Astrocyte-targeted gene delivery of interleukin 2 specifically increases brain-resident regulatory T cell numbers and protects against pathological neuroinflammation.

The ability of immune-modulating biologics to prevent and reverse pathology has transformed recent clinical practice. Full utility in the neuroinflammation space, however, requires identification of both effective targets for local immune modulation and a delivery system capable of crossing the blood-brain barrier. The recent identification and characterization of a small population of regulatory T (Treg) cells resident in the brain presents one such potential therapeutic target. Here, we identified brain interleukin 2 (IL-2) levels as a limiting factor for brain-resident Treg cells. We developed a gene-delivery approach for astrocytes, with a small-molecule on-switch to allow temporal control, and enhanced production in reactive astrocytes to spatially direct delivery to inflammatory sites. Mice with brain-specific IL-2 delivery were protected in traumatic brain injury, stroke and multiple sclerosis models, without impacting the peripheral immune system. These results validate brain-specific IL-2 gene delivery as effective protection against neuroinflammation, and provide a versatile platform for delivery of diverse biologics to neuroinflammatory patients.

Intravoxel incoherent motion as a surrogate marker of perfused vascular density in rat brain.

Intravoxel incoherent motion (IVIM) MRI has emerged as a valuable technique for the assessment of tissue characteristics and perfusion. However, there is limited knowledge about the relationship between IVIM-derived measures and changes at the level of the vascular network. In this study, we investigated the potential use of IVIM MRI as a noninvasive tool for measuring changes in cerebral vascular density. Variations in quantitative immunohistochemical measurements of the vascular density across different regions in the rat brain (cortex, corpus callosum, hippocampus, thalamus, and hypothalamus) were related to the pseudo-diffusion coefficient D* and the flowing blood fraction f in healthy Wistar rats. We assessed whether region-wise differences in the vascular density are reflected by variations in the IVIM measurements and found a significant positive relationship with the pseudo-diffusion coefficient (p < 0.05, ß = 0.24). The effect of cerebrovascular alterations, such as blood-brain barrier (BBB) disruption on the perfusion-related IVIM parameters, is not well understood. Therefore, we investigated the effect of BBB disruption on the IVIM measures in a rat model of metabolic and vascular comorbidities (ZSF1 obese rat) and assessed whether this affects the relationship between the cerebral vascular density and the noninvasive IVIM measurements. We observed increased vascular permeability without detecting any differences in diffusivity, suggesting that BBB leakage is present before changes in the tissue integrity. We observed no significant difference in the relationship between cerebral vascular density and the IVIM measurements in our model of comorbidities compared with healthy normotensive rats.

A multimodal imaging study to highlight elastin-derived peptide pro-tumoral effect in a pancreatic xenograft model.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is highly malignant with a very poor prognosis due to its silent development and metastatic profile with a 5-year survival rate below 10%. PDAC is characterised by an abundant desmoplastic stroma modulation that influences cancer development by extracellular matrix/cell interactions. Elastin is a key element of the extracellular matrix. Elastin degradation products (EDPs) regulate numerous biological processes such as cell proliferation, migration and invasion. The aim of the present study was to characterise for the first time the effect of two EDPs with consensus sequences "GxxPG" and "GxPGxGxG" (VG-6 and AG-9) on PDAC development. The ribosomal protein SA (RPSA) has been discovered recently, acting as a new receptor of EDPs on the surface of tumour cells, contributing to poor prognosis. METHODS: Six week-old female Swiss nude nu/nu (Nu(Ico)-Foxn1nu) mice were subcutaneously injected with human PDAC MIA PaCa-2/eGFP-FLuc+ cells, transduced with a purpose-made lentiviral vector, encoding green fluorescent protein (GFP) and Photinus pyralis (firefly) luciferase (FLuc). Animals were treated three times per week with AG-9 (n = 4), VG-6 (n = 5) or PBS (n = 5). The influence of EDP on PDAC was examined by multimodal imaging (bioluminescence imaging (BLI), fluorescence imaging (FLI) and magnetic resonance imaging (MRI). Tumour volumes were also measured using a caliper. Finally, immunohistology was performed at the end of the in vivo study. RESULTS: After in vitro validation of MIA PaCa-2 cells by optical imaging, we demonstrated that EDPs exacerbate tumour growth in the PDAC mouse model. While VG-6 stimulated tumour growth to some extent, AG-9 had greater impact on tumour growth. We showed that the expression of the RPSA correlates with a possible effect of EDPs in the PDAC model. Multimodal imaging allowed for longitudinal in vivo follow-up of tumour development. In all groups, we showed mature vessels ending in close vicinity of the tumour, except for the AG-9 group where mature vessels are penetrating the tumour reflecting an increase of vascularisation. CONCLUSIONS: Our results suggest that AG-9 strongly increases PDAC progression through an increase in tumour vascularisation.

