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Nanomedicines are sub-micrometer-sized carrier materials designed to improve the biodistribution of i.v. administered (chemo-) therapeutic agents. In recent years, ever more efforts in the nanomedicine field have employed optical imaging (OI) techniques to monitor biodistribution and target site accumulation. Thus far, however, the longitudinal assessment of nanomedicine biodistribution using OI has been impossible, due to limited light penetration (in the case of 2D fluorescence reflectance imaging; FRI) and to the inability to accurately allocate fluorescent signals to nonsuperficial organs (in the case of 3D fluorescence molecular tomography; FMT). Using a combination of high-resolution microcomputed tomography (μCT) and FMT, we have here set out to establish a hybrid imaging protocol for noninvasively visualizing and quantifying the accumulation of near-infrared fluorophore-labeled nanomedicines in tissues other than superficial tumors. To this end, HPMA-based polymeric drug carriers were labeled with Dy750, their biodistribution and tumor accumulation were analyzed using FMT, and the resulting data sets were fused with anatomical μCT data sets in which several different physiologically relevant organs were presegmented. The robustness of 3D organ segmentation was validated, and the results obtained using 3D CT-FMT were compared to those obtained upon standard 3D FMT and 2D FRI. Our findings convincingly demonstrate that combining anatomical μCT with molecular FMT facilitates the noninvasive assessment of nanomedicine biodistribution.

Objectives Here we assess the intrinsic functions of the chemokine receptor CXCR4 in remodeling after myocardial infarction (MI) using Cxcr4 heterozygous (Cxcr(4+/-)) mice. Background Myocardial necrosis triggers complex remodeling and inflammatory changes. The chemokine CXCL12 has been implicated in protection and therapeutic regeneration after MI through recruiting angiogenic outgrowth cells, improving neovascularization and cardiac function, but the endogenous role of its receptor CXCR4 is unknown. Methods MI was induced by ligation of the left descending artery. Langendoff perfusion, echocardiography, quantitative immunohistochemistry, flow cytometry, angiogenesis assays, and cardiomyocyte analysis were performed. Results After 4 weeks, infarct size was reduced in Cxcr4(+/-) mice compared with wild-type mice and in respective bone marrow chimeras compared with controls. This was associated with altered inflammatory cell recruitment, decreased neutrophil content, delayed monocyte infiltration, and a predominance of Gr1(low) over classic Gr1(high) monocytes. Basal coronary flow and its recovery after MI were impaired in Cxcr4(+/-)mice, paralleled by reduced angiogenesis, myocardial vessel density, and endothelial cell count. Notably, no differences in cardiac function were seen in Cxcr4(+/-)mice compared with wild-type mice. Despite defective angiogenesis, Cxcr4(+/-) mouse hearts showed no difference in CXCL12, vascular endothelial growth factor or apoptosis-related gene expression. Electron microscopy revealed lipofuscin-like lipid accumulation in Cxcr4(+/-) mouse hearts and analysis of lipid extracts detected high levels of phosphatidylserine, which protect cardiomyocytes from hypoxic stress in vitro. Conclusions CXCR4 plays a crucial role in endogenous remodeling processes after MI, contributing to inflammatory/progenitor cell recruitment and neovascularization, whereas its deficiency limits infarct size and causes adaptation to hypoxic stress. This should be carefully scrutinized when devising therapeutic strategies involving the CXCL12/CXCR4 axis. (J Am Coll Cardiol 2011;58:2415-23)

Purpose: To develop and evaluate a user-friendly tool to enable efficient, accurate, and reproducible quantification of blood vessel stenosis in computed tomographic (CT) and magnetic resonance (MR) angiographic data sets. Materials and Methods: All clinical experiments were approved by the institutional review board, and informed patient consent was acquired. Animal experiments were approved by the governmental review committee on animal care. A virtual elastic sphere passes through a blood vessel specified by user-provided start and end points, and the adapting diameter over the course of the vessel is recorded. The program was tested in phantoms to determine the accuracy of diameter estimation, and it was applied in micro-CT data sets of mice with induced vessel stenosis. Dual-energy CT angiography and MR angiography were performed in 16 patients with carotid artery stenosis, and reproducibility and required reader time of this automated technique were compared with manual measurements. Additionally, the effect of dual-energy CT-based discrimination between iodine- and calcium-based enhancement was investigated. Differences between carotid artery diameters of mice and between automated and manual measurement durations were assessed with a paired t test. Reproducibility of stenosis scores was evaluated with the Fisher z test. Results: Phantom diameters were determined with an average error of 0.094 mm. Diameters of normal and injured carotid arteries of mice were significantly different (P

