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PurposeTo evaluate the feasibility and sensitivity of multimodality PET/CT and MRI imaging for non-invasive characterization of brain microglial/macrophage activation occurring during the acute phase in a mouse model of relapsing remitting multiple sclerosis (RR-MS) using [18F]DPA-714, a selective radioligand for the 18-kDa translocator protein (TSPO), superparamagnetic iron oxide particles (SPIO), and ex vivo immunohistochemistry.MethodsExperimental autoimmune encephalomyelitis (EAE) was induced in female SJL/J mice by immunization with PLP139–151. Seven symptomatic EAE mice and five controls underwent both PET/CT and MRI studies between 11 and 14 days post-immunization. SPIO was injected i.v. in the same animals immediately after [18F]DPA-714 and MRI acquisition was performed after 24 h. Regional brain volumes were defined according to a mouse brain atlas on co-registered PET and SPIO-MRI images. [18F]DPA-714 standardized uptake value (SUV) ratios (SUVR), with unaffected neocortex as reference, and SPIO fractional volumes (SPIO-Vol) were generated. Both SUVR and SPIO-Vol values were correlated with the clinical score (CS) and among them. Five EAE and four control mice underwent immunohistochemical analysis with the aim of identifying activated microglia/macrophage and TSPO expressions.ResultsSUVR and SPIO-Vol values were significantly increased in EAE compared with controls in the hippocampus (p p p p p p ConclusionsThese preliminary results suggest that both activated microglia and infiltrated macrophages are present in vulnerable brain regions during the acute phase of PLP-EAE and contribute to disease severity. Both [18F]DPA-714-PET and SPIO-MRI appear suitable modalities for preclinical study of neuroinflammation in MS mice models.

While the overall mortality for breast cancer has recently declined, management of triple-negative breast cancer (TNBC) is still challenging because of its aggressive clinical behavior and the lack of targeted therapies. Genomic profiling studies highlighted the high level of heterogeneity of this cancer, which comprises different subtypes with unique phenotypes and response to treatment. Platelet-derived growth factor receptor ? (PDGFR?) is an established mesenchymal/stem cell-specific marker in human glioblastoma and, as recently suggested, it may uniquely mark breast cancer cells with stem-like characteristics and/or that have undergone epithelial-mesenchymal transition. Methods: Immunohistochemical analysis for PDGFR? expression was performed on a human TNBC tissue microarray. Functional assays were conducted on mesenchymal-like TNBC cells to investigate the effect of a previously validated PDGFR? aptamer on invasive cell growth in three-dimensional culture conditions, migration, invasion and tube formation. The aptamer was labeled with a near-infrared (NIR) dye and its binding specificity to PDGFR? was assessed both in vitro (confocal microscopy and flow cytometry analyses) and in vivo (fluorescence molecular tomography in mice bearing TNBC xenografts). A mouse model of TNBC lung metastases formation was established and NIR-labeled PDGFR? aptamer was used to detect lung metastases in mice untreated or intravenously injected with unlabeled aptamer. Results: Here, we present novel data showing that tumor cell expression of PDGFR? identifies a subgroup of mesenchymal tumors with invasive and stem-like phenotype, and propose a previously unappreciated role for PDGFR? in driving TNBC cell invasiveness and metastases formation. We show that the PDGFR? aptamer blocked invasive growth and migration/invasion of mesenchymal TNBC cell lines and prevented TNBC lung metastases formation. Further, upon NIR-labeling, the aptamer specifically bound to TNBC xenografts and detected lung metastases. Conclusions: We propose PDGFR? as a reliable biomarker of a subgroup of mesenchymal TNBCs with invasive and stem-like phenotype as well as the use of the PDGFR? aptamer as a high efficacious tool for imaging and suppression of TNBC lung metastases. This study will allow for the significant expansion of the current repertoire of strategies for managing patients with more aggressive TNBC.

Stem photosynthesis has been suggested to play relevant roles to cope with different biotic and abiotic stress factors, including drought. In the present study, we performed measurements of stem hydraulic conductance and non-structural carbohydrate content in the evergreen Laurus nobilis L. and the deciduous Populusalba L., subjected to inhibition of stem photosynthesis and successive exposure to a drought-recovery cycle in order to check if stem photosynthesis may be involved in allowing hydraulic recovery after drought stress relief. Stem shading affected the growth of L. nobilis but not of P. alba saplings. By contrast, inhibition of stem photosynthesis was coupled to inhibition of hydraulic recovery following embolism build-up under drought in P. alba but not in L. nobilis. The two study species showed a different content and behavior of nonstructural carbohydrates (NSCs). The differences in NSCs’ trend and embolism reversal ability led to a significant relationship between starch content and the corresponding hydraulic conductance values in L. nobilis but not in P. alba. Our findings suggest that stem photosynthesis plays a key role in the maintenance of hydraulic functioning during drought especially in the deciduous species. This, in turn, may increase their vulnerability under current global climate change scenarios.

The optical imaging plays an increasing role in preclinical studies, particularly in cancer biology. The combined ultrasound and optical imaging, named photoacoustic imaging (PAI), is an emerging hybrid technique for real-time molecular imaging in preclinical research and recently expanding into clinical setting. PAI can be performed using endogenous contrast, particularly from oxygenated and deoxygenated hemoglobin and melanin, or exogenous contrast agents, sometimes targeted for specific biomarkers, providing comprehensive morphofunctional and molecular information on tumor microenvironment. Overall, PAI has revealed notable opportunities to improve knowledge on tumor pathophysiology and on the biological mechanisms underlying therapy. The aim of this review is to introduce the principles of PAI and to provide a brief overview of current PAI applications in preclinical research, highlighting also on recent advances in clinical translation for cancer diagnosis, staging, and therapy.

