Quantitative microcomputed tomography analysis of collateral vessel development after ischemic injury

Transgenic mouse models are increasingly being used to investigate the functions of specific growth factors or matrix proteins to design therapeutic strategies for controlling blood vessel growth. However, the available methodologies for evaluating angiogenesis and arteriogenesis in these models are limited by animal size, user subjectivity, the power to visualize the three-dimensional vessel networks, or the capability to employ a vigorous quantitative analysis. In this study, we employed contrast-enhanced microcomputed tomography imaging to assess collateral development after induction of hindlimb ischemia in the mouse. The morphological parameters vessel volume, connectivity, number, thickness, thickness distribution, separation, and degree of anisotropy were evaluated in control and surgery limbs 0, 3, and 14 days postsurgery. Results indicate that the vascular volume of the surgically manipulated limb was reconstituted as early as 3 days after femoral artery excision through development of a series of highly connected, small caliber, closely spaced, and isotropically oriented collateral vessels. Parametric analyses were completed to assess the sensitivity of the calculated morphological parameters to variations in image binarization threshold and voxel size. Images taken at the 36-microm voxel size were found to be optimal for evaluating collateral vessel formation, whereas 8- to 16-microm voxel sizes were needed to resolve smaller vascular structures. This study demonstrates the utility of microcomputed tomography as a robust method for quantitative, three-dimensional analysis of blood vessel networks. Whereas these initial efforts focused on the mouse hindlimb ischemia model, the developed techniques may be applied to a variety of model systems to investigate mechanisms of angiogenesis and arteriogenesis.     

Quantitative measures of femoral fracture repair in rats derived by micro-computed tomography

Although fracture healing is frequently studied in pre-clinical models of long bone fractures using rodents, there is a dearth of objective quantitative techniques to assess successful healing. Biomechanical testing is possibly the most quantitative and relevant to a successful clinical outcome, but it is a destructive technique providing little insight into the cellular mechanisms associated with healing. The advent of X-ray computed tomography (CT) has provided the opportunity to quantitatively and non-destructively assess bone structure and density, but it is unknown how measurements derived using this technology relate to successful healing. To examine possible relationships, we used a pre-clinical model to test for statistically significant correlations between quantitative characteristics of the callus by micro-CT (μCT) and the bending strength, stiffness, and energy-to-failure of the callus as assessed by three-point bending of excised bones. A closed, transverse fracture was generated in the mid-shaft of rat femurs by impact loading. Shortly thereafter, the rats received a one-time, local injection of either the vehicle or one of four doses of lovastatin. Following sacrifice after 4 weeks of healing, fractured femurs were extracted for μCT analysis and then three-point bending. Setting the region of interest to be 3.2 mm above and below the fracture line, we acquired standard and new μCT-derived measurements. The mineralized callus volume and the mineral density of the callus correlated positively with callus strength (r_xy=?0.315, p=0.016 and r_xy=0.444, p<0.0005, respectively) and stiffness (r_xy=?0.271, p=0.040 and r_xy=0.325, p=0.013, respectively), but the fraction of the callus that mineralized and the moment of inertia of the callus did not. This fraction did correlate with energy-to-failure (r_xy=?0.343, p=0.0085). Of the μCT-derived measurements, quantifying defects within the outer bridging cortices of the callus produced the strongest correlation with both callus strength (r_xy=0.557, p<0.0001) and stiffness (r_xy=0.468, p=0.0002). By both reducing structural defects and increasing mineralization, lovastatin appears to increase the callus strength.     

Synchrotron radiation-based microcomputed tomography for three-dimensional growth analysis of Aspergillus niger pellets

Filamentous fungi produce a wide range of relevant biotechnological compounds. The close relationship between fungal morphology and productivity has led to a variety of analytical methods to quantify their macromorphology. Nevertheless, only a µ-computed tomography (µ-CT) based method allows a detailed analysis of the 3D micromorphology of fungal pellets. However, the low sample throughput of a laboratory µ-CT limits the tracking of the micromorphological evolution of a statistically representative number of submerged cultivated fungal pellets over time. To meet this challenge, we applied synchrotron radiation-based X-ray microtomography at the Deutsches Elektronen-Synchrotron [German Electron Synchrotron Research Center], resulting in 19,940 3D analyzed individual fungal pellets that were obtained from 26 sampling points during a 48 h Aspergillus niger submerged batch cultivation. For each of the pellets, we were able to determine micromorphological properties such as number and density of spores, tips, branching points, and hyphae. The computed data allowed us to monitor the growth of submerged cultivated fungal pellets in highly resolved 3D for the first time. The generated morphological database from synchrotron measurements can be used to understand, describe, and model the growth of filamentous fungal cultivations.     