Polyetherketoneketone (PEKK) a high performance thermoplastic polymer that is FDA-approved for cranio- and maxillo-facial as well as spineal surgery. We studied the viability, growth and osteogenic differentiation of bone marrow-derived human and sheep mesenchymal stem cells (MSC) in combination with a 3D scaffold made of PEKK using different cell-based assays. To investigate if autologous MSC, either undifferentiated or osteogenically pre-differentiated, augmented bone formation after implantation, we implanted cell-seeded 3D PEKK scaffolds into calvarial defects in sheep for 12 weeks. The volume and quality of newly formed bone were investigated using micro-computer tomography (micro-CT) and histological stainings. Our results show that the 3D PEKK scaffolds were cyto- and bio-compatible. They allowed for adherence, growth and osteogenic differentiation of human and ovine MSC. However, bone healing seemed unaffected by whether the scaffolds were seeded with MSC. Considerable amounts of newly formed bone were found in all PEKK treated groups, but a fibrous capsule was formed around the implants regardless of cell seeding with MSC. European Association for Cranio-Maxillo-Facial Surgery.

Fluorescence-mediated tomography (FMT) enables longitudinal and quantitative determination of the fluorescence distribution in vivo and can be used to assess the biodistribution of novel probes and to assess disease progression using established molecular probes or reporter genes. The combination with an anatomical modality, e.g., micro computed tomography (µCT), is beneficial for image analysis and for fluorescence reconstruction. We describe a protocol for multimodal µCT-FMT imaging including the image processing steps necessary to extract quantitative measurements. After preparing the mice and performing the imaging, the multimodal data sets are registered. Subsequently, an improved fluorescence reconstruction is performed, which takes into account the shape of the mouse. For quantitative analysis, organ segmentations are generated based on the anatomical data using our interactive segmentation tool. Finally, the biodistribution curves are generated using a batch-processing feature. We show the applicability of the method by assessing the biodistribution of a well-known probe that binds to bones and joints.

Fluorescence-mediated tomography (FMT) enables longitudinal and quantitative determination of the fluorescence distribution in vivo and can be used to assess the biodistribution of novel probes and to assess disease progression using established molecular probes or reporter genes. The combination with an anatomical modality, e.g., micro computed tomography (µCT), is beneficial for image analysis and for fluorescence reconstruction. We describe a protocol for multimodal µCT-FMT imaging including the image processing steps necessary to extract quantitative measurements. After preparing the mice and performing the imaging, the multimodal data sets are registered. Subsequently, an improved fluorescence reconstruction is performed, which takes into account the shape of the mouse. For quantitative analysis, organ segmentations are generated based on the anatomical data using our interactive segmentation tool. Finally, the biodistribution curves are generated using a batch-processing feature. We show the applicability of the method by assessing the biodistribution of a well-known probe that binds to bones and joints.

Aim: To enable multimodal in vivo and ex vivo optical imaging of the biodistribution and tumor accumulation of core-crosslinked polymeric micelles (CCPMs). Materials & methods: mPEG-b-p(HPMAm-Lac)-based polymeric micelles, core-crosslinked via cystamine and covalently labeled with two different fluorophores (Dy-676/488), were synthesized. The CCPMs were intravenously injected into CT26 tumor-bearing mice. Results: Upon intravenous injection, the CCPMs accumulated in CT26 tumors reasonably efficiently, with values reaching approximately 4%ID at 24 h. Ex vivo two-photon laser scanning microscopy confirmed efficient extravasation of the image-guided CCPMs out of tumor blood vessels and relatively deep penetration into the tumor interstitium. Conclusion: CCPMs were labeled with multiple fluorophores, and the results obtained exemplify that combining several different in vivo and ex vivo optical imaging techniques is highly useful for analyzing the biodistribution and tumor accumulation of nanomedicines.

