Amnon Bar-Shir

Reversal of axonal loss and disability in a mouse model of progressive multiple sclerosis

Axonal degeneration is an important determinant of progressive neurological disability in multiple sclerosis (MS). Thus, therapeutic approaches promoting neuroprotection could aid the treatment of progressive MS. Here, we used what we believe is a novel water-soluble fullerene derivative (ABS-75) attached to an NMDA receptor antagonist, which combines antioxidant and anti-excitotoxic properties, to block axonal damage and reduce disease progression in a chronic progressive EAE model. Fullerene ABS-75 treatment initiated after disease onset reduced the clinical progression of chronic EAE in NOD mice immunized with myelin-oligodendrocyte glycoprotein (MOG). Reduced disease progression in ABS-75-treated mice was associated with reduced axonal loss and demyelination in the spinal cord. Fullerene ABS-75 halted oxidative injury, CD11b+ infiltration, and CCL2 expression in the spinal cord of mice without interfering with antigen-specific T cell responses. In vitro, fullerene ABS-75 protected neurons from oxidative and glutamate-induced injury and restored glutamine synthetase and glutamate transporter expression in astrocytes under inflammatory insult. Glutamine synthetase expression was also increased in the white matter of fullerene ABS-75-treated animals. Our data demonstrate the neuroprotective effect of treatment with a fullerene compound combined with a NMDA receptor antagonist, which may be useful in the treatment of progressive MS and other neurodegenerative diseases.

Monitoring enzyme activity using a diamagnetic chemical exchange saturation transfer magnetic resonance imaging contrast agent.

Chemical exchange saturation transfer (CEST) is a new approach for generating magnetic resonance imaging (MRI) contrast that allows monitoring of protein properties in vivo. In this method, a radiofrequency pulse is used to saturate the magnetization of specific protons on a target molecule, which is then transferred to water protons via chemical exchange and detected using MRI. One advantage of CEST imaging is that the magnetizations of different protons can be specifically saturated at different resonance frequencies. This enables the detection of multiple targets simultaneously in living tissue. We present here a CEST MRI approach for detecting the activity of cytosine deaminase (CDase), an enzyme that catalyzes the deamination of cytosine to uracil. Our findings suggest that metabolism of two substrates of the enzyme, cytosine and 5-fluorocytosine (5FC), can be detected using saturation pulses targeted specifically to protons at +2 ppm and +2.4 ppm (with respect to water), respectively. Indeed, after deamination by recombinant CDase, the CEST contrast disappears. In addition, expression of the enzyme in three different cell lines exhibiting different expression levels of CDase shows good agreement with the CDase activity measured with CEST MRI. Consequently, CDase activity was imaged with high-resolution CEST MRI. These data demonstrate the ability to detect enzyme activity based on proton exchange. Consequently, CEST MRI has the potential to follow the kinetics of multiple enzymes in real time in living tissue.

Variable delay multi-pulse train for fast chemical exchange saturation transfer and relayed-nuclear overhauser enhancement MRI

Chemical exchange saturation transfer (CEST) imaging is a new MRI technology allowing the detection of low concentration endogenous cellular proteins and metabolites indirectly through their exchangeable protons. A new technique, variable delay multi‐pulse CEST (VDMP‐CEST), is proposed to eliminate the need for recording full Z‐spectra and performing asymmetry analysis to obtain CEST contrast.

The effect of the diffusion time and pulse gradient duration ratio on the diffraction pattern and the structural information estimated from q-space diffusion MR: Experiments and simulations

