Tolerance of Normal Rabbit Facial Bones and Teeth to Synchrotron X-Ray Microbeam Irradiation.

Microbeam radiation therapy, an alternative radiosurgical treatment under preclinical investigation, aims to safely treat muzzle tumors in pet animals. This will require data on the largely unknown radiation toxicity of microbeam arrays for bones and teeth. To this end, the muzzle of six young adult New Zealand rabbits was irradiated by a lateral array of microplanar beamlets with peak entrance doses of 200, 330 or 500 Gy. The muzzles were examined 431 days postirradiation by computed microtomographic imaging (micro-CT) ex vivo, and extensive histopathology. The boundaries of the radiation field were identified histologically by microbeam tracks in cartilage and other tissues. There was no radionecrosis of facial bones in any rabbit. Conversely, normal incisor teeth exposed to peak entrance doses of 330 Gy or 500 Gy developed marked caries-like damage, whereas the incisors of the two rabbits exposed to 200 Gy remained unscathed. A single, unidirectional array of microbeams with a peak entrance dose ≤200 Gy (valley dose14 Gy) did not damage normal bone, teeth and soft tissues of the muzzle of normal rabbits longer than one year after irradiation. Because of that, Microbeam radiation therapy of muzzle tumors in pet animals is unlikely to cause sizeable damage to normal teeth, bone and soft tissues, if a single array as used here delivers a limited entrance dose of 200 Gy and a valley dose of ≤14 Gy.

Pulmonary acini exhibit complex changes during postnatal rat lung development.

Pulmonary acini represent the functional gas-exchanging units of the lung. Due to technical limitations, individual acini cannot be identified on microscopic lung sections. To overcome these limitations, we imaged the right lower lobes of instillation-fixed rat lungs from postnatal days P4, P10, P21, and P60 at the TOMCAT beamline of the Swiss Light Source synchrotron facility at a voxel size of 1.48 µm. Individual acini were segmented from the three-dimensional data by closing the airways at the transition from conducting to gas exchanging airways. For a subset of acini (N = 268), we followed the acinar development by stereologically assessing their volume and their number of alveoli. We found that the mean volume of the acini increases 23 times during the observed time-frame. The coefficients of variation dropped from 1.26 to 0.49 and the difference between the mean volumes of the fraction of the 20% smallest to the 20% largest acini decreased from a factor of 27.26 (day 4) to a factor of 4.07 (day 60), i.e. shows a smaller dispersion at later time points. The acinar volumes show a large variation early in lung development and homogenize during maturation of the lung by reducing their size distribution by a factor of 7 until adulthood. The homogenization of the acinar sizes hints at an optimization of the gas-exchange region in the lungs of adult animals and that acini of different size are not evenly distributed in the lungs. This likely leads to more homogeneous ventilation at later stages in lung development.

Innovative high-resolution microCT imaging of animal brain vasculature.

Analysis of the angioarchitecture and quantification of the conduit vessels and microvasculature is of paramount importance for understanding the physiological and pathological processes within the central nervous system (CNS). Most of the available in vivo imaging methods lack penetration depth and/or resolution. Some ex vivo methods may provide better resolution, but are mainly destructive, as they are designed for imaging the CNS tissues after their removal from the skull or vertebral column. The removal procedure inevitably alters the in situ relations of the investigated structures and damages the dura mater and leptomeninges. µAngiofil, a polymer-based contrast agent, permits a qualitatively novel postmortem microangio-computed tomography (microangioCT) approach with excellent resolution and, therefore, visualization of the smallest brain capillaries. The datasets obtained empower a rather straightforward quantitative analysis of the vascular tree, including the microvasculature. The µAngiofil has an excellent filling capacity as well as a radio-opacity higher than the one of bone tissue, which allows imaging the cerebral microvasculature even within the intact skull or vertebral column. This permits in situ visualization and thus investigation of the dura mater and leptomeningeal layers as well as their blood supply in their original geometry. Moreover, the methodology introduced here permits correlative approaches, i.e., microangioCT followed by classical histology, immunohistochemistry and even electron microscopy. The experimental approach presented here makes use of common desktop microCT scanners, rendering it a promising everyday tool for the evaluation of the (micro)vasculature of the central nervous system in preclinical and basic research.

