Dose Effect of Mesenchymal Stromal Cell Delivery Through Cardiopulmonary Bypass.

BACKGROUND: Neurologic impairments are a significant concern for survivors after pediatric cardiac surgery with cardiopulmonary bypass (CPB). We have previously shown that mesenchymal stromal cell (MSC) delivery through CPB has the potential to mitigate the effects of CPB on neural stem/progenitor cells. This study assessed the dose effects of MSCs. METHODS: Piglets (n = 20) were randomly assigned to 1 of 4 groups: control, CPB, or CPB followed by MSC administration with low and high doses (10 × 106 and 100 × 106 cells per kilogram). We assessed acute dose effect on cell distribution, multiorgan functions, systemic inflammation, microglia activation, and neural stem/progenitor cell activities. RESULTS: By magnetic resonance imaging, approximately 10 times more MSCs were detected within the entire brain after high-dose delivery than after low-dose delivery. No adverse events affecting hemodynamics, various biomarkers, and neuroimaging were detected after high-dose MSC delivery. High-dose MSCs significantly increased circulating levels of interleukin 4 after CPB. Both MSC groups normalized microglia activation after CPB, demonstrating MSC-induced reduction in cerebral inflammation. There was a significant increase in neuroblasts in the subventricular zone in both treatment groups. The thickness of the most active neurogenic area within the subventricular zone was significantly increased after high-dose treatment compared with CPB and low-dose MSCs, suggesting dose-dependent effects on the neurogenic niche. CONCLUSIONS: MSC delivery through CPB is feasible up to 100 × 106 cells per kilogram. MSC treatment during cardiac surgery has the potential to reduce systemic and cerebral inflammation and to modulate responses of an active neurogenic niche to CPB. Further investigation is necessary to assess the long-term effects and to develop a more complete dose-response curve.

Mesenchymal Stromal Cell Delivery Via Cardiopulmonary Bypass Provides Neuroprotection in a Juvenile Porcine Model.

Oxidative/inflammatory stresses due to cardiopulmonary bypass (CPB) cause prolonged microglia activation and cortical dysmaturation, thereby contributing to neurodevelopmental impairments in children with congenital heart disease (CHD). This study found that delivery of mesenchymal stromal cells (MSCs) via CPB minimizes microglial activation and neuronal apoptosis, with subsequent improvement of cortical dysmaturation and behavioral alteration after neonatal cardiac surgery. Furthermore, transcriptomic analyses suggest that exosome-derived miRNAs may be the key drivers of suppressed apoptosis and STAT3-mediated microglial activation. Our findings demonstrate that MSC treatment during cardiac surgery has significant translational potential for improving cortical dysmaturation and neurological impairment in children with CHD.

Classification of Activated Microglia by Convolutional Neural Networks.

Microglia are the resident macrophages in the central nervous system. Brain injuries, such as traumatic brain injury, hypoxia, and stroke, can induce inflammatory responses accompanying microglial activation. The morphology of microglia is notably diverse and is one of the prominent manifestations during activation. In this study, we proposed to detect the activated microglia in immunohistochemistry images by convolutional neural networks (CNN). 2D Iba1 images (40µm) were acquired from a control and a cardiac arrest treated Sprague-Dawley rat brain by a scanning microscope using a 20X objective. The training data were a collection of 54,333 single-cell images obtained from the cortex and midbrain areas, and curated by experienced neuroscientists. Results were compared between CNNs with different architectures, including Resnet18, Resnet50, Resnet101, and support vector machine (SVM) classifiers. The highest model performance was found by Resnet18, trained after 120 epochs with a classification accuracy of 95.5%. The findings indicate a potential application for using CNN in quantitative analysis of microglial morphology over regional difference in a large brain section.

A Baboon Brain Atlas for Magnetic Resonance Imaging and Positron Emission Tomography Image Analysis.