Progressive kidney diseases and renal fibrosis are associated with endothelial injury and capillary rarefaction. However, our understanding of these processes has been hampered by the lack of tools enabling the quantitative and noninvasive monitoring of vessel functionality. Here, we used micro-computed tomography (µCT) for anatomical and functional imaging of vascular alterations in three murine models with distinct mechanisms of progressive kidney injury: ischemia-reperfusion (I/R, days 1–56), unilateral ureteral obstruction (UUO, days 1–10), and Alport mice (6–8 weeks old). Contrast-enhanced in vivo µCT enabled robust, noninvasive, and longitudinal monitoring of vessel functionality and revealed a progressive decline of the renal relative blood volume in all models. This reduction ranged from −20% in early disease stages to −61% in late disease stages and preceded fibrosis. Upon Microfil perfusion, high-resolution ex vivo µCT allowed quantitative analyses of three-dimensional vascular networks in all three models. These analyses revealed significant and previously unrecognized alterations of preglomerular arteries: a reduction in vessel diameter, a prominent reduction in vessel branching, and increased vessel tortuosity. In summary, using µCT methodology, we revealed insights into macro-to-microvascular alterations in progressive renal disease and provide a platform that may serve as the basis to evaluate vascular therapeutics in renal disease.

Efficient and safe drug delivery across the blood-brain barrier (BBB) remains one of the major challenges of biomedical and (nano-) pharmaceutical research. Here, it is demonstrated that poly(butyl cyanoacrylate)-based microbubbles (MB), carrying ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles within their shell, can be used to mediate and monitor BBB permeation. Upon exposure to transcranial ultrasound pulses, USPIO-MB are destroyed, resulting in acoustic forces inducing vessel permeability. At the same time, USPIO are released from the MB shell, they extravasate across the permeabilized BBB and they accumulate in extravascular brain tissue, thereby providing non-invasive R-2(star)-based magnetic resonance imaging information on the extent of BBB opening. Quantitative changes in R-2(star) relaxometry are in good agreement with 2D and 3D microscopy results on the extravascular deposition of the macromolecular model drug fluorescein isothiocyanate (FITC)-dextran into the brain. Such theranostic materials and methods are considered to be useful for mediating and monitoring drug delivery across the BBB and for enabling safe and efficient treatment of CNS disorders.

Physiologically realistic geometric models of the vasculature in the liver are indispensable for modelling hepatic blood flow, the main connection between the liver and the organism. Current in vivo imaging techniques do not provide sufficiently detailed vascular trees for many simulation applications, so it is necessary to use algorithmic refinement methods. The method of Constrained Constructive Optimization (CCO) ( Schreiner et al., 2006 ) is well suited for this purpose. Its results after calibration have been previously compared to experimentally acquired human vascular trees ( Schwen and Preusser, 2012 ). The goal of this paper is to extend this calibration to the case of rodents (mice and rats), the most commonly used animal models in liver research. Based on in vivo and ex vivo micro-CT scans of rodent livers and their vasculature, we performed an analysis of various geometric features of the vascular trees. Starting from pruned versions of the original vascular trees, we applied the CCO procedure and compared these algorithmic results to the original vascular trees using a suitable similarity measure. The calibration of the postprocessing improved the algorithmic results compared to those obtained using standard CCO. In terms of angular features, the average similarity increased from 0.27 to 0.61, improving the total similarity from 0.28 to 0.40. Finally, we applied the calibrated algorithm to refine measured vascular trees to the (higher) level of detail desired for specific applications. Having successfully adapted the CCO algorithm to the rodent model organism, the resulting individual-specific refined hepatic vascular trees can now be used for advanced modeling involving, e.g., detailed blood flow simulations.

Theranostics 5(8), 905-918 (2015). doi:10.7150/thno.11304, Published by Ivyspring, Wyoming, NSW

A sacrificial templating process using lithographically printed minimal surface structures allows complex de novo geo-metries of delicate hydrogel materials. The hydrogel scaffolds based on cellulose and chitin nanofibrils show differences in terms of attachment of human mesenchymal stem cells, and allow their differentiation into osteogenic outcomes. The approach here serves as a first example toward designer hydrogel scaffolds viable for biomimetic tissue engineering.

