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We found Stat1 promotes Trpm7 transcription by promoting H3K27 acetylation and thus promotes calcium overload of neurons after intracerebral hemorrhage.

Intracerebral hemorrhage (ICH) is classified as a subtype of stroke and calcium (Ca

Engineered proteins provide an interesting template for designing fluorine-19 (19F) magnetic resonance imaging (MRI) contrast agents, yet progress has been hindered by the unpredictable relaxation properties of fluorine. Herein, we present the biosynthesis of a protein block copolymer, termed "fluorinated thermoresponsive assembled protein" (F-TRAP), which assembles into a monodisperse nanoscale micelle with interesting 19F NMR properties and the ability to encapsulate and release small therapeutic molecules, imparting potential as a diagnostic and therapeutic (theranostic) agent. The assembly of the F-TRAP micelle, composed of a coiled-coil pentamer corona and a hydrophobic, thermoresponsive elastin-like polypeptide core, results in a drastic depression in spin-spin relaxation ( T2) times and unaffected spin-lattice relaxation ( T1) times. The nearly unchanging T1 relaxation rates and linearly dependent T2 relaxation rates have allowed for detection via zero echo time 19F MRI, and the in vivo MR potential has been preliminarily explored using 19F magnetic resonance spectroscopy (MRS). This fluorinated micelle has also demonstrated the ability to encapsulate the small-molecule chemotherapeutic doxorubicin and release its cargo in a thermoresponsive manner owing to its inherent stimuli-responsive properties, presenting an interesting avenue for the development of thermoresponsive 19F MRI/MRS-traceable theranostic agents.

Progress in clinical development of magnetic resonance imaging (MRI) substrate-sensors of enzymatic activity has been slow partly due to the lack of human efficacy data. We report here a strategy that may serve as a shortcut from bench to bedside. We tested ultra high-resolution 7T MRI (µMRI) of human surgical histology sections in a 3-year IRB approved, HIPAA compliant study of surgically clipped brain aneurysms. µMRI was used for assessing the efficacy of MRI substrate-sensors that detect myeloperoxidase activity in inflammation. The efficacy of Gd-5HT-DOTAGA, a novel myeloperoxidase (MPO) imaging agent synthesized by using a highly stable gadolinium (III) chelate was tested both in tissue-like phantoms and in human samples. After treating histology sections with paramagnetic MPO substrate-sensors we observed relaxation time shortening and MPO activity-dependent MR signal enhancement. An increase of normalized MR signal generated by ultra-short echo time MR sequences was corroborated by MPO activity visualization by using a fluorescent MPO substrate. The results of µMRI of MPO activity associated with aneurysmal pathology and immunohistochemistry demonstrated active involvement of neutrophils and neutrophil NETs as a result of pro-inflammatory signalling in the vascular wall and in the perivascular space of brain aneurysms.

Engineered proteins provide an interesting template for designing fluorine-19 ((19)F) magnetic resonance imaging (MRI) contrast agents, yet progress has been hindered by the unpredictable relaxation properties of fluorine. Herein, we present the biosynthesis of a protein block copolymer, termed “fluorinated thermoresponsive assembled protein” (F-TRAP), which assembles into a monodisperse nanoscale micelle with interesting (19)F NMR properties and the ability to encapsulate and release small therapeutic molecules, imparting potential as a diagnostic and therapeutic (theranostic) agent. The assembly of the F-TRAP micelle, composed of a coiled-coil pentamer corona and a hydrophobic, thermoresponsive elastin-like polypeptide core, results in a drastic depression in spin-spin relaxation (T(2)) times and unaffected spin-lattice relaxation (T(1)) times. The nearly unchanging T(1) relaxation rates and linearly dependent T(2) relaxation rates have allowed for detection via zero echo time (19)F MRI, and the in vivo MR potential has been preliminarily explored using (19)F magnetic resonance spectroscopy (MRS). This fluorinated micelle has also demonstrated the ability to encapsulate the small-molecule chemotherapeutic doxorubicin and release its cargo in a thermoresponsive manner owing to its inherent stimuli-responsive properties, presenting an interesting avenue for the development of thermoresponsive (19)F MRI/MRS-traceable theranostic agents.

