Evolutionary continuity and divergence of auditory dorsal and ventral pathways in primates revealed by ultra-high field diffusion MRI.

Auditory dorsal and ventral pathways in the human brain play important roles in supporting speech and language processing. However, the evolutionary root of the dual auditory pathways in the primate brain is unclear. By parcellating the auditory cortex of marmosets (a New World monkey species), macaques (an Old World monkey species), and humans using the same individual-based analysis method and tracking the pathways from the auditory cortex based on multi-shell diffusion-weighted MRI (dMRI), homologous auditory dorsal and ventral fiber tracks were identified in these primate species. The ventral pathway was found to be well conserved in all three primate species analyzed but extend to more anterior temporal regions in humans. In contrast, the dorsal pathway showed a divergence between monkey and human brains. First, frontal regions in the human brain have stronger connections to the higher-level auditory regions than to the lower-level auditory regions along the dorsal pathway, while frontal regions in the monkey brain show opposite connection patterns along the dorsal pathway. Second, the left lateralization of the dorsal pathway is only found in humans. Moreover, the connectivity strength of the dorsal pathway in marmosets is more similar to that of humans than macaques. These results demonstrate the continuity and divergence of the dual auditory pathways in the primate brains along the evolutionary path, suggesting that the putative neural networks supporting human speech and language processing might have emerged early in primate evolution.

Soluble PD-L1 reprograms blood monocytes to prevent cerebral edema and facilitate recovery after ischemic stroke.

Acute cerebral ischemia triggers a profound inflammatory response. While macrophages polarized to an M2-like phenotype clear debris and facilitate tissue repair, aberrant or prolonged macrophage activation is counterproductive to recovery. The inhibitory immune checkpoint Programmed Cell Death Protein 1 (PD-1) is upregulated on macrophage precursors (monocytes) in the blood after acute cerebrovascular injury. To investigate the therapeutic potential of PD-1 activation, we immunophenotyped circulating monocytes from patients and found that PD-1 expression was upregulated in the acute period after stroke. Murine studies using a temporary middle cerebral artery (MCA) occlusion (MCAO) model showed that intraperitoneal administration of soluble Programmed Death Ligand-1 (sPD-L1) significantly decreased brain edema and improved overall survival. Mice receiving sPD-L1 also had higher performance scores short-term, and more closely resembled sham animals on assessments of long-term functional recovery. These clinical and radiographic benefits were abrogated in global and myeloid-specific PD-1 knockout animals, confirming PD-1+ monocytes as the therapeutic target of sPD-L1. Single-cell RNA sequencing revealed that treatment skewed monocyte maturation to a non-classical Ly6Clo, CD43hi, PD-L1+ phenotype. These data support peripheral activation of PD-1 on inflammatory monocytes as a therapeutic strategy to treat neuroinflammation after acute ischemic stroke.

A multi-laboratory preclinical trial in rodents to assess treatment candidates for acute ischemic stroke.

Human diseases may be modeled in animals to allow preclinical assessment of putative new clinical interventions. Recent, highly publicized failures of large clinical trials called into question the rigor, design, and value of preclinical assessment. We established the Stroke Preclinical Assessment Network (SPAN) to design and implement a randomized, controlled, blinded, multi-laboratory trial for the rigorous assessment of candidate stroke treatments combined with intravascular thrombectomy. Efficacy and futility boundaries in a multi-arm multi-stage statistical design aimed to exclude from further study highly effective or futile interventions after each of four sequential stages. Six independent research laboratories performed a standard focal cerebral ischemic insult in five animal models that included equal numbers of males and females: young mice, young rats, aging mice, mice with diet-induced obesity, and spontaneously hypertensive rats. The laboratories adhered to a common protocol and efficiently enrolled 2615 animals with full data completion and comprehensive animal tracking. SPAN successfully implemented treatment masking, randomization, prerandomization inclusion and exclusion criteria, and blinded assessment of outcomes. The SPAN design and infrastructure provide an effective approach that could be used in similar preclinical, multi-laboratory studies in other disease areas and should help improve reproducibility in translational science.

Toward accurate cerebral blood flow estimation in mice after accounting for anesthesia.

