Three-dimensional surface texture visualization of bone tissue through epifluorescence-based serial block face imaging.

Serial block face imaging is a microscopy technique in which the top of a specimen is cut or ground away and a mosaic of images is collected of the newly revealed cross-section. Images collected from each slice are then digitally stacked to achieve 3D images. The development of fully automated image acquisition devices has made serial block face imaging more attractive by greatly reducing labour requirements. The technique is particularly attractive for studies of biological activity within cancellous bone as it has the capability of achieving direct, automated measures of biological and morphological traits and their associations with one another. When used with fluorescence microscopy, serial block face imaging has the potential to achieve 3D images of tissue as well as fluorescent markers of biological activity. Epifluorescence-based serial block face imaging presents a number of unique challenges for visualizing bone specimens due to noise generated by sub-surface signal and local variations in tissue autofluorescence. Here we present techniques for processing serial block face images of trabecular bone using a combination of non-uniform illumination correction, precise tiling of the mosaic in each cross-section, cross-section alignment for vertical stacking, removal of sub-surface signal and segmentation. The resulting techniques allow examination of bone surface texture that will enable 3D quantitative measures of biological processes in cancellous bone biopsies.

Ex vivo characterization of human atherosclerotic iliac plaque components using cryo-imaging.

We characterized atherosclerotic plaque components with a novel cryo-imaging system in lieu of standard histological methods commonly used for imaging validation and research endpoints. We aim to accurately identify plaque tissue types from fresh cadaver specimens rapidly (less than 5 h) in three dimensions for large specimens (up to 4 cm vessel segments). A single-blind validation study was designed to determine sensitivity, specificity and inter-rater agreement (Fleiss' Kappa) of cryo-imaging tissue types with histology as the gold standard. Six naïve human raters identified 344 tissue type samples in 36 cryo-image sets after being trained. Tissue type sensitivities are as follows: greater than 90% for adventitia, media-related, smooth muscle cell ingrowth, external elastic lamina, internal elastic lamina, fibrosis, dense calcification and haemorrhage; greater than 80% for lipid and light calcification; and greater than 50% for cholesterol clefts. Specificities were greater than 95% for all tissue types. The results demonstrate convincingly that cryo-imaging can be used to accurately identify most tissue types. If the cryo-imaging data are entered into visualization software, three-dimensional renderings of the plaque can be generated to visualize and quantify plaque components.

Increased atherosclerosis in streptozotocin-induced diabetic mice.

Premature and extensive atheroscleroses involving renal, peripheral, and cardiovascular sites remain major complications of diabetes mellitus. Controversy exists as to the contribution of hyperglycemia versus elevated local or systemic concentrations of insulin to atherosclerosis risk. In this report, we developed the first murine model susceptible to both atherosclerosis and diabetes to determine which diabetogenic factors contribute to vascular disease. C57BL/6 and BALB/c mice were treated with multiple low-dose streptozotocin (STZ) or control citrate buffer and fed rodent chow or an atherogenic-promoting (Ath) diet for 12-20 wk. STZ treatment resulted in sustained hyperglycemia (250-420 mg/dl) and a modest reduction in plasma insulin levels for both strains regardless of diet. Citrate-treated C57BL/6 mice fed the Ath diet showed extensive oil red O-staining fatty streak aortic sinus lesions (20,537+/-2,957 micron2), the size of which did not differ for Ath-fed mice treated with STZ (16,836+/-2,136 micron2). In contrast, hyperglycemic BALB/c mice fed the Ath diet showed a 17-fold increase in atherosclerotic lesion area (7,922+/-2,096 micron2) as compared with citrate-treated mice fed the Ath diet (467+/-318 micron2). Correlations between lesion size and plasma glucose levels were significant for BALB/c (r = 0.741, P < 0.009), but not C57BL/6 (r = 0.314, P<0.3) mice. Lesion size correlated significantly with plasma cholesterol for C57BL/6 (r = 0.612, P<0.03) but not BALB/c (r = 0.630, P<0.1) mice. Immunohistochemistry showed that aortic sinus lesions from both strains contained macrophages, but smooth muscle cells were clearly present in lesions of BALB/c mice. In summary, we present the first small animal model showing accelerated atherosclerosis in response to hyperglycemia. Fatty streaks resembled those of human type II lesions in that both macrophages and smooth muscle cells were evident. In addition, our results support the concept that hyperglycemia as opposed to hyperinsulinemia contributes heavily to risk of atherosclerosis.