Solid tumor microstructure is related to the aggressiveness of the tumor, interstitial pressure and drug delivery pathways, which are closely associated with treatment response, metastatic spread and prognosis. In this study, we introduce a novel diffusion MRI data analysis framework, pulsed and oscillating gradient MRI for assessment of cell size and extracellular space (POMACE), and demonstrate its feasibility in a mouse tumor model. In vivo and ex vivo POMACE experiments were performed on mice bearing the GL261 murine glioma model (n = 8). Since the complete diffusion time dependence is in general non-analytical, the tumor microstructure was modeled in an appropriate time/frequency regime by impermeable spheres (radius Rcell, intracellular diffusivity Dics) surrounded by extracellular space (ECS) (approximated by constant apparent diffusivity Decs in volume fraction ECS). POMACE parametric maps (ECS, Rcell, Dics, Decs) were compared with conventional diffusion-weighted imaging metrics, electron microscopy (EM), alternative ECS determination based on effective medium theory (EMT), and optical microscopy performed on the same samples. It was shown that Decs can be approximated by its long time tortuosity limit in the range [1/(88 Hz)–31 ms]. ECS estimations (44 ± 7% in vivo and 54 ± 11% ex vivo) were in agreement with EMT-based ECS and literature on brain gliomas. Ex vivo, ECS maps correlated well with optical microscopy. Cell sizes (Rcell = 4.8 ± 1.3 in vivo and 4.3 ± 1.4 µm ex vivo) were consistent with EM measurements (4.7 ± 1.8 µm). In conclusion, Rcell and ECS can be quantified and mapped in vivo and ex vivo in brain tumors using the proposed POMACE method. Our experimental results support the view that POMACE provides a way to interpret the frequency or time dependence of the diffusion coefficient in tumors in terms of objective biophysical parameters of neuronal tissue, which can be used for non-invasive monitoring of preclinical cancer studies and treatment efficacy. Copyright © 2016 John Wiley & Sons, Ltd.

Purpose To disentangle the free diffusivity (D0) and cellular membrane restrictions, by means of their surface-to-volume ratio (S/V), using the frequency-dependence of the diffusion coefficient D(ω), measured in brain tumors in the short diffusion-time regime using oscillating gradients (OGSE). Methods In vivo and ex vivo OGSE experiments were performed on mice bearing the GL261 murine glioma model (n = 10) to identify the relevant time/frequency (t/ω) domain where D(ω) linearly decreases with ω−1/2. Parametric maps (S/V, D0) are compared with conventional DWI metrics. The impact of frequency range and temperature (20°C versus 37°C) on S/V and D0 is investigated ex vivo. Results The validity of the short diffusion-time regime is demonstrated in vivo and ex vivo. Ex vivo measurements confirm that the purely geometric restrictions embodied in S/V are independent from temperature and frequency range, while the temperature dependence of the free diffusivity D0 is similar to that of pure water. Conclusion Our results suggest that D(ω) in the short diffusion-time regime can be used to uncouple the purely geometric restriction effect, such as S/V, from the intrinsic medium diffusivity properties, and provides a nonempirical and objective way to interpret frequency/time-dependent diffusion changes in tumors in terms of objective biophysical tissue parameters. Magn Reson Med, 2015. © 2015 Wiley Periodicals, Inc.

