Animal Imaging Research Articles

A versatile depigmentation, clearing, and labeling method for exploring nervous system diversity.

Tissue clearing combined with deep imaging has emerged as a powerful alternative to classical histological techniques. Whereas current techniques have been optimized for imaging selected nonpigmented organs such as the mammalian brain, natural pigmentation remains challenging for most other biological specimens of larger volume. We have developed a fast DEpigmEntation-Plus-Clearing method (DEEP-Clear) that is easily incorporated in existing workflows and combines whole system labeling with a spectrum of detection techniques, ranging from immunohistochemistry to RNA in situ hybridization, labeling of proliferative cells (EdU labeling) and visualization of transgenic markers. With light-sheet imaging of whole animals and detailed confocal studies on pigmented organs, we provide unprecedented insight into eyes, whole nervous systems, and subcellular structures in animal models ranging from worms and squids to axolotls and zebrafish. DEEP-Clear thus paves the way for the exploration of species-rich clades and developmental stages that are largely inaccessible by regular imaging approaches.

Small Animal Shanoir (SAS) A Cloud-Based Solution for Managing Preclinical MR Brain Imaging Studies.

Clinical multicenter imaging studies are frequent and rely on a wide range of existing tools for sharing data and processing pipelines. This is not the case for preclinical (small animal) studies. Animal population imaging is still in infancy, especially because a complete standardization and control of initial conditions in animal models across labs is still difficult and few studies aim at standardization of acquisition and post-processing techniques. Clearly, there is a need of appropriate tools for the management and sharing of data, post-processing and analysis methods dedicated to small animal imaging. Solutions developed for Human imaging studies cannot be directly applied to this specific domain. In this paper, we present the Small Animal Shanoir (SAS) solution for supporting animal population imaging using tools compatible with open data. The integration of automated workflow tools ensures accessibility and reproducibility of research outputs. By sharing data and imaging processing tools, hosted by SAS, we promote data preparation and tools for reproducibility and reuse, and participation in multicenter or replication “open science” studies contributing to the improvement of quality science in preclinical domain. SAS is a first step for promoting open science for small animal imaging and a contribution to the valorization of data and pipelines of reference.

Effects of animal handling on striatal DAT availability in rats.

Positron emission tomography (PET) is a non-invasive technique measuring quantification of physiological and biochemical processes in the living organism. However, there are many considerations including anesthesia and fasting to acquire small animal imaging. We aimed to evaluate the effects of anesthesia and fasting of rats in dopamine transporter (DAT) imaging acquisition. Male Sprague Dawley (SD) rats aged 7weeks and weighing 180-260g were used in this study. Rats were randomly divided by 4 groups. Group A was kept under anesthesia for 40min and fasted over 12h. Group B was only fasted over 12h. Group C was only kept under anesthesia for 40min. Group D was neither kept under anesthesia nor fasted over 12h. PET scans were started at 40min after 18F-FP-CIT injection and obtained for 20min. Volumes-of-interest for striatum and extrastriatal area were used for 18F-FP-CIT PET analysis. Cerebellum was considered as a reference region. Specific binding ratio (SBR) was calculated as follows: [(uptake of target-uptake of cerebellum)]/(uptake of cerebellum). SBR without fasting and anesthesia (group D) was significantly lower than those of other groups (vs group A, p = 0.0004; vs group B, p = 0.0377; vs group C, p = 0.0134). However, SBRs of extrastriatal area (p = 0.5120) were not affected by fasting and anesthesia. In conclusion, the SBR of striatum was increased after anesthesia by isoflurane and fasting. When designing an experiment using DAT imaging, the effects of isoflurane and fasting should be considered.

Tumor-specific near-infrared nanobody probe rapidly labels tumors in an orthotopic mouse model of pancreatic cancer