Background Lead-free hunting bullets are an alternative to lead-containing bullets which cause health risks for humans and endangered scavenging raptors through lead ingestion. However, doubts concerning the effectiveness of lead-free hunting bullets hinder the wide-spread acceptance in the hunting and wildlife management community. Methods We performed terminal ballistic experiments under standardized conditions with ballistic soap as surrogate for game animal tissue to characterize dimensionally stable, partially fragmenting, and deforming lead-free bullets and one commonly used lead-containing bullet. The permanent cavities created in soap blocks are used as a measure for the potential wound damage. The soap blocks were imaged using computed tomography to assess the volume and shape of the cavity and the number of fragments. Shots were performed at different impact speeds, covering a realistic shooting range. Using 3D image segmentation, cavity volume, metal fragment count, deflection angle, and depth of maximum damage were determined. Shots were repeated to investigate the reproducibility of ballistic soap experiments. Results All bullets showed an increasing cavity volume with increasing deposited energy. The dimensionally stable and fragmenting lead-free bullets achieved a constant conversion ratio while the deforming copper and lead-containing bullets showed a ratio, which increases linearly with the total deposited energy. The lead-containing bullet created hundreds of fragments and significantly more fragments than the lead-free bullets. The deflection angle was significantly higher for the dimensionally stable bullet due to its tumbling behavior and was similarly low for the other bullets. The deforming bullets achieved higher reproducibility than the fragmenting and dimensionally stable bullets. Conclusion The deforming lead-free bullet closely resembled the deforming lead-containing bullet in terms of energy conversion, deflection angle, cavity shape, and reproducibility, showing that similar terminal ballistic behavior can be achieved. Furthermore, the volumetric image processing allowed superior analysis compared to methods that involve cutting of the soap blocks.

Background— The aim of this study was to determine the role of the chemokine receptor CXCR7 in atherosclerosis and vascular remodeling. CXCR7 is the alternative receptor of CXCL12, which regulates stem cell–mediated vascular repair and limits atherosclerosis via its receptor, CXCR4. Methods and Results— Wire-induced injury of the carotid artery was performed in mice with a ubiquitous, conditional deletion of CXCR7 and in mice treated with the synthetic CXCR7 ligand CCX771. The effect of CCX771 treatment on atherosclerosis was studied in apolipoprotein E–deficient ( Apoe −/− ) mice fed a high-fat diet for 12 weeks. Lipoprotein fractions were quantified in the plasma of Apoe −/− mice by fast protein liquid chromatography. Uptake of DiI-labeled very low-density lipoprotein to adipose tissue was determined by 2-photon microscopy. We show that genetic deficiency of Cxcr7 increased neointima formation and lesional macrophage accumulation in hyperlipidemic mice after vascular injury. This was related to increased serum cholesterol levels and subsequent hyperlipidemia-induced monocytosis. Conversely, administration of the CXCR7 ligand CCX771 to Apoe −/− mice inhibited lesion formation and ameliorated hyperlipidemia after vascular injury and during atherosclerosis. Treatment with CCX771 reduced circulating very low-density lipoprotein levels but not low-density lipoprotein or high-density lipoprotein levels and increased uptake of very low-density lipoprotein into Cxcr7 -expressing white adipose tissue. This effect of CCX771 was associated with an enhanced lipase activity and reduced expression of Angptl4 in adipose tissue. Conclusions— CXCR7 regulates blood cholesterol by promoting its uptake in adipose tissue. This unexpected cholesterol-lowering effect of CXCR7 is beneficial for atherosclerotic vascular diseases, presumably via amelioration of hyperlipidemia-induced monocytosis, and can be augmented with a synthetic CXCR7 ligand.