Ultrahigh-speed optical coherence tomography imaging and visualization of the embryonic avian heart using a buffered Fourier Domain Mode Locked laser.

The embryonic avian heart is an important model for studying cardiac developmental biology. The mechanisms that govern the development of a four-chambered heart from a peristaltic heart tube are largely unknown due in part to a lack of adequate imaging technology. Due to the small size and rapid motion of the living embryonic avian heart, an imaging system with high spatial and temporal resolution is required to study these models. Here, an optical coherence tomography (OCT) system using a buffered Fourier Domain Mode Locked (FDML) laser is applied for ultrahigh-speed non-invasive imaging of embryonic quail hearts at 100,000 axial scans per second. The high scan rate enables the acquisition of high temporal resolution 2D datasets (195 frames per second or 5.12 ms between frames) and 3D datasets (10 volumes per second). Spatio-temporal details of cardiac motion not resolvable using previous OCT technology are analyzed. Visualization and measurement techniques are developed to non-invasively observe and quantify cardiac motion throughout the brief period of systole (less than 50 msec) and diastole. This marks the first time that the preseptated embryonic avian heart has been imaged in 4D without the aid of gating and the first time it has been viewed in cross section during looping with extremely high temporal resolution, enabling the observation of morphological dynamics of the beating heart during systole.

4D embryonic cardiography using gated optical coherence tomography.

Simultaneous imaging of very early embryonic heart structure and function has technical limitations of spatial and temporal resolution. We have developed a gated technique using optical coherence tomography (OCT) that can rapidly image beating embryonic hearts in four-dimensions (4D), at high spatial resolution (10-15 mum), and with a depth penetration of 1.5 - 2.0 mm that is suitable for the study of early embryonic hearts. We acquired data from paced, excised, embryonic chicken and mouse hearts using gated sampling and employed image processing techniques to visualize the hearts in 4D and measure physiologic parameters such as cardiac volume, ejection fraction, and wall thickness. This technique is being developed to longitudinally investigate the physiology of intact embryonic hearts and events that lead to congenital heart defects.

Molecular weight distribution of proteins synthesized in single, identified neurons of Aplysia.

Parietovisceral ganglia from Aplysia californica were incubated in medium containing leucine-(3)H. Single, identified nerve cell somas were isolated from the ganglia, and their proteins extracted and separated by electrophoresis on 5% SDS-polyacrylamide gels. The distribution of total or newly synthesized proteins from the single neurons was determined by staining or slicing and liquid scintillation counting of the gels. Experiments showed that: (a) a number of proteins were being synthesized in abundance in the nerve cells; (b) different, identified neurons showed reproducibly different labeling patterns in the gels; (c) cells R2 and R15, which showed different distributions of radioactivity in the gels, had similar staining patterns; and (d) there was significant incorporation into material of high (>75,000) molecular weight in most of the cells.

Patterns of proteins synthesized in the R15 neuron of Aplysia. Temporal studies and evidence for processing.