INTRODUCTION:The translocator protein 18kDa (TSPO), a biochemical marker of neuroinflammation, is highly expressed in the brain activated microglia and it is also expressed by peripheral inflammatory cells and normal peripheral tissues. Thus, development of radioligands for the TSPO may contribute to further understanding the in vivo TSPO function in central and peripheral inflammatory processes and other pathologies. Here, we report the biodistribution, the specific binding and the radiometabolites of [(18)F]DPA-714, a promising fluorinated PET radiotracer, in normal mice using a microPET/CT scanner. METHODS:The in vivo biodistribution and kinetics of [(18)F]DPA-714 were measured in mice brain and peripheral tissues. Specific binding to TSPO sites was assessed using pharmacological competitive studies by means of saturation experiments performed by i.v. injection of 1mg/kg of unlabeled DPA-714 or 3mg/kg of unlabeled PK11195. A region of interest analysis was performed to generate time-activity curves in the brain, heart, lung, kidney, spleen and liver. Metabolites assay was performed in the plasma and peripheral organs by radio-HPLC. RESULTS:[(18)F]DPA-714 reached high concentration in lung, heart, kidney and spleen, tissues well known to be rich in TSPO sites. [(18)F]DPA-714 kinetics were faster in the lung and slower in the kidney. Pre-injection of unlabeled DPA-714 or PK11195 inhibited about 80% of [(18)F]DPA-714 uptake in the lung and heart (p CONCLUSION:These results suggest that [(18)F]DPA-714 is a suitable PET ligand for imaging in mice brain and peripheral tissues since it binds with high specificity TSPO binding sites and it is almost unchanged at 60minutes after radiotracer injection in the brain and TSPO-rich regions.

Neuroinflammation (NI) is an adaptive response to different noxious stimuli, involving microglia, astrocytes and peripheral immune cells. NI is a hallmark of several acute and chronic diseases of central nervous system (CNS) and contributes to both damage and repair of CNS tissue. Interventional or genetically modified rodent models mimicking human neuropathologies may provide valuable insights on basic mechanisms of NI, but also for improving the development of new diagnostic and therapeutic strategies. Preclinical positron emission tomography (PET) allows to investigate noninvasively the inflammatory response in CNS of rodent models at a molecular level, validating innovative probes for early diagnosis, and characterizing the time course of neuroinflammatory changes and their relationship with disease progression, as well as the effects of experimental treatments with high translational potential. In particular, recent efforts of preclinical PET field are intended to develop specific and selective radiotracers that target the activation of innate immune system in CNS. Here, we have reviewed the state of art for PET in relevant rodent models of acute and chronic neuropathologies associated with NI, with particular regard on imaging of activated microglia and astrocytes.

The normal growth pattern of female C57BL/6J mice, from 5 to 30 weeks of age, has been investigated in a longitudinal study. Weight, body surface area (BS), and body mass index (BMI) were evaluated in forty mice. Lean mass and fat mass, bone mineral content (BMC), and bone mineral density (BMD) were monitored by dual energy X-ray absorptiometry (DEXA). Weight and BS increased linearly (16.15±0.64–27.64±1.42 g;51.13±0.74–79.57±2.15 cm2,P<0.01), more markedly from 5 to 9 weeks of age(P<0.001). BMD showed a peak at 17 weeks (0.0548±0.0011 g/cm2*m,P<0.01). Lean mass showed an evident gain at 9 (15.8±0.8 g,P<0.001) and 25 weeks (20.5±0.3 g,P<0.01), like fat mass from 13 to 17 weeks (2.0±0.4–3.6±0.7 g,P<0.01). BMI and lean mass index (LMI) reached the highest value at 21 weeks (3.57±0.02–0.284±0.010 g/cm2, resp.), like fat mass index (FMI) at 17 weeks (0.057±0.009 g/cm2) (P<0.01). BMI, weight, and BS showed a moderate positive correlation (0.45–0.85) with lean mass from 5 to 21 weeks. Mixed linear models provided a good prediction for lean mass, fat mass, and BMD. This study may represent a baseline reference for a future comparison of wild-type C57BL/6J mice with models of altered growth.

Bone morphogenetic Proteins (BMPs) are growth factors also involved in ossification and chondrogenesis that have generated interest for their efficiency in inducing bone neo-synthesis. BMPs expression in engineered cells has been successful in stimulating osteoblastic differentiation and ectopic and orthotopic bone formation in vivo. We have previously shown that an adenoviral vector expressing bone morphogenetic protein type-4 (BMP-4) is able to efficiently drive bone formation in a rabbit model of discontinuous bone lesions. However, unregulated secretion of BMPs has also been implicated in bone overproduction and exostosis. We have constructed a replication-defective first generation adenoviral (FG-Ad) vector containing a cassette for the expression of BMP-4 associated with the Herpes Simplex virus thymidine kinase (TK) gene (FG-B4TK) in order to shut down BMP-4 expression and, therefore, regulate bone production. TK expression does not interfere with BMP-4 ability to induce ectopic bone formation in athymic nude mice. Administration of ganciclovir blocks ectopic bone production in quadriceps muscle transduced with the FG-B4TK with no effect on the contralateral muscle transduced with a vector expressing only BMP-4. Histological findings confirmed the pro-apoptotic activity of TK and the reduction of mineralized areas in the quadriceps transduced with FG-B4TK in mice treated with ganciclovir. We have generated a system to block BMP-4 secretion by inducing apoptosis in transduced cells therefore blocking unwanted bone formation. This system is an additional tool to generate regulated amount of bone in discontinuous bone lesions and can be easily coupled with biomaterials capable of recruiting cells and generating a local bioreactor.

