Ultrasound Biomicroscopy in Small Animal Research: Applications in Molecular and Preclinical Imaging

Ultrasound biomicroscopy (UBM) is a noninvasive multimodality technique that allows high-resolution imaging in mice. It is affordable, widely available, and portable. When it is coupled to Doppler ultrasound with color and power Doppler, it can be used to quantify blood flow and to image microcirculation as well as the response of tumor blood supply to cancer therapy. Target contrast ultrasound combines ultrasound with novel molecular targeted contrast agent to assess biological processes at molecular level. UBM is useful to investigate the growth and differentiation of tumors as well as to detect early molecular expression of cancer-related biomarkers in vivo and to monitor the effects of cancer therapies. It can be also used to visualize the embryological development of mice in uterus or to examine their cardiovascular development. The availability of real-time imaging of mice anatomy allows performing aspiration procedures under ultrasound guidance as well as the microinjection of cells, viruses, or other agents into precise locations. This paper will describe some basic principles of high-resolution imaging equipment, and the most important applications in molecular and preclinical imaging in small animal research

Genetic deletion in uncoupling protein 3 augments 18F-fluorodeoxyglucose cardiac uptake in the ischemic heart

BACKGROUND: We investigated the effects of uncoupling protein 3 (UCP3) genetic deletion on 18F-fluorodeoxyglucose (FDG) cardiac uptake by positron emission tomography (PET)/computed tomography (CT) dedicated animal system after permanent coronary artery ligation. METHODS: Cardiac 18F-FDG PET/CT was performed in UCP3 knockout (UCP3-/-) and wild-type (WT) mice one week after induction of myocardial infarction or sham procedure. RESULTS: In sham-operated mice no difference in left ventricular (LV) volume was detectable between WT and UCP3-/-. After myocardial infarction, LV volume was higher in both WT and UCP3-/- compared to sham animals, with a significant interaction (p < 0.05) between genotype and myocardial infarction. In sham-operated animals no difference in FDG standardized uptake value (SUV) was detectable between WT (1.8 ± 0.6) and UCP3-/- (1.8 ± 0.6). After myocardial infarction SUV was significantly higher in remote areas than in infarcted territories in both UCP3-/- and WT mice (both p < 0.01). Moreover, in remote areas, SUV was significantly higher (p < 0.001) in UCP3-/- as compared to WT, while in the infarcted territory SUV was comparable (p = 0.29). A significant relationship (r = 0.68, p < 0.001) between LV volume and SUV was found. CONCLUSIONS: In a mice model of permanent coronary occlusion, UCP3 deficiency results in a metabolic shift that favored glycolytic metabolism and increased FDG uptake in remote areas

PO-038 PDGFRβ as a new biomarker for metastatic triple-negative breast cancer: development of a theranostic anti-PDGFRβ aptamer for imaging and suppression of metastases

Introduction Triple-negative breast cancers (TNBCs) are a heterogeneous group of aggressive tumours lacking oestrogen and progesterone receptors and HER2 receptor, thus excluding the possibility of using targeted therapy against these proteins. Mesenchymal-like (ML) subtype, characterised by a stem-like, undifferentiated phenotype, is more invasive and metastatic than other TNBC subtypes and has a strong tendency to form vasculogenic mimicry (VM). Recently, platelet derived growth factor receptor β (PDGFRβ) has been shown to play a role in VM of TNBC. Regrettably, therapies targeting PDGFRβ with tyrosine kinase inhibitors are not effective in treating TNBCs, thus developing new strategies to target PDGFRβ in TNBC patients is crucial to improve their chances of survival. Here, we describe the characterisation of the Gint4.T anti-PDGFRβ nuclease-resistant RNA aptamer as high efficacious theranostic tool for imaging and suppression of ML TNBC metastases. Material and methods Immunohistochemical analyses on a human TNBC tissue microarray was performed to correlate PDGFRβ expression with clinical and molecular features of different subtypes. Functional assays were conducted on PDGFRβ-positive ML BT-549 and MDA-MB-231 cells to investigate the effect of Gint4.T in interfering with cell growth in 3D conditions, migration, invasion and VM formation. Gint4.T was conjugated with near-infrared (NIR) fluorescent VivoTag-S680 and its binding specificity to receptor was confirmed both in vitro (confocal microscopy and flow cytometry analyses of TNBC cells) and in vivo (fluorescence molecular tomography in mice bearing TNBC xenografts). MDA-MB-231 cells were i.v. injected in nude mice and Gint4.T-NIR was used to detect lung metastases in mice untreated or i.v. injected with Gint4.T or a scrambled aptamer. Results and discussions The expression of PDGFRβ was observed in human TNBC samples characterised by higher metastatic behaviour. Treatment of TNBC cell lines with Gint4.T aptamer blocked their invasive growth and vasculogenic properties in 3D culture conditions, and strongly reduced cell migration/invasion in vitro and metastases formation in vivo. The Gint4.T-NIR was able to specifically bind to TNBC xenografts and detect lung metastases in vivo. Therefore, the aptamer revealed a high efficacious theranostic tool for imaging and suppression of TNBC metastases. Conclusion These studies indicate PDGFRβ as a new biomarker for ML and metastatic TNBC subtype and propose a novel targeting agent for the diagnosis and treatment of metastatic TNBCs

