Noam Shemesh

Protective Effects of Neurotrophic Factor–Secreting Cells in a 6-OHDA Rat Model of Parkinson Disease

Stem cell-based therapy is a promising treatment for neurodegenerative diseases. In our laboratory, a novel protocol has been developed to induce bone marrow-derived mesenchymal stem cells (MSC) into neurotrophic factors- secreting cells (NTF-SC), thus combining stem cell-based therapy with the NTF-based neuroprotection. These cells produce and secrete factors such as brain-derived neurotrophic factor (BDNF) and glial cell-derived neurotrophic factor. Conditioned medium of the NTF-SC that was applied to a neuroblastoma cell line (SH-SY5Y) 1 h before exposure to the neurotoxin 6-hydroxydopamine (6-OHDA) demonstrated marked protection. An efficacy study was conducted on the 6-OHDA-induced lesion, a rat model of Parkinsons disease. The cells, either MSC or NTF-SC, were transplanted on the day of 6-OHDA administration and amphetamine-induced rotations were measured as a primary behavior index. We demonstrated that when transplanted posterior to the 6-OHDA lesion, the NTF-SC ameliorated amphetamine-induced rotations by 45%. HPLC analysis demonstrated that 6-OHDA induced dopamine depletion to a level of 21% compared to the untreated striatum. NTF-SC inhibited dopamine depletion to a level of 72% of the contralateral striatum. Moreover, an MRI study conducted with iron-labeled cells, followed by histological verification, revealed that the engrafted cells migrated toward the lesion. In a histological assessment, we found that the cells induced regeneration in the damaged striatal dopaminergic nerve terminal network. We therefore conclude that the induced MSC have a therapeutic potential for neurodegenerative processes and diseases, both by the NTFs secretion and by the migratory trait toward the diseased tissue.

A general framework to quantify the effect of restricted diffusion on the NMR signal with applications to double pulsed field gradient NMR experiments

Based on a description introduced by Robertson, Grebenkov recently introduced a powerful formalism to represent the diffusion-attenuated NMR signal for simple pore geometries such as slabs, cylinders, and spheres analytically. In this work, we extend this multiple correlation function formalism by allowing for possible variations in the direction of the magnetic field gradient waveform. This extension is necessary, for example, to incorporate the effects of imaging gradients in diffusion-weighted NMR imaging scans and in characterizing anisotropy at different length scales via double pulsed field gradient (PFG) experiments. In cylindrical and spherical pores, respectively, two- and three-dimensional vector operators are employed whose form is deduced from Grebenkovs results via elementary operator algebra for the case of cylinders and the Wigner-Eckart theorem for the case of spheres. The theory was validated by comparison with known findings and with experimental double-PFG data obtained from water-filled microcapillaries.

Migration of Neurotrophic Factors‐Secreting Mesenchymal Stem Cells Toward a Quinolinic Acid Lesion as Viewed by Magnetic Resonance Imaging

Stem cell‐based treatment is a promising frontier for neurodegenerative diseases. We propose a novel protocol for inducing the differentiation of rat mesenchymal stem cells (MSCs) toward neurotrophic factor (NTF)‐secreting cells as a possible neuroprotective agent. One of the major caveats of stem cell transplantation is their fate post‐transplantation. To test the viability of the cells, we tracked the transplanted cells in vivo by magnetic resonance imaging (MRI) scans and validated the results by histology. MSCs went through a two‐step medium‐based differentiation protocol, followed by in vitro characterization using immunocytochemistry and immunoblotting analysis of the cell media. We examined the migratory properties of the cells in the quinolinic acid (QA)‐induced striatal lesion model for Huntingtons disease. The induced cells were labeled and transplanted posterior to the lesion. Rats underwent serial MRI scans to detect cell migration in vivo. On the 19th day, animals were sacrificed, and their brains were removed for immunostaining. Rat MSCs postinduction exhibited both neuronal and astrocyte markers, as well as production and secretion of NTFs. High‐resolution two‐dimensional and three‐dimensional magnetic resonance images revealed that the cells migrated along a distinct route toward the lesion. The in vivo MRI results were validated by the histological study, which demonstrated that phagocytosis had only partially occurred and that MRI could correctly depict the status of the migrating cells. The results show that these cells migrated toward a QA lesion and therefore survived for 19 days post‐transplantation. This gives hope for future research harnessing these cells for treating neurodegenerative diseases.

From single-pulsed field gradient to double-pulsed field gradient MR: gleaning new microstructural information and developing new forms of contrast in MRI.