Osseointegration of ultrafine-grained titanium with a hydrophilic nano-patterned surface: an in vivo examination in miniature pigs.

Advances in biomaterials science and implant surface technology have made dental implants more predictable and implant therapy more attractive to patients. Surgical interventions are becoming less invasive, and patients heal faster and suffer less morbidity. In this preclinical in vivo study, we compared a new ultra-fine grained titanium (ufgTi) implant material with a hydrophilic nano-patterned surface to commercially pure titanium (cpTi) in a well-established animal model. CpTi grade 4 was subjected to Equal Channel Angular Pressing (ECAP), followed by a cold drawing process that provided ultra-fine-grained titanium (ufgTi) with a mean grain size of 300 nm. After metallographic assessment, the surface topography was characterized by laser confocal microscopy and atomic force microscopy. UfgTi and cpTi implants were inserted in the mandible and maxilla of miniature pigs that healed for 4 and for 8 weeks. Osseointegration was assessed by biomechanical torque out analysis, histomorphometric evaluation, and micro-CT analysis. The metallographic properties of UfgTi were significantly better than those of cpTi. Their surface topographies had similar hydrophilic nano-patterned characteristics, with no significant differences in the nanometre range. Histomorphometric and biomechanical torque out analysis revealed no significant differences between ufgTi and cpTi in environments of either low (maxilla) or high (mandible) bone density. We obtained high bone-to-implant contact values irrespective of the bony microarchitecture even when the bone mineral density was low. Overall, this investigation suggests that ufgTi forms a hydrophilic nano-patterned surface with superior metallographic properties compared to cpTi and high levels of osseointegration. Thus, ufgTi has therapeutic potential as a future strategy for the development of small diameter implants to enable less invasive treatment concepts, reduce patient morbidity and may also lower the costs of patient care.

Cutting-edge microangio-CT: new dimensions in vascular imaging and kidney morphometry.

In the last decades, the contrast-enhanced micro-computed tomography (micro-CT) imaging of a whole animal kidney became increasingly important. The visualization was mainly limited to middle-sized vessels. Since modern desktop micro-CT scanners provide the necessary detail resolution, we developed an approach for rapid visualization and consistent assessment of kidney vasculature and glomeruli number. This method is based on µAngiofil, a new polymerizing contrast agent with homogenous X-ray absorption, which provides continuous filling of the complete vasculature and enables correlative imaging approaches. For rapid and reliable kidney morphometry, the microangio-CT (µaCT) data sets from glial cell line-derived neurotrophic factor (GDNF)+/- mice and their wild-type littermates were used. The results were obtained much faster compared with the current gold standard, histology-based stereology, and without processing artifacts. The histology-based morphometry was done afterward on the same kidneys. Both approaches revealed that the GDNF+/- male mice had about 40% fewer glomeruli. Furthermore, our approach allows for the definition of sites of interest for further histological investigation, i.e., correlative morphology. The polymerized µAngiofil stays in perfused vessels and is autofluorescent, which is what greatly facilitates the matching of histological sections with µaCT data. The presented approach is a time-efficient, reliable, qualitative, and quantitative methodology. Besides glomerular morphometry, the µaCT data can be used for qualitative and quantitative analysis of the kidney vasculature and correlative morphology.

Correlative Imaging of the Murine Hind Limb Vasculature and Muscle Tissue by MicroCT and Light Microscopy.

A detailed vascular visualization and adequate quantification is essential for the proper assessment of novel angiomodulating strategies. Here, we introduce an ex vivo micro-computed tomography (microCT)-based imaging approach for the 3D visualization of the entire vasculature down to the capillary level and rapid estimation of the vascular volume and vessel size distribution. After perfusion with µAngiofil®, a novel polymerizing contrast agent, low- and high-resolution scans (voxel side length: 2.58-0.66 µm) of the entire vasculature were acquired. Based on the microCT data, sites of interest were defined and samples further processed for correlative morphology. The solidified, autofluorescent µAngiofil® remained in the vasculature and allowed co-registering of the histological sections with the corresponding microCT-stack. The perfusion efficiency of µAngiofil® was validated based on lectin-stained histological sections: 98 ± 0.5% of the blood vessels were µAngiofil®-positive, whereas 93 ± 2.6% were lectin-positive. By applying this approach we analyzed the angiogenesis induced by the cell-based delivery of a controlled VEGF dose. Vascular density increased by 426% mainly through the augmentation of medium-sized vessels (20-40 µm). The introduced correlative and quantitative imaging approach is highly reproducible and allows a detailed 3D characterization of the vasculature and muscle tissue. Combined with histology, a broad range of complementary structural information can be obtained.