Novel application and validation of in vivo micro‐CT to study bone modelling in 3D

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/149362/1/ocr12265.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/149362/2/ocr12265_am.pd

Metastatic group 3 medulloblastoma is driven by PRUNE1 targeting NME1-TGF-β-OTX2-SNAIL via PTEN inhibition.

Genetic modifications during development of paediatric groups 3 and 4 medulloblastoma are responsible for their highly metastatic properties and poor patient survival rates. PRUNE1 is highly expressed in metastatic medulloblastoma group 3, which is characterized by TGF-β signalling activation, c-MYC amplification, and OTX2 expression. We describe the process of activation of the PRUNE1 signalling pathway that includes its binding to NME1, TGF-β activation, OTX2 upregulation, SNAIL (SNAI1) upregulation, and PTEN inhibition. The newly identified small molecule pyrimido-pyrimidine derivative AA7.1 enhances PRUNE1 degradation, inhibits this activation network, and augments PTEN expression. Both AA7.1 and a competitive permeable peptide that impairs PRUNE1/NME1 complex formation, impair tumour growth and metastatic dissemination in orthotopic xenograft models with a metastatic medulloblastoma group 3 cell line (D425-Med cells). Using whole exome sequencing technology in metastatic medulloblastoma primary tumour cells, we also define 23 common 'non-synonymous homozygous' deleterious gene variants as part of the protein molecular network of relevance for metastatic processes. This PRUNE1/TGF-β/OTX2/PTEN axis, together with the medulloblastoma-driver mutations, is of relevance for future rational and targeted therapies for metastatic medulloblastoma group 3

High-resolution PET/CT imaging of the mouse heart

Different animal models have been used to reproduce coronary heart disease but in the last years mice became the animals of choice, because of their short life cycle and the possibility of genetic manipulation. Various techniques are currently used for cardiovascular imaging in mice, including high-resolution ultrasound, X-ray computed tomography (CT), magnetic resonance imaging and nuclear medicine procedures. In particular, molecular imaging with cardiac positron emission tomography (PET) allows to evaluate noninvasively changes in myocardial perfusion, metabolism, apoptosis, inflammation, and gene expression or to measure changes in left ventricular functional parameters. With technological advancements, dedicated small laboratory PET/CT imaging has emerged in cardiovascular research, providing in vivo a non-invasive, serial and quantitative assessment of left ventricular function, myocardial perfusion and metabolism at a molecular level. This non-invasive methodology might be useful in longitudinal studies monitoring cardiac biochemical parameters and might facilitate studies to assess the effect of different interventions after acute myocardial ischemia

