Microglia protect against age-associated brain pathologies.

Microglia are brain-resident macrophages that contribute to central nervous system (CNS) development, maturation, and preservation. Here, we examine the consequences of permanent microglial deficiencies on brain aging using the Csf1rΔFIRE/ΔFIRE mouse model. In juvenile Csf1rΔFIRE/ΔFIRE mice, we show that microglia are dispensable for the transcriptomic maturation of other brain cell types. By contrast, with advancing age, pathologies accumulate in Csf1rΔFIRE/ΔFIRE brains, macroglia become increasingly dysregulated, and white matter integrity declines, mimicking many pathological features of human CSF1R-related leukoencephalopathy. The thalamus is particularly vulnerable to neuropathological changes in the absence of microglia, with atrophy, neuron loss, vascular alterations, macroglial dysregulation, and severe tissue calcification. We show that populating Csf1rΔFIRE/ΔFIRE brains with wild-type microglia protects against many of these pathological changes. Together with the accompanying study by Chadarevian and colleagues1, our results indicate that the lifelong absence of microglia results in an age-related neurodegenerative condition that can be counteracted via transplantation of healthy microglia.

Tri-modal imaging of gold-dotted magnetic nanoparticles for magnetic resonance imaging, computed tomography and intravascular ultrasound: an in vitro study.

Aim: To examine the multimodal contrasting ability of gold-dotted magnetic nanoparticles (Au*MNPs) for magnetic resonance (MR), computed tomography (CT) and intravascular ultrasound (IVUS) imaging. Materials & methods: Au*MNPs were prepared by adapting an impregnation method, without using surface capping reagents and characterized (transmission electron microscopy, x-ray diffraction and Fourier-transform infrared spectroscopy) with their in vitro cytotoxicity assessed, followed by imaging assessments. Results: The contrast-enhancing ability of Au*MNPs was shown to be concentration-dependent across MR, CT and IVUS imaging. The Au content of the Au*MNP led to evident increases of the IVUS signal. Conclusion: We demonstrated that Au*MNPs showed concentration-dependent contrast-enhancing ability in MRI and CT imaging, and for the first-time in IVUS imaging due to the Au content. These Au*MNPs are promising toward solidifying tri-modal imaging-based theragnostics.

Progression and regression of left ventricular hypertrophy and myocardial fibrosis in a mouse model of hypertension and concomitant cardiomyopathy.

BACKGROUND: Myocardial fibrosis is observed in multiple cardiac conditions including hypertension and aortic stenosis. Excessive fibrosis is associated with adverse clinical outcomes, but longitudinal human data regarding changes in left ventricular remodelling and fibrosis over time are sparse because of the slow progression, thereby making longitudinal studies challenging. The purpose of this study was to establish and characterize a mouse model to study the development and regression of left ventricular hypertrophy and myocardial fibrosis in response to increased blood pressure and to understand how these processes reverse remodel following normalisation of blood pressure. METHODS: We performed a longitudinal study with serial cardiovascular magnetic resonance (CMR) imaging every 2 weeks in mice (n = 31) subjected to angiotensin II-induced hypertension for 6 weeks and investigated reverse remodelling following normalisation of afterload beyond 6 weeks (n = 9). Left ventricular (LV) volumes, mass, and function as well as myocardial fibrosis were measured using cine CMR and the extracellular volume fraction (ECV) s. RESULTS: Increased blood pressure (65 ± 12 vs 85 ± 9 mmHg; p < 0.001) resulted in higher indices of LV hypertrophy (0.09 [0.08, 0.10] vs 0.12 [0.11, 0.14] g; p < 0.001) and myocardial fibrosis (ECV: 0.24 ± 0.03 vs 0.30 ± 0.02; p < 0.001) whilst LV ejection fraction fell (LVEF, 59.3 [57.6, 59.9] vs 46.9 [38.5, 49.6] %; p < 0.001). We found a strong correlation between ECV and histological myocardial fibrosis (r = 0.89, p < 0.001). Following cessation of angiotensin II and normalisation of blood pressure (69 ± 5 vs baseline 65 ± 12 mmHg; p = 0.42), LV mass (0.11 [0.10, 0.12] vs 0.09 [0.08, 0.11] g), ECV (0.30 ± 0.02 vs 0.27 ± 0.02) and LVEF (51.1 [42.9, 52.8] vs 59.3 [57.6, 59.9] %) improved but remained impaired compared to baseline (p < 0.05 for all). There was a strong inverse correlation between LVEF and %ECV during both systemic hypertension (r = - 0.88, p < 0.001) and the increases in ECV observed in the first two weeks of increased blood pressure predicted the reduction in LVEF after 6 weeks (r = - 0.77, p < 0.001). CONCLUSIONS: We have established and characterized angiotensin II infusion and repeated CMR imaging as a model of LV hypertrophy and reverse remodelling in response to systemic hypertension. Changes in myocardial fibrosis and alterations in cardiac function are only partially reversible following relief of hypertension.

