Non-Invasive MRI of Blood-Cerebrospinal Fluid Barrier Function.

The blood-cerebrospinal fluid barrier (BCSFB) is a highly dynamic transport interface that serves brain homeostasis. To date, however, understanding of its role in brain development and pathology has been hindered by the absence of a non-invasive technique for functional assessment. Here we describe a method for non-invasive measurement of BSCFB function by using tracer-free MRI to quantify rates of water delivery from arterial blood to ventricular cerebrospinal fluid. Using this method, we record a 36% decrease in BCSFB function in aged mice, compared to a 13% decrease in parenchymal blood flow, itself a leading candidate biomarker of early neurodegenerative processes. We then apply the method to explore the relationship between BCSFB function and ventricular morphology. Finally, we provide proof of application to the human brain. Our findings position the BCSFB as a promising new diagnostic and therapeutic target, the function of which can now be safely quantified using non-invasive MRI.

CNS distribution, signalling properties and central effects of G-protein coupled receptor 4.

Information on the distribution and biology of the G-protein coupled receptor 4 (GPR4) in the brain is limited. It is currently thought that GPR4 couples to Gs proteins and may mediate central respiratory sensitivity to CO2. Using a knock-in mouse model, abundant GPR4 expression was detected in the cerebrovascular endothelium and neurones of dorsal raphe, retro-trapezoidal nucleus locus coeruleus and lateral septum. A similar distribution was confirmed using RNAscope in situ hybridisation. In HEK293 cells, overexpressing GPR4, it was highly constitutively active at neutral pH with little further increase in cAMP towards acidic pH. The GPR4 antagonist NE 52-QQ57 effectively blocked GPR4-mediated cAMP accumulation (IC50 26.8 nM in HEK293 cells). In HUVEC which natively express GPR4, physiological acidification (pH 7.4-7.0) resulted in a cAMP increase by ∼55% which was completely prevented by 1 µM NE 52-QQ57. The main extracellular organic acid, l-lactic acid (LL; 1-10 mM), suppressed pH dependent activation of GPR4 in HEK293 and HUVEC cells, suggesting allosteric negative modulation. In unanaesthetised mice and rats, NE 52-QQ57 (20 mg kg-1) reduced ventilatory response to 5 and 10% CO2. In anaesthetised rats, systemic administration of NE 52-QQ57 (up to 20 mg kg-1) had no effect on hemodynamics, cerebral blood flow and blood oxygen level dependent responses. Central administration of NE 52-QQ57 (1 mM) in vagotomised anaesthetised rats did not affect CO2-induced respiratory responses. Our results indicate that GPR4 is expressed by multiple neuronal populations and endothelium and that its pH sensitivity is affected by level of expression and LL. NE 52-QQ57 blunts hypercapnic response to CO2 but this effect is absent under anaesthesia, possibly due to the inhibitory effect of LL on GPR4.

Tissue magnetic susceptibility mapping as a marker of tau pathology in Alzheimer's disease.

Alzheimer's disease is connected to a number of other neurodegenerative conditions, known collectively as 'tauopathies', by the presence of aggregated tau protein in the brain. Neuroinflammation and oxidative stress in AD are associated with tau pathology and both the breakdown of axonal sheaths in white matter tracts and excess iron accumulation grey matter brain regions. Despite the identification of myelin and iron concentration as major sources of contrast in quantitative susceptibility maps of the brain, the sensitivity of this technique to tau pathology has yet to be explored. In this study, we perform Quantitative Susceptibility Mapping (QSM) and T2* mapping in the rTg4510, a mouse model of tauopathy, both in vivo and ex vivo. Significant correlations were observed between histological measures of myelin content and both mean regional magnetic susceptibility and T2* values. These results suggest that magnetic susceptibility is sensitive to tissue myelin concentrations across different regions of the brain. Differences in magnetic susceptibility were detected in the corpus callosum, striatum, hippocampus and thalamus of the rTg4510 mice relative to wild type controls. The concentration of neurofibrillary tangles was found to be low to intermediate in these brain regions indicating that QSM may be a useful biomarker for early stage detection of tau pathology in neurodegenerative diseases.

Uranium rhodium bonding in heterometallic complexes.