Brain development is altered in rabbit fetuses with congenital diaphragmatic hernia.

INTRODUCTION: Children with congenital diaphragmatic hernia (CDH) are at risk for neurodevelopmental delay. Some changes are already present prenatally. Herein, we further examined how the brain develops in fetal rabbits with surgically created DH. METHODS: Two fetuses underwent surgical DH creation on day 23 (term = d31). DH pups and littermate controls were harvested at term. Ten DH pups and 11 controls underwent transcardial perfusion for brain fixation and measurement of brain volume, brain folding, neuron and synaptic density, pre-oligodendrocyte count, proliferation, and vascularization. Twelve other DH and 11 controls had echocardiographic assessment of cardiac output and aortic and cerebral blood flow, magnetic resonance imaging (9.4 T) for cerebral volumetry, and molecular assessment of vascularization markers. RESULTS: DH pups had lower lung-to-body weight ratio (1.3 ± 0.3 vs. 2.4 ± 0.3%; p < 0.0001) and lower heart-to-body weight ratio (0.007 ± 0.001 vs. 0.009 ± 0.001; p = 0.0006) but comparable body weight and brain-to-body weight ratio. DH pups had a lower left ventricular ejection fraction, aortic and cerebral blood flow (39 ± 8 vs. 54 ± 15 mm/beat; p = 0.03) as compared to controls but similar left cardiac ventricular morphology. Fetal DH-brains were similar in volume but the cerebellum was less folded (perimeter/surface area: 25.5 ± 1.5 vs. 26.8 ± 1.2; p = 0.049). Furthermore, DH brains had a thinner cortex (143 ± 9 vs. 156 ± 13 µm; p = 0.02). Neuron densities in the white matter were higher in DH fetuses (124 ± 18 vs. 104 ± 14; p = 0.01) with comparable proliferation rates. Pre-oligodendrocyte count was lower, coinciding with the lower endothelial cell count. CONCLUSION: Rabbits with DH had altered brain development compared to controls prenatally, indicating that brain development is already altered prenatally in CDH.

Quantification of regional myocardial mean intracellular water lifetime: A nonhuman primate study in myocardial stress.

Heart failure with preserved ejection fraction (HFpEF) is typically associated with early metabolic remodeling. Noninvasive imaging biomarkers that reflect these changes will be crucial in determining responses to early drug interventions in these patients. Mean intracellular water lifetime (τi ) has been shown to be partially inversely related to Na, K-ATPase transporter activity and may thus provide insight into the metabolic status in HFpEF patients. Here, we aim to perform regional quantification of τi using dynamic contrast-enhanced (DCE) magnetic resonance imaging (MRI) in the nonhuman primate (NHP) heart and evaluate its region-specific variations under conditions of myocardial stress in the context of perturbed myocardial function. Cardiac stress was induced in seven naïve cynomolgus macaques using a dobutamine stepwise infusion protocol. All animals underwent 3 T cardiac dual-bolus DCE and tagging MRI experiments. The shutter-speed model was employed to quantify regional τi from the DCE-MR images. Additionally, τi values were correlated with myocardial strains. During cardiac stress, there was a significant decrease in global τi (192.9 ± 76.3 ms vs 321.6 ± 70 ms at rest, P < 0.05) in the left ventricle, together with an increase in global peak circumferential strain (-15.4% ± 2.7% vs -10.1% ± 2.9% at rest, P < 0.05). Specifically, slice-level analysis further revealed that a greater significant decrease in mean τi was observed in the apical region (ΔτI = 182.4 ms) compared with the basal (Δτi = 113.2 ms) and midventricular regions (Δτi = 108.4 ms). Regional analysis revealed that there was a greater significant decrease in mean τi in the anterior (Δτi = 243.9 ms) and antero-lateral (Δτi = 177.2 ms) regions. In the inferior and infero-septal regions, although a decrease in τi was observed, it was not significant. Whole heart regional quantification of τi is feasible using DCE-MRI. τi is sensitive to regional changes in metabolic state during cardiac stress, and its value correlates with strain.

