Vascular Origins of BOLD and CBV fMRI Signals: Statistical Mapping and Histological Sections Compared

Comparison of 3T blood oxygenation level dependent (BOLD) and cerebral blood volume (CBV) activation maps to histological sections enables the spatial discrimination of functional magnetic resonance imaging (fMRI) signal changes into different vascular compartments. We use a standard gradient echo–echo planar imaging technique to measure BOLD signal changes in the somatosensory cortex in response to whisker stimulation. Corresponding changes in CBV were estimated following the infusion of a super-paramagnetic contrast agent. We imaged in a tangential imaging plane that covered the cortical surface. Images were associated with post mortem histological sections showing both the surface vasculature and cytochrome oxidase stained whisker barrel cortex. We found a significant BOLD signal change in the large draining veins which occurred in the absence of a corresponding CBV change. Results suggest that in the venous drainage system, ~3mm distant from the area of activity, there is a robust change in blood oxygen saturation with little or no volume change. CBV changes are localised over the somatosensory barrel cortex and overlying arterial supply, supporting the theory that CBV changes are greater in the arterial than in the venous vasculature. This work investigating BOLD signal and underlying hemodynamics provides more information on the vascular origins of these important neuroimaging signals

Cortical lamina-dependent blood volume changes in human brain at 7T

Cortical layer-dependent high (sub-millimeter) resolution functional magnetic resonance imaging (fMRI) in human or animal brain can be used to address questions regarding the functioning of cortical circuits, such as the effect of different afferent and efferent connectivities on activity in specific cortical layers. The sensitivity of gradient echo (GE) blood oxygenation level-dependent (BOLD) responses to large draining veins reduces its local specificity and can render the interpretation of the underlying laminar neural activity impossible. The application of the more spatially specific cerebral blood volume (CBV)-based fMRI in humans has been hindered by the low sensitivity of the noninvasive modalities available. Here, a vascular space occupancy (VASO) variant, adapted for use at high field, is further optimized to capture layer-dependent activity changes in human motor cortex at sub-millimeter resolution. Acquired activation maps and cortical profiles show that the VASO signal peaks in gray matter at 0.8–1.6 mm depth, and deeper compared to the superficial and vein-dominated GE-BOLD responses. Validation of the VASO signal change versus well-established iron-oxide contrast agent based fMRI methods in animals showed the same cortical profiles of CBV change, after normalization for lamina-dependent baseline CBV. In order to evaluate its potential of revealing small lamina-dependent signal differences due to modulations of the input-output characteristics, layer-dependent VASO responses were investigated in the ipsilateral hemisphere during unilateral finger tapping. Positive activation in ipsilateral primary motor cortex and negative activation in ipsilateral primary sensory cortex were observed. This feature is only visible in high-resolution fMRI where opposing sides of a sulcus can be investigated independently because of a lack of partial volume effects. Based on the results presented here, we conclude that VASO offers good reproducibility, high sensitivity and lower sensitivity than GE-BOLD to changes in larger vessels, making it a valuable tool for layer-dependent fMRI studies in humans

Real-Time High-Sensitivity Reaction Monitoring of Important Nitrogen-Cycle Synthons by 15N Hyperpolarized Nuclear Magnetic Resonance