The heterotetra- and bimetallic uranium(iv)-rhodium(i) complexes [UIVI2(µ-OArP-1κ1O,2κ1P)2RhI(µ-I)]2 (2) (ArPO- = 2-(diphenylphosphino)-6-tert-butyl-4-methylphenoxide) and UIVI(µ-I)(µ-OArP-1κ1O,2κ1P)3RhI (3) were prepared by treatment of UIVI(OArP-κ2O,P)3 (1) with rhodium(i) iodide olefin complexes. The reaction of 1 with the monodentate cyclooctene (coe) rhodium(i) precursor [(coe)2RhII]2 gives only the bimetallic complex [UIVRhI] 3, and with the diene [(cod)RhII]2 (5) (cod = 1,5-cyclooctadiene), mixtures of [UIVRhI]2 complex 2 and [UIVRhI] 3 along with (cod)RhIOArP-κ2O,P (4), a RhI side-product from the formation of 2. The complexes were characterised by single crystal X-ray diffraction, NMR and UV-vis-NIR spectroscopy, and electrochemistry. The UIV-RhI intermetallic distances in 2 (2.7601(5) Å) and 3 (2.7630(5) Å) are among the shortest between f-elements and transition metals reported to date. Despite almost identical U-Rh bond lengths in the solid state, in solution only weak, and very different interactions between the metal centres are found.

MRI of cerebral micro-vascular flow patterns: A multi-direction diffusion-weighted ASL approach.

The study and clinical assessment of brain disease is currently hindered by a lack of non-invasive methods for the detailed and accurate evaluation of cerebral vascular pathology. Angiography can detect aberrant flow in larger feeding arteries/arterioles but cannot resolve the micro-vascular network. Small vessels are a key site of vascular pathology that can lead to haemorrhage and infarction, which may in turn trigger or exacerbate neurodegenerative processes. In this study, we describe a method to investigate microvascular flow anisotropy using a hybrid arterial spin labelling and multi-direction diffusion-weighted MRI sequence. We present evidence that the technique is sensitive to the mean/predominant direction of microvascular flow in localised regions of the rat cortex. The data provide proof of principle for a novel and non-invasive imaging tool to investigate cerebral micro-vascular flow patterns in healthy and disease states.

Cacidases: caspases can cleave after aspartate, glutamate and phosphoserine residues.

Caspases are a family of proteases found in all metazoans, including a dozen in humans, that drive the terminal stages of apoptosis as well as other cellular remodeling and inflammatory events. Caspases are named because they are cysteine class enzymes shown to cleave after aspartate residues. In the past decade, we and others have developed unbiased proteomic methods that collectively identified ~2000 native proteins cleaved during apoptosis after the signature aspartate residues. Here, we explore non-aspartate cleavage events and identify 100s of substrates cleaved after glutamate in both human and murine apoptotic samples. The extended consensus sequence patterns are virtually identical for the aspartate and glutamate cleavage sites suggesting they are cleaved by the same caspases. Detailed kinetic analyses of the dominant apoptotic executioner caspases-3 and -7 show that synthetic substrates containing DEVD↓ are cleaved only twofold faster than DEVE↓, which is well within the 500-fold range of rates that natural proteins are cut. X-ray crystallography studies confirm that the two acidic substrates bind in virtually the same way to either caspases-3 or -7 with minimal adjustments to accommodate the larger glutamate. Lastly, during apoptosis we found 121 proteins cleaved after serine residues that have been previously annotated to be phosphorylation sites. We found that caspase-3, but not caspase-7, can cleave peptides containing DEVpS↓ at only threefold slower rate than DEVD↓, but does not cleave the unphosphorylated serine peptide. There are only a handful of previously reported examples of proteins cleaved after glutamate and none after phosphorserine. Our studies reveal a much greater promiscuity for cleaving after acidic residues and the name 'cacidase' could aptly reflect this broader specificity.

Application of neurite orientation dispersion and density imaging (NODDI) to a tau pathology model of Alzheimer's disease.

Increased hyperphosphorylated tau and the formation of intracellular neurofibrillary tangles are associated with the loss of neurons and cognitive decline in Alzheimer's disease, and related neurodegenerative conditions. We applied two diffusion models, diffusion tensor imaging (DTI) and neurite orientation dispersion and density imaging (NODDI), to in vivo diffusion magnetic resonance images (dMRI) of a mouse model of human tauopathy (rTg4510) at 8.5months of age. In grey matter regions with the highest degree of tau burden, microstructural indices provided by both NODDI and DTI discriminated the rTg4510 (TG) animals from wild type (WT) controls; however only the neurite density index (NDI) (the volume fraction that comprises axons or dendrites) from the NODDI model correlated with the histological measurements of the levels of hyperphosphorylated tau protein. Reductions in diffusion directionality were observed when implementing both models in the white matter region of the corpus callosum, with lower fractional anisotropy (DTI) and higher orientation dispersion (NODDI) observed in the TG animals. In comparison to DTI, histological measures of tau pathology were more closely correlated with NODDI parameters in this region. This in vivo dMRI study demonstrates that NODDI identifies potential tissue sources contributing to DTI indices and NODDI may provide greater specificity to pathology in Alzheimer's disease.