HEMODYNAMIC EFFECTS OF SOME SEDATIVE DRUGS IN TRASLATIONAL MURINE MODELS

Dexmedetomidine (DEX) and Acepromazine (ACP) are powerful sedatives with remarkable hemodynamic effects, Several phenothiazines and α2-agonist molecules have long been used in translational research separately or in combination. However, their individual or synergistic peripheral hemodynamic effects have not been fully described in different laboratory animal species in spite of their relevance in cardiovascular studies. Some authors reported an attenuation of α2-adrenergic agonist pressor response with the acepromazine-xylazine combination in dogs. Laser Doppler Imaging (LDPI) and High frequency ultrasound echocardiography (HRE) provides best noninvasive measurement of cardiovascular function in mice. The aim of the study was to investigate noninvasively the cardiovascular effects of DEX, of ACP and of their combination in isoflurane (ISO) anesthetized mice. Methods Forty five age-matched and sex-paired CD1 mice, 8 to 10 weeks old, were randomly assigned to one of three experimental groups and underwent 1.5% ISO anesthesia, followed by intraperitoneal injection of either 5 mg/kg ACP, or 1 mg/kg DEX, or by their combination. Body temperature was adjusted to 36 °C. Heart (HR) and respiratory (RR) rates were monitored. Hind paws blood flow (Perfusion Units, volt) was recorded by LDPI apparatus (Periscan®, laser beam power = 1 mV; wavelength = 670 nm; pixel size = 0.25x0.25 mm2; scanner head distance =15 cm; scanning area = 3x2 cm2; scanning time = 2 minutes). An high-resolution ultrasound imaging system (Vevo 770, VisualSonics, transducer frequency 40 MHz, focal length 6 mm, frame rate 30 Hz, spatial resolution 30 μm) synchronized to the electrocardiographic signal was used. A two-dimensional (B-mode) and motion-mode (M-mode) imaging of the left ventricular short axis was taken just at the level of the papillary muscles. Peripheral perfusion (PP), ejection fraction (EF) and fractional shortening (FS) were recorded 10’ and 20’ before and at different intervals after treatments. A further measurement was recorded after reversing dexmedetomidine by the α2-antagonist atipamezole (ATP). We compared PP, HR, RR, EF and FS values at different times by paired non parametric Wilcoxon test and inter-groups differences by One Way Friedman ANOVA. When appropriate, a post hoc analysis by Mann Whitney test was made. Significance was set at P <0.05. Results RR decreased in all groups without significant differences to baseline (P>0.05). ACP increased PP (P=0.005) and reduced FS (P0.05); ATP brought back values such close to baseline (P>0.05). Conclusions In mice ACP+DEX produced more temperate hemodynamic values compared to those following single agents, lessening DEX biphasic hemodynamic pattern. Traslational imaging allows noninvasive and accurate measurements of hemodynamic effects of different sedatives in mice models

Imaging e manipolazione di embrioni di topo in utero guidata dall???ecografia ad alta risoluzione

.La Biomicroscopia ad Ultrasuoni (UBM) rappresenta una metodica di Diagnostica per immagini sperimentale che permette di eseguire studi morfologici e ultrastrutturali in maniera non invasiva e ripetibili nel tempo in modelli animali di malattia umana. Uno dei campi in crescente espansione è quello legato all’ecografia applicata alla diagnosi precoce, alla stadiazione della gravidanza e alla microiniezione in utero. La microiniezione ecoguidata in utero nel periodo embrionale sta diventando unametodologia molto attrattiva per la prevenzione e la cura di malattie genetiche. Inoltre la terapia genica in età fetale offre altri vantaggi, in quanto può indurre nel feto una tolleranza immunitaria contro i transgeni introdottigrazie al fatto che il sistema immunitario del feto non è maturo. Inoltre in età fetale è possible bypassare la barriera emato-encefalica per influenzare la differenziazione delle cellule neuronali e delle staminali. La microiniezione in utero ecoguidata nel topo di laboratorio trova diverseapplicazioni: veicolare a scopo terapeutico marcatori fluorescenti, materiale genico,cellule, farmaci, vettori virali; studi di embriogenesi e/o di lineage cellulare nel l’encefalo, nel cuore, nell’occhio del feto, nella cavità amniotica e nei vasi del cordone ombelicale. Esistono ad oggi, due metodiche descritte in letteratura per l’ersecuzione della microiniezione in utero nel topo di laboratorio