CT- and MRI-Aided Fluorescence Tomography Reconstructions for Biodistribution Analysis.

OBJECTIVES: Optical fluorescence imaging can track the biodistribution of fluorophore-labeled drugs, nanoparticles, and antibodies longitudinally. In hybrid computed tomography-fluorescence tomography (CT-FLT), CT provides the anatomical information to generate scattering and absorption maps supporting a 3-dimensional reconstruction from the raw optical data. However, given the CT's limited soft tissue contrast, fluorescence reconstruction and quantification can be inaccurate and not sufficiently detailed. Magnetic resonance imaging (MRI) can overcome these limitations and extend the options for tissue characterization. Thus, we aimed to establish a hybrid CT-MRI-FLT approach for whole-body imaging and compared it with CT-FLT. MATERIALS AND METHODS: The MRI-based hybrid imaging approaches were established first by scanning a water and coconut oil-filled phantom, second by quantifying Cy7 concentrations of inserts in dead mice, and finally by analyzing the biodistribution of AF750-labeled immunoglobulins (IgG, IgA) in living SKH1 mice. Magnetic resonance imaging, acquired with a fat-water-separated mDixon sequence, CT, and FLT were co-registered using markers in the mouse holder frame filled with white petrolatum, which was solid, stable, and visible in both modalities. RESULTS: Computed tomography-MRI fusion was confirmed by comparing the segmentation agreement using Dice scores. Phantom segmentations showed good agreement, after correction for gradient linearity distortion and chemical shift. Organ segmentations in dead and living mice revealed adequate agreement for fusion. Marking the mouse holder frame and the successful CT-MRI fusion enabled MRI-FLT as well as CT-MRI-FLT reconstructions. Fluorescence tomography reconstructions supported by CT, MRI, or CT-MRI were comparable in dead mice with 60 pmol fluorescence inserts at different locations. Although standard CT-FLT reconstruction only considered general values for soft tissue, skin, lung, fat, and bone scattering, MRI's more versatile soft tissue contrast enabled the additional consideration of liver, kidneys, and brain. However, this did not change FLT reconstructions and quantifications significantly, whereas for extending scattering maps, it was important to accurately segment the organs and the entire mouse body. The various FLT reconstructions also provided comparable results for the in vivo biodistribution analyses with fluorescent immunoglobulins. However, MRI additionally enabled the visualization of gallbladder, thyroid, and brain. Furthermore, segmentations of liver, spleen, and kidney were more reliable due to better-defined contours than in CT. Therefore, the improved segmentations enabled better assignment of fluorescence signals and more differentiated conclusions with MRI-FLT. CONCLUSIONS: Whole-body CT-MRI-FLT was implemented as a novel trimodal imaging approach, which allowed to more accurately assign fluorescence signals, thereby significantly improving pharmacokinetic analyses.

Dye labeling for optical imaging biases drug carriers' biodistribution and tumor uptake.

Biodistribution analyses of nanocarriers are often performed with optical imaging. Though dye tags can interact with transporters, e.g., organic anion transporting polypeptides (OATPs), their influence on biodistribution was hardly studied. Therefore, this study compared tumor cell uptake and biodistribution (in A431 tumor-bearing mice) of four near-infrared fluorescent dyes (AF750, IRDye750, Cy7, DY-750) and dye-labeled poly(N-(2-hydroxypropyl)methacrylamide)-based nanocarriers (dye-pHPMAs). Tumor cell uptake of hydrophobic dyes (Cy7, DY-750) was higher than that of hydrophilic dyes (AF750, IRDye750), and was actively mediated but not related to OATPs. Free dyes' elimination depended on their hydrophobicity, and tumor uptake correlated with blood circulation times. Dye-pHPMAs circulated longer and accumulated stronger in tumors than free dyes. Dye labeling significantly influenced nanocarriers' tumor accumulation and biodistribution. Therefore, low-interference dyes and further exploration of dye tags are required to achieve the most unbiased results possible. In our assessment, AF750 and IRDye750 best qualified for labeling hydrophilic nanocarriers.