The olive baboon (Papio anubis) is phylogenetically proximal to humans. Investigation into the baboon brain has shed light on the function and organization of the human brain, as well as on the mechanistic insights of neurological disorders such as Alzheimer's and Parkinson's. Non-invasive brain imaging, including positron emission tomography (PET) and magnetic resonance imaging (MRI), are the primary outcome measures frequently used in baboon studies. PET functional imaging has long been used to study cerebral metabolic processes, though it lacks clear and reliable anatomical information. In contrast, MRI provides a clear definition of soft tissue with high resolution and contrast to distinguish brain pathology and anatomy, but lacks specific markers of neuroreceptors and/or neurometabolites. There is a need to create a brain atlas that combines the anatomical and functional/neurochemical data independently available from MRI and PET. For this purpose, a three-dimensional atlas of the olive baboon brain was developed to enable multimodal imaging analysis. The atlas was created on a population-representative template encompassing 89 baboon brains. The atlas defines 24 brain regions, including the thalamus, cerebral cortex, putamen, corpus callosum, and insula. The atlas was evaluated with four MRI images and 20 PET images employing the radiotracers for [11C]benzamide, [11C]metergoline, [18F]FAHA, and [11C]rolipram, with and without structural aids like [18F]flurodeoxyglycose images. The atlas-based analysis pipeline includes automated segmentation, registration, quantification of region volume, the volume of distribution, and standardized uptake value. Results showed that, in comparison to PET analysis utilizing the "gold standard" manual quantification by neuroscientists, the performance of the atlas-based analysis was at >80 and >70% agreement for MRI and PET, respectively. The atlas can serve as a foundation for further refinement, and incorporation into a high-throughput workflow of baboon PET and MRI data. The new atlas is freely available on the Figshare online repository (https://doi.org/10.6084/m9.figshare.16663339), and the template images are available from neuroImaging tools & resources collaboratory (NITRC) (https://www.nitrc.org/projects/haiko89/).

Comparison of in vivo and in situ detection of hippocampal metabolites in mouse brain using 1 H-MRS.

The study of cerebral metabolites relies heavily on detection methods and sample preparation. Animal experiments in vivo require anesthetic agents that can alter brain metabolism, whereas ex vivo experiments demand appropriate fixation methods to preserve the tissue from rapid postmortem degradation. In this study, the metabolic profiles of mouse hippocampi using proton magnetic resonance spectroscopy (1 H-MRS) were compared in vivo and in situ with or without focused beam microwave irradiation (FBMI) fixation. Ten major brain metabolites, including lactate (Lac), N-acetylaspartate (NAA), total choline (tCho), myo-inositol (mIns), glutamine (Gln), glutamate (Glu), aminobutyric acid (GABA), glutathione (GSH), total creatine (tCr) and taurine (Tau), were analyzed using LCModel. After FBMI fixation, the concentrations of Lac, tCho and mIns were comparable with those obtained in vivo under isoflurane, whereas other metabolites were significantly lower. Except for a decrease in NAA and an increase in Tau, all the other metabolites remained stable over 41 hours in FBMI-fixed brains. Without FBMI, the concentrations of mIns (before 2 hours), tCho and GABA were close to those measured in vivo. However, higher Lac (P < .01) and lower NAA, Gln, Glu, GSH, tCr and Tau were observed (P < .01). NAA, Gln, Glu, GSH, tCr and Tau exhibited good temporal stability for at least 20 hours in the unfixed brain, whereas a linear increase of tCho, mIns and GABA was observed. Possible mechanisms of postmortem degradation are discussed. Our results indicate that a proper fixation method is required for in situ detection depending on the targeted metabolites of specific interests in the brain.

Nano-therapeutic cancer immunotherapy using hyperthermia-induced heat shock proteins: insights from mathematical modeling.

BACKGROUND: Nano-therapeutic utilizing hyperthermia therapy in combination with chemotherapy, surgery, and radiation is known to treat various types of cancer. These cancer treatments normally focus on reducing tumor burden. Nevertheless, it is still challenging to confine adequate thermal energy in a tumor and obtain a complete tumor ablation to avoid recurrence and metastasis while leaving normal tissues unaffected. Consequently, it is critical to attain an alternative tumor-killing mechanism to circumvent these challenges. Studies have demonstrated that extracellular heat shock proteins (HSPs) activate antitumor immunity during tumor cell necrosis. Such induced immunity was further shown to assist in regressing tumor and reducing recurrence and metastasis. However, only a narrow range of thermal dose is reported to be able to acquire the optimal antitumor immune outcome. Consequently, it is crucial to understand how extracellular HSPs are generated. MATERIALS AND METHODS: In this work, a predictive model integrating HSP synthesis mechanism and cell death model is proposed to elucidate the HSP involvement in hyperthermia cancer immune therapy and its relation with dead tumor cells. This new model aims to provide insights into the thermally released extracellular HSPs by dead tumor cells for a more extensive set of conditions, including various temperatures and heating duration time. RESULTS: Our model is capable of predicting the optimal thermal parameters to generate maximum HSPs for stimulating antitumor immunity, promoting tumor regression, and reducing metastasis. The obtained nonlinear relation between extracellular HSP concentration and increased dead cell number, along with rising temperature, shows that only a narrow range of thermal dose is able to generate the optimal antitumor immune result. CONCLUSION: Our predictive model is capable of predicting the optimal temperature and exposure time to generate HSPs involved in the antitumor immune activation, with a goal to promote tumor regression and reduce metastasis.

Dendrimer- and copolymer-based nanoparticles for magnetic resonance cancer theranostics.