One of the hallmarks of diffusion NMR and MRI is its ability to utilize restricted diffusion to probe compartments much smaller than the excited volume or the MRI voxel, respectively, and to extract microstructural information from them. Single‐pulsed field gradient (s‐PFG) MR methodologies have been employed with great success to probe microstructures in various disciplines, ranging from chemistry to neuroscience. However, s‐PFG MR also suffers from inherent shortcomings, especially when specimens are characterized by orientation or size distributions: in such cases, the microstructural information available from s‐PFG experiments is limited or lost. Double‐pulsed field gradient (d‐PFG) MR methodology, an extension of s‐PFG MR, has attracted attention owing to recent theoretical studies predicting that it can overcome certain inherent limitations of s‐PFG MR. In this review, we survey the microstructural features that can be obtained from conventional s‐PFG methods in the different q regimes, and highlight its limitations. The experimental aspects of d‐PFG methodology are then presented, together with an overview of its theoretical underpinnings and a general framework for relating the MR signal decay and material microstructure, affording new microstructural parameters. We then discuss recent studies that have validated the theory using phantoms in which the ground truth is well known a priori, a crucial step prior to the application of d‐PFG methodology in neuronal tissue. The experimental findings are in excellent agreement with the theoretical predictions and reveal, inter alia, zero‐crossings of the signal decay, robustness towards size distributions and angular dependences of the signal decay from which accurate microstructural parameters, such as compartment size and even shape, can be extracted. Finally, we show some initial findings in d‐PFG MR imaging. This review lays the foundation for future studies, in which accurate and novel microstructural information could be extracted from complex biological specimens, eventually leading to new forms of contrast in MRI. Copyright

Pore diameter mapping using double pulsed-field gradient MRI and its validation using a novel glass capillary array phantom

Double pulsed-field gradient (d-PFG) MRI can provide quantitative maps of microstructural quantities and features within porous media and tissues. We propose and describe a novel MRI phantom, consisting of wafers of highly ordered glass capillary arrays (GCA), and its use to validate and calibrate a d-PFG MRI method to measure and map the local pore diameter. Specifically, we employ d-PFG Spin-Echo Filtered MRI in conjunction with a recently introduced theoretical framework, to estimate a mean pore diameter in each voxel within the imaging volume. This simulation scheme accounts for all diffusion and imaging gradients within the diffusion weighted MRI (DWI) sequence, and admits the violation of the short gradient pulse approximation. These diameter maps agree well with pore sizes measured using both optical microscopy and single PFG diffusion diffraction NMR spectroscopy using the same phantom. Pixel-by-pixel analysis shows that the local pore diameter can be mapped precisely and accurately within a specimen using d-PFG MRI.

Measuring small compartmental dimensions with low-q angular double-PGSE NMR: The effect of experimental parameters on signal decay.

In confined geometries, the MR signal attenuation obtained from single pulsed gradient spin echo (s-PGSE) experiments reflects the dimension of the compartment, and in some cases, its geometry. However, to measure compartment size, high q-values must be applied, requiring high gradient strengths and/or long pulse durations and diffusion times. The angular double PGSE (d-PGSE) experiment has been proposed as a means to extract dimensions of confined geometries using low q-values. In one realization of the d-PGSE experiment, the first gradient pair is fixed along the x-axis, and the orientation of the second gradient pair is varied in the X-Y plane. Such a measurement is sensitive to microscopic anisotropy induced by the boundaries of the restricting compartment, and allows extraction of the compartment dimension. In this study, we have juxtaposed angular d-PGSE experiments and simulations to extract sizes from well-characterized NMR phantoms consisting of water filled microcapillaries. We are able to accurately extract sizes of small compartments (5mum) using the angular d-PGSE experiment even when the short gradient pulse (SGP) approximation is violated and over a range of mixing and diffusion times. We conclude that the angular d-PGSE experiment may fill an important niche in characterizing compartment sizes in which restricted diffusion occurs.

Conventions and nomenclature for double diffusion encoding NMR and MRI

Stejskal and Tanners ingenious pulsed field gradient design from 1965 has made diffusion NMR and MRI the mainstay of most studies seeking to resolve microstructural information in porous systems in general and biological systems in particular. Methods extending beyond Stejskal and Tanners design, such as double diffusion encoding (DDE) NMR and MRI, may provide novel quantifiable metrics that are less easily inferred from conventional diffusion acquisitions. Despite the growing interest on the topic, the terminology for the pulse sequences, their parameters, and the metrics that can be derived from them remains inconsistent and disparate among groups active in DDE. Here, we present a consensus of those groups on terminology for DDE sequences and associated concepts. Furthermore, the regimes in which DDE metrics appear to provide microstructural information that cannot be achieved using more conventional counterparts (in a model‐free fashion) are elucidated. We highlight in particular DDEs potential for determining microscopic diffusion anisotropy and microscopic fractional anisotropy, which offer metrics of microscopic features independent of orientation dispersion and thus provide information complementary to the standard, macroscopic, fractional anisotropy conventionally obtained by diffusion MR. Finally, we discuss future vistas and perspectives for DDE. Magn Reson Med 75:82–87, 2016.