Preclinical molecular imaging is an emerging field. Improving the ability of scientists to study the molecular basis of human pathology in animals is of the utmost importance for future advances in all fields of human medicine. Moreover, the possibility of developing new imaging techniques or of implementing old ones adapted to the clinic is a significant area. Cardiology, neurology, immunology and oncology have all been studied with preclinical molecular imaging. The functional techniques of photoacoustic imaging (PAI), fluorescence molecular tomography (FMT), positron emission tomography (PET), and single photon emission computed tomography (SPECT) in association with each other or with the anatomic reference provided by computed tomography (CT) as well as with anatomic and functional information provided by magnetic resonance (MR) have all been proficiently applied to animal models of human disease. All the above-mentioned imaging techniques have shown their ability to explore the molecular mechanisms involved in animal models of disease. The clinical translatability of most of the techniques motivates the ongoing study of their possible fields of application. The ability to combine two or more techniques allows obtaining as much information as possible on the molecular processes involved in pathologies, reducing the number of animals necessary in each experiment. Merging molecular probes compatible with various imaging technique will further expand the capability to achieve the best results.

PURPOSE: To evaluate the feasibility and sensitivity of 18F-DPA-714 for the study of microglial activation in the brain and spinal cord of transgenic SOD1G93A mice using high-resolution PET/CT and to evaluate the Iba1 and TSPO expression with immunohistochemistry. METHODS: Nine symptomatic SOD1G93A mice (aged 117 ± 12.7days, clinical score range 1-4) and five WT SOD1 control mice (aged 108 ± 28.5days) underwent 18F-DPA-714 PET/CT. SUV ratios were calculated by normalizing the cerebellar (rCRB), brainstem (rBS), motor cortex (rMCX) and cervical spinal cord (rCSC) activities to that of the frontal association cortex. Two WT SOD1 and six symptomatic SOD1G93A mice were studied by immunohistochemistry. RESULTS: In the symptomatic SOD1G93A mice, rCRB, rBS and rCSC were increased as compared to the values in WT SOD1 mice, with a statistically significantly difference in rBS (2.340 ± 0.784 vs 1.576 ± 0.287, p = 0.014). Immunofluorescence studies showed that TSPO expression was increased in the trigeminal, facial, ambiguus and hypoglossal nuclei, as well as in the spinal cord, of symptomatic SOD1G93A mice and was colocalized with increased Iba1 staining. CONCLUSION: Increased 18F-DPA-714 uptake can be detected with high-resolution PET/CT in the brainstem of transgenic SOD1G93A mice, a region known to be a site of degeneration and increased microglial activation in amyotrophic lateral sclerosis, in agreement with increased TSPO expression in the brainstem nuclei shown by immunostaining. Therefore, 18F-DPA-714 PET/CT might be a suitable tool to evaluate microglial activation in the SOD1G93A mouse model.

New Findings • What is the topic of this review?To discuss the mouse models of myocardial ischaemia and infarction and the applications of dedicated hybrid positron emission tomography (PET)/computed tomography (CT) systems technology for small laboratory animals, including radiotracers and image postprocessing. • What advances does it highlight?In a mouse model of coronary occlusion, non-invasive measurement of infarct size with high-resolution PET/CT systems has excellent reproducibility and high accuracy, supporting the use of this non-invasive methodology in longitudinal studies to monitor cardiac biochemical parameters and to assess the effect of different interventions after acute myocardial ischaemia. Different animal models have been used to reproduce coronary heart disease, but in recent years mice have become 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 non-invasive evaluation of changes in myocardial perfusion, metabolism, apoptosis, inflammation and gene expression or measurement of changes in left ventricular functional parameters. With technological advances, 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 to monitor cardiac biochemical parameters and might facilitate studies to assess the effect of different interventions after acute myocardial ischaemia.

Germline CDKN2A mutations are observed in 20-50% of melanoma-prone families. We identified melanoma patients that were heterozygous for non-coding germline variants in the 5'-UTR of CDKN2A (c.-21C > T; c.-25C > Tc c.-56G > T; c.-67G > C) and examined their impact on the p16(INK4a) 5'-UTR activity using two luciferase-based reporter vectors that differ in basal transcription level and that were transfected into the melanoma-derived WM266-4 and in the breast cancer-derived MCF7 cells. The wild-type 5'-UTR sequence, containing a reported SNP (c.-33G > C) and a known melanoma-predisposing mutation (c.-34G > T), was included as controls. Results revealed that the variants at -21 and -34 severely reduced the reporter activity. The variants at -56 and at -25&-180 exhibited a milder impact, while results with c.-67G > C were dependent on the plasmid type. Quantification of the luciferase mRNA indicated that the effects of the variants were mainly post-transcriptional. Using a bicistronic dual-luciferase reporter plasmid, we confirmed that c.-21C > T and c.-34G > T had a severe negative impact in both cell lines. We also applied a polysomal profiling technique to samples heterozygous for the 5'-UTR variants, including patient-derived lymphoblasts. Analysis of allelic imbalance indicated that in addition to the c.-21C > T variant, the c.-56T > G and c.-67G > C variants also reduced mRNA translation efficiency. Overall, our results suggest that the c.-21C > T sequence variant is a melanoma-predisposing mutation. The c.-25C > T&c.-180G > A and particularly the c.-56G > T variants showed a range of intermediate functional defects in the different assays, and were not observed in the control population. We propose that these variants should be considered as potential mutations.