Morphological Aspects of Tumor Angiogenesis.

The tumor vasculature is a chaotic mixture of abnormal, hierarchically disorganized vessels that differ from those of normal tissues with respect to organization, structure and function. Firstly, tumor vessel wall structure is abnormal and heterogeneous within the tumor. Besides contractile wall components, the perivascular compartment is often lacking pericytes, what makes the tumor vessels fragile and leaky. Secondly, another group of abnormalities involves distortions in angioarchitecture and vasculature as network. Common features of tumor vessels, irrespective of their origin, size and growth pattern, are absence of hierarchical organization, formation of vessels with irregular contours and their heterogeneous distribution within the tumor.

The total number of acini remains constant throughout postnatal rat lung development.

The pulmonary airways are subdivided into conducting and gas-exchanging airways. The small tree of gas-exchanging airways which is fed by the most distal conducting airway represents an acinus. Very little is known about the development of the number of acini. The goal of this study was to estimate their number throughout rat postnatal development. Right middle rat lung lobes were obtained at postnatal day 4-60, stained with heavy metals, paraffin embedded, and scanned by synchrotron radiation-based X-ray tomographic microscopy or imaged with micro computed tomography after critical point drying. The acini were counted by detection of the transitional bronchioles [bronchioalveolar duct junction (BADJ)] by using morphological criteria (thickness of the walls of airways and appearance of alveoli) during examination of the resulting three-dimensional (3D) image stacks. Between postnatal days 4-60, the number of acini per lung remained constant (5,840 ± 547 acini), but their volume increased significantly. We concluded that the acini are formed before the end of the saccular stage (before postnatal day 4) and that the developmental increase of the lung volume is achieved by an increase of the acinar volume and not by an increase of their number. Furthermore, our results propose that the bronchioalveolar stem cells, which are residing in the BADJ, are as constant in their location as the BADJ itself.

A Motion-Based Feature for Event-Based Pattern Recognition.

This paper introduces an event-based luminance-free feature from the output of asynchronous event-based neuromorphic retinas. The feature consists in mapping the distribution of the optical flow along the contours of the moving objects in the visual scene into a matrix. Asynchronous event-based neuromorphic retinas are composed of autonomous pixels, each of them asynchronously generating "spiking" events that encode relative changes in pixels' illumination at high temporal resolutions. The optical flow is computed at each event, and is integrated locally or globally in a speed and direction coordinate frame based grid, using speed-tuned temporal kernels. The latter ensures that the resulting feature equitably represents the distribution of the normal motion along the current moving edges, whatever their respective dynamics. The usefulness and the generality of the proposed feature are demonstrated in pattern recognition applications: local corner detection and global gesture recognition.

Efficient estimation of the total number of acini in adult rat lung.

Pulmonary airways are subdivided into conducting and gas-exchanging airways. An acinus is defined as the small tree of gas-exchanging airways, which is fed by the most distal purely conducting airway. Until now a dissector of five consecutive sections or airway casts were used to count acini. We developed a faster method to estimate the number of acini in young adult rats. Right middle lung lobes were critical point dried or paraffin embedded after heavy metal staining and imaged by X-ray micro-CT or synchrotron radiation-based X-rays tomographic microscopy. The entrances of the acini were counted in three-dimensional (3D) stacks of images by scrolling through them and using morphological criteria (airway wall thickness and appearance of alveoli). Segmentation stopper were placed at the acinar entrances for 3D visualizations of the conducting airways. We observed that acinar airways start at various generations and that one transitional bronchiole may serve more than one acinus. A mean of 5612 (±547) acini per lung and a mean airspace volume of 0.907 (±0.108) µL per acinus were estimated. In 60-day-old rats neither the number of acini nor the mean acinar volume did correlate with the body weight or the lung volume.