Here, we show how signal amplification by reversible exchange hyperpolarization of a range of 15N-containing synthons can be used to enable studies of their reactivity by 15N nuclear magnetic resonance (NO 2 - (28% polarization), ND 3 (3%), PhCH 2NH 2 (5%), NaN 3 (3%), and NO 3 - (0.1%)). A range of iridium-based spin-polarization transfer catalysts are used, which for NO 2 - work optimally as an amino-derived carbene-containing complex with a DMAP- d 2 coligand. We harness long 15N spin-order lifetimes to probe in situ reactivity out to 3 × T 1. In the case of NO 2 - ( T 1 17.7 s at 9.4 T), we monitor PhNH 2 diazotization in acidic solution. The resulting diazonium salt ( 15N- T 1 38 s) forms within 30 s, and its subsequent reaction with NaN 3 leads to the detection of hyperpolarized PhN 3 ( T 1 192 s) in a second step via the formation of an identified cyclic pentazole intermediate. The role of PhN 3 and NaN 3 in copper-free click chemistry is exemplified for hyperpolarized triazole ( T 1 < 10 s) formation when they react with a strained alkyne. We also demonstrate simple routes to hyperpolarized N 2 in addition to showing how utilization of 15N-polarized PhCH 2NH 2 enables the probing of amidation, sulfonamidation, and imine formation. Hyperpolarized ND 3 is used to probe imine and ND 4 + ( T 1 33.6 s) formation. Furthermore, for NO 2 -, we also demonstrate how the 15N-magnetic resonance imaging monitoring of biphasic catalysis confirms the successful preparation of an aqueous bolus of hyperpolarized 15NO 2 - in seconds with 8% polarization. Hence, we create a versatile tool to probe organic transformations that has significant relevance for the synthesis of future hyperpolarized pharmaceuticals

Investigation of the neurovascular coupling in positive and negative BOLD responses in human brain at 7T

Decreases in stimulus-dependent blood oxygenation level dependent (BOLD) signal and their underlying neurovascular origins have recently gained considerable interest. In this study a multi-echo, BOLD-corrected vascular space occupancy (VASO) functional magnetic resonance imaging (fMRI) technique was used to investigate neurovascular responses during stimuli that elicit positive and negative BOLD responses in human brain at 7 T. Stimulus-induced BOLD, cerebral blood volume (CBV), and cerebral blood flow (CBF) changes were measured and analyzed in ‘arterial’ and ‘venous’ blood compartments in macro- and microvasculature. We found that the overall interplay of mean CBV, CBF and BOLD responses is similar for tasks inducing positive and negative BOLD responses. Some aspects of the neurovascular coupling however, such as the temporal response, cortical depth dependence, and the weighting between ‘arterial’ and ‘venous’ contributions, are significantly different for the different task conditions. Namely, while for excitatory tasks the BOLD response peaks at the cortical surface, and the CBV change is similar in cortex and pial vasculature, inhibitory tasks are associated with a maximum negative BOLD response in deeper layers, with CBV showing strong constriction of surface arteries and a faster return to baseline. The different interplays of CBV, CBF and BOLD during excitatory and inhibitory responses suggests different underlying hemodynamic mechanisms

Physiological and pathological brain activation in the anesthetized rat produces hemodynamic-dependent cortical temperature increases that can confound the BOLD fMRI signal

Anesthetized rodent models are ubiquitous in pre-clinical neuroimaging studies. However, because the associated cerebral morphology and experimental methodology results in a profound negative brain-core temperature differential, cerebral temperature changes during functional activation are likely to be principally driven by local inflow of fresh, core-temperature, blood. This presents a confound to the interpretation of blood-oxygenation level-dependent (BOLD) functional magnetic resonance imaging (fMRI) data acquired from such models, since this signal is also critically temperature-dependent. Nevertheless, previous investigation on the subject is surprisingly sparse. Here, we address this issue through use of a novel multi-modal methodology in the urethane anesthetized rat. We reveal that sensory stimulation, hypercapnia and recurrent acute seizures induce significant increases in cortical temperature that are preferentially correlated to changes in total hemoglobin concentration (Hbt), relative to cerebral blood flow and oxidative metabolism. Furthermore, using a phantom-based evaluation of the effect of such temperature changes on the BOLD fMRI signal, we demonstrate a robust inverse relationship between both variables. These findings suggest that temperature increases, due to functional hyperemia, should be accounted for to ensure accurate interpretation of BOLD fMRI signals in pre-clinical neuroimaging studies

Optimised and Rapid Pre-clinical Screening in the SOD1G93A Transgenic Mouse Model of Amyotrophic Lateral Sclerosis (ALS)