Lynch syndrome is an inherited cancer syndrome caused by germline mutations in mismatch repair (MMR) genes MLH1, MSH2, MSH6 and PMS2. LS predisposes to high risk of early-onset colorectal, endometrial and other tumors. Patients with Lynch syndrome have also been shown to have an elevated risk for pancreatic cancer (PC). In this study, we aimed to estimate the frequency of suspected Lynch syndrome among a series of 135 PC patients. Further, we wanted to determine the frequency of MMR gene mutations in the suspected Lynch syndrome cases. We also aimed to verify the pathogenicity of any novel non-truncating variants we might detect with a functional assay. Based on personal and/or familial cancer history, 19 patients were classified as suspected Lynch syndrome cases. DNA material for mutation analysis was available for eleven of them. Four patients were found to carry a total of five MLH1 or MSH2 variants. Of these, MSH2-Q402X, MSH2-G322D, and MLH1-K618A had been previously reported, while the MSH2-E205Q and MSH2-V367I variants were novel. MSH2-Q402X is a known stop mutation and reported here for the first time here in association with PC. MLH1-K618A was found in the unaffected branch of a kindred, suggesting that it may be a polymorphism or a low penetrance variant. MSH2-G322D likely does not cause a MMR defect, although this variant has also been associated with breast cancer as indeed seen in our patient. The novel variants MSH2-E205Q and MSH2-V367I were found in the same patient. Both novel variants were however functional in the applied MMR assay. Our findings suggest that only a small subset of pancreatic cancer patients carry pathogenic MMR mutations.

We evaluated the contribution of germline CDKN2A mutations and MC1R variants to the development of melanoma in a hospital-based study of single (SPM, n = 398) and multiple primary melanoma (MPM, n = 95). The overall frequency of CDKN2A mutations was 15.2%, and four-fold higher in MPM than in SPM cases (OR = 4.27; 95% CI 2.43-7.53). The likelihood of identifying a CDKN2A mutation increased with family history of melanoma and younger age at diagnosis in MPM cases. Compared to SPM patients, the risk of harboring a CDKN2A mutation rose as the number of primary melanomas increased and was not influenced by family history. The G101W and E27X founder mutations were the most common. Several other mutations (W15X, Q50X, R58X, A68L, A127P and H142R) were detected for the first time in Italian patients. One novel mutation, T77A, was identified. Several non-coding variants with unknown functional significance were also found (5'UTR -25C > T, -21C > T, -67G > C, IVS1 +37G > C); the novel 5'UTR -21C > T variant was not detected in controls. The CDKN2A A148T polymorphism was more frequent in MPM patients than in the control population (15.7% versus 6.6%). Compared to the SPM patients, MPM cases had a 2-fold increased probability of being MC1R variant carriers and a higher probability of carrying two or more variants. No specific association was observed between the type of variant and the number of melanomas, suggesting that the number rather than the type of MC1R variant increases the risk of MPM. We observed no interaction between CDKN2A status and the presence of MC1R variants. The high frequency of CDKN2A mutations in our MPM cases, independent of their family history, is of relevance to genetic counseling and testing in our population.

Objectives. To present a practical approach that combines biomechanical tests, microcomputed tomography (mu CT) and histomorphometry, providing quantitative results on bone structure and mechanical properties in a minipig model, in order to investigate the specific response to an innovative dental biomaterial. Methods. Titanium implants with innovative three-dimensional scaffolds were inserted in the tibias of 4 minipigs. Primary stability and osseointegration were investigated by means of insertion torque (IT) values, resonance frequency analysis (RFA), bone-to-implant contact (BIC), bone mineral density (BMD) and stereological measures of trabecular bone. Results. A significant positive correlation was found between IT and RFA (r = 0.980, p = 0.0001). BMD at the implant sites was 18% less than the reference values (p = 0.0156). Pefi-implant Tb.Th was 50% higher, while Tb.N was 50% lower than the reference zone (p

BACKGROUND: We investigated the effects of uncoupling protein 3 (UCP3) genetic deletion on (18)F-fluorodeoxyglucose (FDG) cardiac uptake by positron emission tomography (PET)/computed tomography (CT) dedicated animal system after permanent coronary artery ligation. METHODS: Cardiac (18)F-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.

We have recently generated knockout mice for the Cbx7 gene, coding for a polycomb group protein that is downregulated in human malignant neoplasias. These mice develop liver and lung adenomas and carcinomas, which confirms a tumour suppressor role for CBX7. The CBX7 ability to downregulate CCNE1 expression likely accounts for the phenotype of the Cbx7-null mice. Unexpectedly, Cbx7-knockout mice had a higher fat tissue mass than wild-type, suggesting a role of CBX7 in adipogenesis. Consistently, we demonstrate that Cbx7-null mouse embryonic fibroblasts go towards adipocyte differentiation more efficiently than their wild-type counterparts, and this effect is Cbx7 dose-dependent. Similar results were obtained when Cbx7-null embryonic stem cells were induced to differentiate into adipocytes. Conversely, mouse embryonic fibroblasts and human adipose-derived stem cells overexpressing CBX7 show an opposite behaviour. These findings support a negative role of CBX7 in the control of adipocyte cell growth and differentiation.