BackgroundNanobodies, derived from camelid antibodies made of only heavy chains, are the smallest, biologic, antigen-binding fragments (~15kDa) with faster pharmacokinetics and better tumor penetration efficiency than standard antibodies. The present study evaluates the efficacy of a fluorescent, anti-carcinoembryonic antigen (CEA) nanobody for rapid tumor labeling in an orthotopic mouse model of pancreatic cancer. MethodsAnti-CEA or control nanobodies were conjugated with the near-infrared fluorophore IRDye 800CW. Fragments of BxPC-3 (high-CEA expressing) or MiaPACA-2 (low-CEA expressing) human pancreatic cancer cell lines were orthotopically implanted into the pancreatic tail of nude mice. After tumors reached 7 to 10 mm in size, 2 nmol anti-CEA or control nanobody-IRDye800CW were injected intravenously. Mice were imaged at various time points hours post-injection. ResultsAnti-CEA nanobodies clearly labeled BxPC3 orthotopic pancreatic tumors 3 hours after injection. The signal was present as early as 15 minutes after injection and was robust at 1 to 3 hours after injection with a tumor-to-background ratio of 2.66. In contrast, there was very low accumulation in the low CEA-expressing, MiaPACA2 pancreatic orthotopic tumors. The fluorophore-conjugated nanobody was specific for CEA-expressing tumors, while the control nanobody did not show any tumor-specific signal. Both nanobodies had strong kidney uptake as expected for small-molecule probes. The fluorescence signal was detectable using 2 clinical, Food and Drug Administration-approved, 800 nm imaging devices as well as small animal imaging systems. ConclusionThis anti-CEA, nanobody-based, fluorescent probe labeled pancreatic orthotopic tumors within 15 minutes of intravenous injection. Fluorescent anti-CEA nanobodies have labeling kinetics that approach the speed of nonspecific dyes such as indocyanine green but with the specificity of antibodies. The use of fluorescently-labeled, intact antibodies leads to a labeling delay of 48 to 96 hours between probe administration and the necessarily delayed time of operation, which can be avoided with nanobodies. The kinetics of a nanobody-based probe makes it a practical agent for same-day, patient administration and fluorescence-guided surgery.

Conditional ablation of H-2Kb and H-2Db on discrete APC subsets modulate outcomes in experimental cerebral malaria

Abstract Cerebral malaria (CM) is a severe complication arising from Plasmodium falciparum infection which results in high mortality. Lack of mechanistic insight into this pathology has resulted in ineffective therapeutic strategies. Using the Plasmodium berghei ANKA model of experimental cerebral malaria (ECM) our lab recapitulated hallmarks of human CM using small animal imaging, including blood-brain barrier (BBB) permeability, severe edema and microhemorrhage visible via T1 and T2-weighted MRI. Previously it had been demonstrated that infiltration of CD8 T cells with cytotoxic capacity is required to induce BBB permeability and ECM. The observation that not only infiltration, but cytotoxic killing is required provoked us to analyze differential MHC class I-restricted priming among discrete antigen presenting cell (APC) types. Using new MHC class I conditional knockout mice generated by our team, we addressed CD8 T cell priming and capacity to disrupt the BBB in ECM. Our findings revealed that mice lacking the H-2Kb MHC class I molecule expression in CD11c+ cells, but not LysM+ cells, were protected from the development of lethal ECM. Subsequently, conditional ablation of the H-2Db on these same subsets yielded similar results. Therefore, we demonstrate that both Db and Kb class I molecule expression by CD11c+ cells enable CD8 T cell priming that results in BBB permeability during ECM. In contrast, CD8 T cells primed by H-2Kb or H-2Db expressing LysM+ APCs are not capable of inducing BBB disruption. These results imply discrete APC subsets prime functionally distinct CD8 T cell responses in the CNS and modulate the outcome of cerebral malaria. This work begins to bridge the mechanistic gap and will impact future therapeutic strategies.

Prevention of Type 1 Diabetes via a lipid Nanoparticle-Based Oral Immunotherapy that Targets Selective Lymphoid Tissues

Abstract Type 1 diabetes (T1D) is an untreatable autoimmune disease caused by the destruction of pancreatic beta cells by autoreactive T cells. Antigen presenting cells (APCs) malfunctions and aberrant accumulation of type 1 interferons contribute to T1D, and the inhibition of inflammatory cytokines signaling via JAK inhibitors like Tofacitinib (Tofa) has shown promise in the prevention of the disease. However, maintenance of a therapeutic concentration of these drugs is challenging and their prolonged use leads to significant side effects. Our objective was to design a controlled and localized delivery of Tofa via biocompatible lipid nanoparticles, Nanostructured Lipid Carrier (NLC), and assess if, via this strategy, a short-term administration would impact T1D development. We identified a formulation of NLC that had negligible toxicity, could be readily taken up by multiple immune cells, and had a favorable Tofa encapsulation efficiency. Live animal imaging confirmed these particles have the unique property of accumulating in lymphoid tissues, particularly in spleen, pancreatic and mesenteric lymph nodes when administrated via oral gavage. Ex-vivo, Tofa-NLC rapidly delivered Tofa to mouse APCs preventing their maturation and the release of inflammatory cytokines. Importantly, short-term administration of Tofa-NLC via oral gavage at early (3-week-old) or late (10-week-old) stage in disease prone NOD mice promoted a significant delay of T1D onset. Cell profiling and in vivo challenges indicated that this therapeutic effect is likely due to localized inhibition of APCs maturation and promotion of anergy in diabetogenic T cells. These results highlight the versatility of this lipid nanoparticle-based drug delivery strategy.