Effects of alcohol exposure on the glutamatergic system: a combined longitudinal 18 F-FPEB and 1 H-MRS study in rats.

In a longitudinal rat model of alcohol consumption, we showed that exposure to alcohol decreased the concentration of glutamate in the prefrontal cortex, whereas a normalization occurred during abstinence. 18F-FPEB PET scans revealed that pre-exposure mGluR5 availability in the nucleus accumbens was associated with future alcohol preference. Finally, alcohol exposure induced a decrease in mGluR5 availability in the bilateral hippocampus and amygdala compared with baseline, and in the hippocampus and striatum compared with saccharin (Figure).

A magnetic resonance multi-atlas for the neonatal rabbit brain.

The rabbit model has become increasingly popular in neurodevelopmental studies as it is best suited to bridge the gap in translational research between small and large animals. In the context of preclinical studies, high-resolution magnetic resonance imaging (MRI) is often the best modality to investigate structural and functional variability of the brain, both in vivo and ex vivo. In most of the MRI-based studies, an important requirement to analyze the acquisitions is an accurate parcellation of the considered anatomical structures. Manual segmentation is time-consuming and typically poorly reproducible, while state-of-the-art automated segmentation algorithms rely on available atlases. In this work we introduce the first digital neonatal rabbit brain atlas consisting of 12 multi-modal acquisitions, parcellated into 89 areas according to a hierarchical taxonomy. Delineations were performed iteratively, alternating between segmentation propagation, label fusion and manual refinements, with the aim of controlling the quality while minimizing the bias introduced by the chosen sequence. Reliability and accuracy were assessed with cross-validation and intra- and inter-operator test-retests. Multi-atlas, versioned controlled segmentations repository and supplementary materials download links are available from the software repository documentation at https://github.com/gift-surg/SPOT-A-NeonatalRabbit.

Quantitative MRI biomarkers to characterize regional left ventricular perfusion and function in nonhuman primates during dobutamine-induced stress: A reproducibility and reliability study.

PURPOSE: To identify reproducible and reliable noninvasive regional imaging biomarkers of cardiac function and perfusion at rest and under stress in healthy nonhuman primates (NHPs) that may be used in the future for the early characterization of preclinical heart failure models, to evaluate therapy, and for clinical translation. MATERIALS AND METHODS: Seven naive cynomolgus macaques underwent test-retest 3T cardiac MRI tagging and dual-bolus perfusion experiments. Regional cardiac function biomarkers, such as peak circumferential strain (CS), average diastolic strain-rate (DSR), contractile reserve (CR), diastolic reserve, peak torsion, and torsion reserve were quantified. Further, regional myocardial blood flow (MBF), myocardial perfusion reserve (MPR), and myocardial perfusion reserve-to-contractile reserve (MPR/CR) were also derived. Inter- and intraobserver reproducibility and test-retest reliability analyses were conducted using the reliability and generalizability coefficients including correlation coefficient (CC) and intraclass correlation coefficient (ICC). RESULTS: Overall, peak CS, DSR, and MBF are robust biomarkers at both rest and stress with moderate-good inter- and intraobserver reproducibility and test-retest reliability. At rest: intra-/interobserver reproducibility (CC): peak CS (0.81/0.81), DSR (0.81/0.81), MBF (0.72/0.57), peak torsion (0.79/0.79); test-retest reliability: (CC/ICC): peak CS (0.62/0.75), DSR (0.24/0.55), MBF (0.66/0.62), and peak torsion (0.79/0.78). Under stress: intra-/interobserver reproducibility (CC): peak CS (0.61/0.60), DSR (0.50/0.50), MBF (0.63/0.61), MPR (0.43/0.43), and peak torsion (0.38/0.38); test-retest reliability: (CC/ICC): peak CS (0.58/0.58), DSR (0.24/0.43), MBF (0.58/0.58), MPR (0.43/0.38), and peak torsion (0.38/0.38). CONCLUSION: We demonstrated the feasibility of using cardiac MRI to characterize left ventricular functional and perfusion responses to stress in an NHP species, and specific robust biomarkers such as peak CS, DSR, MBF, diastolic reserve, and MPR have been identified for clinical translation and drug research. LEVEL OF EVIDENCE: 1 J. Magn. Reson. Imaging 2017;45:556-569.