Manganese-enhanced T1 mapping to quantify myocardial viability: validation with 18F-fluorodeoxyglucose positron emission tomography.

Gadolinium chelates are widely used in cardiovascular magnetic resonance imaging (MRI) as passive intravascular and extracellular space markers. Manganese, a biologically active paramagnetic calcium analogue, provides novel intracellular myocardial tissue characterisation. We previously showed manganese-enhanced MRI (MEMRI) more accurately quantifies myocardial infarction than gadolinium delayed-enhancement MRI (DEMRI). Here, we evaluated the potential of MEMRI to assess myocardial viability compared to gold-standard 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET) viability. Coronary artery ligation surgery was performed in male Sprague-Dawley rats (n = 13) followed by dual MEMRI and 18F-FDG PET imaging at 10-12 weeks. MEMRI was achieved with unchelated (EVP1001-1) or chelated (mangafodipir) manganese. T1 mapping MRI was followed by 18F-FDG micro-PET, with tissue taken for histological correlation. MEMRI and PET demonstrated good agreement with histology but native T1 underestimated infarct size. Quantification of viability by MEMRI, PET and MTC were similar, irrespective of manganese agent. MEMRI showed superior agreement with PET than native T1. MEMRI showed excellent agreement with PET and MTC viability. Myocardial MEMRI T1 correlated with 18F-FDG standard uptake values and influx constant but not native T1. Our findings indicate that MEMRI identifies and quantifies myocardial viability and has major potential for clinical application in myocardial disease and regenerative therapies.

Non-invasive assessment of liver disease in rats using multiparametric magnetic resonance imaging: a feasibility study.

Non-invasive quantitation of liver disease using multiparametric magnetic resonance imaging (MRI) could refine clinical care pathways, trial design and preclinical drug development. The aim of this study was to evaluate the use of multiparametric MRI in experimental models of liver disease. Liver injury was induced in rats using 4 or 12 weeks of carbon tetrachloride (CCl4) intoxication and 4 or 8 weeks on a methionine and choline deficient (MCD) diet. Liver MRI was performed using a 7.0 Tesla small animal scanner at baseline and specified timepoints after liver injury. Multiparametric liver MRI parameters [T1 mapping, T2* mapping and proton density fat fraction (PDFF)] were correlated with gold standard histopathological measures. Mean hepatic T1 increased significantly in rats treated with CCl4 for 12 weeks compared to controls [1122±78 ms versus 959±114 ms; d=162.7, 95% CI (11.92, 313.4), P=0.038] and correlated strongly with histological collagen content (rs=0.717, P=0.037). In MCD diet-treated rats, hepatic PDFF correlated strongly with histological fat content (rs=0.819, P<0.0001), steatosis grade (rs=0.850, P<0.0001) and steatohepatitis score (rs=0.818, P<0.0001). Although there was minimal histological iron, progressive fat accumulation in MCD diet-treated livers significantly shortened T2*. In preclinical models, quantitative MRI markers correlated with histopathological assessments, especially for fatty liver disease. Validation in longitudinal studies is required.This article has an associated First Person interview with the first author of the paper.

Serelaxin as a potential treatment for renal dysfunction in cirrhosis: Preclinical evaluation and results of a randomized phase 2 trial.