Ultrastructure of mouse incisor ameloblasts after vascular perfusion with colchicine

Summary In order to revalue the effects of colchicine on incisor secretory ameloblasts, entire mice were perfused with Krebs solution supplemented with a buffer and amino acids, through the right common carotid artery. The normal ultrastructure of the cells was maintained for 2 h with the perfusate alone. When colchicine (0.3–3.0 g/ml) was added to the perfusate, it induced ultrastructural changes, such as the loss of cytoplasmic microtubules, the loss of secretory granules in Tomes' process, the abnormal accumulation and secretion of secretory granules, disarranged Golgi apparatus and the fragmentation of rough endoplasmic reticulum. Vesicles (150–400 nm in diameter) resembling immature secretory granules also accumulated, the degree of accumulation depending on the duration of colchicine treatment. The accumulation of secretory granules and these vesicles suggests that the intracellular transport system was affected by colchicine but that the production of secretory granules was continuous throughout the experimental period. The present perfusion system has enabled us to treat ameloblasts with an agent that is a useful experimental tool for elucidating cell functions, despite being lethal to animals in vivo.     

Automated classification of urinary stones based on microcomputed tomography images using convolutional neural network

PurposeThe classification of urinary stones is important prior to treatment because the treatments depend on three types of urinary stones, i.e., calcium, uric acid, and mixture stones. We have developed an automatic approach for the classification of urinary stones into the three types based on microcomputed tomography (micro-CT) images using a convolutional neural network (CNN).Materials and methodsThirty urinary stones from different patients were scanned in vitro using micro-CT (pixel size: 14.96 μm; slice thickness: 15 μm); a total of 2,430 images (micro-CT slices) were produced. The slices (227 × 227 pixels) were classified into the three categories based on their energy dispersive X-ray (EDX) spectra obtained via scanning electron microscopy (SEM). The images of urinary stones from each category were divided into three parts; 66%, 17%, and 17% of the dataset were assigned to the training, validation, and test datasets, respectively. The CNN model with 15 layers was assessed based on validation accuracy for the optimization of hyperparameters such as batch size, learning rate, and number of epochs with different optimizers. Then, the model with the optimized hyperparameters was evaluated for the test dataset to obtain classification accuracy and error.ResultsThe validation accuracy of the developed approach with CNN with optimized hyperparameters was 0.9852. The trained CNN model achieved a test accuracy of 0.9959 with a classification error of 1.2%.ConclusionsThe proposed automated CNN-based approach could successfully classify urinary stones into three types, namely calcium, uric acid, and mixture stones, using micro-CT images.     

Quantitative analysis of DNA-damage response factors after sequential ion microirradiation

Several proteins are known to form foci at DNA sites damaged by ionizing radiation. We study DNA damage response by immunofluorescence microscopy after microirradiation of cells with energetic ions. By using microirradiation, it is possible to irradiate different regions on a single dish at different time-points and to differentiate between cells irradiated earlier and later. This allows to directly compare immunofluorescence intensities in both subsets of cells with little systematic error because both subsets are cultivated and stained under identical conditions. In addition, by using irradiation patterns such as crossing lines, it is possible to irradiate individual cells twice and to differentiate between immunofluorescence signals resulting from the cellular response to the earlier and to the later irradiation event. Here, we describe the quantitative evaluation of immunofluorescence intensities after sequential irradiation.     