Preclinical Imaging and Spectroscopy in the NIR-II Window with Single-Walled Carbon Nanotubes

We developed a near-infrared small animal imaging system, IR VIVO, that provides real-time images, videos, and spectral imaging in the NIR-I and shortwave infrared (SWIR or NIR-II) regions. We found that SWIR wavelengths can give optimal resolution for in vivo optical imaging of deep organs in mice due to the low tissue autofluorescence, scattering and absorption of light at these wavelengths. We also show how this higher penetration depth can enable the detection of small wavelength changes in the emission of carbon nanotube-based sensors implanted in vivo using a continuously tunable filter.First, we demonstrated superior image clarity and penetration depth at NIR-II wavelengths (1000-1700 nm) in vivo using an FDA approved dye, indocyanine green (ICG). We used 780 nm excitation and detection using a Zephir 1.7 InGaAs camera and 1250 nm long-pass emission filter. We found low tissue autofluorescence at 1250 nm. The ICG dye imaging resulted in superior visualization of microvasculature, perfusion, measurement of heart rate, respiratory rate, hepatobiliary, and intestinal contractions. Further anatomical and functional imaging was carried out by looking at the kinetics of ICG, resulting in identification of different organs.In vivo near-infrared hyperspectral imaging of carbon nanotubes was conducted using a continuously-tunable filter in the imager. A diffraction volume Bragg grating (VBG), was used to provide narrow-band wavelength selection. Coupled with a homogeneous global illumination, spectrally-defined images were acquired in the entire field of view. The result was a dataset containing both 2D spatial information and the full spectrum for each point in the image. We conducted near-infrared hyperspectral imaging of single-walled carbon nanotubes to measure small wavelength changes of the nanotubes implanted into live mice. Hyperspectral measurements of carbon nanotube sensors for lipids in the liver as well as implantable sensors for doxorubicin were conducted, facilitating liver disease monitoring and drug pharmacokinetics measurements.We believe wide-area NIR-II imaging and spectral/hyperspectral measurements have broad potential applications for the use of carbon nanotubes and other NIR-I/II materials in basic materials/biology, translational, and clinical work, including sensor arrays, point-of-care measurements, implants, whole-animal imaging, and intraoperative/surgical imaging.

Utilising nanoparticles to enhance the vaccine induced T cell immune response

Abstract Vaccines are a powerful and cost-effective tool to limit the spread of disease, however, there are still some complex diseases for which we don’t yet have a successful and long-lasting vaccine, for example malaria and cancers. One reason for this is the difficult choice of target antigen, though there is also a need for potent adjuvants/delivery systems capable of inducing both antibody and T cell responses. Current licensed adjuvants, for example Alum, mainly elicit strong antibody responses, therefore, novel vaccine delivery systems need to be developed. One such system is by utilising nanoparticles to increase the vaccine induced immune response. Inorganic carboxylated polystyrene nanoparticles in the viral size range (40–50nm) are non-inflammatory, stable in solution, and target dendritic cells. Importantly, these nanoparticles elicit potent antibody, CD4+ T cell and CD8+ T cell responses to a range of antigens in murine studies, including to malaria and cancer peptides. Using fluorescent nanoparticles allows us to assess the interaction of these nanoparticles with specific cell subsets, such as antigen presenting cells, by flow cytometry. Furthermore, we can analyse the biodistribution of the nanoparticles using small animal imaging via the IVIS. Ideally, for maximum translation capacity into human vaccines these nanoformulations would be biodegradable. In addition to our model polystyrene nanovaccines we have been investigating biodegradable nanoparticles made from other materials, i.e. iron oxide. Comparable to polystyrene nanoparticles, biodegradable nanovaccines are also endocytosed by dendritic cells and are capable of inducing antibodies and cytokine secretion from both CD4+ T cells and CD8+ T cells.