BACKGROUND: Chronic liver scarring from any cause leads to cirrhosis, portal hypertension, and a progressive decline in renal blood flow and renal function. Extreme renal vasoconstriction characterizes hepatorenal syndrome, a functional and potentially reversible form of acute kidney injury in patients with advanced cirrhosis, but current therapy with systemic vasoconstrictors is ineffective in a substantial proportion of patients and is limited by ischemic adverse events. Serelaxin (recombinant human relaxin-2) is a peptide molecule with anti-fibrotic and vasoprotective properties that binds to relaxin family peptide receptor-1 (RXFP1) and has been shown to increase renal perfusion in healthy human volunteers. We hypothesized that serelaxin could ameliorate renal vasoconstriction and renal dysfunction in patients with cirrhosis and portal hypertension. METHODS AND FINDINGS: To establish preclinical proof of concept, we developed two independent rat models of cirrhosis that were characterized by progressive reduction in renal blood flow and glomerular filtration rate and showed evidence of renal endothelial dysfunction. We then set out to further explore and validate our hypothesis in a phase 2 randomized open-label parallel-group study in male and female patients with alcohol-related cirrhosis and portal hypertension. Forty patients were randomized 1:1 to treatment with serelaxin intravenous (i.v.) infusion (for 60 min at 80 µg/kg/d and then 60 min at 30 µg/kg/d) or terlipressin (single 2-mg i.v. bolus), and the regional hemodynamic effects were quantified by phase contrast magnetic resonance angiography at baseline and after 120 min. The primary endpoint was the change from baseline in total renal artery blood flow. Therapeutic targeting of renal vasoconstriction with serelaxin in the rat models increased kidney perfusion, oxygenation, and function through reduction in renal vascular resistance, reversal of endothelial dysfunction, and increased activation of the AKT/eNOS/NO signaling pathway in the kidney. In the randomized clinical study, infusion of serelaxin for 120 min increased total renal arterial blood flow by 65% (95% CI 40%, 95%; p < 0.001) from baseline. Administration of serelaxin was safe and well tolerated, with no detrimental effect on systemic blood pressure or hepatic perfusion. The clinical study's main limitations were the relatively small sample size and stable, well-compensated population. CONCLUSIONS: Our mechanistic findings in rat models and exploratory study in human cirrhosis suggest the therapeutic potential of selective renal vasodilation using serelaxin as a new treatment for renal dysfunction in cirrhosis, although further validation in patients with more advanced cirrhosis and renal dysfunction is required. TRIAL REGISTRATION: ClinicalTrials.gov NCT01640964.

Functionalized superparamagnetic iron oxide nanoparticles provide highly efficient iron-labeling in macrophages for magnetic resonance-based detection in vivo.

BACKGROUND AIMS: Tracking cells during regenerative cytotherapy is crucial for monitoring their safety and efficacy. Macrophages are an emerging cell-based regenerative therapy for liver disease and can be readily labeled for medical imaging. A reliable, clinically applicable cell-tracking agent would be a powerful tool to study cell biodistribution. METHODS: Using a recently described chemical design, we set out to functionalize, optimize and characterize a new set of superparamagnetic iron oxide nanoparticles (SPIONs) to efficiently label macrophages for magnetic resonance imaging-based cell tracking in vivo. RESULTS: A series of cell health and iron uptake assays determined that positively charged SPIONs (+16.8 mV) could safely label macrophages more efficiently than the formerly approved ferumoxide (-6.7 mV; Endorem) and at least 10 times more efficiently than the clinically approved SPION ferumoxytol (-24.2 mV; Rienso). An optimal labeling time of 4 h at 25 µg/mL was demonstrated to label macrophages of mouse and human origin without any adverse effects on cell viability whilst providing substantial iron uptake (>5 pg Fe/cell) that was retained for 7 days in vitro. SPION labeling caused no significant reduction in phagocytic activity and a shift toward a reversible M1-like phenotype in bone marrow-derived macrophages (BMDMs). Finally, we show that SPION-labeled BMDMs delivered via the hepatic portal vein to mice are localized in the hepatic parenchyma resulting in a 50% drop in T2* in the liver. Engraftment of exogenous cells was confirmed via immunohistochemistry up to 3 weeks posttransplantation. DISCUSSION: A positively charged dextran-coated SPION is a promising tool to noninvasively track hepatic macrophage localization for therapeutic monitoring.

Multicenter Evaluation of Geometric Accuracy of MRI Protocols Used in Experimental Stroke.

It has recently been suggested that multicenter preclinical stroke studies should be carried out to improve translation from bench to bedside, but the accuracy of magnetic resonance imaging (MRI) scanners routinely used in experimental stroke has not yet been evaluated. We aimed to assess and compare geometric accuracy of preclinical scanners and examine the longitudinal stability of one scanner using a simple quality assurance (QA) protocol. Six 7 Tesla animal scanners across six different preclinical imaging centers throughout Europe were used to scan a small structural phantom and estimate linear scaling errors in all orthogonal directions and volumetric errors. Between-scanner imaging consisted of a standard sequence and each center's preferred sequence for the assessment of infarct size in rat models of stroke. The standard sequence was also used to evaluate the drift in accuracy of the worst performing scanner over a period of six months following basic gradient calibration. Scaling and volumetric errors using the standard sequence were less variable than corresponding errors using different stroke sequences. The errors for one scanner, estimated using the standard sequence, were very high (above 4% scaling errors for each orthogonal direction, 18.73% volumetric error). Calibration of the gradient coils in this system reduced scaling errors to within ±1.0%; these remained stable during the subsequent 6-month assessment. In conclusion, despite decades of use in experimental studies, preclinical MRI still suffers from poor and variable geometric accuracy, influenced by the use of miscalibrated systems and various types of sequences for the same purpose. For effective pooling of data in multicenter studies, centers should adopt standardized procedures for system QA and in vivo imaging.