27例下颌骨缺损重建钛板修复术后并发症临床探讨

目的:本文通过27例下颌骨缺损重建钛板修复术后并发症的探讨,分析并发症发生的原因,及临床减少或者避免产生上述并发症的可能性。方法:收集因下颌骨切除后植入钛板91例中发生并发症的27例临床病例资料.进行回顾性分析研究。结果:术后出现感染导致钛板取出的8例,固位螺钉松动的9例,钛板浅化外露的4例,钛板折断的3例,咬合关系严重错乱的3例。结论:术后感染是钛板植入后较为常见的并发症,术后放疗更加剧了感染的危险性。钛螺钉松脱是另一发生率较高的并发症,主要是由于颌力负担及钛板桡曲力度大小有关,钛板浅化外露及折断移位的发生主要与缺损部分及钛板植入的形状有关,放疗同样会增加钛板外露的几率。     

Three-dimensional characterization of the vascular bed in bone metastasis of the rat by microcomputed tomography (MicroCT)

Background

Angiogenesis contributes to proliferation and metastatic dissemination of cancer cells. Anatomy of blood vessels in tumors has been characterized with 2D techniques (histology or angiography). They are not fully representative of the trajectories of vessels throughout the tissues and are not adapted to analyze changes occurring inside the bone marrow cavities.

Methodology/Principal Findings

We have characterized the vasculature of bone metastases in 3D at different times of evolution of the disease. Metastases were induced in the femur of Wistar rats by a local injection of Walker 256/B cells. Microfil®, (a silicone-based polymer) was injected at euthanasia in the aorta 12, 19 and 26 days after injection of tumor cells. Undecalcified bones (containing the radio opaque vascular casts) were analyzed by microCT, and a first 3D model was reconstructed. Bones were then decalcified and reanalyzed by microCT; a second model (comprising only the vessels) was obtained and overimposed on the former, thus providing a clear visualization of vessel trajectories in the invaded metaphysic allowing quantitative evaluation of the vascular volume and vessel diameter. Histological analysis of the marrow was possible on the decalcified specimens. Walker 256/B cells induced a marked osteolysis with cortical perforations. The metaphysis of invaded bones became progressively hypervascular. New vessels replaced the major central medullar artery coming from the diaphyseal shaft. They sprouted from the periosteum and extended into the metastatic area. The newly formed vessels were irregular in diameter, tortuous with a disorganized architecture. A quantitative analysis of vascular volume indicated that neoangiogenesis increased with the development of the tumor with the appearance of vessels with a larger diameter.

Conclusion

This new method evidenced the tumor angiogenesis in 3D at different development times of the metastasis growth. Bone and the vascular bed can be identified by a double reconstruction and allowed a quantitative evaluation of angiogenesis upon time.     

Site specific effects of zoledronic acid during tibial and mandibular fracture repair

Numerous factors can affect skeletal regeneration, including the extent of bone injury, mechanical loading, inflammation and exogenous molecules. Bisphosphonates are anticatabolic agents that have been widely used to treat a variety of metabolic bone diseases. Zoledronate (ZA), a nitrogen-containing bisphosphonate (N-BP), is the most potent bisphosphonate among the clinically approved bisphosphonates. Cases of bisphosphonate-induced osteonecrosis of the jaw have been reported in patients receiving long term N-BP treatment. Yet, osteonecrosis does not occur in long bones. The aim of this study was to compare the effects of zoledronate on long bone and cranial bone regeneration using a previously established model of non-stabilized tibial fractures and a new model of mandibular fracture repair. Contrary to tibial fractures, which heal mainly through endochondral ossification, mandibular fractures healed via endochondral and intramembranous ossification with a lesser degree of endochondral ossification compared to tibial fractures. In the tibia, ZA reduced callus and cartilage formation during the early stages of repair. In parallel, we found a delay in cartilage hypertrophy and a decrease in angiogenesis during the soft callus phase of repair. During later stages of repair, ZA delayed callus, cartilage and bone remodeling. In the mandible, ZA delayed callus, cartilage and bone remodeling in correlation with a decrease in osteoclast number during the soft and hard callus phases of repair. These results reveal a more profound impact of ZA on cartilage and bone remodeling in the mandible compared to the tibia. This may predispose mandible bone to adverse effects of ZA in disease conditions. These results also imply that therapeutic effects of ZA may need to be optimized using time and dose-specific treatments in cranial versus long bones.     