Building functional vascular grafts from human pluripotent stem cells-derived endothelial cells

Background & Aim Stem cell therapy may transform the treatment of cardiovascular disease, with an anticipated impact rivalling the results of revascularization and device therapies. Human pluripotent stem cells are currently developed as sources of tissue-specific cells for organ repair and regeneration. In clinically relevant small and large animal studies, we sought to investigate the feasibility of human embryonic and induced pluripotent stem cell-derived endothelial cell (hESC-EC, hiPSC-EC) engraftments. Methods, Results & Conclusion To generate endothelial cells, human pluripotent stem cells were treated with endothelial growth factors and CD31+ cell population was isolated from differentiating cultures. We obtained highly expandable populations of hESC-EC and hiPSC-EC which expressed mature endothelial cell markers. We implanted cell-laden hydrogel Matrigel subcutaneously into athymic nude rats. We studied angiogenesis in a small animal imaging system equipped with a high-resolution SPECT/CT and PET/MRI to acquire whole-body images. At two weeks after axillary or inguinal implantation in rats, a robust increase in local perfusion was detected by radiolabeled albumin at the engraftment sites, suggesting the functional incorporation of human hPSC-EC into the host microvasculature. Histology confirmed survival, successful engraftment, and formation of capillary-like structures. In a canine model, hiPSC-EC-seeded onto decellularised vascular segments were functional as infrarenal aortic interposition grafts. As assessed by quantitative PCR during follow-up, the expression of human angiogenic factors was induced, suggesting that endothelial cells may undergo maturation in vivo. Similar to the rodent model, we showed retention of hiPSC-EC and dynamic remodelling of the vessel wall with good maintenance of vascular patency in dogs. Combination of pluripotent stem cell-derived endothelial cells and biomatrices may be a promising tissue engineering approach to repair ischemic tissues and induce the formation of blood vessel networks.

The cellular and molecular basis of in vivo synaptic plasticity in rodents.

Plasticity within the neuronal networks of the brain underlies the ability to learn and retain new information. The initial discovery of synaptic plasticity occurred by measuring synaptic strength in vivo, applying external stimulation and observing an increase in synaptic strength termed long-term potentiation (LTP). Many of the molecular pathways involved in LTP and other forms of synaptic plasticity were subsequently uncovered in vitro. Over the last few decades, technological advances in recording and imaging in live animals have seen many of these molecular mechanisms confirmed in vivo, including structural changes both pre- and postsynaptically, changes in synaptic strength, and changes in neuronal excitability. A well-studied aspect of neuronal plasticity is the capacity of the brain to adapt to its environment, gained by comparing the brains of deprived and experienced animals in vivo, and in direct response to sensory stimuli. Multiple in vivo studies have also strongly linked plastic changes to memory by interfering with the expression of plasticity and by manipulating memory engrams. Plasticity in vivo also occurs in the absence of any form of external stimulation, i.e., during spontaneous network activity occurring with brain development. However, there is still much to learn about how plasticity is induced during natural learning and how this is altered in neurological disorders.

Physiological Animal Imaging with 68Ga-Citrate.

Gallium-68 is an ideal research and hospital-based PET radioisotope. The uptake mechanism of Gallium citrate is a combination of specific and non-specific processes, for example, vasodilatation, increased vascular permeability, plasma transferrin binding and lactoferrin and siderophores. In this study, by applying the 68Ge/68Ga generator product, a simple technique for the synthesis and quality control of 68Ga-citrate was introduced and was followed by preliminary animal studies. The synthesis of 68Ga-citrate was performed with a cationic method using the Scintomics automated synthesis system (Scintomics GmbH GRP module 4V). Since the standard procedure for quality control (QC) was not available, the definition of chemical and radiochemical purity of 68Ga-citrate was carried out according to the ICH Q2(R1) guideline. The standard QC tests were analysed with Scintomics 8100 radio-HPLC system equipped with a radioactivity detector. In this study, a New Zealand rabbit weighing 2520 g was used for PET/CT images. 68Ga-citrate synthesis was performed by a cationic method without using organic solvents. The labelling efficiency was found to be >98%. The HPLC method used to assess the radiochemical purity of 68Ga -citrate was validated as rapid, accurate and reproducible enough to apply it to patients safely. The physiological distribution of 68Ga-citrate was investigated in a healthy rabbit. The blood pool, liver, spleen, kidneys and growth plates were the most common sites of 68Ga-citrate involvement.

