Anti-resorptive agents reduce the size of resorption cavities: a three-dimensional dynamic bone histomorphometry study.

Alterations in resorption cavities and bone remodeling events during anti-resorptive treatment are believed to contribute to reductions in fracture risk. Here, we examine changes in the size of individual remodeling events associated with treatment with a selective estrogen receptor modulator (raloxifene) or a bisphosphonate (risedronate). Adult female rats (6months of age) were submitted to ovariectomy (n=17) or sham surgery (SHAM, n=5). One month after surgery, the ovariectomized animals were separated into three groups: untreated (OVX, n=5), raloxifene treated (OVX+Ral, n=6) and risedronate treated (OVX+Ris, n=6). At 10months of age, the lumbar vertebrae were submitted to three-dimensional dynamic bone histomorphometry to examine the size (depth, breadth and volume) of individual resorption cavities and formation events. Maximum resorption cavity depth did not differ between the SHAM (23.66±1.87µm, mean±SD) and OVX (22.88±3.69µm) groups but was smaller in the OVX+Ral (14.96±2.30µm) and OVX+Ris (14.94±2.70µm) groups (p<0.01). Anti-resorptive treatment was associated with reductions in the surface area of resorption cavities and the volume occupied by each resorption cavity (p<0.01 each). The surface area and volume of individual formation events (double-labeled events) in the OVX+Ris group were reduced as compared to other groups (p<0.02). Raloxifene treated animals showed similar amounts of bone remodeling (ES/BS and dLS/BS) compared to sham-operated controls but smaller cavity size (depth, breadth and volume). The current study shows that anti-resorptive agents influence the size of resorption cavities and individual remodeling events and that the effect of anti-resorptives on individual remodeling events may not always be directly related to the degree of suppression of bone remodeling.

Obstructive sleep apnea and pregnancy: the effect on perinatal outcomes.

Obstructive sleep apnea (OSA) is characterized by repeated episodes of upper airway obstruction, resulting in hypoxemia, hypercapnia and sleep fragmentation. Pathophysiological sequelae include sympathetic activation, increased oxidative stress and a generalized inflammatory response, culminating in endothelial dysfunction. These are the proposed mechanisms that mediate the increased risk of hypertension and cardiovascular disease among patients with OSA outside of pregnancy. It is intriguing to consider the consequences of these events on pregnancy outcomes. There is a growing literature on the impact of maternal OSA on hypertensive disorders of pregnancy, gestational diabetes and impaired fetal growth. The data, while promising, require confirmation with larger numbers to verify the findings. OSA may be an important mediator of the poor perinatal outcomes associated with maternal obesity; moreover, one which may be amenable to treatment. This review discusses OSA and summarizes the current literature linking OSA with adverse perinatal outcomes.

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.

C57BL/6 and congenic interleukin-10-deficient mice can serve as models of Campylobacter jejuni colonization and enteritis.

Campylobacter jejuni is a globally distributed cause of human food-borne enteritis and has been linked to chronic joint and neurological diseases. We hypothesized that C. jejuni 11168 colonizes the gastrointestinal tract of both C57BL/6 mice and congenic C57BL/6 interleukin-10-deficient (IL-10(-/-)) mice and that C57BL/6 IL-10(-/-) mice experience C. jejuni 11168-mediated clinical signs and pathology. Individually housed mice were challenged orally with C. jejuni 11168, and the course of infection was monitored by clinical examination, bacterial culture, C. jejuni-specific PCR, gross pathology, histopathology, immunohistochemistry, and anti-C. jejuni-specific serology. Ceca of C. jejuni 11168-infected mice were colonized at high rates: ceca of 50/50 wild-type mice and 168/170 IL-10(-/-) mice were colonized. In a range from 2 to 35 days after infection with C. jejuni 11168, C57BL/6 IL-10(-/-) mice developed severe typhlocolitis best evaluated at the ileocecocolic junction. Rates of colonization and enteritis did not differ between male and female mice. A dose-response experiment showed that as little as 10(6) CFU produced significant disease and pathological lesions similar to responses seen in humans. Immunohistochemical staining demonstrated C. jejuni antigens within gastrointestinal tissues of infected mice. Significant anti-C. jejuni plasma immunoglobulin levels developed by day 28 after infection in both wild-type and IL-10-deficient animals; antibodies were predominantly T-helper-cell 1 (Th1)-associated subtypes. These results indicate that the colonization of the mouse gastrointestinal tract by C. jejuni 11168 is necessary but not sufficient for the development of enteritis and that C57BL/6 IL-10(-/-) mice can serve as models for the study of C. jejuni enteritis in humans.

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.

Entropy based method to correct intensity inhomogeneity in MR images.