Non-invasive imaging holds significant potential for implementation in tissue engineering. It can e.g. be used to monitor the localization and function of tissue-engineered implants, as well as their resorption and remodelling. Thus far, however, the vast majority of efforts in this area of research have focused on the use of ultrasmall super-paramagnetic iron oxide (USPIO) nanoparticle-labeled cells, colonizing the scaffolds, to indirectly image the implant material. Reasoning that directly labeling scaffold materials might be more beneficial (enabling imaging also in case of non-cellularized implants), more informative (enabling the non-invasive visualization and quantification of scaffold degradation) and more easy to translate into the clinic (since cell-free materials are less complex from a regulatory point-of-view), we here prepared three different types of USPIO nanoparticles, and incorporated them both passively and actively (via chemical conjugation; during collagen crosslinking) into collagen-based scaffold materials. We furthermore optimized the amount of USPIO incorporated into the scaffolds, correlated the amount of entrapped USPIO with MR signal intensity, showed that the labeled scaffolds are highly biocompatible, demonstrated that scaffold degradation can be visualized using MRI and provided initial proof-of-principle for the in vivo visualization of the scaffolds. Consequently, USPIO-labeled scaffold materials seem to be highly suitable for image-guided tissue engineering applications.

Objectives In chronic liver injury, angiogenesis, the formation of new blood vessels from pre-existing ones, may contribute to progressive hepatic fibrosis and to development of hepatocellular carcinoma. Although hypoxia-induced expression of vascular endothelial growth factor (VEGF) occurs in advanced fibrosis, we hypothesised that inflammation may endorse hepatic angiogenesis already at early stages of fibrosis. Design Angiogenesis in livers of c57BL/6 mice upon carbon tetrachloride- or bile duct ligation-induced chronic hepatic injury was non-invasively monitored using in vivo contrast-enhanced micro computed tomography (µCT) and ex vivo anatomical µCT after hepatic Microfil perfusion. Functional contributions of monocyte-derived macrophage subsets for angiogenesis were explored by pharmacological inhibition of CCL2 using the Spiegelmer mNOX-E36. Results Contrast-enhanced in vivo µCT imaging allowed non-invasive monitoring of the close correlation of angiogenesis, reflected by functional hepatic blood vessel expansion, with experimental fibrosis progression. On a cellular level, inflammatory monocyte-derived macrophages massively accumulated in injured livers, colocalised with newly formed vessels in portal tracts and exhibited pro-angiogenic gene profiles including upregulated VEGF and MMP9. Functional in vivo and anatomical ex vivo µCT analyses demonstrated that inhibition of monocyte infiltration by targeting the chemokine CCL2 prevented fibrosis-associated angiogenesis, but not fibrosis progression. Monocyte-derived macrophages primarily fostered sprouting angiogenesis within the portal vein tract. Portal vein diameter as a measure of portal hypertension depended on fibrosis, but not on angiogenesis. Conclusions Inflammation-associated angiogenesis is promoted by CCL2-dependent monocytes during fibrosis progression. Innovative in vivo µCT methodology can accurately monitor angiogenesis and antiangiogenic therapy effects in experimental liver fibrosis.

Theranostics 4(10), 960-971 (2014). doi:10.7150/thno.9293, Published by Ivyspring, Wyoming, NSW

The Enhanced Permeability and Retention (EPR) effect is extensively used in drug delivery research. Taking into account that EPR is a highly variable phenomenon, we have here set out to evaluate if contrast-enhanced functional ultrasound (ceUS) imaging can be employed to characterize EPR-mediated passive drug targeting to tumors. Using standard fluorescence molecular tomography (FMT) and two different protocols for hybrid computed tomography-fluorescence molecular tomography (CT-FMT), the tumor accumulation of a ~ 10 nm-sized near-infrared-fluorophore-labeled polymeric drug carrier (pHPMA-Dy750) was evaluated in CT26 tumor-bearing mice. In the same set of animals, two different ceUS techniques (2D MIOT and 3D B-mode imaging) were employed to assess tumor vascularization. Subsequently, the degree of tumor vascularization was correlated with the degree of EPR-mediated drug targeting. Depending on the optical imaging protocol used, the tumor accumulation of the polymeric drug carrier ranged from 5 to 12% of the injected dose. The degree of tumor vascularization, determined using ceUS, varied from 4 to 11%. For both hybrid CT-FMT protocols, a good correlation between the degree of tumor vascularization and the degree of tumor accumulation was observed, within the case of reconstructed CT-FMT, correlation coefficients of ~ 0.8 and p-values of < 0.02. These findings indicate that ceUS can be used to characterize and predict EPR, and potentially also to pre-select patients likely to respond to passively tumor-targeted nanomedicine treatments.