Enhancing the magnetic relaxivity of MRI contrast agents via the localized superacid microenvironment of graphene quantum dots

The design of contrast agents (CAs) with high magnetic relaxivities is a key issue in the field of magnetic resonance imaging (MRI). The traditional strategy employed is aimed at optimizing the structural design of the magnetic atoms in the CA. However, it is difficult to obtain an agent with magnetic relaxivity over 100 mM−1 s−1 using this approach. In this work, we demonstrate that modulation of the localized superacid microenvironment of certain CAs (Gd3+ loaded polyethylene glycol modified graphene oxide quantum dots or ‘GPG’ for short) can effectively enhance the longitudinal magnetic relaxivities (r1) by accelerating proton exchange. r1 values of a series of GPGs are significantly increased by 20–30 folds compared to commercially available CAs over a wide range of static magnetic field strengths (e.g. 210.9 mM−1 s−1vs. 12.3 mM−1 s−1 at 114 μT, 127.0 mM−1 s−1vs. 4.9 mM−1 s−1 at 7.0 T). GPG aided MRI images is then acquired both in vitro and in vivo with low biotoxicities. Furthermore, folic-acid-modified GPG is demonstrated suitable for MRI-fluorescence dual-modal tumor targeting imaging in animals with more than 98.3% specific cellular uptake rate.

Versatile Single-Element Ultrasound Imaging Platform using a Water-Proofed MEMS Scanner for Animals and Humans

Single-element transducer based ultrasound (US) imaging offers a compact and affordable solution for high-frequency preclinical and clinical imaging because of its low cost, low complexity, and high spatial resolution compared to array-based US imaging. To achieve B-mode imaging, conventional approaches adapt mechanical linear or sector scanning methods. However, due to its low scanning speed, mechanical linear scanning cannot achieve acceptable temporal resolution for real-time imaging, and the sector scanning method requires specialized low-load transducers that are small and lightweight. Here, we present a novel single-element US imaging system based on an acoustic mirror scanning method. Instead of physically moving the US transducer, the acoustic path is quickly steered by a water-proofed microelectromechanical (MEMS) scanner, achieving real-time imaging. Taking advantage of the low-cost and compact MEMS scanner, we implemented both a tabletop system for in vivo small animal imaging and a handheld system for in vivo human imaging. Notably, in combination with mechanical raster scanning, we could acquire the volumetric US images in live animals. This versatile US imaging system can be potentially used for various preclinical and clinical applications, including echocardiography, ophthalmic imaging, and ultrasound-guided catheterization.

Focus-tunable fiber-laser ultrasound sensor for high-resolution linear-scanning photoacoustic computed tomography

In this study, we presented a high-resolution linear-scanning photoacoustic computed tomography (PACT) system with a focus-tunable fiber-laser ultrasound sensor. By bending the fiber laser into a curvature radius of 25 mm, an 85 μm × 150 μm in-plane resolution and a 300 μm elevational resolution were achieved. The elevational resolution was ∼3 times better than the previously reported value. Hair phantoms at multiple depths from 2 cm to 6 cm were clearly observed by tuning the bending curvature. Additionally, the fiber-laser-based PACT system showed its capability for high-resolution imaging of small animals based on the images of abdominal subcutaneous vasculatures from a rat. With the excitation light delivered by a fiber bundle and the ultrasound detected by a fiber laser, this all-fiber linear-scanning PACT system holds great potential for photoacoustic imaging in preclinical and clinical applications.

Multi-parametric MRI of kidney disease progression for autosomal recessive polycystic kidney disease: mouse model and initial patient results.