Imaging learned fear circuitry in awake mice using fMRI.

Functional magnetic resonance imaging (fMRI) of learned behaviour in 'awake rodents' provides the opportunity for translational preclinical studies into the influence of pharmacological and genetic manipulations on brain function. fMRI has recently been employed to investigate learned behaviour in awake rats. Here, this methodology is translated to mice, so that future fMRI studies may exploit the vast number of genetically modified mouse lines that are available. One group of mice was conditioned to associate a flashing light (conditioned stimulus, CS) with foot shock (PG; paired group), and another group of mice received foot shock and flashing light explicitly unpaired (UG; unpaired group). The blood oxygen level-dependent signal (proxy for neuronal activation) in response to the CS was measured 24 h later in awake mice from the PG and UG using fMRI. The amygdala, implicated in fear processing, was activated to a greater degree in the PG than in the UG in response to the CS. Additionally, the nucleus accumbens was activated in the UG in response to the CS. Because the CS signalled an absence of foot shock in the UG, it is possible that this region is involved in processing the safety aspect of the CS. To conclude, the first use of fMRI to visualise brain activation in awake mice that are completing a learned emotional task is reported. This work paves the way for future preclinical fMRI studies to investigate genetic and environmental influences on brain function in transgenic mouse models of disease and aging.

Complement inhibition and statins prevent fetal brain cortical abnormalities in a mouse model of preterm birth.

Premature babies are particularly vulnerable to brain injury. In this study we focus on cortical brain damage associated with long-term cognitive, behavioral, attentional or socialization deficits in children born preterm. Using a mouse model of preterm birth (PTB), we demonstrated that complement component C5a contributes to fetal cortical brain injury. Disruption of cortical dendritic and axonal cytoarchitecture was observed in PTB-mice. Fetuses deficient in C5aR (-/-) did not show cortical brain damage. Treatment with antibody anti-C5, that prevents generation of C5a, also prevented cortical fetal brain injury in PTB-mice. C5a also showed a detrimental effect on fetal cortical neuron development and survival in vitro. Increased glutamate release was observed in cortical neurons in culture exposed to C5a. Blockade of C5aR prevented glutamate increase and restored neurons dendritic and axonal growth and survival. Similarly, increased glutamate levels - measured by (1)HMRS - were observed in vivo in PTB-fetuses compared to age-matched controls. The blockade of glutamate receptors prevented C5a-induced abnormal growth and increased cell death in isolated fetal cortical neurons. Simvastatin and pravastatin prevented cortical fetal brain developmental and metabolic abnormalities -in vivo and in vitro. Neuroprotective effects of statins were mediated by Akt/PKB signaling pathways. This study shows that complement activation plays a crucial role in cortical fetal brain injury in PTL and suggests that complement inhibitors and statins might be good therapeutic options to improve neonatal outcomes in preterm birth.

An anatomically unbiased approach for analysis of renal BOLD magnetic resonance images.

Oxygenation defects may contribute to renal disease progression, but the chronology of events is difficult to define in vivo without recourse to invasive methodologies. Blood oxygen level-dependent magnetic resonance imaging (BOLD MRI) provides an attractive alternative, but the R2* signal is physiologically complex. Postacquisition data analysis often relies on manual selection of region(s) of interest. This approach excludes from analysis significant quantities of biological information and is subject to selection bias. We present a semiautomated, anatomically unbiased approach to compartmentalize voxels into two quantitatively related clusters. In control F344 rats, low R2* clustering was located predominantly within the cortex and higher R2* clustering within the medulla (70.96 ± 1.48 vs. 79.00 ± 1.50; 3 scans per rat; n = 6; P < 0.01) consistent anatomically with a cortico-medullary oxygen gradient. An intravenous bolus of acetylcholine caused a transient reduction of the R2* signal in both clustered segments (P < 0.01). This was nitric oxide dependent and temporally distinct from the hemodynamic effects of acetylcholine. Rats were then chronically infused with angiotensin II (60 ng/min) and rescanned 3 days later. Clustering demonstrated a disruption of the cortico-medullary gradient, producing less distinctly segmented mean R2* clusters (71.30 ± 2.00 vs. 72.48 ± 1.27; n = 6; NS). The acetylcholine-induced attenuation of the R2* signal was abolished by chronic angiotensin II infusion, consistent with reduced nitric oxide bioavailability. This global map of oxygenation, defined by clustering individual voxels on the basis of quantitative nearness, might be more robust in defining deficits in renal oxygenation than the absolute magnitude of R2* in small, manually selected regions of interest defined exclusively by anatomical nearness.