In biomedical research, the use of animal models of human disease offers the opportunity to test and optimize magnetic resonance imaging (MRI) pulse sequences, and to compare image features with corresponding histopathology, the current gold standard for disease assessment. Despite these great advantages, the coregistration steps between MRI and histological sections obtained through light microscopy have presented important practical challenges (1–5). The main difficulties encountered during the validation steps stem from both the slice section misalignments and the significant difference in slice thicknesses between sub-millimetric in vivo MRI (ranging from 100 μm to 1 mm thickness) and histology sections (thicknesses commonly ranging from 5 to 100 μm). These limitations have been partially addressed by acquiring three-dimensional (3D) ex vivo images of excised and perfused organs of interest. The scanning is usually assessed over long imaging times using a dedicated coil closely fitting the sample to improve the sensitivity (6–20). The accumulation of repeated scans results in motion-free and highly resolved 3D MRI datasets enabling a precise virtual realignment of the image slice of interest to closely match the physical histology section (21–26). However, important discrepancies remain between these two analyses (22). These notable differences can be attributed to sample changes associated with postmortem tissue processing due to fixation and dehydration resulting in tissue deformation, as well as artifacts caused by sectioning and the chemicals used during the staining process (27–33). These discrepancies have been addressed using time-consuming postprocessing techniques when establishing 3D atlases (21–24). But this approach may be more difficult to systematically implement for every existing transgenic mouse line or murine disease model due to limited resources or lack of image postprocessing expertise. Alternatively, histological slides can conceivably be scanned directly by both MRI and light microscopy (34–36). Coregistration would then be easily achievable provided that MRI sensitivity and the experimental conditions allow direct imaging with adequate spatial resolution of histological sections ranging from 5 to 100 μm. Along these lines, efforts have been devoted to the design of dedicated radiofrequency (RF) coil geometries that closely matches the shape of the tissue of interest. Nabuurs and coworkers (36) proposed the use of an inductively coupled loop combined with a commercial linear birdcage mouse head coil capable of housing the slide setup. The insertion of the coupled loop was aimed at driving the effective RF coverage toward the tissue of interest thereby reciprocally increasing the resulting sensitivity. This solution led to a less than fourfold gain in sensitivity under the best geometrical inductive coupling conditions compared with results obtained using the linear birdcage mouse head coil alone. Alternatively, Meadowcroft et al. (35) proposed a unique RF coil design based on a flat U-shaped structure that closely matches the planar nature of a histological tissue sample providing the most optimal filling factor. The authors demonstrated excellent RF homogeneity throughout the region of interest (24 × 24 mm) within the coil cavity designed to house a sample encased on dual glass coverslips. The length (L) of the coil was doubled (L = 48 mm) by inserting an equal-sized Teflon spacer between the two flat copper strips next to the U-shaped cavity leading to the driving port. The presence of this spacer both provided some distributed capacitance and allowed for the current to be spread evenly throughout the strip before reaching the sample cavity from the connections to the tuning capacitor. Although the sensitivity of this new probe design was not compared with other commonly available RF coil structures, the authors demonstrated their ability to acquire highly detailed MRI from 60 μm sections of freshly mounted tissue sandwiched between two coverslips. This setup enabled the accurate correlation between MRI and light microscopy by direct imaging of postmortem tissue samples from either human Alzheimer’s Disease (AD) subjects or mice of a strain exhibiting some characteristics of AD (34). Here, we build on the work of Meadowcroft et al. to accommodate both wider and thicker tissue slides by devising an in-house set of five histology MRI probes. All newly designed RF histology resonators were systemically characterized by mapping their RF homogeneity and examining how each performed against commercially available surface and whole mouse head coils. In addition, we examined the effect of the spacer on the resulting coil sensitivity by replacing the Teflon with either air or glass as alternative insulators. We intended to image both freshly mounted tissue and premounted specimens originating from either paraffin embedding or cryosections having thicknesses ranging from 5 to 1000 μm. To this effect, our study investigated the best conditions for sample preparation for either case in order to minimize MRI artifacts. We demonstrated that our new five-coil set can accommodate off-the-shelf standardized histology preparations ranging from dual coverslips to glass slides. MRI examples of tissue derived from whole mouse organs and human specimens illustrate both the performance of each coil, as well as the near perfect coregistration with histology.

Solid tumor microstructure is related to the aggressiveness of the tumor, interstitial pressure and drug delivery pathways, which are closely associated with treatment response, metastatic spread and prognosis. In this study, we introduce a novel diffusion MRI data analysis framework, pulsed and oscillating gradient MRI for assessment of cell size and extracellular space (POMACE), and demonstrate its feasibility in a mouse tumor model. In vivo and ex vivo POMACE experiments were performed on mice bearing the GL261 murine glioma model (n = 8). Since the complete diffusion time dependence is in general non-analytical, the tumor microstructure was modeled in an appropriate time/frequency regime by impermeable spheres (radius Rcell , intracellular diffusivity Dics ) surrounded by extracellular space (ECS) (approximated by constant apparent diffusivity Decs in volume fraction ECS). POMACE parametric maps (ECS, Rcell , Dics , Decs ) were compared with conventional diffusion-weighted imaging metrics, electron microscopy (EM), alternative ECS determination based on effective medium theory (EMT), and optical microscopy performed on the same samples. It was shown that Decs can be approximated by its long time tortuosity limit in the range [1/(88 Hz)-31 ms]. ECS estimations (44 ± 7% in vivo and 54 ± 11% ex vivo) were in agreement with EMT-based ECS and literature on brain gliomas. Ex vivo, ECS maps correlated well with optical microscopy. Cell sizes (Rcell = 4.8 ± 1.3 in vivo and 4.3 ± 1.4 µm ex vivo) were consistent with EM measurements (4.7 ± 1.8 µm). In conclusion, Rcell and ECS can be quantified and mapped in vivo and ex vivo in brain tumors using the proposed POMACE method. Our experimental results support the view that POMACE provides a way to interpret the frequency or time dependence of the diffusion coefficient in tumors in terms of objective biophysical parameters of neuronal tissue, which can be used for non-invasive monitoring of preclinical cancer studies and treatment efficacy. Copyright © 2016 John WileySons, Ltd.