Quantitative contrast-enhanced ultrasound plays an important role in tumor characterization and treatment assessment. Besides established functional ultrasound techniques, ultrasound molecular imaging using microbubbles targeted to disease-associated markers is increasingly being applied in pre-clinical studies. Often, repeated injections of non-targeted or targeted microbubbles during the same imaging session are administered. However, the influence of repeated injections on the accuracy of the quantitative data is unclear. Therefore, in tumor-bearing mice, we investigated the influence of multiple injections of non-targeted microbubbles (SonoVue) on time to peak and peak enhancement in liver and tumor tissue and of vascular endothelial growth factor receptor 2 (VEGFR2)-targeted contrast agents (MicroMarker) on specific tumor accumulation. We found significantly decreasing values for time to peak and a tendency for increased values for peak enhancement after multiple injections. Repeated injections of VEGFR2-targeted microbubbles led to significantly increased tumor accumulation, which may result from the exposure of additional binding sites at endothelial surfaces caused by mechanical forces from destroyed microbubbles.

Despite the considerable progress made in the stent development in the last decades, cardiovascular diseases remain the main cause of death in western countries. Beside the benefits offered by the development of different drug-eluting stents, the coronary revascularization bears also the life-threatening risks of in-stent thrombosis and restenosis. Research on new therapeutic strategies is impaired by the lack of appropriate methods to study stent implantation and restenosis processes. Here, we describe a rapid and accessible procedure of stent implantation in mouse carotid artery, which offers the possibility to study in a convenient way the molecular mechanisms of vessel remodeling and the effects of different drug coatings.

The combination of target-specific drugs like bevacizumab with chemotherapeutics has improved treatment efficacy in advanced colorectal cancer (CRC). However, the clinical prognosis of metastatic CRCs is still poor, and novel drugs are currently assessed with respect to their efficacies in patients with CRCs. In a phase III study, the multikinase inhibitor regorafenib (BAY 73-4506) has recently been shown to prolong survival of patients with CRCs after standard therapies failed. In the present study, the activity of regorafenib was investigated in comparison with the angiogenesis inhibitor DC101 in the highly aggressive, murine CT26 metastatic colon cancer model. While a treatment for 10 days with DC101 given at a dose of 34 mg/kg every third day significantly delayed tumor growth compared with vehicle-treated animals, regorafenib completely suppressed tumor growth at a daily oral dose of 30 mg/kg. Regorafenib also induced a stronger reduction in tumor vascularization, as longitudinally assessed in vivo by dynamic contrast-enhanced MRI (DCE-MRI) and confirmed by immunohistochemistry. In addition, regorafenib inhibited the angiogenic activity more strongly and induced a three times higher apoptosis rate than DC101. Even more important, regorafenib completely prevented the formation of liver metastases, whereas in DC101-treated animals, the metastatic rate was only reduced by 33% compared with the vehicle group. In addition, regorafenib significantly reduced the amount of infiltrating macrophages. These data show that the multikinase inhibitor regorafenib exerts strong antiangiogenic, antitumorigenic, and even antimetastatic effects on highly aggressive colon carcinomas indicative for its high potential in the treatment of advanced CRCs. Mol Cancer Ther; 12(7); 1322–31. ©2013 AACR.