Purpose: To improve the accuracy of cerebral blood flow (CBF) measurement in mice by accounting for the anesthesia effects. Methods: The dependence of CBF on anesthesia dose and time was investigated by simultaneously measuring respiration rate (RR) and heart rate (HR) under four different anesthetic regimens. Quantitative CBF was measured by a phase-contrast (PC) MRI technique. RR was evaluated with a mouse monitoring system (MouseOX) while HR was determined using an ultrashort-TE MRI sequence. CBF, RR, and HR were recorded dynamically with a temporal resolution of 1 min in a total of 19 mice. Linear regression models were used to investigate the relationships among CBF, anesthesia dose, RR, and HR. Results: CBF, RR, and HR all showed a significant dependence on anesthesia dose (p < 0.0001). However, the dose in itself was insufficient to account for the variations in physiological parameters, in that they showed a time-dependent change even for a constant dose. RR and HR together can explain 52.6% of the variations in CBF measurements, which is greater than the amount of variance explained by anesthesia dose (32.4%). Based on the multi-parametric regression results, a model was proposed to correct the anesthesia effects in mouse CBF measurements, specifically C B F c o r r e c t e d = C B F + 0.58 R R - 0.41 H R - 32.66 D o s e . We also reported awake-state CBF in mice to be 142.0 ± 8.8 mL/100 g/min, which is consistent with the model-predicted value. Conclusion: The accuracy of CBF measurement in mice can be improved by using a correction model that accounts for respiration rate, heart rate, and anesthesia dose.

The Stroke Preclinical Assessment Network: Rationale, Design, Feasibility, and Stage 1 Results.

Cerebral ischemia and reperfusion initiate cellular events in brain that lead to neurological disability. Investigating these cellular events provides ample targets for developing new treatments. Despite considerable work, no such therapy has translated into successful stroke treatment. Among other issues-such as incomplete mechanistic knowledge and faulty clinical trial design-a key contributor to prior translational failures may be insufficient scientific rigor during preclinical assessment: nonblinded outcome assessment; missing randomization; inappropriate sample sizes; and preclinical assessments in young male animals that ignore relevant biological variables, such as age, sex, and relevant comorbid diseases. Promising results are rarely replicated in multiple laboratories. We sought to address some of these issues with rigorous assessment of candidate treatments across 6 independent research laboratories. The Stroke Preclinical Assessment Network (SPAN) implements state-of-the-art experimental design to test the hypothesis that rigorous preclinical assessment can successfully reduce or eliminate common sources of bias in choosing treatments for evaluation in clinical studies. SPAN is a randomized, placebo-controlled, blinded, multilaboratory trial using a multi-arm multi-stage protocol to select one or more putative stroke treatments with an implied high likelihood of success in human clinical stroke trials. The first stage of SPAN implemented procedural standardization and experimental rigor. All participating research laboratories performed middle cerebral artery occlusion surgery adhering to a common protocol and rapidly enrolled 913 mice in the first of 4 planned stages with excellent protocol adherence, remarkable data completion and low rates of subject loss. SPAN stage 1 successfully implemented treatment masking, randomization, prerandomization inclusion/exclusion criteria, and blinded assessment to exclude bias. Our data suggest that a large, multilaboratory, preclinical assessment effort to reduce known sources of bias is feasible and practical. Subsequent SPAN stages will evaluate candidate treatments for potential success in future stroke clinical trials using aged animals and animals with comorbid conditions.

Unlabeled aspirin as an activatable theranostic MRI agent for breast cancer.

Rationale: Chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) is emerging as an alternative to gadolinium-based contrast MRI. We have evaluated the possibility of CEST MRI of orthotopic breast tumor xenografts with unlabeled aspirin's conversion to salicylic acid (SA) through various enzymatic activities, most notably inhibition of cyclooxygenase (COX)-1/-2 enzymes. Methods: We measured the COX-1/-2 expression in four breast cancer cell lines by Western Blot analysis and selected the highest and lowest expressing cell lines. We then performed CEST MRI following aspirin treatment to detect SA levels and ELISA to measure levels of downstream prostaglandin E2 (PGE2). We also injected aspirin into the tail vein of mice growing orthotopic tumor xenografts which expressed high and low COX-1/-2 and acquired SA CEST MR images of these tumor xenografts for up to 70 minutes. Tumors were then harvested to perform Western Blot and ELISA experiments to measure COX-1/-2 expression and PGE2 levels, respectively. Results: Western Blots determined that SUM159 cells contained significantly higher COX-1/-2 expression levels than MDA-MB-231 cells, in line with higher levels of downstream PGE2. SA CEST MRI yielded similar contrast at approximately 3% for both cell lines, independent of COX-1/-2 expression level. PGE2 levels decreased by about 50% following aspirin treatment. Results from our mouse study aligned with cultured cells, the overall SA CEST MRI contrast in both MDA-MB-231 and SUM159 tumor xenograft models was 5~8% at one hour post injection. PGE2 levels were ten times higher in SUM159 than MDA-MB-231 and decreased by 50%. The CEST contrast directly depended on the injected dose, with ~6%, ~3% and ~1.5% contrast observed following injection of 100 µL of 300 mM, 200 mM and 150 mM aspirin, respectively. Conclusions: Our data demonstrate the feasibility of using aspirin as a noninvasive activatable CEST MRI contrast agent for breast tumor detection.