Nuclear factor erythroid 2-related factor 2 (Nrf2) deficiency causes age-dependent progression of female osteoporosis.

BACKGROUND: Nuclear factor erythroid 2-related factor 2 (Nrf2) is a crucial transcription factor for cellular redox homeostasis. The association of Nrf2 with elderly female osteoporotic has yet to be fully described. The aim was to elucidate a potential age-dependent Nrf2 contribution to female osteoporosis in mice. METHODS: Eighteen female wild type (WT) and 16 Nrf2-knockout (KO) mice were sacrificed at different ages (12 weeks = young mature adult and 90 weeks = old) to analyze their femurs. The morphological properties (trabecular and cortical) were evaluated by micro-computed tomography (µCT) and compared to gold standard histochemistry analysis. The quasi-static compression tests were performed to calculate the mechanical properties of bones. Additionally, the population of bone resorbing cells and aromatase expression by osteocytes was immunohistochemically evaluated and empty osteocyte lacunae was counted in cortical bone. RESULTS: Old Nrf2-KO mice revealed a significantly reduced trabecular bone mineral density (BMD), cortical thickness, cortical area, and bone fraction compared to old WT mice, regardless of no significant difference in skeletally mature young adult mice between WT and KO. Specifically, while all old WT mice showed thin metaphyseal trabeculae, trabecular bone was completely absent in 60% of old KO mice. Additionally, old KO mice showed significantly more osteoclast-like cells and fewer aromatase-positive osteocytes than WT mice, whereas the occurrence of empty osteocyte lacunae did not differ between both groups. Nrf2-KO mice further showed an age-dependently reduced fracture resilience compared to age-matched WT mice. CONCLUSION: Our results suggest that chronic Nrf2 loss can lead to age-dependent progression of female osteoporosis.

Intrinsic Respiratory Gating for Simultaneous Multi-Mouse µCT Imaging to Assess Liver Tumors.

Small animal micro computed tomography (µCT) is an important tool in cancer research and is used to quantify liver and lung tumors. A type of cancer that is intensively investigated with µCT is hepatocellular carcinoma (HCC). µCT scans acquire projections from different angles of the gantry which rotates X-ray source and detector around the animal. Motion of the animal causes inconsistencies between the projections which lead to artifacts in the resulting image. This is problematic in HCC research, where respiratory motion affects the image quality by causing hypodense intensity at the liver edge and smearing out small structures such as tumors. Dealing with respiratory motion is particularly difficult in a high throughput setting when multiple mice are scanned together and projection removal by retrospective respiratory gating may compromise image quality and dose efficiency. In mice, inhalation anesthesia leads to a regular respiration with short gasps and long phases of negligible motion. Using this effect and an iterative reconstruction which can cope with missing angles, we discard the relatively few projections in which the gasping motion occurs. Moreover, since gated acquisition, i.e., acquiring multiple projections from a single gantry angle is not a requirement, this method can be applied to existing scans. We applied our method in a high throughput setting in which four mice with HCC tumors were scanned simultaneously in a multi-mouse bed. To establish a ground truth, we manually selected projections with visible respiratory motion. Our automated intrinsic breathing projection selection achieved an accordance of 97% with manual selection. We reconstructed volumetric images and demonstrated that our intrinsic gating method significantly reduces the hypodense depiction at the cranial liver edge and improves the detectability of small tumors. Furthermore, we show that projection removal in a four mice scan discards only 7.5% more projections than in a single-mouse setting, i.e., four mouse scanning does not substantially compromise dose efficiency or image quality. To the best of our knowledge, no comparable method that combines multi-mouse scans for high throughput, intrinsic respiratory gating, and an available iterative reconstruction has been described for liver tumor imaging before.

Mixing Matrix-corrected Whole-body Pharmacokinetic Modeling Using Longitudinal Micro-computed Tomography and Fluorescence-mediated Tomography.