The time-course of changes in the pattern of newly synthesized proteins in the R15 neuron of the parietovisceral ganglion of Aplysia californica has been studied at 14 degrees C. 5% polyacrylamide gels containing sodium dodecyl sulfate (SDS) have been used to separate newly synthesized (leucine-labeled) proteins from the neuron. We have demonstrated that the pattern of newly synthesized proteins from the R15 neuron does not change significantly if 5-h pulses of labeled leucine are given during the first 72 h of in vitro incubation of the excised ganglion. However, the level of leucine incorporation begins to decline somewhere between 17 and 43 h after the ganglion is isolated; at 43 and 69 h the levels of incorporation fell to 29 and 10% of the initial level, respectively. A number of conclusions have been drawn from the use of a sequential, double-label type of experiment in the same cell. There is processing of SDS-soluble, 12,000-dalton (12k) material to 6,000-9,000-dalton (6-9k) material. These materials are the two major peaks on gels after long labeling periods and together account for about 35% of all newly synthesized proteins. After synthesis of 12k material, there is a gradual disappearance of 12k (half-life about 8 h) and simultaneous appearance of 6-9k material on the gels, as the postsynthesis "chase" period of ganglia incubation is increased. The processing of 12k to 6-9k material occurs even in the presence of anisomycin, a protein syntehsis inhibitor, during the chase period. While the rate of 12k to 6-9k conversion can vary from cell to cell, it appears to remain consistent within, and is characteristic of, any individual R15. We detect no circadian rhythm in either the rate of 12k synthesis or the rate of 12k to 6-9k processing with 5-h label periods. These results are discussed in relation to the roles of 12k and 6-9k material in the R15 neuron.

Cardiac Na currents and the inactivating, reopening, and waiting properties of single cardiac Na channels.

Tetrodotoxin (TTX)-sensitive Na currents were examined in single dissociated ventricular myocytes from neonatal rats. Single channel and whole cell currents were measured using the patch-clamp method. The channel density was calculated as 2/micron 2, which agreed with our usual finding of four channels per membrane patch. At 20 degrees C, the single channel conductance was 20 pS. The open time distributions were fit by a single-exponential function with a mean open time of approximately 1.0 ms at membrane potentials from -60 to -40 mV. Averaged single channel and whole cell currents were similar when scaled and showed both fast and slow rates of inactivation. The inactivation and activation gating shifted quickly to hyperpolarized potentials for channels in cell-attached as well as excised patches, whereas a much slower shift occurred in whole cells. Slowly inactivating currents were present in both whole cell and single channel current measurements at potentials as positive as -40 mV. In whole cell measurements, the potential range could be extended, and slow inactivation was present at potentials as positive as -10 mV. The curves relating steady state activation and inactivation to membrane potential had very little overlap, and slow inactivation occurred at potentials that were positive to the overlap. Slow inactivation is in this way distinguishable from the overlap or window current, and the slowly inactivating current may contribute to the plateau of the rat cardiac action potential. On rare occasions, a second set of Na channels having a smaller unit conductance and briefer duration was observed. However, a separate set of threshold channels, as described by Gilly and Armstrong (1984. Nature [Lond.]. 309:448), was not found. For the commonly observed Na channels, the number of openings in some samples far exceeded the number of channels per patch and the latencies to first opening or waiting times were not sufficiently dispersed to account for the slowly inactivating currents: the slow inactivation was produced by channel reopening. A general model was developed to predict the number of openings in each sample. Models in which the number of openings per sample was due to a dispersion of waiting times combined with a rapid transition from an open to an absorbing inactivated state were unsatisfactory and a model that was more consistent with the results was identified.

Activation and inactivation of single calcium channels in snail neurons.

Activation and inactivation properties of Ca currents were investigated by studying the behavior of single Ca channels in snail neurons. The methods described in the previous paper were used. In addition, a zero-phase digital filter has been incorporated to improve the analysis of latencies to first opening, or waiting times. It was found that a decrease in the probability of single channel opening occurred with time. This was especially marked at 29 degrees C and paralleled the inactivation observed in macroscopic currents. The fact that a single channel was observed means that there is a significant amount of reopening from the "inactivated" state. Small depolarizations at 18 degrees C showed little inactivation. From these measurements, histograms of single channel open, closed, and waiting times were analyzed to estimate the rate constants of a three-state model of activation. Two serious discrepancies with the model were found. First, waiting time distributions at -20 mV were slower than those predicted by parameters obtained from an analysis of the single channel closed times. Second, it was shown that the time and the magnitude of the peak of the waiting time histogram were inconsistent with a three-state model. It is concluded that a minimum of four states are involved in activation. Some four-state models may be eliminated from further consideration. However, a comprehensive model of Ca channel kinetics must await further measurements.