Background Ultrasound is a valuable non-invasive tool used in obstetrics and gynecology to monitor the growth and well being of the human fetus. The laboratory mouse has recently emerged as an appropriate model for fetal and perinatal studies because morphogenetic processes in mice exhibit adequate homology to those in humans, and genetic manipulations are relatively simple to perform in mice. High-frequency ultrasound (HFUS) has recently become available for small animal preclinical imaging and can be used to study pregnancy and development in the mouse. The objective of the current study was to assess the main applications of HFUS in the evaluation of fetal growth and placental function and to better understand human congenital diseases. Methodology/Principal Findings On each gestational day, at least 5 dams were monitored with HFUS; a total of ∼200 embryos were examined. Because it is not possible to measure each variable for the entire duration of the pregnancy, the parameters were divided into three groups as a function of the time at which they were measured. Univariate analysis of the relationship between each measurement and the embryonic day was performed using Spearman’s rank correlation (Rs). Continuous linear regression was adopted for multivariate analysis of significant parameters. All statistical tests were two-sided, and a p value of 0.05 was considered statistically significant. Conclusions/Significance The study describes the main applications of HFUS to assess changes in phenotypic parameters in the developing CD1 mouse embryo and fetus during pregnancy and to evaluating physiological fetal and placental growth and the development of principal organs such as the heart, kidney, liver, brain and eyes in the embryonic mouse. A database of normal structural and functional parameters of mouse development will provide a useful tool for the better understanding of morphogenetic and cardiovascular anomalies in transgenic and mutant mouse models.

Animal experiments are necessary for a better understanding of diseases and for developing new therapeutic strategies. The mouse (Mus musculus) is currently the most popular laboratory animal in biomedical research. Mice imaging procedures are increasingly used in preclinical research because they allow in vivo monitoring and they are readily available for longitudinal and noninvasive studies as well as investigations into the evolution of diseases and the effects of new therapies. New imaging techniques and sophisticated laboratory animal imaging tools are currently producing a large body of evidence about the possible interference of anesthesia with different imaging methods that have the potential to compromise the results of in vivo studies. The purpose of this article is to review the existing literature on molecular imaging studies in mice, to describe the effects of different anesthetic protocols on their outcome, and to report our own experience with such studies.

Gastrointestinal stromal tumors (GISTs) are mesenchymal tumors of the gastrointestinal tract. Most (80 %) contain activating mutations in the KIT receptor tyrosine kinase, roughly 10 % in platelet-derived growth factor receptor-alpha (PDGFRA). In a small subset, BRAF mutations are an alternative molecular pathway. GISTs respond well to imatinib, but low response is seen in patients with wild-type KIT or PDGFRA. Resistance has also been reported as a result of mutations in downstream effectors such as BRAF. We provide here a molecular characterization of a series of primary GISTs from Italian patients. Of 121 GIST cases diagnosed between 2000 and 2012, 83 were evaluated by PCR amplification and direct sequencing for mutations in KIT exons 8, 9, 11, 13, and 17, PDGFRA exons 12, 14, and 18, and BRAF exon 15. Eighty-one GISTs also underwent K-RAS testing. Sixty-four GISTs were positive: 55 had mutations in KIT and 9 in PDGFRA; 16 patients were mutation negative. Three samples came from NF1 patients and were KIT- and PDGFRA negative. Overall, we identified six novel mutations in KIT (p.K550_M552delinsL, p.Q556_W557delinsG p.Q556_G575del, p.W557_V559delinsQ p.P573_R588dup, p.G592_K593dup) and one novel mutation in PDGFRA (p.D842_N848delinsVDV), thus contributing to widening the spectrum of known mutations in GIST tumors and confirming the most frequently altered regions underlying GIST development. Among the 64 KIT- and PDGFRA-positive sporadic patients in our series, no BRAF or KRAS mutations were identified, suggesting that co-occurrence of these mutations is likely to be rare in the northwestern Italian population and not a frequent cause of primary resistance to imatinib in KIT-positive GIST patients.

Background: Anesthetic agents alter microcirculation, influencing tissue oxygenation and delivery of vital substrates. Laser Doppler perfusion imaging is a widespread technique in the field of microvascular research that can evaluate noninvasively and in real time the effects of environmental conditions, physical manipulations, diseases and treatments on peripheral perfusion. This study aims to evaluate laser Doppler perfusion imaging as a means to detect changes in skin microcirculation induced by some popular anesthetic agents in a murine model. Twenty-four age- and gender-matched healthy CD1 mice were examined by laser Doppler perfusion imaging. The skin microcirculatory response was measured at the level of plantar surfaces during isoflurane anesthesia with or without subsequent dexmedetomidine or acepromazine. At the end of the procedure, dexmedetomidine was reversed by atipamezole administration. Results: In all mice, skin blood flow under isoflurane anesthesia did not show significant differences over time (P = 0.1). The serial perfusion pattern and values following acepromazine or dexmedetomidine administration differed significantly (P < 0.05). Conclusions: We standardized a reliable laser Doppler perfusion imaging protocol to non-invasively assess changes in skin microcirculation induced by anesthesia in mice, considering the advantages and drawbacks of this technique and its translational value. © 2013 Gargiulo et al.; licensee BioMed Central Ltd.

Background. Preclinical perfusion studies are useful for the improvement of diagnosis and therapy in dermatologic, cardiovascular and rheumatic human diseases. The Laser Doppler Perfusion Imaging (LDPI) technique has been used to evaluate superficial alterations of the skin microcirculation in surgically induced murine hindlimb ischemia. We assessed the reproducibility and the accuracy of LDPI acquisitions and identified several critical factors that could affect LDPI measurements in mice. Methods. Twenty mice were analysed. Statistical standardisation and a repeatability and reproducibility analysis were performed on mouse perfusion signals with respect to differences in body temperature, the presence or absence of hair, the type of anaesthesia used for LDPI measurements and the position of the mouse body. Results. We found excellent correlations among measurements made by the same operator (i.e., repeatability) under the same experimental conditions and by two different operators (i.e., reproducibility). A Bland-Altman analysis showed the absence of bias in repeatability (p = 0.29) or reproducibility (p = 0.89). The limits of agreement for repeatability were –0.357 and –0.033, and for reproducibility, they were –0.270 and 0.238. Significant differences in perfusion values were observed in different experimental groups. Conclusions. Different experimental conditions must be considered as a starting point for the evaluation of new drugs and strategic therapies.