Effects of red blood cells with reduced deformability on cerebral blood flow and vascular water transport: measurements in rats using time-resolved pulsed arterial spin labelling at 9.4 T.

BACKGROUND: Our aim was to introduce damaged red blood cells (RBCs) as a tool for haemodynamic provocation in rats, hypothesised to cause decreased cerebral blood flow (CBF) and prolonged water capillary transfer time (CTT), and to investigate whether expected changes in CBF could be observed and if haemodynamic alterations were reflected by the CTT metric. METHODS: Damaged RBCs exhibiting a mildly reduced deformability were injected to cause aggregation of RBCs. Arterial spin labelling (ASL) magnetic resonance imaging experiments were performed at 9.4 T. Six datasets (baseline plus five datasets after injection) were acquired for each animal in a study group and a control group (13 and 10 female adult Wistar rats, respectively). For each dataset, ASL images at ten different inversion times were acquired. The CTT model was adapted to the use of a measured arterial input function, implying the use of a realistic labelling profile. Repeated measures ANOVA was used (alpha error = 0.05). RESULTS: After injection, significant differences between the study group and control group were observed for relative CBF in white matter (up to 20 percentage points) and putamen (up to 18-20 percentage points) and for relative CTT in putamen (up to 35-40 percentage points). CONCLUSIONS: Haemodynamic changes caused by injection of damaged RBCs were observed by ASL-based CBF and CTT measurements. Damaged RBCs can be used as a tool for test and validation of perfusion imaging modalities. CTT model fitting was challenging to stabilise at experimental signal-to-noise ratio levels, and the number of free parameters was minimised.

Classifications of atherosclerotic plaque components with T1 and T2* mapping in 11.7 T MRI.

BACKGROUND AND AIMS: Histopathology is the gold standard for analysis of atherosclerotic plaques but has drawbacks due to the destructive nature of the method. Ex vivo MRI is a non-destructive method to image whole plaques. Our aim was to use quantitative high field ex vivo MRI to classify plaque components, with histology as gold standard. METHODS: Surgically resected carotid plaques from 12 patients with recent TIA or stroke were imaged at 11.7 T MRI. Quantitative T1/T2* mapping sequences and qualitative T1/T2* gradient echo sequences with voxel size of 30 × 30 × 60 µm3 were obtained prior to histological preparation, sectioning and staining for lipids, inflammation, hemorrhage, and fibrous tissue. Regions of interest (ROI) were selected based on the histological staining at multiple levels matched between histology and MRI. The MRI parameters of each ROI were then analyzed with quadratic discriminant analysis (QDA) for classification. RESULTS: A total of 965 ROIs, at 70 levels matched between histology and MRI, were registered based on histological staining. In the nine plaques where three or more plaque components were possible to co-localize with MRI, the mean degree of misclassification by QDA was 16.5 %. One of the plaques contained mostly fibrous tissue and lipids and had no misclassifications, and two plaques mostly contained fibrous tissue. QDA generally showed good classification for fibrous tissue and lipids, whereas plaques with hemorrhage and inflammation had more misclassifications. CONCLUSION: 11.7 T ex vivo high field MRI shows good visual agreement with histology in carotid plaques. T1/T2* maps analyzed with QDA is a promising non-destructive method to classify plaque components, but with a higher degree of misclassifications in plaques with hemorrhage or inflammation.

Mutant G2019S-LRRK2 Induces Abnormalities in Arteriolar Cerebral Blood Volume in Mouse Brains: An MRI Study.