The human SOD1G93A transgenic mouse has been used extensively since its development in 1994 as a model for amyotrophic lateral sclerosis (ALS). In that time, a great many insights into the toxicity of mutant SOD1 have been gained using this and other mutant SOD transgenic mouse models. They all demonstrate a selective toxicity towards motor neurons and in some cases features of the pathology seen in the human disease. These models have two major drawbacks. Firstly the generation of robust preclinical data in these models has been highlighted as an area for concern. Secondly, the amount of time required for a single preclinical experiment in these models (3–4 months) is a hurdle to the development of new therapies. We have developed an inbred C57BL/6 mouse line from the original mixed background (SJLxC57BL/6) SOD1G93A transgenic line and show here that the disease course is remarkably consistent and much less prone to background noise, enabling reduced numbers of mice for testing of therapeutics. Secondly we have identified very early readouts showing a large decline in motor function compared to normal mice. This loss of motor function has allowed us to develop an early, sensitive and rapid screening protocol for the initial phases of denervation of muscle fibers, observed in this model. We describe multiple, quantitative readouts of motor function that can be used to interrogate this early mechanism. Such an approach will increase throughput for reduced costs, whilst reducing the severity of the experimental procedures involved

Hyperpolarizing Pyruvate through Signal Amplification By Reversible Exchange (SABRE)

Hyperpolarization methods that premagnetise agents such as pyruvate are currently receiving significant attention. This is because they produce sensitivity gains that allow disease tracking and interrogation of cellular metabolism by magnetic resonance. Here, we communicate how Signal Amplification by Reversible Exchange (SABRE) can provide strong ¹³C pyruvate signal enhancements in seconds through the formation of the novel polarization transfer catalyst [Ir(H)₂(η²-pyruvate)(DMSO)(IMes)]. By harnessing SABRE, strong signals for 1-¹³C and 2-¹³C pyruvate in addition to a long-lived singlet state in the 1,2-¹³C₂ form are readily created, the latter can be observed five minutes after the initial hyperpolarization step. We also demonstrate how this development may help with future studies of chemical reactivity

SABRE hyperpolarized anticancer agents for use in 1 H MRI

PURPOSE: Enabling drug tracking (distribution/specific pathways) with magnetic resonance spectroscopy requires manipulation (via hyperpolarization) of spin state populations and targets with sufficiently long magnetic lifetimes to give the largest possible window of observation. Here, we demonstrate how the proton resonances of a group of thienopyridazines (with known anticancer properties), can be amplified using the para-hydrogen (p-H2 ) based signal amplification by reversible exchange (SABRE) hyperpolarization technique. METHODS: Thienopyridazine isomers, including a 2 H version, were synthesized in house. Iridium-based catalysts dissolved in a methanol-d4 solvent facilitated polarization transfer from p-H2 gas to the target thienopyridazines. Subsequent SABRE 1 H responses of hyperpolarized thienopyridazines were completed (400 MHz NMR). Pseudo-singlet state approaches were deployed to extend magnetic state lifetimes. Proof of principle spectral-spatial images were acquired across a range of field strengths (7T-9.4T MRI). RESULTS: 1 H-NMR signal enhancements of -10,130-fold at 9.4T (~33% polarization) were achieved on thieno[2,3-d]pyridazine (T[2,3-d]P), using SABRE under optimal mixing/field transfer conditions. 1 H T1 lifetimes for the thienopyridazines were ~18-50 s. Long-lived state approaches extended the magnetic lifetime of target proton sites in T[2,3-d]P from an average of 25-40 seconds. Enhanced in vitro imaging (spatial and chemical shift based) of target T[2,3-d]P was demonstrated. CONCLUSION: Here, we demonstrate the power of SABRE to deliver a fast and cost-effective route to hyperpolarization of important chemical motifs of anticancer agents. The SABRE approach outlined here lays the foundations for realizing continuous flow, hyperpolarized tracking of drug delivery/pathways