Atherosclerosis is characterized by intimal plaques of the arterial vessels that develop slowly and, in some cases, may undergo spontaneous rupture with subsequent heart attack or stroke. Currently, noninvasive diagnostic tools are inadequate to screen atherosclerotic lesions at high risk of acute complications. Therefore, the attention of the scientific community has been focused on the use of molecular imaging for identifying vulnerable plaques. Genetically engineered murine models such as ApoE(-/-) and ApoE(-/-)Fbn1C1039G(+/-) mice have been shown to be useful for testing new probes targeting biomarkers of relevant molecular processes for the characterization of vulnerable plaques, such as vascular endothelial growth factor receptor (VEGFR)-1, VEGFR-2, intercellular adhesion molecule (ICAM)-1, P-selectin, and integrins, and for the potential development of translational tools to identify high-risk patients who could benefit from early therapeutic interventions. This review summarizes the main animal models of vulnerable plaques, with an emphasis on genetically altered mice, and the state-of-the-art preclinical molecular imaging strategies.

Different animal models have been used to reproduce coronary heart disease, but in recent years mice have become 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 non-invasive evaluation of changes in myocardial perfusion, metabolism, apoptosis, inflammation and gene expression or measurement of changes in left ventricular functional parameters. With technological advances, 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 to monitor cardiac biochemical parameters and might facilitate studies to assess the effect of different interventions after acute myocardial ischaemia.

Different species have been used to reproduce myocardial infarction models but in the last years mice became the animals of choice for the analysis of several diseases, due to their short life cycle and the possibility of genetic manipulation. Many techniques are currently used for cardiovascular imaging in mice, including X-ray computed tomography (CT), high-resolution ultrasound, magnetic resonance imaging, and nuclear medicine procedures. Cardiac positron emission tomography (PET) allows to examine noninvasively, on a molecular level and with high sensitivity, regional changes in myocardial perfusion, metabolism, apoptosis, inflammation, and gene expression or to measure changes in anatomical and functional parameters in heart diseases. Currently hybrid PET/CT scanners for small laboratory animals are available, where CT adds high-resolution anatomical information. This paper reviews mouse models of myocardial infarction and discusses the applications of dedicated PET/CT systems technology, including animal preparation, anesthesia, radiotracers, and images postprocessing.

BACKGROUND: This study assessed the reproducibility and accuracy of 2-deoxy-2[(18)F]fluoro-D: -glucose ((18)F-FDG) for non-invasive quantification of myocardial infarct size in mice by a high-resolution positron emission tomography (PET)/computed tomography (CT) system. METHODS AND RESULTS: Mice were studied by (18)F-FDG PET/CT 1 week after induction of myocardial infarction by permanent coronary occlusion or sham procedure. In a subset of mice, PET/CT was repeated 2 days apart to assess the reproducibility of infarct size measurements. Histological analysis was used as reference method to validate imaging data. The average difference in infarct size measurements between the first and the second study was -0.42% ± 2.07% (95% confidence interval -2.6 to 1.75) with a repeatability coefficient of 4.05%. At Bland-Altman analysis, the lower and upper limits of agreement between the two repeated studies were -4.46% and 3.63%, respectively, and no correlation between difference and mean was found (P = .89). The concordance correlation coefficient was 0.99 (P < .001) and the intraclass coefficient of correlation was 0.99. A high correlation between PET/CT and histology was found for measurement of infarct size (P < .001). Using Bland-Altman analysis, the mean difference in infarct size measurement (PET/CT minus histology) was 1.9% (95% confidence interval 0.94% to 2.86%). CONCLUSIONS: In a mice model of permanent coronary occlusion non-invasive measurement of infarct size with high-resolution (18)F-FDG, PET/CT has excellent reproducibility and accuracy. These findings support the use of this methodology in serial studies.

Animal experiments are necessary for a better understanding of diseases and for developing new therapeutic strategies. The mouse (Mus musculus) is currently the most popular laboratory animal in biomedical research. Experimental procedures on animals often require anesthesia and/or analgesia to obtain adequate immobilization and to reduce stress or pain. Mice anesthesia is challenging for several reasons including the animals’ size, metabolic rate, and the high risk of hypothermia and hypoglycemia. Moreover, anesthetic agents influence physiological parameters, further interfering with experimental results. Small animal imaging procedures are increasingly used in biomedical research both because the animals allow in vivo monitoring and because they are readily available for longitudinal and noninvasive studies as well as investigations into the evolution of diseases and the effects of new therapies. Anesthesia must adapt to the imaging technique, the procedure length, and the aim of the study. The purpose of this article is to review the existing literature on anesthetic protocols adopted in mice for molecular imaging studies and to report our experience.

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 biomarkersin vivoand 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.