q-Space diffusion MRI (QSI) provides a means of obtaining microstructural information about porous materials and neuronal tissues from diffusion data. However, the accuracy of this structural information depends on experimental parameters used to collect the MR data. q-Space diffusion MR performed on clinical scanners is generally collected with relatively long diffusion gradient pulses, in which the gradient pulse duration, delta, is comparable to the diffusion time, Delta. In this study, we used phantoms, consisting of ensembles of microtubes, and mathematical models to assess the effect of the ratio of the diffusion time and the duration of the diffusion pulse gradient, i.e., Delta/delta, on the MR signal attenuation vs. q, and on the measured structural information extracted therefrom. We found that for Delta/delta approximately 1, the diffraction pattern obtained from q-space MR data are shallower than when the short gradient pulse (SGP) approximation is satisfied. For long delta the estimated compartment size is, as expected, smaller than the real size. Interestingly, for Delta/delta approximately 1 the diffraction peaks are shifted to even higher q-values, even when delta is kept constant, giving the impression that the restricted compartments are even smaller than they are. When phantoms composed of microtubes of different diameters are used, it is more difficult to estimate the diameter distribution in this regime. Excellent agreement is found between the experimental results and simulations that explicitly account for the use of long duration gradient pulses. Using such experimental data and this mathematical framework, one can estimate the true compartment dimensions when long and finite gradient pulses are used even when Delta/delta approximately 1.

QSI and DTI of excised brains of the myelin-deficient rat.

High b-value q-space diffusion imaging (QSI) and conventional DTI methodologies were used to study the MRI diffusion characteristics of excised brains of 21-day-old myelin-deficient (md) rats and their age-matched controls. Three different indices were calculated from the QSI data, i.e., Displacement, Probability and Kurtosis, for the purpose of evaluating the effect of the myelin sheaths on the MR diffusion characteristics in white matter (WM) ROIs of the md versus control brains. The examined WM ROIs were the corpus callosum, the external capsule, and the internal capsule. In all examined WM ROIs, significant differences were observed between the md and control brains for all QSI indices. These differences reveal that myelin sheaths surrounding the axons in WM ROIs mostly affect the component exhibiting restricted diffusion, which is manifested by low mean displacement values and high probability and kurtosis values. Such differences were found to be more pronounced in long diffusion times, i.e., Delta=200 ms. Conventional DTI performed with relatively low b-values (b<1500 s/mm2) was also used to study md versus control brains. Interestingly, the fractional anisotropy (FA) index, which was calculated from DTI data, did not reveal any significant difference between the groups in the examined WM ROIs. However, some distinctions were revealed by the three eigenvalues (lambda1, lambda2, and lambda3) obtained from the tensor analysis. These findings were supported by Voxel-based analysis using SPM. Finally, MRI-guided histology showed very good agreement between myelin-stained regions and regions with highly restricted diffusion detected by QSI.

CEST phase mapping using a length and offset varied saturation (LOVARS) scheme.

Chemical exchange saturation transfer MRI is a promising new technique for cellular and molecular imaging. This contrast allows the detection of tumors and ischemia without the use of gadolinium as well as the design of microenvironment‐sensitive probes that can be discriminated based on their exchange contrast properties and saturation frequency. Current acquisition schemes to detect and analyze this contrast suffer from sensitivity to spatial B0 inhomogeneity and low contrast‐to‐noise‐ratio, which is an obstacle to widespread adoption of the technology. A new method to detect chemical exchange saturation transfer contrast is proposed here, termed “length and offset varied saturation” which acquires a set of images with the saturation parameters varied so as to modulate the exchange contrast. Either fast fourier transform or the general linear model can be employed to decompose the modulation patterns into separate sources of water signal loss. After transformation, a length and offset varied saturation phase map is generated, which is insensitive to B0 inhomogeneity. When collected on live mice bearing 9L gliosarcomas, and compared to the conventional asymmetry in the magnetization transfer ratio map using offset increment correction, the results show that length and offset varied saturation phase mapping obtains about three to four times contrast‐to‐noise‐ratio and exhibits less B0 artifacts. Magn Reson Med, 2012.

Human Protamine‑1 as an MRI Reporter Gene Based on Chemical Exchange

Genetically engineered reporters have revolutionized the understanding of many biological processes. MRI-based reporter genes can dramatically improve our ability to monitor dynamic gene expression and allow coregistration of subcellular genetic information with high-resolution anatomical images. We have developed a biocompatible MRI reporter gene based on a human gene, the human protamine-1 (hPRM1). The arginine-rich hPRM1 (47% arginine residues) generates high MRI contrast based on the chemical exchange saturation transfer (CEST) contrast mechanism. The 51 amino acid-long hPRM1 protein was fully synthesized using microwave-assisted technology, and the CEST characteristics of this protein were compared to other CEST-based contrast agents. Both bacterial and human cells were engineered to express an optimized hPRM1 gene and showed higher CEST contrast compared to controls. Live cells expressing the hPRM1 reporter gene, and embedded in three-dimensional culture, also generated higher CEST contrast compared to wild-type live cells.