BACKGROUND: Parkinson's disease (PD) is the second most common neurodegenerative disease and the most common movement disorder characterized by motor impairments resulting from midbrain dopamine neuron loss. Abnormalities in small pial arteries and arterioles, which are the primary pathways of local delivery of nutrients and oxygen in brain tissue, have been reported in many neurodegenerative diseases including PD. Mutations in LRRK2 cause genetic PD and contribute to sporadic PD. The most common PD-linked mutation LRRK2 G2019S contributes 20-47% of genetic forms of PD in Caucasian populations. The human LRRK2 G2019S transgenic mouse model displays PD-like movement impairment and was used to identify novel LRRK2 inhibitors, which provides a useful model for studying microvascular abnormalities in PD. OBJECTIVES: To investigate abnormalities in arteriolar cerebral blood volume (CBVa) in various brain regions using the inflow-based vascular-space occupancy (iVASO) MRI technique in LRRK2 mouse models of PD. METHODS: Anatomical and iVASO MRI scans were performed in 5 female and 7 male nontransgenic (nTg), 3 female and 4 male wild-type (WT) LRRK2, and 5 female and 7 male G2019S-LRRK2 mice of 9 months of age. CBVa was calculated and compared in the substantia nigra (SN), olfactory cortex, and prefrontal cortex. RESULTS: Compared to nTg mice, G2019S-LRRK2 mice showed decreased CBVa in the SN, but increased CBVa in the olfactory and prefrontal cortex in both male and female groups, whereas WT-LRRK2 mice showed no change in CBVa in the SN (male and female), the olfactory (female), and prefrontal (female) cortex, but a slight increase in CBVa in the olfactory and prefrontal cortex in the male group only. CONCLUSIONS: Alterations in the blood volume of small arteries and arterioles (CBVa) were detected in the G2019S-LRRK2 mouse model of PD. The opposite changes in CBVa in the SN and the cortex indicate that PD pathology may have differential effects in different brain regions. Our results suggest the potential value of CBVa as a marker for clinical PD studies.

White Matter Brain Development after Exposure to Circulating Cell-Free Hemoglobin and Hyperoxia in a Rat Pup Model.

Neonates born with critical congenital heart defects are at risk of diffuse white matter injuries and neurodevelopmental impairments. This study aimed to determine the impact of circulating cell-free hemoglobin and hyperoxia, both present during cardiopulmonary bypass circulation, on white matter brain development. Postnatal day 6 rat pups were injected intraperitoneally with cell-free Hb or vehicle and exposed to hyperoxia (fiO2 = 0.8) or normoxia (fiO2 = 0.21) for 24 h. We evaluated apoptosis, myelination, and oligodendrocyte maturation with immunohistochemistry, gene and protein analyses, and in vivo diffusion tensor magnetic resonance imaging (MRI). Consistent with previous studies, we found an increase in apoptosis of oligodendrocytes as determined by TUNEL+ staining in Olig2+ cells in white matter, cortex, thalamus, and hippocampus following exposure to hyperoxia with no additional effect of cell-free Hb. A transient increase in the mRNA expression of intercellular adhesion molecule 1 at 6 h was observed following combined exposure to cell-free Hb and hyperoxia. No indications of oligodendrocyte maturational delay or hypomyelination were observed after either insult, delivered separately or combined, as determined by immunohistochemistry, Western blot, and diffusion tensor MRI. In our model, exposure to circulatory cell-free Hb, with or without concomitant hyperoxia, did not significantly alter brain white matter development.

Measurement of vascular water transport in human subjects using time-resolved pulsed arterial spin labelling.

Most approaches to arterial spin labelling (ASL) data analysis aim to provide a quantitative measure of the cerebral blood flow (CBF). This study, however, focuses on the measurement of the transfer time of blood water through the capillaries to the parenchyma (referred to as the capillary transfer time, CTT) as an alternative parameter to characterise the haemodynamics of the system. The method employed is based on a non-compartmental model, and no measurements need to be added to a common time-resolved ASL experiment. Brownian motion of labelled spins in a potential was described by a one-dimensional general Langevin equation as the starting point, and as a Fokker-Planck differential equation for the averaged distribution of labelled spins at the end point, which takes into account the effects of flow and dispersion of labelled water by the pseudorandom nature of the microvasculature and the transcapillary permeability. Multi-inversion time (multi-TI) ASL data were acquired in 14 healthy subjects on two occasions in a test-retest design, using a pulsed ASL sequence and three-dimensional gradient and spin echo (3D-GRASE) readout. Based on an error analysis to predict the size of a region of interest (ROI) required to obtain reasonably precise parameter estimates, data were analysed in two relatively large ROIs, i.e. the occipital lobe (OC) and the insular cortex (IC). The average values of CTT in OC were 260 ± 60 ms in the first experiment and 270 ± 60 ms in the second experiment. The corresponding IC values were 460 ± 130 ms and 420 ± 139 ms, respectively. Information related to the water transfer time may be important for diagnostics and follow-up of cerebral conditions or diseases characterised by a disrupted blood-brain barrier or disturbed capillary blood flow.