World journal of hepatology 9(24), 1030-1039(2017). doi:10.4254/wjh.v9.i24.1030, Published by Beijing Baishideng BioMed Scientific Co., Beijing

The putative presence of the erythropoietin receptor (EpoR) on human cancer cells has given rise to controversial discussion about the use of recombinant human erythropoietin (rhuEpo) for treatment of patients with chemotherapy-induced anemia. In vivo analysis of the EpoR status in tumors could help in elucidating the role of erythropoietin in cancer. Thus, the aim of this study was to develop a targeted EpoR probe for the investigation of EpoR expression in human lung cancer xenografts by fluorescence-mediated tomography.Epo-Cy5.5 was generated by coupling Cy5.5 to rhuEpo. In vitro binding assays were performed using the EpoR-positive non-small cell lung cancer (NSCLC) cell lines A549 (lower EpoR expression) and H838 (higher EpoR expression), the EpoR-negative cell line H2030, and EpoR/EGFP-overexpressing HeLa cells. In vivo specificity of Epo-Cy5.5 was confirmed by competition analyses using micro-CT/fluorescence-mediated tomography fusion imaging. Biodistribution was analyzed over 50 h after injection. Binding of Epo-Cy5.5 was validated on tumor cryosections.After intravenous injection, the probe was rapidly cleared from the circulation. An accumulation was observed in liver and kidneys, with a maximum at 7 h after injection followed by a decline, indicating renal excretion. Almost constant accumulation of Epo-Cy5.5 was found in bone marrow and tumors, indicating specific receptor binding. The probe allowed the discrimination between H838 with higher EpoR expression (89.54 ± 15.91 nM at 25 h) and A549 tumors with lower EpoR expression (60.45 ± 14.59 nM at 25 h, P0.05). Tumor accumulation of Epo-Cy5.5 could be significantly reduced by adding unlabeled rhuEpo (P0.05 at 4, 7, and 24 h). In vitro validation confirmed specific binding of Epo-Cy5.5 to the tumor cells, and this binding correlated with the EpoR expression level. Binding was also observed on endothelial cells. Vessel density and Epo-Cy5.5 binding on endothelial cells were comparable.Epo-Cy5.5 allows the longitudinal analysis of EpoR expression in tumors and thereby can investigate the influence of erythropoietin on EpoR expression, tumor growth, and angiogenesis.

To compare magnetic resonance imaging (MRI), micro-computed tomography (μCT), optical reflectance imaging (ORI) and caliper measurements for subcutaneous tumor detection and size assessment.HCT 116-green- (GFP)-red-fluorescent protein (RFP) tumor volumes were measured in vivo by calipers and by ORI, MRI and μCT over 15 days and validated ex vivo. The method correlating best with the ex vivo tumor volumes was used as reference for longitudinal in vivo correlations.MRI and ORI detected tumors at day 1 post-injection, μCT after 3 days. The in vivo MRI data correlated best with the ex vivo tumor volumes (r²=0.96), followed by μCT (r²=0.93). Thus, MRI was chosen as the reference. μCT-(r²=0.90), in vivo caliper data (r²=0.80) and fluorescence intensities (GFP:r²=0.71; RFP:r²=0.75) highly correlated with MRI-data, whereas fluorescent areas (GFP:r²=0.26; RFP:r²=0.30) poorly correlated.MRI sensitively detects tumors and precisely determines their size; μCT is an accurate alternative for larger tumors; ORI is as sensitive as MRI, but overestimates small tumor sizes; and fluorescence intensity correlates better with tumor volume than fluorescence area.

The aim of most preclinical imaging studies is to derive qualitative statements about the matter under investigation, such as novel probes, therapies, pathologies, biologic mechanisms, mouse models, devices, or analysis methods. Multiple steps are commonly required to achieve this goal. First, appropriate scanning protocols need to be selected because this affects relevant image properties such as noise and resolution. After scanning and reconstruction, post-processing may be required, e.g., to fuse multimodal data sets or to extract quantitative maps using parametric fitting. Then, regions of interest (ROIs) are often defined, resembling tissue types, organs, or lesions. This may be achieved using interactive or even automated segmentation tools which can reduce the user-dependent variability. The ROIs are used to extract quantitative or at least comparable measurements such as volumes, diameters, densities, or amounts of accumulated probes. Finally, an appropriate statistical test is selected and applied to turn the measurements into a qualitative, or “significant” statement, e.g., a significant difference between tumor sizes or a strong correlation between a novel imaging approach and an ex vivo validation procedure. All these steps are connected, and interdisciplinary expertise is required to successfully plan and execute small animal imaging studies. The aim of this chapter is to introduce problems, concepts, and approaches that should be considered during these steps and are related to qualitative and quantitative data analysis.