PURPOSE: Pharmacokinetic modeling can be applied to quantify the kinetics of fluorescently labeled compounds using longitudinal micro-computed tomography and fluorescence-mediated tomography (µCT-FMT). However, fluorescence blurring from neighboring organs or tissues and the vasculature within tissues impede the accuracy in the estimation of kinetic parameters. Contributions of elimination and retention activities of fluorescent probes inside the kidneys and liver can be hard to distinguish by a kinetic model. This study proposes a deconvolution approach using a mixing matrix to model fluorescence contributions to improve whole-body pharmacokinetic modeling. PROCEDURES: In the kinetic model, a mixing matrix was applied to unmix the fluorescence blurring from neighboring tissues and blood vessels and unmix the fluorescence contributions of elimination and retention in the kidney and liver compartments. Accordingly, the kinetic parameters of the hepatobiliary and renal elimination routes and five major retention sites (the kidneys, liver, bone, spleen, and lung) were investigated in simulations and in an in vivo study. In the latter, the pharmacokinetics of four fluorescently labeled compounds (indocyanine green (ICG), HITC-iodide-microbubbles (MB), Cy7-nanogels (NG), and OsteoSense 750 EX (OS)) were evaluated in BALB/c nude mice. RESULTS: In the simulations, the corrected modeling resulted in lower relative errors and stronger linear relationships (slopes close to 1) between the estimated and simulated parameters, compared to the uncorrected modeling. For the in vivo study, MB and NG showed significantly higher hepatic retention rates (P<0.05 and P<0.05, respectively), while OS had smaller renal and hepatic retention rates (P<0.01 and P<0.01, respectively). Additionally, the bone retention rate of OS was significantly higher (P<0.01). CONCLUSIONS: The mixing matrix correction improves pharmacokinetic modeling and thus enables a more accurate assessment of the biodistribution of fluorescently labeled pharmaceuticals by µCT-FMT.

An Anatomical Thermal 3D Model in Preclinical Research: Combining CT and Thermal Images.

Even though animal trials are a controversial topic, they provide knowledge about diseases and the course of infections in a medical context. To refine the detection of abnormalities that can cause pain and stress to the animal as early as possible, new processes must be developed. Due to its noninvasive nature, thermal imaging is increasingly used for severity assessment in animal-based research. Within a multimodal approach, thermal images combined with anatomical information could be used to simulate the inner temperature profile, thereby allowing the detection of deep-seated infections. This paper presents the generation of anatomical thermal 3D models, forming the underlying multimodal model in this simulation. These models combine anatomical 3D information based on computed tomography (CT) data with a registered thermal shell measured with infrared thermography. The process of generating these models consists of data acquisition (both thermal images and CT), camera calibration, image processing methods, and structure from motion (SfM), among others. Anatomical thermal 3D models were successfully generated using three anesthetized mice. Due to the image processing improvement, the process was also realized for areas with few features, which increases the transferability of the process. The result of this multimodal registration in 3D space can be viewed and analyzed within a visualization tool. Individual CT slices can be analyzed axially, sagittally, and coronally with the corresponding superficial skin temperature distribution. This is an important and successfully implemented milestone on the way to simulating the internal temperature profile. Using this temperature profile, deep-seated infections and inflammation can be detected in order to reduce animal suffering.

Disturbed gut microbiota and bile homeostasis in Giardia-infected mice contributes to metabolic dysregulation and growth impairment.

Although infection with the human enteropathogen Giardia lamblia causes self-limited diarrhea in adults, infant populations in endemic areas experience persistent pathogen carriage in the absence of diarrhea. The persistence of this protozoan parasite in infants has been associated with reduced weight gain and linear growth (height-for-age). The mechanisms that support persistent infection and determine the different disease outcomes in the infant host are incompletely understood. Using a neonatal mouse model of persistent G. lamblia infection, we demonstrate that G. lamblia induced bile secretion and used the bile constituent phosphatidylcholine as a substrate for parasite growth. In addition, we show that G. lamblia infection altered the enteric microbiota composition, leading to enhanced bile acid deconjugation and increased expression of fibroblast growth factor 15. This resulted in elevated energy expenditure and dysregulated lipid metabolism with reduced adipose tissue, body weight gain, and growth in the infected mice. Our results indicate that this enteropathogen's modulation of bile acid metabolism and lipid metabolism in the neonatal mouse host led to an altered body composition, suggesting how G. lamblia infection could contribute to growth restriction in infants in endemic areas.