B-cell lymphoma is a clonal expansion of neoplastic cells that may result in fatal outcomes. Here, we report the in vivo targeting and growth inhibition of aggressive A20 murine B-cell lymphoma by idiotype-specific peptide pA20-36. pA20-36 was selected from random peptide libraries and bound specifically to the B-cell receptor (BCR) of A20 cells in mice engrafted with A20 lymphoma, as shown by histology and positron emission tomographic analysis. BCR cross-linking of A20 cells with pA20-36 resulted in massive apoptosis of targeted tumor cells and in an increased survival of the diseased animals without any detectable evidence of toxicity. The pA20-36 treatment reverted the immune suppression of the tumor microenvironment as shown by reduced expression of vascular endothelial growth factor, interleukin-10, and transforming growth factor-β cytokines together with a lower number of CD11b+Gr-1+ inhibitor myeloid-derived suppressor cells and Foxp3+CD4+ Treg cells. Furthermore, pA20-36 treatment was associated with an increased number of tumor-infiltrating, activated CD8+ T cells that exerted a tumor-specific cytolytic activity. These findings show that a short peptide that binds specifically to the complementarity-determining regions of the A20 BCR allows in vivo detection of neoplastic cells together with significant inhibition of tumor growth in vivo.

Background: Through negative regulation of gene expression, microRNAs (miRNAs) can function in cancers as oncosuppressors, and they can show altered expression in various tumor types. Here we have investigated medulloblastoma tumors (MBs), which arise from an early impairment of developmental processes in the cerebellum, where Notch signaling is involved in many cell-fate-determining stages. MBs occur bimodally, with the peak incidence seen between 3-4 years and 8-9 years of age, although it can also occur in adults. Notch regulates a subset of the MB cells that have stem-cell-like properties and can promote tumor growth. On the basis of this evidence, we hypothesized that miRNAs targeting the Notch pathway can regulated these phenomena, and can be used in anti-cancer therapies. Methodology/Principal Findings: In a screening of MB cell lines, the miRNA miR-199b-5p was seen to be a regulator of the Notch pathway through its targeting of the transcription factor HES1. Down-regulation of HES1 expression by miR-199b-5p negatively regulates the proliferation rate and anchorage-independent growth of MB cells. MiR-199b-5p over-expression blocks expression of several cancer stem-cell genes, impairs the engrafting potential of MB cells in the cerebellum of athymic/nude mice, and of particular interest, decreases the MB stem-cell-like (CD133+) subpopulation of cells. In our analysis of 61 patients with MB, the expression of miR-199b-5p in the non-metastatic cases was significantly higher than in the metastatic cases (P = 0.001). Correlation with survival for these patients with high levels of miR-199b expression showed a positive trend to better overall survival than for the low-expressing patients. These data showing the down-regulation of miR-199b-5p in metastatic MBs suggest a potential silencing mechanism through epigenetic or genetic alterations. Upon induction of de-methylation using 5-aza-deoxycytidine, lower miR-199b-5p expression was seen in a panel of MB cell lines, supported an epigenetic mechanism of regulation. Furthermore, two cell lines (Med8a and UW228) showed significant upregulation of miR-199b-5p upon treatment. Infection with MB cells in an induced xenograft model in the mouse cerebellum and the use of an adenovirus carrying miR-199b-5p indicate a clinical benefit through this negative influence of miR-199b-5p on tumor growth and on the subset of MB stem-cell-like cells, providing further proof of concept. Conclusions/Significance: Despite advances in our understanding of the pathogenesis of MB, one-third of these patients remain incurable and current treatments can significantly damage long-term survivors. Here we show that miR-199b-5p expression correlates with metastasis spread, identifying a new molecular marker for a poor-risk class in patients with MB. We further show that in a xenograft model, MB tumor burden can be reduced, indicating the use of miR199b-5p as an adjuvant therapy after surgery, in combination with radiation and chemotherapy, for the improvement of anti-cancer MB therapies and patient quality of life. To date, this is the first report that expression of a miRNA can deplete the tumor stem cells, indicating an interesting therapeutic approach for the targeting of these cells in brain tumors. © 2009 Garzia et al.

GenoMEL, comprising major familial melanoma research groups from North America, Europe, Asia, and Australia has created the largest familial melanoma sample yet available to characterize mutations in the high-risk melanoma susceptibility genes CDKN2A/alternate reading frames (ARF), which encodes p16 and p14ARF, and CDK4 and to evaluate their relationship with pancreatic cancer (PC), neural system tumors (NST), and uveal melanoma (UM). This study included 466 families (2,137 patients) with at least three melanoma patients from 17 GenoMEL centers. Overall, 41% (n = 190) of families had mutations; most involved p16 (n = 178). Mutations in CDK4 (n = 5) and ARF (n = 7) occurred at similar frequencies (2-3%). There were striking differences in mutations across geographic locales. The proportion of families with the most frequent founder mutation(s) of each locale differed significantly across the seven regions (P = 0.0009). Single founder CDKN2A mutations were predominant in Sweden (p.R112_L113insR, 92% of family's mutations) and the Netherlands (c.225_243del19, 90% of family's mutations). France, Spain, and Italy had the same most frequent mutation (p.G101W). Similarly, Australia and United Kingdom had the same most common mutations (p.M53I, c.IVS2-105A>G, p.R24P, and p.L32P). As reported previously, there was a strong association between PC and CDKN2A mutations (P < 0.0001). This relationship differed by mutation. In contrast, there was little evidence for an association between CDKN2A mutations and NST (P = 0.52) or UM (P = 0.25). There was a marginally significant association between NST and ARF (P = 0.05). However, this particular evaluation had low power and requires confirmation. This GenoMEL study provides the most extensive characterization of mutations in high-risk melanoma susceptibility genes in families with three or more melanoma patients yet available. (Cancer Res 2006; 66(20): 9818-28)

Mutations in the CDKN2A gene underlie melanoma susceptibility in as many as 50% of melanoma kindreds in selected populations, and several CDKN2A founder mutations have been described. Inherited mutations in CDKN2A have been found to be associated with other, non-melanoma cancers including pancreatic cancer (PC) and neural system tumors (NST). Here we report a novel germline mutation in exon 1 of the CDKN2A gene, E27X, which we first detected in melanoma patients living in or originally from a small geographic area bordering Liguria in north-western Italy. A subset of melanoma kindreds positive for this mutation displayed PC and neuroblastoma. E27X generates a premature stop codon, leading to dramatically reduced protein levels of p16 and leaving p14ARF unaltered. As PC and NSTs have been postulated to be preferentially associated with CDKN2A mutations located in exon 2 and/or affecting p14ARF alone, the position of E27X in exon 1alpha provides interesting insights towards clarifying the mechanisms by which the CDKN2A/ARF locus is involved in cancer predisposition.