Cancer theranostics is one of the most important approaches for detecting and treating patients at an early stage. To develop such a technique, accurate detection, specific targeting, and controlled delivery are the key components. Various kinds of nanoparticles have been proposed and demonstrated as potential nanovehicles for cancer theranostics. Among them, polymer-like dendrimers and copolymer-based core-shell nanoparticles could potentially be the best possible choices. At present, magnetic resonance imaging (MRI) is widely used for clinical purposes and is generally considered the most convenient and noninvasive imaging modality. Superparamagnetic iron oxide (SPIO) and gadolinium (Gd)-based dendrimers are the major nanostructures that are currently being investigated as nanovehicles for cancer theranostics using MRI. These structures are capable of specific targeting of tumors as well as controlled drug or gene delivery to tumor sites using pH, temperature, or alternating magnetic field (AMF)-controlled mechanisms. Recently, Gd-based pseudo-porous polymer-dendrimer supramolecular nanoparticles have shown 4-fold higher T1 relaxivity along with highly efficient AMF-guided drug release properties. Core-shell copolymer-based nanovehicles are an equally attractive alternative for designing contrast agents and for delivering anti-cancer drugs. Various copolymer materials could be used as core and shell components to provide biostability, modifiable surface properties, and even adjustable imaging contrast enhancement. Recent advances and challenges in MRI cancer theranostics using dendrimer- and copolymer-based nanovehicles have been summarized in this review article, along with new unpublished research results from our laboratories.

Effective heating of magnetic nanoparticle aggregates for in vivo nano-theranostic hyperthermia.

Magnetic resonance (MR) nano-theranostic hyperthermia uses magnetic nanoparticles to target and accumulate at the lesions and generate heat to kill lesion cells directly through hyperthermia or indirectly through thermal activation and control releasing of drugs. Preclinical and translational applications of MR nano-theranostic hyperthermia are currently limited by a few major theoretical difficulties and experimental challenges in in vivo conditions. For example, conventional models for estimating the heat generated and the optimal magnetic nanoparticle sizes for hyperthermia do not accurately reproduce reported in vivo experimental results. In this work, a revised cluster-based model was proposed to predict the specific loss power (SLP) by explicitly considering magnetic nanoparticle aggregation in in vivo conditions. By comparing with the reported experimental results of magnetite Fe3O4 and cobalt ferrite CoFe2O4 magnetic nanoparticles, it is shown that the revised cluster-based model provides a more accurate prediction of the experimental values than the conventional models that assume magnetic nanoparticles act as single units. It also provides a clear physical picture: the aggregation of magnetic nanoparticles increases the cluster magnetic anisotropy while reducing both the cluster domain magnetization and the average magnetic moment, which, in turn, shift the predicted SLP toward a smaller magnetic nanoparticle diameter with lower peak values. As a result, the heating efficiency and the SLP values are decreased. The improvement in the prediction accuracy in in vivo conditions is particularly pronounced when the magnetic nanoparticle diameter is in the range of ~10-20 nm. This happens to be an important size range for MR cancer nano-theranostics, as it exhibits the highest efficacy against both primary and metastatic tumors in vivo. Our studies show that a relatively 20%-25% smaller magnetic nanoparticle diameter should be chosen to reach the maximal heating efficiency in comparison with the optimal size predicted by previous models.

Magnetic Resonance Nano-Theranostics for Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) is one of the most challenging diseases to treat in clinical oncology due to its high mortality rates and inefficient conventional treatment methods. Difficulties with early detection, post-surgical recurrences, and resistance to chemotherapy and/or radiotherapy are important reasons for the poor prognosis of those with GBM. Over the past few decades, magnetic resonance (MR) theranostics using magnetic nanoparticles has shown unique advantages and great promises for the diagnosis and treatment of cancers. Magnetic nanoparticles not only serve as "molecular beacons" to enhance tumor contrast in magnetic resonance imaging (MRI), but also serve as "molecular bullets" for targeted drug delivery, controlled release, and induced hyperthermia. Moreover, multiple functions of magnetic nanoparticles can be synergistically engineered into a single nanoplatform, making it possible to simultaneously image, treat, target, and monitor the targeted lesions. The multi-functionality of nanoparticles, also called nano-theranostics, gives rises to effective new approaches for combating GBM. In this work, recent research and progress concerning the applications of MR nano-theranostics on GBM using magnetic nanoparticles will be highlighted, focusing on topics such as diagnosis, therapy, targeting, and hyperthermia, as well as outstanding challenges for MR nanotheranostics in treating GBM. The conclusions are generally applicable to other types of brain tumors.

Sensitive imaging of magnetic nanoparticles for cancer detection by active feedback MR.