Current clinical Gd3+-based T1 magnetic resonance imaging (MRI) contrast agents (CAs) are suboptimal or unsuitable, especially at higher magnetic fields (>1.5 Tesla) for advanced MRI applications such as blood pool, cellular and molecular imaging. Herein, towards the goal of developing a safe and more efficacious high field T1 MRI CA for these applications, we report the sub-acute toxicity and contrast enhancing capabilities of a novel nanoparticle MRI CA comprising of manganese (Mn2+) intercalated graphene nanoparticles functionalized with dextran (hereafter, Mangradex) in rodents. Sub-acute toxicology performed on rats intravenously injected with Mangradex at 1, 50 or 100 mg/kg dosages 3 times per week for three weeks indicated that dosages ≤50 mg/kg could serve as potential diagnostic doses. Whole body 7 Tesla MRI performed on mice injected with Mangradex at a potential diagnostic dose (25 mg/kg or 455 nanomoles Mn2+/kg; ~2 orders of magnitude lower than the paramagnetic ion concentration in a typical clinical dose) showed persistent (up to at least 2 hours) contrast enhancement in the vascular branches (Mn2+ concentration in blood at steady state = 300 ppb, per voxel = 45 femtomoles). The results lay the foundations for further development of Mangradex as a vascular and cellular/ molecular MRI probe.

To disentangle the free diffusivity (D0 ) and cellular membrane restrictions, by means of their surface-to-volume ratio (S/V), using the frequency-dependence of the diffusion coefficient D(ω), measured in brain tumors in the short diffusion-time regime using oscillating gradients (OGSE).In vivo and ex vivo OGSE experiments were performed on mice bearing the GL261 murine glioma model (n = 10) to identify the relevant time/frequency (t/ω) domain where D(ω) linearly decreases with ω(-1/2) . Parametric maps (S/V, D0 ) are compared with conventional DWI metrics. The impact of frequency range and temperature (20°C versus 37°C) on S/V and D0 is investigated ex vivo.The validity of the short diffusion-time regime is demonstrated in vivo and ex vivo. Ex vivo measurements confirm that the purely geometric restrictions embodied in S/V are independent from temperature and frequency range, while the temperature dependence of the free diffusivity D0 is similar to that of pure water.Our results suggest that D(ω) in the short diffusion-time regime can be used to uncouple the purely geometric restriction effect, such as S/V, from the intrinsic medium diffusivity properties, and provides a nonempirical and objective way to interpret frequency/time-dependent diffusion changes in tumors in terms of objective biophysical tissue parameters. Magn Reson Med 76:237-247, 2016. © 2015 Wiley Periodicals, Inc.

Recent studies showed that synaptic Ab can further cause distal axonopathy and neurodegeneration leading to neuronal loss. We explored an in vivo setup to demonstrate the synaptic Ab to cause pre-synaptic axonal degeneration. Methods: Ab1-42 (4 n mole, N 1⁄4 6) or saline (N 1⁄4 6) were injected at the right optic tract axonal terminals in 12-week-old C57BL/6 mice. In 1 and 3 months after Ab injection, mice were placed in a Bruker 4.7T small animal MRI for Diffusion Tensor Imaging (DTI) with slice thickness 0.5 mm, field of view of 2cm x 2cm and matrix 128 x 128 (zero filling to 256 x 256) to cover the visual system from eyes to superior colliculus. Animals were then sacrificed. The optic tract and nerve were examined using a primary antibody against phosphorylated neurofilament (pNF, SMI-31) and myelin basic protein (MBP). Results: In optic tract, the Ab-injected side (right side) of the tracts showed a 12-16% decrease of axial diffusivity (P