Quantitative evaluation of rigid and elastic registrations for abdominal perfusion imaging with X-ray computed tomography

ObjectivesDynamic X-ray computed tomography with contrast agent injection allows quantifying the tumor response to an antiangiogenic treatment. For hepatic tumors, it is crucial to correct for respiratory movements to ensure a good quantification of perfusion parameters.Material and MethodsTwo registration models were tested on eight dynamic exams: 1) rigid model and 2) elastic model based on Free Form Deformation. Each exam included about 50 volumes acquired under free-breathing conditions. The registration methods were quantitatively and automatically evaluated by defining a global score. This score was based on a Dice index and measured the superimposition rate between corresponding bony structures of different frames of the temporal exam.ResultsBefore registration, the mean value of the score was 0.78. After rigid registration, these values were globally lower with an average of 0.73. After elastic registration, a mean score of 0.90 was reached which was much higher than before registration.ConclusionThis automatic evaluation showed its interest for controlling the quality of dynamic CT registrations. It also helped proving that elastic methods are much more efficient than rigid methods for registering abdominal dynamic volumes, when considering the whole field of view.     

Quantitative analysis of ceramides using a novel lipidomics approach with three dimensional response modelling

In the outermost layer of the skin, the stratum corneum (SC), ceramides form a diverse and essential pool of lipids. Due to their diversity and the limited availability of synthetic standards it is challenging to quantitatively analyse all SC ceramides independently. We aim to perform a detailed analysis of ceramides on SC harvested from in vivo and ex vivo skin, therefore, a LC/MS method was developed in which all steps from sample acquisition until data analysis were examined and optimized. Improving extraction efficiency of ceramides resulted in an increase in efficiency from 71.5% to 99.3%. It was shown that sample harvesting by tape-stripping in vivo was accurate and precise. A full scan MS method was developed, compatible with all sample types, enabling simultaneously qualitative and quantitative data analysis. A novel three dimensional response model was constructed to quantify all detected ceramides from full scan data using a limited amount of synthetic ceramides. The application is demonstrated on various SC sample types. When ex vivo SC was regenerated during human skin culture, increases are observed in the amount of the ceramide sphingosine subclasses, in mono unsaturated ceramides (which have an cis-double bond in the acyl chain), and ceramides with a short C34 carbon chain (ceramides with a total carbon chain of 34 carbon atoms), compared with native human skin. These changes in ceramide levels are also often encountered in diseased skin.     

High density culture of hybridoma cells using a perfusion culture vessel with an external centrifuge

The influence of centrifugal force on the growth of cells was examined by exposing the cells of the mouse-human hybridoma X87 line to centrifugal force (100–500 G) for ten minutes twice a day and comparing the static culture with that of unexposed cells. In this experiment, both cell proliferation and specific antibody productivity were independent of the centrifugal effect, and gave the same results as in the case of no exposure to centrifugal force. High density cultivation of the mouse-human hybridoma X87 line was obtained by a perfusion system where the cells were separated from the culture medium by continuous centrifugation. In the serum-free culture, the maximum viable cell density exceeded 10⁷ cells/ml, and monoclonal antibody was stably produced for 37 days. The results in this culture were equivalent to those obtained by intermittent centrifugal cell separation from the culture medium, and separation by gravitational settlement.     

Gender dimorphic tissue perfusion response after acute hemorrhage and resuscitation: role of vascular endothelial cell function

Proestrous female rodents are protected from the deleterious effects of trauma-hemorrhage that are observed in males. We hypothesized that the gender dimorphic outcome after trauma-hemorrhage might be related to gender differences in endothelial function and organ perfusion under such conditions. Male and cycle-matched proestrous female Sprague-Dawley rats underwent a midline laparotomy, hemorrhagic shock (40 mmHg for approximately 90 min), and resuscitation (Ringer lactate, 4x shed blood volume over 60 min). Various parameters were measured 2 h after completion of resuscitation. In the first set of animals, the left ventricle was cannulated and heart performance (maximal rate of left ventricular pressure increase) as well as cardiac output and organ perfusion rates were determined with (85)Sr microspheres. In the second set of animals, aortic vessel rings were harvested and relaxation in response to acetylcholine and nitroglycerin was measured. In the third set of animals, in situ isolated small intestine was perfused to measure the response of the splanchnic vessel bed to acetylcholine and nitroglycerin. After trauma-hemorrhage and resuscitation, females maintained cardiac output and demonstrated increased splanchnic and cardiac perfusion compared with males. Moreover, female intestines did not manifest the endothelial dysfunction that was observed in male intestines after hemorrhagic shock. We conclude that proestrous females show improved endothelial function and tissue perfusion patterns after hemorrhagic shock and that this gender-specific response might be a potential mechanism contributing to the beneficial effects of the proestrus stage under such conditions.     