Heterogeneity in lung (18)FDG uptake in pulmonary arterial hypertension: potential of dynamic (18)FDG positron emission tomography with kinetic analysis as a bridging biomarker for pulmonary vascular remodeling targeted treatments.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a disease of progressive vascular remodeling, characterized by dysregulated growth of pulmonary vascular cells and inflammation. A prevailing view is that abnormal cellular metabolism, notably aerobic glycolysis that increases glucose demand, underlies the pathogenesis of PAH. Increased lung glucose uptake has been reported in animal models. Few data exist from patients with PAH. METHODS AND RESULTS: Dynamic positron emission tomography imaging with fluorine-18-labeled 2-fluoro-2-deoxyglucose ((18)FDG) ligand with kinetic analysis demonstrated increased mean lung parenchymal uptake in 20 patients with PAH, 18 with idiopathic PAH (IPAH) (FDG score: 3.27±1.22), and 2 patients with connective tissue disease (5.07 and 7.11) compared with controls (2.02±0.71; P<0.05). Further compartment analysis confirmed increased lung glucose metabolism in IPAH. Lung (18)FDG uptake and metabolism varied within the IPAH population and within the lungs of individual patients, consistent with the recognized heterogeneity of vascular pathology in this disease. The monocrotaline rat PAH model also showed increased lung (18)FDG uptake, which was reduced along with improvements in vascular pathology after treatment with dicholoroacetate and 2 tyrosine kinase inhibitors, imatinib and sunitinib. Hyperproliferative pulmonary vascular fibroblasts isolated from IPAH patients exhibited upregulated glycolytic gene expression, along with increased cellular (18)FDG uptake; both were reduced by dicholoroacetate and imatinib. CONCLUSIONS: Some patients with IPAH exhibit increased lung (18)FDG uptake. (18)FDG positron emission tomography imaging is a tool to investigate the molecular pathology of PAH and its response to treatment.

In vivo mapping of vascular inflammation using the translocator protein tracer 18F-FEDAA1106.

Noninvasive imaging methods are required to monitor the inflammatory content of atherosclerotic plaques. FEDAA1106 (N-(5-fluoro-2-phenoxyphenyl)-N-(2-(2-fluoroethoxy)-5-methoxybenzyl) acetamide) is a selective ligand for TSPO-18kDa (also known as peripheral benzodiazepine receptor), which is expressed by activated macrophages. We compared 18F-FEDAA1106 and 2-deoxy-2-[18F]fluoro-d-glucose (18F-FDG, a marker of glucose metabolism) for positron emission tomographic (PET) imaging of vascular inflammation. This was tested using a murine model in which focal inflammation was induced in the carotid artery via placement of a constrictive cuff. Immunostaining revealed CD68-positive cells (macrophages) at a disturbed flow site located downstream from the cuff. Dynamic PET imaging using 18F-FEDAA1106 or 18F-FDG was registered to anatomic data generated by computed tomographic (CT)/CT angiography. Standardized uptake values were significantly increased at cuffed compared to contralateral arteries using either 18F-FEDAA1106 (p < .01) or FDG (p < .05). However, the 18F-FEDAA1106 signal was significantly higher at the inflamed disturbed flow region compared to the noninflamed uniform flow regions, whereas differences in FDG uptake were less distinct. We conclude that 18F-FEDAA1106 can be used in vivo for detection of vascular inflammation. Moreover, the signal pattern of 18F-FEDAA1106 corresponded with vascular inflammation more specifically than FDG uptake.