Optical imaging of the whole-body to cellular biodistribution of clinical-stage PEG-b-pHPMA-based core-crosslinked polymeric micelles.

Core-crosslinked polymeric micelles (CCPM) based on PEG-b-pHPMA-lactate are clinically evaluated for the treatment of cancer. We macroscopically and microscopically investigated the biodistribution and target site accumulation of CCPM. To this end, fluorophore-labeled CCPM were intravenously injected in mice bearing 4T1 triple-negative breast cancer (TNBC) tumors, and their localization at the whole-body, tissue and cellular level was analyzed using multimodal and multiscale optical imaging. At the organism level, we performed non-invasive 3D micro-computed tomography-fluorescence tomography (µCT-FLT) and 2D fluorescence reflectance imaging (FRI). At the tissue and cellular level, we performed extensive immunohistochemistry, focusing primarily on cancer, endothelial and phagocytic immune cells. The CCPM achieved highly efficient tumor targeting in the 4T1 TNBC mouse model (18.6 %ID/g), with values twice as high as those in liver and spleen (9.1 and 8.9 %ID/g, respectively). Microscopic analysis of tissue slices revealed that at 48 h post injection, 67% of intratumoral CCPM were localized extracellularly. Phenotypic analyses on the remaining 33% of intracellularly accumulated CCPM showed that predominantly F4/80+ phagocytes had taken up the nanocarrier formulation. Similar uptake patterns were observed for liver and spleen. The propensity of CCPM to primarily accumulate in the extracellular space in tumors suggests that the anticancer efficacy of the formulation mainly results from sustained release of the chemotherapeutic payload in the tumor microenvironment. In addition, their high uptake by phagocytic immune cells encourages potential use for immunomodulatory anticancer therapy. Altogether, the beneficial biodistribution, efficient tumor targeting and prominent engagement of PEG-b-pHPMA-lactate-based CCPM with key cell populations underline the clinical versatility of this clinical-stage nanocarrier formulation.

Effects of Strontium-Doped ß-Tricalcium Scaffold on Longitudinal Nuclear Factor-Kappa Beta and Vascular Endothelial Growth Factor Receptor-2 Promoter Activities during Healing in a Murine Critical-Size Bone Defect Model.

It was hypothesized that strontium (Sr)-doped ß-tricalcium phosphate (TCP)-based scaffolds have a positive effect on the regeneration of large bone defects (LBD). Readouts in our mice models were nuclear factor-kappa beta (NF-κB) activity and vascular endothelial growth factor receptor-2 (VEGFR-2) promoter activity during the healing process. A 2-mm critical-size femoral fracture was performed in transgenic NF-κB- and VEGFR-2-luciferase reporter mice. The fracture was filled with a 3D-printed ß-TCP scaffold with or without Sr. A bioluminescence in-vivo imaging system was used to sequentially investigate NF-κB and VEGFR-2 expression for two months. After sacrifice, soft and osseous tissue formation in the fracture sites was histologically examined. NF-κB activity increased in the ß-TCP + Sr group in the latter stage (day 40-60). VEGFR-2 activity increased in the + Sr group from days 0-15 but decreased and showed significantly less activity than the ß-TCP and non-scaffold groups from days 40-60. The new bone formation and soft tissue formation in the + Sr group were significantly higher than in the ß-TCP group, whereas the percentage of osseous tissue formation in the ß-TCP group was significantly higher than in the ß-TCP + Sr group. We analyzed longitudinal VEGFR-2 promoter activity and NF-κB activity profiles, as respective agents of angiogenesis and inflammation, during LBD healing. The extended inflammation phase and eventually more rapid resorption of scaffold caused by the addition of strontium accelerates temporary bridging of the fracture gaps. This finding has the potential to inform an improved treatment strategy for patients who suffer from osteoporosis.

L-Selectin/CD62L is a Key Driver of Non-Alcoholic Steatohepatitis in Mice and Men.