Transgenic mice are used increasingly to model brain amyloidosis, mimicking the pathogenic processes involved in Alzheimer's disease (AD). In this chapter, an in vivo strategy is described that has been successfully used to map amyloid-β deposits in transgenic mouse models of AD with magnetic resonance imaging (MRI), utilizing both the endogenous contrast induced by the plaques attributed to their iron content and by selectively enhancing the signal from amyloid-β plaques using molecular-targeting vectors labeled with MRI contrast agents. To obtain sufficient spatial resolution for effective and sensitive mouse brain imaging, magnetic fields of 7-Tesla (T) or more are required. These are higher than the 1.5-T field strength routinely used for human brain imaging. The higher magnetic fields affect contrast agent efficiency and dictate the choice of pulse sequence parameters for in vivo MRI, all addressed in this chapter. Two-dimensional (2D) multi-slice and three-dimensional (3D) MRI acquisitions are described and their advantages and limitations are discussed. The experimental setup required for mouse brain imaging is explained in detail, including anesthesia, immobilization of the mouse's head to reduce motion artifacts, and anatomical landmarks to use for the slice alignment procedure to improve image co-registration during longitudinal studies and for subsequent matching of MRI with histology.

Amyloid plaques are one of the pathological hallmarks of Alzheimer's disease (AD). The visualization of amyloid plaques in the brain is important to monitor AD progression and to evaluate the efficacy of therapeutic interventions. Our group has developed several contrast agents to detect amyloid plaques in vivo using magnetic resonance microimaging (μMRI) in AD transgenic mice, where we used intra-carotid mannitol to enhance blood brain barrier (BBB) permeability. In the present study, we used ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles, chemically coupled with Aβ 1-42 peptide to detect amyloid deposition along with mannitol for in vivo μMRI by femoral intravenous injection. A 3D gradient multi-echo sequence was used for imaging with a 100 μm isotropic resolution. The amyloid plaques detected by T2*-weighted μMRI were confirmed with matched histological sections. Furthermore, two different quantitative analyses were used. The region of interest-based quantitative measurement of T2* values showed contrast injected APP/PS1 mice had significantly reduced T2* values compared to wild-type mice. In addition, the scans were examined with voxel-based morphometry (VBM) using statistical parametric mapping (SPM) for comparison of contrast injected AD transgenic and wild-type mice. The regional differences seen in VBM comparing USPIO-Aβ1-42 injected APP/PS1 and wild type mice correlated with the amyloid plaque distribution histologically, contrasting with no differences between the two groups of mice without contrast agent injection in regions of the brain with amyloid deposition. Our results demonstrated that both approaches were able to identify the differences between AD transgenic mice and wild type mice, after injected with USPIO-Aβ1-42. The feasibility of using less invasive intravenous femoral injections for amyloid plaque detection in AD transgenic mice facilitates using this method for longitudinal studies in the pathogenesis of AD.

Glioblastoma stem cells (GSCs) have the ability to self-renew, differentiate into tumor lineages, and give rise to tumors in immunodeficient animal models. More importantly, GSCs are resistant to current therapies, leading to tumor recurrence. Glioblastoma (GBM) is highly heterogeneous at the molecular and cellular level. This heterogeneity also exhibits itself within the stem cell compartment, further impairing the development of effective therapies. CD133+ GSCs cells have increased tumorigenicity. However, the facts that CD133- cells also give rise to tumors in animal models and that some tumors do not have a CD133+ cell population suggest that CD133 is not a universal GSC marker. The Notch signaling pathway, previously believed to be activated in CD133+ GSCs, is important for GSC self-renewal and therapy resistance. To study GSC heterogeneity, we used genetic reporters for Notch activation and made the surprising observation that Notch signaling is active in small populations of cells that do not express CD133 (CD133-/Notch + ; n = 3 primary GBM cultures). Our in vitro tumorsphere formation and in vivo tumor formation assays showed that both CD133 + /Notch- and CD133-/Notch+ cells have stem cell properties. Global transcriptome analysis revealed that CD133 + /Notch- and CD133-/Notch+ GSCs utilize differential transcriptional programs, which prime CD133 + /Notch- GSCs to rely on anaerobic glycolysis. Furthermore, CD133/-Notch+ cells are able to give rise to CD133 + /Notch- cells, although the converse does not occur. This finding suggests that the two cell types have distinct differentiation potentials. Finally, preliminary data suggest that these two GSC subtypes behave differentially in response to temozolomide and radiation treatment, which are the standard of care for GBM. We are currently testing the contribution of distinct GSCs to tumor heterogeneity in vivo, using lineage-tracing systems. Understanding the biology of GSCs and the functional importance of heterogeneity within GSC populations is critical for the design of novel therapies.