Optogenetic-guided cortical plasticity after nerve injury

Peripheral nerve injury causes sensory dysfunctions that are thought to be attributable to changes in neuronal activity occurring in somatosensory cortices both contralateral and ipsilateral to the injury. Recent studies suggest that distorted functional response observed in deprived primary somatosensory cortex (S1) may be the result of an increase in inhibitory interneuron activity and is mediated by the transcallosal pathway. The goal of this study was to develop a strategy to manipulate and control the transcallosal activity to facilitate appropriate plasticity by guiding the cortical reorganization in a rat model of sensory deprivation. Since transcallosal fibers originate mainly from excitatory pyramidal neurons somata situated in laminae III and V, the excitatory neurons in rat S1 were engineered to express halorhodopsin, a light-sensitive chloride pump that triggers neuronal hyperpolarization. Results from electrophysiology, optical imaging, and functional MRI measurements are concordant with that within the deprived S1, activity in response to intact forepaw electrical stimulation was significantly increased by concurrent illumination of halorhodopsin over the healthy S1. Optogenetic manipulations effectively decreased the adverse inhibition of deprived cortex and revealed the major contribution of the transcallosal projections, showing interhemispheric neuroplasticity and thus, setting a foundation to develop improved rehabilitation strategies to restore cortical functions.

Observation of restricted diffusion in the presence of a free diffusion compartment: Single- and double-PFG experiments

Theoretical and experimental studies of restricted diffusion have been conducted for decades using single pulsed field gradient (s-PFG) diffusion experiments. In homogenous samples, the diffusion-diffraction phenomenon arising from a single population of diffusing species has been observed experimentally and predicted theoretically. In this study, we introduce a composite bi-compartmental model which superposes restricted diffusion in microcapillaries with free diffusion in an unconfined compartment, leading to fast and slow diffusing components in the NMR signal decay. Although simplified (no exchange), the superposed diffusion modes in this model may exhibit features seen in more complex porous materials and biological tissues. We find that at low q-values the freely diffusing component masks the restricted diffusion component, and that prolongation of the diffusion time shifts the transition from free to restricted profiles to lower q-values. The effect of increasing the volume fraction of freely diffusing water was also studied; we find that the transition in the signal decay from the free mode to the restricted mode occurs at higher q-values when the volume fraction of the freely diffusing water is increased. These findings were then applied to a phantom consisting of crossing fibers, which demonstrated the same qualitative trends in the signal decay. The angular d-PGSE experiment, which has been recently shown to be able to measure small compartmental dimensions even at low q-values, revealed that microscopic anisotropy is lost at low q-values where the fast diffusing component is prominent. Our findings may be of importance in studying realistic systems which exhibit compartmentation.

Advances in using MRI probes and sensors for in vivo cell tracking as applied to regenerative medicine

The field of molecular and cellular imaging allows molecules and cells to be visualized in vivo non-invasively. It has uses not only as a research tool but in clinical settings as well, for example in monitoring cell-based regenerative therapies, in which cells are transplanted to replace degenerating or damaged tissues, or to restore a physiological function. The success of such cell-based therapies depends on several critical issues, including the route and accuracy of cell transplantation, the fate of cells after transplantation, and the interaction of engrafted cells with the host microenvironment. To assess these issues, it is necessary to monitor transplanted cells non-invasively in real-time. Magnetic resonance imaging (MRI) is a tool uniquely suited to this task, given its ability to image deep inside tissue with high temporal resolution and sensitivity. Extraordinary efforts have recently been made to improve cellular MRI as applied to regenerative medicine, by developing more advanced contrast agents for use as probes and sensors. These advances enable the non-invasive monitoring of cell fate and, more recently, that of the different cellular functions of living cells, such as their enzymatic activity and gene expression, as well as their time point of cell death. We present here a review of recent advancements in the development of these probes and sensors, and of their functioning, applications and limitations.