Introduction Acute graft versus host disease of the gut (agGVHD) is caused by a T-cell mediated attack against the gut epithelia and remains one of the complications with the highest mortality after allogenic stem cell transplantation (allo-SCT). In particular, patients not responding to the initial immunosuppressive therapy during the first weeks have a poor outcome and frequently do not show complete recovery of their symptoms. The underlying mechanisms of refractory agGVHD are not fully understood. Telomere length (TL) reflects the replicative history of a cell. Reaching a critically short TL, cells stop dividing and enter cellular senescence. The aim of the study was to elucidate the role of telomere-mediated replicative exhaustion of enterocytes in affected patients with agGVHD compared to unaffected control patients after allo-SCT. Methods The study was carried out in a single-blinded fashion including 13 patients with refractory agGVHD and 7 patients undergoing allo-SCT, but without clinical history of GVHD. Proliferation score of gut enterocytes was assessed in a semi quantitative manner using MIB-1 immunohistochemistry staining. The TL of the enterocytes obtained from paraffin-embedded diagnostic colonoscopy/sigmoidoskopy biopsies were analyzed using confocal quantitative telomere FISH (cqFISH). Patients were matched according to age, clinical characteristics and onset of GVHD. Results Patients with agGVHD showed a significantly increased proliferation score (median: 2.0, range: 0.5-3.0, n= y, p=0.04) compared to the control group (median: 1.0, range: 0.5-2.0, n=y). TL of initial samples obtained during the first 100 days after transplantation did not differ significantly between patients with (mean±SE: 8.1±0.8 kb, n=13) and without GVHD (7.4±1.4 kb, n=7, p=0.65). Of 10 patients, longitudinal analysis was carried out. Five out of six patients with acute GVHD showed massive telomere shortening during the first weeks after development of GVHD (-2.9 kb, p=0.03), whereas no changes were observed in unaffected controls (-0.1 kb, n=3, p=0.75). This translates into a loss of 0.209 kb per week in patients with acute GVHD, compared to 0.004 kb in the control group. Conclusion In this preliminary study, we found that enterocytes of patients with refractory acute gut GVHD show increased proliferation and dramatically shortened TL in affected gut endothelium during the course of the disease. Increased cellular turnover due to T-cell mediated attack on the enteral stem cell pool and subsequent replicative exhaustion might provide a mechanism explaining eventual refractoriness of gut GVHD to immunosuppressive therapy. Based on our findings, TL measurement should be further explored prospectively as a prognostic biomarker in patients with agGVHD. Disclosures: No relevant conflicts of interest to declare.

EJNMMI Research 1(1), 26 (2011). doi:10.1186/2191-219X-1-26, Published by Springer, Berlin [u.a.]

d Pathological deposition of collagen is a hallmark of kidney fibrosis. To illustrate this process we employed multimodal optical imaging to visualize and quantify collagen deposition in murine models of kidney fibrosis (ischemia-reperfusion or unilateral ureteral obstruction) using the collagen-binding adhesion protein CNA35. For in vivo imaging, we used hybrid computed tomography-fluorescence molecular tomography and CNA35 labeled with the near-infrared fluorophore Cy7. Upon intravenous injection, CNA35-Cy7 accumulation was significantly higher in fibrotic compared to non-fibrotic kidneys. This difference was not detected for a non-specific scrambled version of CNA35-Cy7. Ex vivo, on kidney sections of mice and patients with renal fibrosis, CNA35-FITC co-localized with fibrotic collagen type I and III, but not with the basement membrane collagen type IV. Following intravenous injection, CNA35-FITC bound to both interstitial and perivascular fibrotic areas. In line with this perivascular accumulation, we observed significant perivascular fibrosis in the mouse models and in biopsy sections from patients with chronic kidney disease using computer-based morphometry quantification. Thus, molecular imaging of collagen using CNA35 enabled specific non-invasive quantification of kidney fibrosis. Collagen imaging revealed significant perivascular fibrosis as a consistent component next to the more commonly assessed interstitial fibrosis. Our results lay the basis for further probe and protocol optimization towards the clinical translation of molecular imaging of kidney fibrosis.