We are involved in a comprehensive program to characterize atherosclerotic disease using multiple MR images having different contrast mechanisms (T1W, T2W, PDW, magnetization transfer, etc.) of human carotid and animal model arteries. We use specially designed intravascular and surface array coils that give high signal-to-noise but suffer from sensitivity inhomogeneity and significant noise. We present here a new non-parametric method for correcting the images without assumption of the number of different tissues. Intensity inhomogeneity is modeled with cubic spline and is locally optimized using an entropy criterion. Validation has been performed on a specially design neck phantom as well as actual MR scans on patient neck. The steep bias is corrected sufficiently to aid human interpretation of gray scales. It should also make possible computerized tissue classification.

MRI-guided laser thermal ablation: model to predict cell death from MR thermometry images for real-time therapy monitoring.

Solid tumors and other pathologies can be treated using laser thermal ablation under interventional magnetic resonance imaging (iMRI) guidance. We developed a model to predict cell death from MR thermometry measurements and applied it to in vivo rabbit brain data. To align post-ablation T2-weighted spin-echo MR lesion images to gradient echo MR images, from which temperature is derived, we used a registration method that aligned fiducials placed near the thermal lesion. We used the outer boundary of the hyperintense rim in the post-ablation MR lesion image as the boundary for cell death, as verified from histology. Model parameters were simultaneously estimated using an iterative optimization algorithm applied to every interesting pixel in 328 images from multiple experiments having various temperature histories. For a necrotic region of 766 voxels across all lesions, the model gave a voxel specificity and sensitivity of 98.1% and 78.4%, respectively. Median distance between the segmented necrotic boundary and the mislabeled voxels was within one MR voxel. Furthermore, our model predicted fewer errors as compared to the critical temperature cell death model. This is good evidence that iMRI temperature maps can be used with our model to predict therapeutic regions in real-time.

New normalization methods for cDNA microarray data.

MOTIVATION: The focus of this paper is on two new normalization methods for cDNA microarrays. After the image analysis has been performed on a microarray and before differentially expressed genes can be detected, some form of normalization must be applied to the microarrays. Normalization removes biases towards one or other of the fluorescent dyes used to label each mRNA sample allowing for proper evaluation of differential gene expression. RESULTS: The two normalization methods that we present here build on previously described non-linear normalization techniques. We extend these techniques by firstly introducing a normalization method that deals with smooth spatial trends in intensity across microarrays, an important issue that must be dealt with. Secondly we deal with normalization of a new type of cDNA microarray experiment that is coming into prevalence, the small scale specialty or 'boutique' array, where large proportions of the genes on the microarrays are expected to be highly differentially expressed. AVAILABILITY: The normalization methods described in this paper are available via http://www.pi.csiro.au/gena/ in a software suite called tRMA: tools for R Microarray Analysis upon request of the authors. Images and data used in this paper are also available via the same link.

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.

Surface organization and nanopatterning of collagen by dip-pen nanolithography.

Collagen is a key fibrous protein in biological systems, characterized by a complex structural hierarchy as well as the ability to self-assemble into liquid crystalline mesophases. The structural features of collagen influence cellular responses and material properties, with importance for a wide range of biomaterials and tissue architectures. The mechanism by which fibrillar collagen structures form from liquid crystalline mesophases is not well characterized. We report positive printing of collagen and a collagen-like peptide down to 30-50-nm line widths, using the atomic force microscopy technique of dip-pen nanolithography. The method preserved the triple-helical structure and biological activity of collagen and even fostered the formation of characteristic higher levels of structural organization. The "direct-write" capability of biologically relevant molecules, while preserving their structure and functionality, provides tremendous flexibility in future biological device applications and in proteomics arrays, as well as a new strategy to study the important hierarchical assembly processes of biological systems.

Commissioning of a mobile electron accelerator for intraoperative radiotherapy.

Radiation performance characteristics of a dedicated intraoperative accelerator were determined to prepare the unit for clinical use. The linear accelerator uses standing wave X-band technology (wavelength approximately 3 centimeters) in order to minimize the mass of the accelerator. The injector design, smaller accelerator components, and low electron beam currents minimize radiation leakage. The unit may be used in a standard operating room without additional shielding. The mass of the accelerator gantry is 1250 Kg (weight approximately 2750 lbs) and the unit is transportable between operating rooms. Nominal electron energies are 4, 6, 9, and 12 MeV, and operate at selectable dose rates of 2.5 or 10 Gray per minute. D(max) depths in water for a 10 cm applicator are 0.7, 1.3, 1.7, and 2.0 for these energies, respectively. The depths of 80% dose are 1.2, 2.1, 3.1, and 3.9 cm, respectively. Absolute calibration using the American Association of Physicists in Medicine TG-51 protocol was performed for all electron energies using the 10 cm applicator. Applicator sizes ranged from 3 to 10 cm diameter for flat applicators, and 3 to 6 cm diameter for 30 degrees beveled applicators. Output factors were determined for all energies relative to the 10 cm flat applicator. Central axis depth dose profiles and isodose plots were determined for every applicator and energy combination. A quality assurance protocol, performed each day before patient treatment, was developed for output and energy constancy.

Home healthcare nurses--why they leave and why they stay.