Dysregulation of intercellular communication in vitro and in vivo via extracellular vesicles secreted by pancreatic duct adenocarcinoma cells and generated under the influence of the AG9 elastin peptide-conditioned microenvironment.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with poor prognosis due to its highly metastatic profile. Intercellular communication between cancer and stromal cells via extracellular vesicles (EVs) is crucial for the premetastatic microenvironment preparation leading to tumour metastasis. This study shows that under the influence of bioactive peptides derived from the extracellular matrix microenvironment, illustrated here by the AG-9 elastin-derived peptide (EDP), PDAC cells secrete more tumour-derived EVs. Compared to PDAC-derived EVs, tumour-derived EVs resulting from AG-9 treatment (PDAC AG-9-derived EVs) significantly stimulated cell proliferation. At constant amount, tumour-derived EVs were similarly taken up by PDAC and HMEC-1 cells. Tumour-derived EVs stimulated cell proliferation, migration, proteinase secretion, and angiogenesis. Bioluminescence imaging allowed tumour-derived EV/FLuc+ tracking in vivo in a PDAC mouse model. The biodistribution of PDAC AG-9-derived EVs was different to PDAC-derived EVs. Our results demonstrate that the microenvironment, through EDP release, may not only influence the genesis of EVs but may also affect tumour progression (tumour growth and angiogenesis), and metastatic homing by modifying the in vivo biodistribution of tumour-derived EVs. They are potential candidates for targeted drug delivery and modulation of tumour progression, and they constitute a new generation of therapeutic tools, merging oncology and genic therapy.

Remotely Triggered LAD Occlusion Using a Balloon Catheter in Spontaneously Breathing Mice.

Acute myocardial infarction (AMI) is a prevalent and high-mortality cardiovascular condition. Despite advancements in revascularization strategies for AMI, it frequently leads to myocardial ischemia-reperfusion injury (IRI), amplifying cardiac damage. Murine models serve as vital tools for investigating both acute injury and chronic myocardial remodeling in vivo. This study presents a unique closed-chest technique for remotely inducing myocardial IRI in mice, enabling the investigation of the very early phase of occlusion and reperfusion using in-vivo imaging such as MRI or PET. The protocol utilizes a remote occlusion method, allowing precise control over ischemia initiation after chest closure. It reduces surgical trauma, enables spontaneous breathing, and enhances experimental consistency. What sets this technique apart is its potential for simultaneous noninvasive imaging, including ultrasound and magnetic resonance imaging (MRI), during occlusion and reperfusion events. It offers a unique opportunity to analyze tissue responses in almost real-time, providing critical insights into processes during ischemia and reperfusion. Extensive systematic testing of this innovative approach was conducted, measuring cardiac necrosis markers for infarction, assessing the area at risk using contrast-enhanced MRI, and staining infarcts at the scar maturation stage. Through these investigations, emphasis was placed on the value of the proposed tool in advancing research approaches to myocardial ischemia-reperfusion injury and accelerating the development of targeted interventions. Preliminary findings demonstrating the feasibility of combining the proposed innovative experimental protocol with noninvasive imaging techniques are presented herein. These initial results highlight the benefit of utilizing the purpose-built animal cradle to remotely induce myocardial ischemia while simultaneously conducting MRI scans.

3D printing of ferromagnetic passive shims for field shaping in magnetic resonance imaging.