CD62L (L-Selectin) dependent lymphocyte infiltration is known to induce inflammatory bowel disease (IBD), while its function in the liver, especially in non-alcoholic steatohepatitis (NASH), remains unclear. We here investigated the functional role of CD62L in NASH in humans as well as in two mouse models of steatohepatitis. Hepatic expression of a soluble form of CD62L (sCD62L) was measured in patients with steatosis and NASH. Furthermore, CD62L-/- mice were fed with a methionine and choline deficient (MCD) diet for 4 weeks or with a high fat diet (HFD) for 24 weeks. Patients with NASH displayed increased serum levels of sCD62L. Hepatic CD62L expression was higher in patients with steatosis and increased dramatically in NASH patients. Interestingly, compared to wild type (WT) mice, MCD and HFD-treated CD62L-/- mice were protected from diet-induced steatohepatitis. This was reflected by less fat accumulation in hepatocytes and a dampened manifestation of the metabolic syndrome with an improved insulin resistance and decreased cholesterol and triglyceride levels. Consistent with ameliorated disease, CD62L-/- animals exhibited an enhanced hepatic infiltration of Treg cells and a strong activation of an anti-oxidative stress response. Those changes finally resulted in less fibrosis in CD62L-/- mice. Additionally, this effect could be reproduced in a therapeutic setting by administrating an anti-CD62L blocking antibody. CD62L expression in humans and mice correlates with disease activity of steatohepatitis. CD62L knockout and anti-CD62L-treated mice are protected from diet-induced steatohepatitis suggesting that CD62L is a promising target for therapeutic interventions in NASH.

Data Curation for Preclinical and Clinical Multimodal Imaging Studies.

PURPOSE: In biomedical research, imaging modalities help discover pathological mechanisms to develop and evaluate novel diagnostic and theranostic approaches. However, while standards for data storage in the clinical medical imaging field exist, data curation standards for biomedical research are yet to be established. This work aimed at developing a free secure file format for multimodal imaging studies, supporting common in vivo imaging modalities up to five dimensions as a step towards establishing data curation standards for biomedical research. PROCEDURES: Images are compressed using lossless compression algorithm. Cryptographic hashes are computed on the compressed image slices. The hashes and compressions are computed in parallel, speeding up computations depending on the number of available cores. Then, the hashed images with digitally signed timestamps are cryptographically written to file. Fields in the structure, compressed slices, hashes, and timestamps are serialized for writing and reading from files. The C++ implementation is tested on multimodal data from six imaging sites, well-documented, and integrated into a preclinical image analysis software. RESULTS: The format has been tested with several imaging modalities including fluorescence molecular tomography/x-ray computed tomography (CT), positron emission tomography (PET)/CT, single-photon emission computed tomography/CT, and PET/magnetic resonance imaging. To assess performance, we measured the compression rate, ratio, and time spent in compression. Additionally, the time and rate of writing and reading on a network drive were measured. Our findings demonstrate that we achieve close to 50 % reduction in storage space for µCT data. The parallelization speeds up the hash computations by a factor of 4. We achieve a compression rate of 137 MB/s for file of size 354 MB. CONCLUSIONS: The development of this file format is a step to abstract and curate common processes involved in preclinical and clinical multimodal imaging studies in a standardized way. This work also defines better interface between multimodal imaging modalities and analysis software.

Bone regeneration in rabbit calvarial critical-sized defects filled with composite in situ formed xenogenic dentin and biphasic tricalcium phosphate/hyroxyapatite mixture.