This is a second article in a series of two reports on the National Association for Home Care's (NAHC) study of nursing recruitment and retention. The first article presented the results of the quantitative analysis. This article presents results of the qualitative analysis.

Comparison of algorithms for combining X-ray angiography images.

Using a one-dimensional convective-dispersive model of contrast agent flow in a blood vessel, we optimized and compared algorithms for combining a temporal sequence of X-ray angiography images, each with incomplete arterial filling, into a single-output image with fully opacified arteries. The four algorithms were: maximum opacity (MO) with a maximum over time at each spatial location; matched filtering (MAT); recursive filtering (REC) with a maximum opacity; and an approximate matched filter (AMF) consisting of a correlation with a kernel that approximates the matched filter kernel followed by a maximum opacity operation. Based on the contrast-to-noise ratio (CNR), MAT is theoretically the best algorithm. However, with spatially varying clinical images, a poorly matched MAT kernel greatly degraded CNR to the point of even inverting artery contrast. The practical AMF method maintained uniform CNR values over the entire field of view and gave >90% of the theoretical limit set by MAT. REC and MO created fully opacified arteries, but provided little CNR enhancement. By holding CNR at a nominal reference value, simulations predicted that AMF could be used with a contrast agent volume reduced by as much as 66%. Alternatively, X-ray exposure rate could be lowered. Although MO and REC are more easily implemented, the contrast enhancement with AMF makes it attractive for processing diagnostic angiography images acquired with a reduced contrast agent dose.

Digital subtraction peripheral angiography using image stacking: initial clinical results.

Using clinically acquired x-ray angiography image sequences, we compared three algorithms for creating a single diagnostic quality image that combined input images containing flowing contrast agent. These image-stacking algorithms were: maximum opacity with the minimum gray-scale value across time recorded at each spatial location, (REC) recursive temporal filtering followed by a maximum opacity operation, and (AMF) an approximate matched filter consisting of a convolution with a kernel approximating the matched filter followed by a maximum opacity operation. Eighteen clinical exams of the peripheral arteries of the legs were evaluated. AMF gave 2.7 times greater contrast to noise ratio than the single best subtraction image and 1.3 times improvement over REC, the second best stacking algorithm. This is consistent with previous simulations showing that AMF performs nearly equal to the optimal result from matched filtering without the well-known limitations. For example, unlike matched filtering, AMF filter coefficients were obtained automatically using an image-processing algorithm. AMF effectively brought out small collateral arteries, otherwise difficult to see, without degrading artery sharpness or stenosis grading. Comparing results using reduced and full contrast agent volumes demonstrated that contrast agent load could be reduced to one-third of the conventional amount with AMF processing. By simulating reduced x-ray exposures on clinical exams, we determined that x-ray exposure could be reduced by 80% with AMF processing. We conclude that AMF is a promising, potential technique for reducing contrast agent load and for improving vessel visibility, both very important characteristics for vascular imaging.

Hearing-aid outcome measured following one month of hearing aid use by the elderly.

This study reports the results of a large number of hearing-aid outcome measures obtained from 173 elderly hearing-aid wearers following one month of hearing-aid use. All participants in this study were fit binaurally with identical full-concha in-the-ear (ITE) hearing aids having linear Class-D amplifiers with output-limiting compression. Outcome measures included several measures of speech recognition, as well as several self-report measures of hearing-aid performance, benefit, satisfaction, and use. Comparison of mean data from this sample of hearing-aid wearers to other larger sets of data, obtained previously for several of these measures of hearing-aid outcome evaluated in isolation, indicated that the participants in this study were representative of the participants in other larger-scale studies. Subsequent principal-components factor analysis of the data from this study indicated that there were seven distinct dimensions of hearing-aid outcome. Attempts to document the effectiveness and efficacy of hearing aids for elderly persons with impaired hearing will be most complete when assessing performance along all seven dimensions of hearing-aid outcome. Clinically efficient procedures for doing so are discussed.

Hyperefficient detection of targets in noisy images.

We compared human detection of visual targets in noisy images with that of a theoretically optimum matched filter. Using a small thin target with vertically aligned markers, we obtained hyperefficient detection as high as 91% as compared with the theoretical optimum, a value far exceeding the 30-50% value typically reported. When the markers were removed, detection efficiencies degraded to an average of 27%, even though subjects were aware that the target was always placed in the center of a reasonably small panel. Using a nine-alternative forced-choice experiment, we compared detection by human observers with a matched-filter computational observer on a trial-by-trial basis. With the markers present, when humans missed the correct panel, they most often chose the panel with the second-highest decision variable output from the computational observer, suggesting that the template-matching model is a good one. To model results without the markers, we included location uncertainty and additional noise sources in the template matching of the computational observer. A location uncertainty of only 1 pixel, corresponding to a retinal distance of approximately 12 microm, a dimension of the order of the size of the receptive field of photoreceptors, explained the psychometric data. With the marker present, the model suggests that hyperefficient detection is obtained by limiting target location uncertainty to <6 microm. Together these results give important new insights into human visual detection mechanisms.