Magnetic Resonance Imaging (MRI) often encounters image quality degradation due to magnetic field inhomogeneities. Conventional passive shimming techniques involve the manual placement of discrete magnetic materials, imposing limitations on correcting complex inhomogeneities. To overcome this, we propose a novel 3D printing method utilizing binder jetting technology to enable precise deposition of a continuous range of concentrations of ferromagnetic ink. This approach grants complete control of the magnitude of the magnetic moment within the passive shim enabling tailored corrections of B0 field inhomogeneities. By optimizing the magnetic field distribution using linear programming and an in-house written Computer-Aided Design (CAD) generation software, we printed shims with promising results in generating low spherical harmonic corrections. Experimental evaluations demonstrate feasibility of these 3D printed passive shims to induce target magnetic fields corresponding to second-order spherical harmonic, as evidenced by acquired B0 maps. The electrically insulating properties of the printed shims eliminate the risk of eddy currents and heating, thus ensuring safety. The dimensional fabrication accuracy of the printed shims surpasses previous methods, enabling more precise and localized correction of subject-specific inhomogeneities. The findings highlight the potential of binder-jetted 3D printed passive shims in MRI shimming as a versatile and efficient solution for fabricating passive shims, with the potential to enhance the quality of MRI imaging while also being applicable to other types of Magnetic Resonance systems.

Pleiotropic effects of trisomy and pharmacologic modulation on structural, functional, molecular, and genetic systems in a Down syndrome mouse model.

Down syndrome (DS) is characterized by skeletal and brain structural malformations, cognitive impairment, altered hippocampal metabolite concentration and gene expression imbalance. These alterations were usually investigated separately, and the potential rescuing effects of green tea extracts enriched in epigallocatechin-3-gallate (GTE-EGCG) provided disparate results due to different experimental conditions. We overcame these limitations by conducting the first longitudinal controlled experiment evaluating genotype and GTE-EGCG prenatal chronic treatment effects before and after treatment discontinuation. Our findings revealed that the Ts65Dn mouse model reflected the pleiotropic nature of DS, exhibiting brachycephalic skull, ventriculomegaly, neurodevelopmental delay, hyperactivity, and impaired memory robustness with altered hippocampal metabolite concentration and gene expression. GTE-EGCG treatment modulated most systems simultaneously but did not rescue DS phenotypes. On the contrary, the treatment exacerbated trisomic phenotypes including body weight, tibia microarchitecture, neurodevelopment, adult cognition, and metabolite concentration, not supporting the therapeutic use of GTE-EGCG as a prenatal chronic treatment. Our results highlight the importance of longitudinal experiments assessing the co-modulation of multiple systems throughout development when characterizing preclinical models in complex disorders and evaluating the pleiotropic effects and general safety of pharmacological treatments.

Q-VAT: Quantitative Vascular Analysis Tool.

As our imaging capability increase, so does our need for appropriate image quantification tools. Quantitative Vascular Analysis Tool (Q-VAT) is an open-source software, written for Fiji (ImageJ), that perform automated analysis and quantification on large two-dimensional images of whole tissue sections. Importantly, it allows separation of the vessel measurement based on diameter, allowing the macro- and microvasculature to be quantified separately. To enable analysis of entire tissue sections on regular laboratory computers, the vascular network of large samples is analyzed in a tile-wise manner, significantly reducing labor and bypassing several limitations related to manual quantification. Double or triple-stained slides can be analyzed, with a quantification of the percentage of vessels where the staining's overlap. To demonstrate the versatility, we applied Q-VAT to obtain morphological read-outs of the vasculature network in microscopy images of whole-mount immuno-stained sections of various mouse tissues.

Myocardial ischemia-reperfusion injury and the influence of inflammation.