The purpose of this study was to evaluate bone healing in calvarial defects using two bone graft substitute materials; biphasic beta-tricalcium phosphate/hydroxyapatite in hydrogel (ß-TCP/HA) versus composite non-demineralized xenogenic dentin with ß-TCP/HA mixture. Full thickness critical-sized defects were created bilaterally in 10 New Zealand male rabbits. Seven defects were left empty, six filled with biphasic tricalcium phosphate putty, and seven were filled with composite non-demineralized xenogenic dentin with biphasic tricalcium phosphate. Animals were sacrificed at eight weeks postoperatively and the healing of the biomaterial-filled defects was compared radiographically and by histomorphometry. Micro-computed tomography (µCT) was utilized to analyze the osteogenesis and healing patterns of the defects. Quantitative analysis of volume fraction (%) of the newly formed bone and remaining graft material (FV=filling volume/TV=tissue volume) and mean intensity [HU] in the defects were evaluated. Defects filled with composite dentin with biphasic tri-calcium phosphate showed volume fraction (FV/TV) in the order of 55.81% ± 17.72%, whereas defects filled with only biphasic tricalcium phosphate showed a fraction of 39.84% ± 16.06%, which represent the ratio of remaining graft material and new bone formation to the tissue volume. The empty negative control defects showed a volume fraction of 19.14% ± 8.787%. Histological analysis showed significant percentage increase in bone formation and residual graft with the composite Dentin/ß-TCP group after 8 weeks. The findings suggest that composite xenogenic dentin with biphasic tricalcium phosphate showed improved osteogenesis when compared to biphasic tricalcium phosphate without the addition of non-demineralized dentin. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 773-782, 2019.

A preclinical micro-computed tomography database including 3D whole body organ segmentations.

The gold-standard of preclinical micro-computed tomography (µCT) data processing is still manual delineation of complete organs or regions by specialists. However, this method is time-consuming, error-prone, has limited reproducibility, and therefore is not suitable for large-scale data analysis. Unfortunately, robust and accurate automated whole body segmentation algorithms are still missing. In this publication, we introduce a database containing 225 murine 3D whole body µCT scans along with manual organ segmentation of most important organs including heart, liver, lung, trachea, spleen, kidneys, stomach, intestine, bladder, thigh muscle, bone, as well as subcutaneous tumors. The database includes native and contrast-enhanced, regarding spleen and liver, µCT data. All scans along with organ segmentation are freely accessible at the online repository Figshare. We encourage researchers to reuse the provided data to evaluate and improve methods and algorithms for accurate automated organ segmentation which may reduce manual segmentation effort, increase reproducibility, and even reduce the number of required laboratory animals by reducing a source of variability and having access to a reliable reference group.

Comparison of Bone Grafts From Various Donor Sites in Human Bone Specimens.

The objective of the current study was to compare the three-dimensional (3D) morphometric microstructure in human cadaveric bone specimens taken from various commonly utilized donor sites for autogenous bone grafting. Autogenous bone grafts can be harvested from various anatomic sites and express heterogeneous bone quality with a specific 3D microstructure for each site. The long-term structural integrity and susceptibility to resorption of the graft depend on the selected donor bone. Micro-computed tomography generates high-resolution datasets of bone structures and calcifications making this modality versatile for microarchitecture analysis and quantification of the bone. Six bone specimens, 10 mm in length, where anatomically possible, were obtained from various anatomical sites from 10 human dentate cadavers (4 men, 6 women, mean age 69.5 years). Specimens were scanned using a micro-computed tomography device and volumetrically reconstructed. A virtual cylindrical inclusion was reconstructed to analyze the bone mineral density and structural morphometric analysis using bone indices: relative bone volume, surface density, trabecular thicknesses, and trabecular separation. Calvarial bone specimens showed the highest mineral density, followed by the chin, then mandibular ramus then the tibia, whereas iliac crest and maxillary tuberosity had lower bone mineral densities. The pairwise comparison revealed statistically significant differences in the bone mineral density and relative bone volume index in the calvaria, mandibular ramus, mandibular symphysis groups when compared with those in the iliac crest and maxillary tuberosity, suggesting higher bone quality in the former groups than in the latter; tibial specimens expressed variable results.

Bone regeneration using composite non-demineralized xenogenic dentin with beta-tricalcium phosphate in experimental alveolar cleft repair in a rabbit model.