Although the presence of multiple cases of melanoma on the same side of a family is the best predictor of germline CDKN2A mutation, other features (i.e. early age at onset) may be useful to identify carriers. We analysed the records of 682 hospital-based Ligurian melanoma patients. Of these, 238 cases (34 familial, 14 non-familial multiple primary and 190 non-familial single primary melanomas) were consecutively enrolled for screening of the CDKN2A and CDK4 genes. Screening of the 34 familial patients revealed that nine were carriers of the CDKN2A G101W founder mutation. Of the 14 non-familial multiple primary melanoma patients, three carried the G101W founder mutation and one the P48T mutation. For the non-familial patients with a single melanoma, 17 of 190 carried germline CDKN2A mutations, with most (16/17) carrying the G101W Ligurian founder mutation and one a novel single base pair substitution, D74Y. The effect of mutation on age at diagnosis was significant (P=0.012) after correcting for melanoma type (familial or non-familial), number of primaries (single or multiple), gender and disease occurrence (incident or prevalent). Early age at onset may be a good predictor of CDKN2A mutation in Liguria, where the G101W founder mutation is prevalent among melanoma patients, independent of family history.

Expression of p16INK4A, the product of the melanoma susceptibility gene CDKN2A, has been shown to decrease in correlation with tumor progression. P16INK4A is a key regulator of cell-cycle function, and likely interacts with a variety of targets alongside cyclin-dependent kinases (CDKs). One such target is nuclear factor KB (NF-kappaB), a pleiotropic transcription factor that plays a crucial role in apoptosis, oncogenesis and cell cycle control. NF-kappaB p65 has been shown to be activated in melanoma cell lines but few studies decribe its expression in the tissue. In the present study we focused on synchronous expression of p16INK4A and NF-kappaB p65 and their functional activation in melanoma cell lines and biopsy tissue. Activation of NF-kappaB p65, as observed by electrophoretic mobility shift assay in cell lines, was correlated with expression and cellular localization of the active and inactive forms of its inhibitor, IkappaB-alpha. In melanocytic lesions, p16INK4A and NF-kappaB p65 expression were inversely correlated with levels of the nuclear component of NF-kappaB p65 increasing from nevi to primary melanomas and metastases.

10543 Background: Mutations in the CDKN2A gene underlie melanoma susceptibility in as many as 50% of melanoma kindreds in selected populations, and several CDKN2A founder mutations have been described. Inherited mutations in CDKN2A have been found to be associated with other non-melanoma cancers including pancreatic cancer and neural system tumours. Here we report a novel germline mutation in exon 1 of the CDKN2A gene, E27X, which we first detected in melanoma patients living in or originally from a small geographic area bordering Liguria, in north-western Italy. A subset of melanoma kindreds positive for this mutation displayed pancreatic cancer and neuroblastoma. Methods: We investigated 70 pancreatic cancer patients from a case-control study living in or originally coming from a small geographic area bordering Liguria, in north-western Italy, for CDKN2A mutations and ARF. Results: 2 out of 70 patients displayed the E27X mutation and 1 of the 2 patients had a first degree relative affected by pancreatic cancer. Conclusions: E27X generates a premature stop codon, leading to dramatically reduced protein levels of p16 and leaving p14ARF unaltered. As pancreatic cancer and neural system tumours have been postulated to be preferentially associated with CDKN2A mutations located in exon 2 and/or affecting p14ARF alone, the position of E27X in exon 1a provides interesting insights towards clarifying the mechanisms by which the CDKN2A/ARF locus is involved in cancer predisposition. Its finding in pancreatic cancer patients confirms common susceptibility to melanoma and pancreatic cancer due to this CDKN2A exon 1 mutation. No significant financial relationships to disclose.

Bone marrow-derived mesenchymal stem cells (BM-MSCs) are shown to participate in tumor progression by establishing a favorable tumor microenvironment (TME) that promote metastasis through a cytokine networks. However, the mechanism of homing and recruitment of BM-MSCs into tumors and their potential role in malignant tissue progression is poorly understood and controversial. Here we show that BM-MSCs increase aggressiveness of triple-negative breast cancer (TNBC) cell lines evaluated as capability to migrate, invade and acquire stemness markers. Importantly, we demonstrate that the treatment of BM-MSCs with a nuclease-resistant RNA aptamer against platelet-derived growth factor receptor ? (PDGFR?) causes the inhibition of receptor-dependent signaling pathways thus drastically hampering BM-MSC recruitment towards TNBC cell lines and BM-MSCs trans-differentiation into carcinoma-associated fibroblast (CAF)-like cells. Moreover, in vivo molecular imaging analysis demonstrated the aptamer ability to prevent BM-MSCs homing to TNBC xenografts. Collectively, our results indicate the anti-PDGFR? aptamer as a novel therapeutic tool to interfere with BM-MSCs attraction to TNBC providing the rationale to further explore the aptamer in more complex pre-clinical settings.