Acute myocardial infarction is caused by a sudden coronary artery occlusion and leads to ischemia in the corresponding myocardial territory which generally results in myocardial necrosis. Without restoration of coronary perfusion, myocardial scar formation will cause adverse remodelling of the myocardium and heart failure. Successful introduction of percutaneous coronary intervention and surgical coronary artery bypass grafting made it possible to achieve early revascularisation/reperfusion, hence limiting the ischemic zone of myocardium. However, reperfusion by itself paradoxically triggers an exacerbated and accelerated injury in the myocardium, called ischemia-reperfusion (I/R) injury. This mechanism is partially driven by inflammation through multiple interacting pathways. In this review we summarize the current insights in mechanisms of I/R injury and the influence of altered inflammation. Multiple pharmacological and interventional therapeutic strategies (ischemic conditioning) have proven to be beneficial during I/R in preclinical models but were notoriously unsuccessful upon clinical translation. In this review we focus on common mechanisms of I/R injury, altered inflammation and potential therapeutic strategies. We hypothesize that a dual approach may be of value because I/R injury patients are predestined with multiple comorbidities and systemic low-grade inflammation, which requires targeted intervention before other strategies can be fully effective.

Radiation dose-escalation and dose-fractionation modulate the immune microenvironment, cancer stem cells and vasculature in experimental high-grade gliomas.

BACKGROUND: In the context of high-grade gliomas (HGGs), very little evidence is available concerning the optimal radiotherapy (RT) schedule to be used in radioimmunotherapy combinations. This studied was aimed at shedding new light in this field by analyzing the effects of RT dose escalation and dose fractionation on the tumor microenvironment of experimental HGGs. METHODS: Neurospheres (NS) CT-2A HGG-bearing C57BL/6 mice were treated with stereotactic RT. For dose-escalation experiments, mice received 2, 4 or 8 Gy as single administrations. For dose-fractionation experiments, mice received 4 Gy as a single fraction or multiple (1.33x3 Gy) fractions. The impact of the RT schedule on murine survival and tumor immunity was evaluated. Modifications of glioma stem cells (GSCs), tumor vasculature and tumor cell replication were also assessed. RESULTS: RT dose-escalation was associated with an improved immune profile, with higher CD8+ T cells and CD8+ T cells/regulatory T cells (Tregs) ratio (P=0.0003 and P=0.0022, respectively) and lower total tumor associated microglia/macrophages (TAMs), M2 TAMs and monocytic myeloid derived suppressor cells (mMDSCs) (P=0.0011, P=0.0024 and P<0.0001, respectively). The progressive increase of RT dosages prolonged survival (P<0.0001) and reduced tumor vasculature (P=0.069), tumor cell proliferation (P<0.0001) and the amount of GSCs (P=0.0132 or lower). Compared to the unfractionated regimen, RT dose-fractionation negatively affected tumor immunity by inducing higher total TAMs, M2 TAMs and mMDSCs (P=0.0051, P=0.0036 and P=0.0436, respectively). Fractionation also induced a shorter survival (P=0.0078), a higher amount of GSCs (P=0.0015 or lower) and a higher degree of tumor cell proliferation (P=0.0003). CONCLUSIONS: This study demonstrates that RT dosage and fractionation significantly influence survival, tumor immunity and GSCs in experimental HGGs. These findings should be taken into account when aiming at designing more synergistic and effective radio-immunotherapy combinations.

Magnetic resonance imaging as a non-invasive tool to assess gastric emptying in mice.