In vivo imaging of tau pathology using multi-parametric quantitative MRI.

As the number of people diagnosed with Alzheimer's disease (AD) reaches epidemic proportions, there is an urgent need to develop effective treatment strategies to tackle the social and economic costs of this fatal condition. Dozens of candidate therapeutics are currently being tested in clinical trials, and compounds targeting the aberrant accumulation of tau proteins into neurofibrillary tangles (NFTs) are the focus of substantial current interest. Reliable, translatable biomarkers sensitive to both tau pathology and its modulation by treatment along with animal models that faithfully reflect aspects of the human disease are urgently required. Magnetic resonance imaging (MRI) is well established as a valuable tool for monitoring the structural brain changes that accompany AD progression. However the descent into dementia is not defined by macroscopic brain matter loss alone: non-invasive imaging measurements sensitive to protein accumulation, white matter integrity and cerebral haemodynamics probe distinct aspects of AD pathophysiology and may serve as superior biomarkers for assessing drug efficacy. Here we employ a multi-parametric array of five translatable MRI techniques to characterise the in vivo pathophysiological phenotype of the rTg4510 mouse model of tauopathy (structural imaging, diffusion tensor imaging (DTI), arterial spin labelling (ASL), chemical exchange saturation transfer (CEST) and glucose CEST). Tau-induced pathological changes included grey matter atrophy, increased radial diffusivity in the white matter, decreased amide proton transfer and hyperperfusion. We demonstrate that the above markers unambiguously discriminate between the transgenic group and age-matched controls and provide a comprehensive profile of the multifaceted neuropathological processes underlying the rTg4510 model. Furthermore, we show that ASL and DTI techniques offer heightened sensitivity to processes believed to precede detectable structural changes and, as such, provides a platform for the study of disease mechanisms and therapeutic intervention.

Hepatic arterial spin labelling MRI: an initial evaluation in mice.

The development of strategies to combat hepatic disease and augment tissue regeneration has created a need for methods to assess regional liver function. Liver perfusion imaging has the potential to fulfil this need, across a range of hepatic diseases, alongside the assessment of therapeutic response. In this study, the feasibility of hepatic arterial spin labelling (HASL) was assessed for the first time in mice at 9.4 T, its variability and repeatability were evaluated, and it was applied to a model of colorectal liver metastasis. Data were acquired using flow-sensitive alternating inversion recovery-arterial spin labelling (FAIR-ASL) with a Look-Locker readout, and analysed using retrospective respiratory gating and a T1 -based quantification. This study shows that preclinical HASL is feasible and exhibits good repeatability and reproducibility. Mean estimated liver perfusion was 2.2 ± 0.8 mL/g/min (mean ± standard error, n = 10), which agrees well with previous measurements using invasive approaches. Estimates of the variation gave a within-session coefficient of variation (CVWS) of 7%, a between-session coefficient of variation (CVBS) of 9% and a between-animal coefficient of variation (CVA) of 15%. The within-session Bland-Altman repeatability coefficient (RCWS) was 18% and the between-session repeatability coefficient (RCBS) was 29%. Finally, the HASL method was applied to a mouse model of liver metastasis, in which significantly lower mean perfusion (1.1 ± 0.5 mL/g/min, n = 6) was measured within the tumours, as seen by fluorescence histology. These data indicate that precise and accurate liver perfusion estimates can be achieved using ASL techniques, and provide a platform for future studies investigating hepatic perfusion in mouse models of disease.

Evaluation of skeletal tissue repair, part 1: assessment of novel growth-factor-releasing hydrogels in an ex vivo chick femur defect model.