IL-4 and interferon gamma regulate expression of inducible nitric oxide synthase in chronic lymphocytic leukemia cells.

Chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of long-lived non-dividing CD5(+) B cells. Nitric oxide (NO) is an important regulator of apoptosis, and the viability of cultured B-CLL cells may be dependent on the autocrine production of nitric oxide by inducible nitric oxide synthase (NOS2). We performed this study to determine whether cytokine factors that prevent spontaneous in vitroapoptosis of B-CLL cells induce B-CLL cell NOS2 enzyme activity. B-CLL cells expressed NOS enzyme activity and NOS2 protein and mRNA. IL-4 and IFN-gamma increased B-CLL cell NOS2 enzyme activity and protein expression during in vitro culture. IFN-gamma, but not IL-4, increased NOS2 mRNA expression in cultured B-CLL cells suggesting that IL-4-mediated changes of NOS2 protein expression occurred at the post-transcriptional level. We were unable to detect increased concentrations of nitrite or nitrate (NO(x)) as surrogate markers of NO production in B-CLL cell cultures treated with IL-4 or IFN-gamma. IL-4 and IFN-gamma diminished NOS inhibitor-induced B-CLL cell death. In summary, we found that B-CLL cells expressed NOS2 and that IL-4 and IFN-gamma increased B-CLL NOS2 expression. Cytokine-mediated expression of NOS2 by B-CLL cells may promote their survival, and therapeutic strategies that target NOS2 or quench NO may be beneficial in patients with B-CLL.

The analysis of survival (mortality) data: fitting Gompertz, Weibull, and logistic functions.

Survival functions are fitted to survival data from several large populations. The Gompertz survival function corresponds to exponential mortality rate increases with time. The Weibull survival function corresponds to mortality rates that increase as a power function of time. A two-parameter, logistic survival function is introduced, and corresponds to mortality rates that increase, and then decrease, with time. A three-parameter logistic-mortality function also is examined. It reflects mortality rates that rise, and then plateau, with age. Data are from published studies of medflies, Drosophila, house flies, flour beetles, and humans. Some survival data are better fit by a logistic survival function than by the more traditionally used Gompertz or Weibull functions. Gompertz, Weibull, or logistic survival functions often fit the survival of 95+% of a population, and the 'tails' of the survival curves usually appear to fall between the values predicted by the three functions. For some populations, such 'tails' appear to be too complex to be fit well by any simple function. Survival data for males and females in some populations are best fit by different functions. Populations of 100 or more are needed to distinguish among the functions. When testing effects of environmental or genetic manipulations on survival, it has been common to determine the changes in parameter values for a given function, such as Gompertz. It may be equally important to determine whether the best-fit function has changed as well.

A comparison of methods for estimating mortality parameters from survival data.

The Gompertz mortality function, Rm = R0e alpha t, is frequently used to describe changes in mortality rate (Rm) with time (t). In this paper, four methods for determining the best fit values of the two parameters, R0 and alpha, are compared. Three of the four methods use the Gompertz mortality function with mortality rate estimates derived from survival data to determine the best fit values for the two parameters. All three confront problems. The fourth method uses the Gompertz survival function, which can be derived from the Gompertz mortality function and which allows one to use survival data directly. It thereby avoids the problems and generally gives the best estimates for the two parameters. The use of the mortality function, with mortality rate estimates, confronts four distinct problems. One of these is caused by time intervals when zero organisms die. A second is caused by errors produced in estimating mortality rates from survival data. If too high a proportion of a population die in a given time interval, the mortality rate estimates are too low. A third problem is the sensitivity of the mortality-equation-based analyses to values at the end of the survival curve, where scatter in mortality values tends to be greater. A final problem occurs when time intervals greater than one time unit (day, week, year, etc.) are used in the analysis. Such problems with the use of mortality rates to estimate parameter values are revealed when the calculated parameters are used to produce a survival curve, or when known values of R0 and alpha are used to generate survival data. This paper introduces a non-linear regression analysis, using a Simplex algorithm to fit parameters R0 and alpha in the Gompertz Survival function and concludes that it gives more reliable and consistent results with a variety of data than do three methods that use the mortality function.