BACKGROUND: Methods to study gastric emptying in rodents are time consuming or terminal, preventing repetitive assessment in the same animal. Magnetic resonance imaging (MRI) is a non-invasive technique increasingly used to investigate gastrointestinal function devoid of these shortcomings. Here, we evaluated MRI to measure gastric emptying in control animals and in two different models of gastroparesis. METHODS: Mice were scanned using a 9.4 Tesla MR scanner. Gastric volume was measured by delineating the stomach lumen area. Control mice were scanned every 30 min after ingestion of a 0.2 g meal and stomach volume was quantified. The ability of MRI to detect delayed gastric emptying was evaluated in models of morphine-induced gastroparesis and streptozotocin-induced diabetes. KEY RESULTS: Magnetic resonance imaging reproducibly detected increased gastric volume following ingestion of a standard meal and progressively decreased with a half emptying time of 59 ± 5 min. Morphine significantly increased gastric volume measured at t = 120 min (saline: 20 ± 2 vs morphine: 34 ± 5 mm3 ; n = 8-10; p < 0.001) and increased half emptying time using the breath test (saline: 85 ± 22 vs morphine: 161 ± 46 min; n = 10; p < 0.001). In diabetic mice, gastric volume assessed by MRI at t = 60 min (control: 23 ± 2 mm3 ; n = 14 vs diabetic: 26 ± 5 mm3 ; n = 18; p = 0.014) but not at t = 120 min (control: 21 ± 3 mm3 ; n = 13 vs diabetic: 18 ± 5 mm3 ; n = 18; p = 0.115) was significantly increased compared to nondiabetic mice. CONCLUSIONS AND INFERENCES: Our data indicate that MRI is a reliable and reproducible tool to assess gastric emptying in mice and represents a useful technique to study gastroparesis in disease models or for evaluation of pharmacological compounds.

Multimodal in vivo Imaging of the Integrated Postnatal Development of Brain and Skull and Its Co-modulation With Neurodevelopment in a Down Syndrome Mouse Model.

The brain and skeletal systems are intimately integrated during development through common molecular pathways. This is evidenced by genetic disorders where brain and skull dysmorphologies are associated. However, the mechanisms underlying neural and skeletal interactions are poorly understood. Using the Ts65Dn mouse model of Down syndrome (DS) as a case example, we performed the first longitudinal assessment of brain, skull and neurobehavioral development to determine alterations in the coordinated morphogenesis of brain and skull. We optimized a multimodal protocol combining in vivo micro-computed tomography (µCT) and magnetic resonance imaging (µMRI) with morphometric analyses and neurodevelopmental tests to longitudinally monitor the different systems' development trajectories during the first postnatal weeks. We also explored the impact of a perinatal treatment with green tea extracts enriched in epigallocatechin-3-gallate (GTE-EGCG), which can modulate cognition, brain and craniofacial development in DS. Our analyses quantified alterations associated with DS, with skull dysmorphologies appearing before brain anomalies, reduced integration and delayed acquisition of neurodevelopmental traits. Perinatal GTE-EGCG induced disparate effects and disrupted the magnitude of integration and covariation patterns between brain and skull. Our results exemplify how a longitudinal research approach evaluating the development of multiple systems can reveal the effect of morphological integration modulating the response of pathological phenotypes to treatment, furthering our understanding of complex genetic disorders.

Arteriovenous Fistulae in Chronic Kidney Disease and the Heart: Physiological, Histological, and Transcriptomic Characterization of a Novel Rat Model.

Background Arteriovenous fistulae (AVFs) are the gold standard for vascular access in those requiring hemodialysis but may put an extra hemodynamic stress on the cardiovascular system. The complex interactions between the heart, kidney, and AVFs remain incompletely understood. Methods and Results We characterized a novel rat model of five-sixths partial nephrectomy (NX) and AVFs. NX induced increases in urea, creatinine, and hippuric acid. The addition of an AVF (AVF+NX) further increased urea and a number of uremic toxins such as trimethylamine N-oxide and led to increases in cardiac index, left and right ventricular volumes, and right ventricular mass. Plasma levels of uremic toxins correlated well with ventricular morphology and function. Heart transcriptomes identified altered expression of 8 genes following NX and 894 genes following AVF+NX, whereas 290 and 1431 genes were altered in the kidney transcriptomes, respectively. Gene ontology and Kyoto Encyclopedia of Genes and Genomes analysis revealed gene expression changes related to cell division and immune activation in both organs, suppression of ribosomes and transcriptional activity in the heart, and altered renin-angiotensin signaling as well as chronodisruption in the kidney. All except the latter were worsened in AVF+NX compared with NX. Conclusions Inflammation and organ dysfunction in chronic kidney disease are exacerbated following AVF creation. Furthermore, our study provides important information for the discovery of novel biomarkers and therapeutic targets in the management of cardiorenal syndrome.