Current clinical treatments for skeletal conditions resulting in large-scale bone loss include autograft or allograft, both of which have limited effectiveness. In seeking to address bone regeneration, several tissue engineering strategies have come to the fore, including the development of growth factor releasing technologies and appropriate animal models to evaluate repair. Ex vivo models represent a promising alternative to simple in vitro systems or complex, ethically challenging in vivo models. We have developed an ex vivo culture system of whole embryonic chick femora, adapted in this study as a critical size defect model to investigate the effects of novel bone extracellular matrix (bECM) hydrogel scaffolds containing spatio-temporal growth factor-releasing microparticles and skeletal stem cells on bone regeneration, to develop a viable alternative treatment for skeletal degeneration. Alginate/bECM hydrogels combined with poly (d,l-lactic-co-glycolic acid) (PDLLGA)/triblock copolymer (10-30% PDLLGA-PEG-PDLLGA) microparticles releasing VEGF, TGF-ß3 or BMP-2 were placed, with human adult Stro-1+ bone marrow stromal cells, into 2mm central segmental defects in embryonic chick femurs. Alginate/bECM hydrogels loaded with HSA/VEGF or HSA/TGF-ß3 demonstrated a cartilage-like phenotype, with minimal collagen I deposition, comparable to HSA-only control hydrogels. The addition of BMP-2 releasing microparticles resulted in enhanced structured bone matrix formation, evidenced by increased Sirius red-stained matrix and collagen expression within hydrogels. This study demonstrates delivery of bioactive growth factors from a novel alginate/bECM hydrogel to augment skeletal tissue formation and the use of an organotypic chick femur defect culture system as a high-throughput test model for scaffold/cell/growth factor therapies for regenerative medicine.

Evaluation of skeletal tissue repair, part 2: enhancement of skeletal tissue repair through dual-growth-factor-releasing hydrogels within an ex vivo chick femur defect model.

There is an unmet need for improved, effective tissue engineering strategies to replace or repair bone damaged through disease or injury. Recent research has focused on developing biomaterial scaffolds capable of spatially and temporally releasing combinations of bioactive growth factors, rather than individual molecules, to recapitulate repair pathways present in vivo. We have developed an ex vivo embryonic chick femur critical size defect model and applied the model in the study of novel extracellular matrix (ECM) hydrogel scaffolds containing spatio-temporal combinatorial growth factor-releasing microparticles and skeletal stem cells for bone regeneration. Alginate/bovine bone ECM (bECM) hydrogels combined with poly(d,l-lactic-co-glycolic acid) (PDLLGA)/triblock copolymer (10-30% PDLLGA-PEG-PLDLGA) microparticles releasing dual combinations of vascular endothelial growth factor (VEGF), chondrogenic transforming growth factor beta 3 (TGF-ß3) and the bone morphogenetic protein BMP2, with human adult Stro-1+bone marrow stromal cells (HBMSCs), were placed into 2mm central segmental defects in embryonic day 11 chick femurs and organotypically cultured. Hydrogels loaded with VEGF combinations induced host cell migration and type I collagen deposition. Combinations of TGF-ß3/BMP2, particularly with Stro-1+HBMSCs, induced significant formation of structured bone matrix, evidenced by increased Sirius red-stained matrix together with collagen expression demonstrating birefringent alignment within hydrogels. This study demonstrates the successful use of the chick femur organotypic culture system as a high-throughput test model for scaffold/cell/growth factor therapies in regenerative medicine. Temporal release of dual growth factors, combined with enriched Stro-1+HBMSCs, improved the formation of a highly structured bone matrix compared to single release modalities. These studies highlight the potential of a unique alginate/bECM hydrogel dual growth factor release platform for bone repair.

Conservation of caspase substrates across metazoans suggests hierarchical importance of signaling pathways over specific targets and cleavage site motifs in apoptosis.

Caspases, cysteine proteases with aspartate specificity, are key players in programmed cell death across the metazoan lineage. Hundreds of apoptotic caspase substrates have been identified in human cells. Some have been extensively characterized, revealing key functional nodes for apoptosis signaling and important drug targets in cancer. But the functional significance of most cuts remains mysterious. We set out to better understand the importance of caspase cleavage specificity in apoptosis by asking which cleavage events are conserved across metazoan model species. Using N-terminal labeling followed by mass spectrometry, we identified 257 caspase cleavage sites in mouse, 130 in Drosophila, and 50 in Caenorhabditis elegans. The large majority of the caspase cut sites identified in mouse proteins were found conserved in human orthologs. However, while many of the same proteins targeted in the more distantly related species were cleaved in human orthologs, the exact sites were often different. Furthermore, similar functional pathways are targeted by caspases in all four species. Our data suggest a model for the evolution of apoptotic caspase specificity that highlights the hierarchical importance of functional pathways over specific proteins, and proteins over their specific cleavage site motifs.

Imaging the paediatric lung: what does nanotechnology have to offer?

This review will provide an overview of current research into lung imaging with nanoparticles, with a focus on the use of nanoparticles as molecular imaging agents to observe pathological processes and to monitor the effectiveness of nanoparticulate drug delivery systems. Various imaging modalities together with their advantages and limitations for lung imaging will be discussed. We will also explore the range of nanoparticles used, as well as active or passive targeting of nanoparticles.

The importance of RF bandwidth for effective tagging in pulsed arterial spin labeling MRI at 9.4T.