Isolating aging mutants: a novel method yields three strains of the nematode Caenorhabditis elegans with extended life spans.

We designed a novel procedure for the isolation of mutant strains with significantly increased life spans in the nematode Caenorhabditis elegans. This procedure involves using heat-shock to screen a large number of animals and isolate a few which are more resistant to heat-shock stress. From the heat-shock-resistant animals, three mutant strains, HG25, HG96, and HG246, all exhibiting increased life span, were isolated. One mutant strain (HG246) develops more slowly than the wild-type strain, N2. Two mutant strains, HG96 and HG246, exhibit lower fertility than the wild-type. Each of the three mutant strains has a normal appearance. Their locomotive behavior also appears normal; only HG246 shows slightly slower movement. Their feeding behavior appears normal, and the males of HG25 and HG96 show normal mating behavior. However, the males of HG246, either are defective in their mating ability or their sperm are defective. The results indicate that heat-shock can be used as a means to facilitate the isolation of mutants which have longer life expectancy.

C57BL/6 mice fed high fat diets as models for diabetes-accelerated atherosclerosis.

Non-insulin-dependent diabetes mellitus (NIDDM) is a major risk factor for the development of atherosclerosis in humans. The development of an animal model that displays accelerated atherosclerosis associated with NIDDM will aid in elucidating the mechanisms that associate these disorders. C57BL/6 mice may provide such a model system. This strain becomes obese, hyperglycemic and insulin resistant when fed a high fat diet (diabetogenic diet) and is susceptible to atherosclerotic lesion development when fed a separate high fat diet containing cholesterol and bile acids (atherogenic diet). This report tests whether a diet commonly used to induce atherosclerosis also provokes a diabetic phenotype and whether a diet used to induce diabetes provokes the development of aortic fatty streak lesions. Mice of strains C57BL/6, C3H/He, BALB/c and seven recombinant inbred (RI) strains were fed an atherogenic diet for 14 weeks and glucose parameters were measured. No correlation was observed between atherosclerosis susceptibility and fasting insulin or glucose levels, or glucose clearance following short-term insulin or glucose treatment. Analysis of the RI strains suggested that multiple genes control these glucose metabolic parameters. Feeding the diabetogenic diet for 14 weeks to C57BL/6 mice induced obesity and diabetes and 2-fold increases in plasma lipoprotein concentrations. Also, small aortic sinus lipid deposits were observed in 40% of the mice. Thus, analysis of the diabetogenic diet fed C57BL/6 mouse may provide an important tool for further studies of diabetes accelerated vascular disease.

An intracavitary cone system for electron beam therapy using a Therac 20 linear accelerator.

The Therac 20 is an AECL medical linear accelerator that produces electron and photon beams. Electron fields are produced by a scanned beam; collimation is provided by two sets of primary collimators and further collimated by external electron trimmers located 11 cm above the plane of isocenter (100 cm). These collimators are not suitable for intracavitary treatment. To overcome this limitation, we have designed an intracavitary cone system that attaches to the electron trimmers. Since the trimmers do not have to be removed while this system is in use, there is no need to bypass the associated interlock system. The apparatus consists of a platform which slides onto the lower set of trimmers, onto which a lead insert is attached. Dosimetry measurements for 9, 13, and 17 MeV electron beams are reported for three different treatment cones.