Background:Autosomal recessive polycystic kidney disease (ARPKD) is a rare but potentially lethal genetic disorder typically characterized by diffuse renal microcysts. Clinical trials for patients with ARPKD are not currently possible due to the absence of sensitive measures of ARPKD kidney disease progression and/or therapeutic efficacy.Methods:In this study, animal and human MRI scanners were used to obtain quantitative kidney T1 and T2 relaxation time maps for both excised kidneys from bpk and wild type (WT) mice as well as for a pediatric patient with ARPKD and a healthy adult volunteer.Results:Mean kidney T1 and T2 relaxation times showed significant increases with age (p<0.05) as well as significant increases in comparison to WT mice (p<2 × 10−10). Significant or nearly significant linear correlations were observed for mean kidney T1 (p=0.030) and T2 (p=0.054) as a function of total kidney volume, respectively. Initial MRF assessments in a patient with ARPKD showed visible increases in both kidney T1 and T2 in comparison to the healthy volunteer.Conclusions:These preclinical and initial clinical MRI studies suggest that renal T1 and T2 relaxometry may provide an additional outcome measure to assess cystic kidney disease progression in patients with ARPKD.

Imaging of bremsstrahlung X-rays from tritium water in a plastic bag using a LaGPS radiation imaging system

Tritium (H-3) is a pure beta-emitting radionuclide and beta particles have extremely low energy (maximum energy: 18.6 keV). Thus the in-vivo imaging of H-3 is thought to be impossible. However, beta particles emit bremsstrahlung X-rays in subjects that may be imaged from outside of the subjects. We tried to image the bremsstrahlung X-rays from H-3 water using a newly developed radiation imaging system. The developed imaging system used a pixelated Ce-doped (Gd, La)2Si2O7 (LaGPS) scintillator plate optically coupled to a flat-panel position-sensitive photomultiplier tube (FP-PMT). Using the imaging system, we conducted bremsstrahlung X-ray imaging from H-3 water in a plastic bag with 37-MBq radioactivity. We obtained tungsten slit mask images with a spatial resolution of ∼3 mm full width at half maximum (FWHM). The energy spectrum of the bremsstrahlung X-rays from the H-3 water showed a broad distribution with an average energy of ∼10 keV. The measured sensitivities of the LaGPS imaging system for bremsstrahlung X-rays from H-3 water in a plastic bag were 1.8 × 10−7. We conclude that the imaging of bremsstrahlung X-rays from H-3 water was really possible and it has a potential to be a new method for the in-vivo H-3 imaging of small animals, plants, or materials.