Evaluation of the in vitro degradation of macroporous hydrogels using gravimetry,confined compression testing,and microcomputed tomography

This study investigated the in vitro degradation characteristics of macroporous hydrogels based on poly(propylene fumarate-co-ethylene glycol) (P(PF-co-EG)). Four formulations were fabricated to test the effect of porosity and cross-linking density on the degradation of the resulting macroporous hydrogels. Macroporosity was introduced by the addition of sodium bicarbonate and ascorbic acid, the precursors of the carbon dioxide porogen, in the initiation system for the hydrogel cross-linking. Macroporous hydrogels with porosities of 0.80 +/- 0.03 and 0.89 +/- 0.03 were synthesized by the addition of sodium bicarbonate of concentrations 40 and 80 mg/mL and ascorbic acid of concentrations 0.05 and 0.1 mol/L, respectively. Poly(ethylene glycol) diacrylate (PEG-DA) was utilized as a cross-linker. The molecular weight between cross-links had a significant effect on weight loss after 12 weeks, where samples with M(C) of 1,880 +/- 320 synthesized with a P(PF-co-EG):PEG-DA ratio of 3:1 had a significantly greater mass loss due to degradation than those with M(C) of 1,000 +/- 100 synthesized with a P(PF-co-EG):PEG-DA ratio of 1:1. In contrast, porosity played a minimal role in determining the weight loss. Mechanical testing of the hydrogels under confined compression showed a decrease in compressive modulus over the degradation time for all formulations. In addition, an increase in hydrogel equilibrium water content and pore wall thickness was observed with degradation time, whereas the hydrogel porosity and surface area density remained invariant. The results from microcomputed tomography corroborated with the rest of the measurements and indicated a bulk degradation mechanism of the macroporous hydrogels.     

RooTrak: automated recovery of three-dimensional plant root architecture in soil from x-ray microcomputed tomography images using visual tracking

X-ray microcomputed tomography (μCT) is an invaluable tool for visualizing plant root systems within their natural soil environment noninvasively. However, variations in the x-ray attenuation values of root material and the overlap in attenuation values between roots and soil caused by water and organic materials represent major challenges to data recovery. We report the development of automatic root segmentation methods and software that view μCT data as a sequence of images through which root objects appear to move as the x-y cross sections are traversed along the z axis of the image stack. Previous approaches have employed significant levels of user interaction and/or fixed criteria to distinguish root and nonroot material. RooTrak exploits multiple, local models of root appearance, each built while tracking a specific segment, to identify new root material. It requires minimal user interaction and is able to adapt to changing root density estimates. The model-guided search for root material arising from the adoption of a visual-tracking framework makes RooTrak less sensitive to the natural ambiguity of x-ray attenuation data. We demonstrate the utility of RooTrak using μCT scans of maize (Zea mays), wheat (Triticum aestivum), and tomato (Solanum lycopersicum) grown in a range of contrasting soil textures. Our results demonstrate that RooTrak can successfully extract a range of root architectures from the surrounding soil and promises to facilitate future root phenotyping efforts.     

Quantitative discrimination of NPC cell lines using optical coherence tomography

We tried to explore the intrinsic differences in the optical properties of the four representative NPC cell lines on the models of radiobiology and metastasis by OCT. The scattering coefficients and anisotropies were extracted by fitting the average a‐scan attenuation curves based on the multiple scatter effect. The values of scattering coefficients and anisotropy factors were 5.21 ± 0.11, 5.30 ± 0.09, 5.92 ± 0.21, 6.97 ± 0.22, and 0.892 ± 0.009, 0.886 ± 0.006, 0.884 ± 0.009, 0.86 ± 0.01 for CNE1, CNE2, 5‐8F and 6‐10B pellets (p < 0.05, P = 0.07 for CNE1 and CNE2), respectively. The results showed that the radiobiology and metastasis cell's model could be distinguished obviously; which implied that the corresponding types of NPC tissue might be potentially differentiated by OCT. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)