The movement towards MRI at higher field strengths (>7T) has enhanced the appeal of arterial spin labeling (ASL) for many applications due to improved SNR of the measurements. Greater field strength also introduces increased magnetic susceptibility effects resulting in marked B(0) field inhomogeneity. Although B(0) field perturbations can be minimised by shimming over the imaging volume, marked field inhomogeneity is likely to remain within the labeling region for pulsed ASL (PASL). This study highlights a potential source of error in cerebral blood flow quantification using PASL at high field. We show that labeling efficiency in flow-sensitive alternating inversion recovery (FAIR) displayed marked sensitivity to the RF bandwidth of the inversion pulse in a rat model at 9.4T. The majority of preclinical PASL studies have not reported the bandwidth of the inversion pulse. We show that a high bandwidth pulse of > = 15 kHz was required to robustly overcome the field inhomogeneity in the labeling region at high field strength, which is significantly greater than the inversion bandwidth ~2-3 kHz used in previous studies. Unless SAR levels are at their limit, we suggest the use of a high bandwidth labeling pulse for most PASL studies.

Cerebral blood flow changes during pilocarpine-induced status epilepticus activity in the rat hippocampus.

INTRODUCTION: There is a known relationship between convulsive status epilepticus (SE) and hippocampal injury. Although the precise causes of this hippocampal vulnerability remains uncertain, potential mechanisms include excitotoxicity and ischaemia. It has been hypothesised that during the early phase of seizures, cerebral blood flow (CBF) increases in the cortex to meet energy demand, but it is unclear whether these compensatory mechanisms occur in the hippocampus. In this study we investigated CBF changes using perfusion MRI during SE in the pilocarpine rat. METHODS: First, we determined whether SE could be induced under anaesthesia. Two anaesthetic protocols were investigated: isoflurane (n=6) and fentanyl/medetomidine (n=7). Intrahippocampal EEG electrodes were used to determine seizure activity and reflex behaviours were used to assess anaesthesia. Pilocarpine was administered to induce status epilepticus. For CBF measurements, MRI arterial spin labelling was performed continuously for up to 3h. Either pilocarpine (375 mg/kg) (n=7) for induction of SE or saline (n=6) was administered. Diazepam (10mg/kg) was administered i.p. 90 min after the onset of SE. RESULTS AND DISCUSSION: We demonstrated time-dependent significant (p<0.05) differences between the CBF responses in the parietal cortex and the hippocampus during SE. This regional response indicates a preferential distribution of flow to certain regions of the brain and may contribute to the selective vulnerability observed in the hippocampus in humans.

Site-directed ligand discovery.

We report a strategy (called "tethering") to discover low molecular weight ligands ( approximately 250 Da) that bind weakly to targeted sites on proteins through an intermediary disulfide tether. A native or engineered cysteine in a protein is allowed to react reversibly with a small library of disulfide-containing molecules ( approximately 1,200 compounds) at concentrations typically used in drug screening (10 to 200 microM). The cysteine-captured ligands, which are readily identified by MS, are among the most stable complexes, even though in the absence of the covalent tether the ligands may bind very weakly. This method was applied to generate a potent inhibitor for thymidylate synthase, an essential enzyme in pyrimidine metabolism with therapeutic applications in cancer and infectious diseases. The affinity of the untethered ligand (K(i) approximately 1 mM) was improved 3,000-fold by synthesis of a small set of analogs with the aid of crystallographic structures of the tethered complex. Such site-directed ligand discovery allows one to nucleate drug design from a spatially targeted lead fragment.

Mutational analysis of the major coat protein of M13 identifies residues that control protein display.

We have reported variants of the M13 bacteriophage major coat protein (P8) that enable high copy display of monomeric and oligomeric proteins, such as human growth hormone and steptavidin, on the surface of phage particles (Sidhu SS, Weiss GA, Wells JA. 2000. High copy display of large proteins on phage for functional selections. J Mol Biol 296:487-495). Here, we explore how an optimized P8 variant (opti-P8) could evolve the ability to efficiently display a protein fused to its N-terminus. Reversion of individual opti-P8 residues back to the wild-type P8 residue identifies a limited set of hydrophobic residues responsible for the high copy protein display. These hydrophobic amino acids bracket a conserved hydrophobic face on the P8 alpha helix thought to be in contact with the phage coat. Mutations additively combine to promote high copy protein display, which was further enhanced by optimization of the linker between the phage coat and the fusion protein. These data are consistent with a model in which protein display-enhancing mutations allow for better packing of the fusion protein into the phage coat. The high tolerance for phage coat protein mutations observed here suggests that filamentous phage coat proteins could readily evolve new capabilities.