MP16-18 THE ROLE OF CANCER-ASSOCIATED FIBROBLASTS AND THEIR EXTRACELLULAR VESICLES IN PROSTATE CANCER PROGRESSION

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology I (MP16)1 Apr 2020MP16-18 THE ROLE OF CANCER-ASSOCIATED FIBROBLASTS AND THEIR EXTRACELLULAR VESICLES IN PROSTATE CANCER PROGRESSION Johannes Linxweiler*, Angela Zaccagnino, Dirk Himbert, Turkan Hajili, Philip Zeuschner, Christina Körbel, Michael Stöckle, Michael D. Menger, Matthias Saar, and Kerstin Junker Johannes Linxweiler*Johannes Linxweiler* More articles by this author , Angela ZaccagninoAngela Zaccagnino More articles by this author , Dirk HimbertDirk Himbert More articles by this author , Turkan HajiliTurkan Hajili More articles by this author , Philip ZeuschnerPhilip Zeuschner More articles by this author , Christina KörbelChristina Körbel More articles by this author , Michael StöckleMichael Stöckle More articles by this author , Michael D. MengerMichael D. Menger More articles by this author , Matthias SaarMatthias Saar More articles by this author , and Kerstin JunkerKerstin Junker More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000841.018AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The interaction between cancer cells und cancer-associated fibroblasts (CAFs) plays an important role in the biology of prostate cancer (PCa) and seems to be also mediated by the mutual exchange of extracellular vesicles (EVs). In this project, we analyzed the functional effect of CAFs and their EVs on PCa cells in vitro and in vivo. METHODS: Primary cultures of CAFs, not-cancer-associated (NCAFs) and benign prostate hyperplasia-associated (BPHFs) fibroblasts were established from human samples and characterized by immunofluorescence staining (IF; Pan-CK, Vimentin, αSMA). Their effect on the proliferation of PCa cells (LuCaP136 spheroids, LNCaP cells) was analyzed in vitro via a MTS Assay. In an orthotopic xenograft model, we performed a coinjection of CAFs/NCAFs/BPHFs with PCa cells (LuCaP136 spheroids, LNCaP cells) and subsequently monitored tumor burden for 10 weeks via serum PSA measurements and small animal imaging. Fibroblast-secreted EVs were isolated via ultracentrifugation and characterized by Western Blot, Nanoparticle Tracking Analysis and transmission electron microscopy. The uptake of fibroblast-secreted EVs by PCa cells was analyzed in vitro by fluorescence labeling experiments. RESULTS: CAF, NCAF and BPHF primary cultures could be established successfully and were characterized by IF (all Pan-CK- and Vimentin+; only CAFs αSMA+). The viability of LuCaP136 spheroids and LNCaP cells was significantly stimulated by cocultivation with fibroblasts in vitro (LuCaP136 spheroids by CAFs, LNCaP cells by all fibroblast types). In the xenograft experiments, an intraprostatic (orthotopic) coinjection of cancer cells together with CAFs resulted in significantly larger primary tumors, more lymph node and lung metastases compared to the coinjection of cancer cells together with NCAFs or BPHFs. EVs secreted by primary prostate fibroblasts could be successfully isolated by ultracentrifugation from conditioned media as confirmed by the expression of EV markers (CD9, Syntenin), typical round shape with lipid bilayer and a median size of about 100nm. EVs from CAFs were successfully taken up by PCa cells in fluorescence labeling experiments. CONCLUSIONS: The isolation and characterization of prostate fibroblast primary cultures and their EVs was successfully established. CAFs stimulated the growth of PCa cells in vitro and in vivo and their EV´s were taken up by PCa cells. The molecular mechanisms being involved in this interaction, especially the role of EVs, will be elucidated in future work. Source of Funding: Braun-Stiftung, Else Kröner-Fresenius-Stiftung © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e222-e222 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Johannes Linxweiler* More articles by this author Angela Zaccagnino More articles by this author Dirk Himbert More articles by this author Turkan Hajili More articles by this author Philip Zeuschner More articles by this author Christina Körbel More articles by this author Michael Stöckle More articles by this author Michael D. Menger More articles by this author Matthias Saar More articles by this author Kerstin Junker More articles by this author Expand All Advertisement PDF downloadLoading ...

Transient ischemic stroke induces substantial leukocyte recruitment to both the ischemic and non‐ischemic cerebrovasculature as visualized through live animal imaging.

Ischemic stroke is a leading cause of a death and disability worldwide. Current treatment strategies rely on early restoration of blood flow; however, injury persists despite successful reperfusion. The innate immune response plays a pivotal role in reperfusion injury, but the mechanisms and time‐course of neutrophil and monocyte recruitment to the cerebrovasculature (CBV) during ischemic stroke remain unclear. We sought to characterize the real‐time leukocyte response at the CBV in a mouse stroke model with reperfusion.To visualize leukocyte recruitment induced by ischemia, Ly6G‐tdTomato mice (which have fluorescent red neutrophils) were subjected to 90 minutes of transient middle cerebral artery occlusion (tMCAO) followed by reperfusion. Real‐time spinning‐disc confocal intravital microscopy of the CBV was performed at 24 and 72 hours following reperfusion. Intracranial windows were placed in both ischemic and non‐ischemic hemispheres; and tMCAO treated mice were compared to mice that underwent sham surgery. Neutrophil recruitment (adhesion, rolling) and extravasation were quantified in vivo on both the ischemic and contralateral non‐ischemic hemispheres. Postmortem staining with 2,3,5‐triphenyltetrazolium chloride was used to confirm induction of the stroke and to quantify infarct volume. In addition, wide‐field microscopy was used to examine neutrophil extravasation across the parenchyma.Animals subjected to sham surgery showed negligible leukocyte recruitment to the CBV. However, marked increases in leukocyte rolling, adhesion, and extravasation were observed at 24 and 72 hours following the tMCAO procedure. Interestingly, non‐ischemic hemispheres also demonstrated increased leukocyte rolling and adhesion, but not extravasation tMCAO group. Also, substantially more neutrophil extravasation was observed in the core of the infarct at 72 hours. These findings are consistent with an early recruitment of neutrophils to the cortical surface at early time points and a delayed infiltration into the core, as reported by others.These results show that a significant leukocyte response persists after reperfusion following tMCAO. This response is present both on the ischemic and, to a lesser extent, the non‐ischemic cortical CBV. Acute leukocyte recruitment observed on the non‐ischemic hemisphere suggests that ischemic stroke triggers a global inflammatory response within the CBV. Further reducing inflammation through immunomodulatory therapy in conjunction with reperfusion therapy may be beneficial in not only reducing stroke volume but potentially mitigating inflammation mediated secondary neuronal damage.Support or Funding InformationNIH R01 HL046849 and R37 HL064774 to W.A.M.