BACKGROUND: Alveolar cleft repair is performed via bone grafting procedure to restore the dental arch continuity. A suitable bone substitute materials should possess osteoinductive and osteoconductive properties, to promote new bone formation, along with a slowly resorbable scaffold that is subsequently replaced with functionally viable bone. Calcium phosphate biomaterials have long proved their efficacy as bone replacement materials. Dentin in several forms has also demonstrated its possibility to be used as bone graft replacement material in several studies. The purpose of this study was to evaluate bone regeneration pattern and quantify bone formation after grafting pre-established experimental alveolar clefts defects model in rabbits using composite xenogenic dentin and ß-TCP in comparison to ß-TCP alone. METHODS: Unilateral alveolar cleft defects were created in 16 New Zealand rabbits according to previously described methodology. Alveolar clefts were allowed 8 weeks healing period. 8 defects were filled with ß-TCP, whereas 8 defects filled with composite xenogenic dentin with ß-TCP. Bone regeneration of the healed defects was compared at the 8 weeks after intervention. Quantification of bone formation was analyzed using micro-computed tomography (µCT) and histomorphometric analysis. RESULTS: µCT and histomorphometric analysis revealed that defects filled with composite dentin/ß-TCP showed statistically higher bone volume fraction, bone mineral density and percentage residual graft volume when compared to ß-TCP alone. An improved surgical handling of the composite dentin/ß-TCP graft was also noted. CONCLUSIONS: Composite xenogenic dentin/ß-TCP putty expresses enhanced bone regeneration compared to ß-TCP alone in the reconstruction of rabbit alveolar clefts defects.

Sensitivity and accuracy of hybrid fluorescence-mediated tomography in deep tissue regions.

Fluorescence-mediated tomography (FMT) enables noninvasive assessment of the three-dimensional distribution of near-infrared fluorescence in mice. The combination with micro-computed tomography (µCT) provides anatomical data, enabling improved fluorescence reconstruction and image analysis. The aim of our study was to assess sensitivity and accuracy of µCT-FMT under realistic in vivo conditions in deeply-seated regions. Accordingly, we acquired fluorescence reflectance images (FRI) and µCT-FMT scans of mice which were prepared with rectal insertions with different amounts of fluorescent dye. Default and high-sensitivity scans were acquired and background signal was analyzed for three FMT channels (670 nm, 745 nm, and 790 nm). Analysis was performed for the original and an improved FMT reconstruction using the µCT data. While FRI and the original FMT reconstruction could detect 100 pmol, the improved FMT reconstruction could detect 10 pmol and significantly improved signal localization. By using a finer sampling grid and increasing the exposure time, the sensitivity could be further improved to detect 0.5 pmol. Background signal was highest in the 670 nm channel and most prominent in the gastro-intestinal tract and in organs with high relative amounts of blood. In conclusion, we show that µCT-FMT allows sensitive and accurate assessment of fluorescence in deep tissue regions.

Detection of rotavirus species A, B and C in domestic mammalian animals with diarrhoea and genotyping of bovine species A rotavirus strains.

Rotaviruses (RVs) are a major cause of neonatal diarrhoea in humans and animals worldwide. In this study, 425 faecal samples were collected between 1999 and 2013 from diarrhoeic livestock and companion animals at different locations in Germany and tested for RVs. A previously published real-time RT-PCR assay was optimized for detection of a larger variety of RV species A (RVA) strains, and real-time RT-PCR assays for detection of RV species B (RVB) and C (RVC) were newly developed. The detection limits of the assays were 1.54×10(2), 3.95×10(2) and 3.60×10(3) genome copies for RVA, RVB and RVC, respectively. RVA was identified in 85.2% of bovine samples, 51.2% of porcine samples, 50.0% of feline samples, 43.2% of equine samples and 39.7% of canine samples. RVB was found in 3.0% of bovine samples, 2.7% of equine samples and 1.6% of porcine samples. RVC was detected in 31.0% of porcine samples, 21.7% of feline samples, 9.0% of canine samples and 6.0% of bovine samples. For genotyping, 101 RVA-positive bovine samples were further analysed by semi-nested RT-PCR. Genotype combination G6P[5] was most frequently detected (67.3% of samples), followed by G6P[11] (13.9%), G10P[5] (4.0%), G8P[11] (3.0%), G6P[1] (1.0%), and G10P[11] (1.0%). Mixed RVA infections were detected in 5.9% of samples; no or incomplete typing was possible in 4.0% of the samples. This first overview on RV species and RVA genotypes in diarrhoeic livestock and companion animals from Germany indicates a broad circulation of a large variety of RVs.