Rat lungs show a biphasic formation of new alveoli during postnatal development.

Roughly 90% of the gas-exchange surface is formed by alveolarization of the lungs. To the best of our knowledge, the formation of new alveoli has been followed in rats only by means of morphological description or interpretation of semiquantitative data until now. Therefore, we estimated the number of alveoli in rat lungs between postnatal days 4 and 60 by unambiguously counting the alveolar openings. We observed a bulk formation of new alveoli between days 4 and 21 (17.4 times increase from 0.8 to 14.3 millions) and a second phase of continued alveolarization between days 21 and 60 (1.3 times increase to 19.3 million). The (number weighted) mean volume of the alveoli decreases during the phase of bulk alveolarization from ∼593,000 µm(3) at day 4 to ∼141,000 µm(3) at day 21, but increases again to ∼298,000 µm(3) at day 60. We conclude that the "bulk alveolarization" correlates with the mechanism of classical alveolarization (alveolarization before the microvascular maturation is completed) and that the "continued alveolarization" follows three proposed mechanisms of late alveolarization (alveolarization after microvascular maturation). The biphasic pattern is more evident for the increase in alveolar number than for the formation of new alveolar septa (estimated as the length of the free septal edge). Furthermore, a striking negative correlation between the estimated alveolar size and published data on retention of nanoparticles was detected.

Dose optimization approach to fast X-ray microtomography of the lung alveoli.

A basic prerequisite for in vivo X-ray imaging of the lung is the exact determination of radiation dose. Achieving resolutions of the order of micrometres may become particularly challenging owing to increased dose, which in the worst case can be lethal for the imaged animal model. A framework for linking image quality to radiation dose in order to optimize experimental parameters with respect to dose reduction is presented. The approach may find application for current and future in vivo studies to facilitate proper experiment planning and radiation risk assessment on the one hand and exploit imaging capabilities on the other.

Visualization and stereological characterization of individual rat lung acini by high-resolution X-ray tomographic microscopy.

The small trees of gas-exchanging pulmonary airways, which are fed by the most distal purely conducting airways, are called acini and represent the functional gas-exchanging units. The three-dimensional architecture of the acini has a strong influence on ventilation and particle deposition. Due to the difficulty in identifying individual acini on microscopic lung sections, the knowledge about the number of acini and their biological parameters, like volume, surface area, and number of alveoli per acinus, are limited. We developed a method to extract individual acini from lungs imaged by high-resolution synchrotron radiation-based X-ray tomographic microscopy and estimated their volume, surface area, and number of alveoli. Rat acini were isolated by semiautomatically closing the airways at the transition from conducting to gas-exchanging airways. We estimated a mean internal acinar volume of 1.148 mm(3), a mean acinar surface area of 73.9 mm(2), and a mean of 8,470 alveoli/acinus. Assuming that the acini are similarly sized throughout different regions of the lung, we calculated that a rat lung contains 5,470 ± 833 acini. We conclude that our novel approach is well suited for the fast and reliable characterization of a large number of individual acini in healthy, diseased, or transgenic lungs of different species, including humans.

Integrating segmentation methods from the Insight Toolkit into a visualization application.

The Insight Toolkit (ITK) initiative from the National Library of Medicine has provided a suite of state-of-the-art segmentation and registration algorithms ideally suited to volume visualization and analysis. A volume visualization application that effectively utilizes these algorithms provides many benefits: it allows access to ITK functionality for non-programmers, it creates a vehicle for sharing and comparing segmentation techniques, and it serves as a visual debugger for algorithm developers. This paper describes the integration of image processing functionalities provided by the ITK into VolView, a visualization application for high performance volume rendering. A free version of this visualization application is publicly available and is available in the online version of this paper. The process for developing ITK plugins for VolView according to the publicly available API is described in detail, and an application of ITK VolView plugins to the segmentation of Abdominal Aortic Aneurysms (AAAs) is presented. The source code of the ITK plugins is also publicly available and it is included in the online version.