Three dimensional structure of human fibrinogen under aqueous conditions visualized by atomic force microscopy.

Fibrinogen plays a central role in surface-induced thrombosis. However, the interactions of fibrinogen with different substrata remain poorly understood because of the difficulties involved in imaging globular proteins under aqueous conditions. We present detailed three dimensional molecular scale images of fibrinogen molecules on a hydrophobic surface under aqueous conditions obtained by atomic force microscopy. Hydrated fibrinogen monomers are visualized as overlapping ellipsoids; dimers and trimers have linear conformations predominantly, and increased affinity for the hydrophobic surface compared with monomeric fibrinogen. The results demonstrate the importance of hydration on protein structure and properties that affect surface-dependent interactions.

Aging hypotheses, aging markers and the concept of biological age.

The following two points are made in this article: (1) the likely existence of more than one underlying cause of senescence strengthens the case for the development of a number of reliable markers of aging and (2) the concept of a single biological or functional age for an organism should be used with great caution, if at all.

In vivo effects of interleukin-1 beta on blood leukocytes in BB rats prone or resistant to diabetes.

Previous studies have determined that daily low dose injections of the potent cytokine interleukin-1 beta (IL-1 beta) decreased the frequency of insulin-dependent diabetes mellitus (IDDM) in diabetes-prone (DP) BB rats. In contrast, high dose injections induced an earlier than normal onset. In this study we tested whether the effects of daily human recombinant IL-1 beta injections on leukocyte subsets were associated with its modulation of IDDM onset in BB rats. Prior to the onset of IDDM in DP BB rats, high dose IL-1 beta induced leukocytosis (P less than 0.05), neutrophilia (P less than 0.01), and monocytosis (P less than 0.001). At the onset of IDDM, lymphocyte (P less than 0.01) and neutrophil (P less than 0.001) numbers were increased in high dose treated DP rats but not in rats given saline or low dose IL-1 beta. In 60-day-old diabetes-resistant (DR) BB rats, neurophilia was induced by both low (P less than 0.05) and high (P less than 0.001) dose IL-1 beta without the development of IDDM. At 130 days of age, when the rats were killed, it was discovered that 14/22 (64%) IL-1 beta injected DR rats developed neutralizing IL-1 beta antibodies. Significantly lower neutrophil numbers were observed in high dose DR rats which developed IL-1 beta antibodies compared with those which did not (P = 0.032). Thus, neutrophilia was dissociated from high IL-1 beta acceleration of IDDM onset.(ABSTRACT TRUNCATED AT 250 WORDS)

A suture-reinforced scleral sling. Technique for suspension of the ptotic upper lid.

A frontalis sling procedure for ptosis repair, employing preserved human bank sclera and polyfilament, cable-type, ophthalmic (Supramid Extra) suture in combination has been devised and tried. Results indicate that these two readily available materials that are now used separately may be combined for lid suspension. They are easy to work with and produce lasting results.

Some hypotheses concerning axon regeneration.

We review evidence related to several hypotheses concerning the mechanism of axon regeneration and present new data addressing one hypothesis. That one hypothesis concerns the signal that initiates changes in the cell bodies of neurons after axon damage. We identify a molecule that has a number of the properties expected of such a signal. We also review the hypothesis that induction of some genes is tightly correlated with nerve regeneration, and conclude that such a correlation is not so 'tight'. Nevertheless, proteins whose rate of synthesis or transport is increased in some systems are good candidates for playing important roles in regrowth. A third hypothesis, that mammalian CNS neurons fail to regenerate because of a failure to induce growth-associated proteins, is probably not true. Growth-associated proteins appear to be induced, at least transiently, in some cases where regeneration is abortive. The state of the neuron undoubtedly is important in regeneration, but many neurons, even in the CNS, appear to be able to support axon regrowth given the proper environment. Thus, support seems stronger for the view that the environment at the site of damage (including surfaces and growth factors) determines whether significant regrowth occurs in most cases.