NEMA-2008 and In-Vivo Animal and Plant Imaging Performance of the Large FOV Preclinical Digital PET/CT System Discoverist 180

The RAYCAN Discoverist 180 (RAYCAN D180) is a novel preclinical positron emission tomography (PET) and computed tomography (CT) integrated system for the imaging of small- and medium-sized animals and plants. We measured a system resolution of (2.56±0.04) mm (radial), (2.46±0.05) mm (transverse), and (1.67 ± 0.03) mm (axial) at the center of the field of view using a filtered backprojection reconstruction algorithm. An OSEM-PSF-3D reconstruction algorithm improves the spatial resolution to (1.34±0.04) mm (radial), (1.35±0.05) mm (transverse), and (1.64±0.04) mm (axial). The peak noise equivalent count rate of the system is (713 ± 2) kcps, (207 ± 2) kcps, and (47 ± 1) kcps reached at approximately 97 MBq for the mouse-, rat- and monkey-like phantoms, respectively. This result is approximately $2\times $ higher and is reached at approximately $3\times $ higher activity in comparison with most of the existing commercial and research scanners. The peak absolute system sensitivity is (4.00±0.02)% and is competitive with state-of-the-art preclinical PET scanners. We found an image uniformity of (4.86±0.04)%. Finally, we report the static PET/CT scan of a mouse and of a leaf of Epipremnum aureum, and the time-activity curves of the [18F]-FDG metabolism in mouse left myocardial ventricle and kidneys obtained with a PET/CT scan in dynamic modality.

Thermal management proposal for a low-profile positron emission tomography fully pixelated front-end for submillimetric resolution MRI compatible insert dedicated to small animals

The LabPET II is a positron emission tomography technology platform developed for high resolution imaging of small and mid-sized animals. In its original configuration, the electronics includes many printed circuit boards assembled with right angle connectors creating cavities difficult to cool down by air convection. To alleviate this problem and also to reduce the required space radially, a new low-profile electronic architecture is developed. It consists of a Carrier Board on which four printed circuit interposers, based on a ball grid array technology, are soldered. The aim of this work is to study different thermal management scenarios for a PET insert based on this new LabPET II low-profile electronics. A prototype assembly of 12 carrier boards was developed with the thermal model of the LabPET II ASIC built in the Flow Simulation module of SolidWorks. First, a thermal comparison between the typical LabPET II scanner and the simulated low-profile prototype was performed in similar conditions. Second, simulations were used to evaluate three thermal management approaches on the scanner with low-profile electronics: no thermal management, forced airflow (with a fan) and water-cooling. In the latter, three simulations were conducted to maintain the top face of the heat sink located on the carrier board at temperatures of 15, 20 or 30 degrees Celsius. The comparison between the original LabPET II and a scanner with low-profile electronics has shown an ASIC mean temperature of 55 degrees Celsius and 40 degrees Celsius, respectively. In the exploration studies, without any thermal management, the ASIC temperature reached up to 205 degrees Celsius. With an airflow, created with a fan blowing air axially on the extremity of the heatsinks outside the FoV, the ASIC temperature decreases below 100 degrees Celsius. With an applied surface condition of 15 degrees Celsius on the heat sinks extremity parts, the ASIC maximum temperature is 65 degrees Celsius. When surface conditions of 20 and 30 degrees Celsius are applied on the top of the heat sink, the ASIC temperature is reduced to 28 and 38 degrees Celsius, respectively. The new low-profile architecture demonstrates that heat sinks with a simple water-cooling solution reduce the temperature down to 25 degrees Celsius, a real improvement compared to the 55 degrees in the current LabPET II architecture. This improvement will be useful inside an MRI system where eddy currents are induced in all conductive structures and generate heat. Thus, thermal cooling capabilities must be sufficient to compensate for this additional heat generation process to obtain an adequate operating temperature and optimal performance of LabPET II detectors.