Detecting diffusion-diffraction patterns in size distribution phantoms using double-pulsed field gradient NMR: Theory and experiments

NMR observable nuclei undergoing restricted diffusion within confining pores are important reporters for microstructural features of porous media including, inter-alia, biological tissues, emulsions and rocks. Diffusion NMR, and especially the single-pulsed field gradient (s-PFG) methodology, is one of the most important noninvasive tools for studying such opaque samples, enabling extraction of important microstructural information from diffusion-diffraction phenomena. However, when the pores are not monodisperse and are characterized by a size distribution, the diffusion-diffraction patterns disappear from the signal decay, and the relevant microstructural information is mostly lost. A recent theoretical study predicted that the diffusion-diffraction patterns in double-PFG (d-PFG) experiments have unique characteristics, such as zero-crossings, that make them more robust with respect to size distributions. In this study, we theoretically compared the signal decay arising from diffusion in isolated cylindrical pores characterized by lognormal size distributions in both s-PFG and d-PFG methodologies using a recently presented general framework for treating diffusion in NMR experiments. We showed the gradual loss of diffusion-diffraction patterns in broadening size distributions in s-PFG and the robustness of the zero-crossings in d-PFG even for very large standard deviations of the size distribution. We then performed s-PFG and d-PFG experiments on well-controlled size distribution phantoms in which the ground-truth is well-known a priori. We showed that the microstructural information, as manifested in the diffusion-diffraction patterns, is lost in the s-PFG experiments, whereas in d-PFG experiments the zero-crossings of the signal persist from which relevant microstructural information can be extracted. This study provides a proof of concept that d-PFG may be useful in obtaining important microstructural features in samples characterized by size distributions.

Mesenchymal stem cells induced to secrete neurotrophic factors attenuate quinolinic acid toxicity: A potential therapy for Huntington's disease

Huntingtons disease (HD) is a hereditary, progressive and ultimately fatal neurodegenerative disorder. Excitotoxicity and reduced availability of neurotrophic factors (NTFs) likely play roles in HD pathogenesis. Recently we developed a protocol that induces adult human bone marrow derived mesenchymal stem cells (MSCs) into becoming NTF secreting cells (NTF(+) cells). Striatal transplantation of such cells represents a promising autologous therapeutic approach whereby NTFs are delivered to damaged areas. Here, the efficacy of NTF(+) cells was evaluated using the quinolinic acid (QA) rat model for excitotoxicity. We show that NTF(+) cells transplanted into rat brains after QA injection survive transplantation (19% after 6 weeks), maintain their NTF secreting phenotype and significantly reduce striatal volume changes associated with QA lesions. Moreover, QA-injected rats treated with NTF(+) cells exhibit improved behavior; namely, perform 80% fewer apomorphine induced rotations than PBS-treated QA-injected rats. Importantly, we found that MSCs derived from HD patients can be induced to become NTF(+) cells and exert efficacious effects similarly to NTF(+) cells derived from healthy donors. To our knowledge, this is the first study to take adult bone marrow derived mesenchymal stem cells from patients with an inherited disease, transplant them into an animal model and evidence therapeutic benefit. Using MRI we demonstrate in vivo that PBS-treated QA-injected striatae exhibit increasing T(2) values over time in lesioned regions, whereas T(2) values decrease in equivalent regions of QA-injected rats treated with NTF(+) cells. We conclude that NTF cellular treatment could serve as a novel therapy for managing HD.

Noninvasive bipolar double-pulsed-field-gradient NMR reveals signatures for pore size and shape in polydisperse, randomly oriented, inhomogeneous porous media

Noninvasive characterization of pore size and shape in opaque porous media is a formidable challenge. NMR diffusion-diffraction patterns were found to be exceptionally useful for obtaining such morphological features, but only when pores are monodisperse and coherently placed. When locally anisotropic pores are randomly oriented, conventional diffusion NMR methods fail. Here, we present a simple, direct, and general approach to obtain both compartment size and shape even in such settings and even when pores are characterized by internal field gradients. Using controlled porous media, we show that the bipolar-double-pulsed-field-gradient (bp-d-PFG) methodology yields diffusion-diffraction patterns from which pore size can be directly obtained. Moreover, we show that pore shape, which cannot be obtained by conventional methods, can be directly inferred from the modulation of the signal in angular bp-d-PFG experiments. This new methodology significantly broadens the types of porous media that can be studied using noninvasive diffusion-diffraction NMR.