparations. Displaced amacrine cells had small rounded cell bodies (mean diameter 6.2 microns) increasing in size with eccentricity, and a unistratified dendritic tree of fine, nearly radial, varicose dendrites in sublamina 4 of the inner plexiform layer. They had elliptical dendritic fields (mean diameter 66 micron) aligned parallel to the retina's horizontal meridian. A population of amacrine cells was found with somas at the inner margin of the inner nuclear layer and soma and dendritic morphology matching those of displaced amacrines. These amacrine cells had unistratified dendritic trees at the junction of sublaminae 1 and 2 of the inner plexiform layer. Pigeon displaced amacrine cells and their matching amacrines are similar to starburst cells of the rabbit retina.(ABSTRACT TRUNCATED AT 250 WORDS)

The anatomy of two functional types of mechanoreceptive 'free' nerve-ending in the head skin of Xenopus embryos.

The structure of the two functional types of 'free' nerve-ending in the head skin of late Xenopus embryos has been examined by horseradish peroxidase staining through their cells in the trigeminal ganglion and by electron microscopy. Type I neurites are identified as the 'movement' detectors by their purely homolateral innervation. They have many fine branches between the superficial skin cells, bearing numerous large varicosities. Type II neurites cross the midline to innervate both sides of the head as do the 'rapid transient' detectors found by physiology. They have a few fairly straight branches between the skin cell layers with few elongated varicosities.