(68)Ga-labeled superparamagnetic iron oxide nanoparticles (SPIONs) for multi-modality PET/MR/Cherenkov luminescence imaging of sentinel lymph nodes.

The aim of this study was to develop (68)Ga-SPIONs for use as a single contrast agent for dynamic, quantitative and high resolution PET/MR imaging of Sentinel Lymph Node (SLN). In addition (68)Ga enables Cherenkov light emission which can be used for optical guidance during resection of SLN. SPIONs were labeled with (68)Ga in ammonium acetate buffer, pH 5.5. The labeling yield and stability in human serum were determined using instant thin layer chromatography. An amount of 0.07-0.1 mL (~5-10 MBq, 0.13 mg Fe) of (68)Ga-SPIONs was subcutaneously injected in the hind paw of rats. The animals were imaged at 0-3 h and 25 h post injection with PET/CT, 9.4 T MR and CCDbased Cherenkov optical systems. A biodistribution study was performed by dissecting and measuring the radioactivity in lymph nodes, kidneys, spleen, liver and the injection site. The labeling yield was 97.3 ± 0.05% after 15 min and the (68)Ga-SPIONs were stable in human serum. PET, MR and Cherenkov luminescence imaging clearly visualized the SLN. Biodistribution confirmed a high uptake of the (68)Ga-SPIONs within the SLN. We conclude that generator produced (68)Ga can be labeled to SPIONs. Subcutaneously injected (68)Ga-SPIONs can enhance the identification of the SLNs by combining sensitive PET and high resolution MR imaging. Clinically, hybrid PET/MR cameras are already in use and (68)Ga-SPIONs have a great potential as a single-dose, tri-modality agent for diagnostic imaging and potential Cherenkov luminescent guided resection of SLN.

Denoising of arterial spin labeling data: wavelet-domain filtering compared with Gaussian smoothing.

PURPOSE: To investigate a wavelet-based filtering scheme for denoising of arterial spin labeling (ASL) data, potentially enabling reduction of the required number of averages and the acquisition time. METHODS: ASL magnetic resonance imaging (MRI) provides quantitative perfusion maps by using arterial water as an endogenous tracer. The signal difference between a labeled image, where inflowing arterial spins are inverted, and a control image is proportional to blood perfusion. ASL perfusion maps suffer from low SNR, and the experiment must be repeated a number of times (typically more than 40) to achieve adequate image quality. In this study, systematic errors introduced by the proposed wavelet-domain filtering approach were investigated in simulated and experimental image datasets and compared with conventional Gaussian smoothing. RESULTS: Application of the proposed method enabled a reduction of the number of averages and the acquisition time by at least 50% with retained standard deviation, but with effects on absolute CBF values close to borders and edges. CONCLUSIONS: When the ASL perfusion maps showed moderate-to-high SNRs, wavelet-domain filtering was superior to Gaussian smoothing in the vicinity of borders between gray and white matter, while Gaussian smoothing was a better choice for larger homogeneous areas, irrespective of SNR.

Denoising of complex MRI data by wavelet-domain filtering: application to high-b-value diffusion-weighted imaging.

The Rician distribution of noise in magnitude magnetic resonance (MR) images is particularly problematic in low signal-to-noise ratio (SNR) regions. The Rician noise distribution causes a nonzero minimum signal in the image, which is often referred to as the rectified noise floor. True low signal is likely to be concealed in the noise, and quantification is severely hampered in low-SNR regions. To address this problem we performed noise reduction (or denoising) by Wiener-like filtering in the wavelet domain. The filtering was applied to complex MRI data before construction of the magnitude image. The noise-reduction algorithm was applied to simulated and experimental diffusion-weighted (DW) images. Denoising considerably reduced the signal standard deviation (SD, by up to 87% in simulated images) and decreased the background noise floor (by approximately a factor of 6 in simulated and experimental images).