PURPOSE: Sensitive imaging of superparamagnetic nanoparticles or aggregates is of great importance in MR molecular imaging and medical diagnosis. For this purpose, a conceptually new approach, termed active feedback magnetic resonance, was developed. METHODS: In the presence of the Zeeman field, a dipolar field is induced by the superparamagnetic nanoparticles or aggregates. Such dipolar field creates spatial and temporal (due to water diffusion) variations to the precession frequency of the nearby water 1 H magnetization. Sensitive imaging of magnetic nanoparticles or aggregates can be achieved by manipulating the intrinsic spin dynamics by selective self-excitation and fixed-point dynamics under active feedback fields. RESULTS: Phantom experiments of superparamagnetic nanoparticles; in vitro experiments of brain tissue with blood clots; and in vivo mouse images of colon cancers, with and without labeling by magnetic nanoparticles, suggest that this new approach provides enhanced, robust, and positive contrast in imaging magnetic nanoparticles or aggregates for cancer detection. CONCLUSION: The spin dynamics originated from selective self-excitation and fixed-point dynamics under active feedback fields have been shown to be sensitive to dipolar fields generated by magnetic nanoparticles. Magn Reson Med 74:33-41, 2015. © 2014 Wiley Periodicals, Inc.

Unibody core-shell smart polymer as a theranostic nanoparticle for drug delivery and MR imaging.

Developing novel multifunctional nanoparticles (NPs) with robust preparation, low cost, high stability, and flexible functionalizability is highly desirable. This study provides an innovative platform, termed unibody core-shell (UCS), for this purpose. UCS is comprised of two covalent-bonded polymers differed only by the functional groups at the core and the shell. By conjugating Gd(3+) at the stable core and encapsulating doxorubicin (Dox) at the shell in a pH-sensitive manner, we developed a theranostic NPs (UCS-Gd-Dox) that achieved a selective drug release (75% difference between pH 7.4 and 5.5) and MR imaging (r1 = 0.9 and 14.5 mm(-1) s(-1) at pH 7.4 and 5.5, respectively). The anti-cancer effect of UCS-Gd-Dox is significantly better than free Dox in tumor-bearing mouse models, presumably due to enhanced permeability and retention effect and pH-triggered release. To the best of our knowledge, this is the simplest approach to obtain the theranostic NPs with Gd-conjugation and Dox doping.

A robust approach to correct for pronounced errors in temperature measurements by controlling radiation damping feedback fields in solution NMR.

Accurate temperature measurement is a requisite for obtaining reliable thermodynamic and kinetic information in all NMR experiments. A widely used method to calibrate sample temperature depends on a secondary standard with temperature-dependent chemical shifts to report the true sample temperature, such as the hydroxyl proton in neat methanol or neat ethylene glycol. The temperature-dependent chemical shift of the hydroxyl protons arises from the sensitivity of the hydrogen-bond network to small changes in temperature. The frequency separation between the alkyl and the hydroxyl protons are then converted to sample temperature. Temperature measurements by this method, however, have been reported to be inconsistent and incorrect in modern NMR, particularly for spectrometers equipped with cryogenically-cooled probes. Such errors make it difficult or even impossible to study chemical exchange and molecular dynamics or to compare data acquired on different instruments, as is frequently done in biomolecular NMR. In this work, we identify the physical origins for such errors to be unequal amount of dynamical frequency shifts on the alkyl and the hydroxyl protons induced by strong radiation damping (RD) feedback fields. Common methods used to circumvent RD may not suppress such errors. A simple, easy-to-implement solution was demonstrated that neutralizes the RD effect on the frequency separation by a "selective crushing recovery" pulse sequence to equalize the transverse magnetization of both spin species. Experiments using cryoprobes at 500 MHz and 800 MHz demonstrated that this approach can effectively reduce the errors in temperature measurements from about ±4.0 K to within ±0.4 K in general.

Novel MRI contrast development by lock-in suppression.

PURPOSE: The goal of this study is to develop novel MR contrast by frequency lock-in technique. METHODS: An electronic feedback device that can control the frequency and bandwidth of the feedback RF field is presented. In this study, the effects of lock-in suppressed imaging are discussed both theoretically and experimentally. RESULTS: Two important imaging experiments were performed. The first experiment used magnetizations with the same central frequency but different frequency distributions and was compared with MR images obtained with T2 contrast agents. Lock-in suppressed images showed an improvement in contrast relative to the conventional imaging method. The second experiment used magnetizations with small shifts in frequency and a broad frequency distribution. This is helpful for differentiating between small structural variations in biological tissues. The contrast achieved in in vivo tumor imaging using the lock-in suppressed technique provide higher spatial resolutions and discriminate the regimes of necrosis and activation consistent with pathologic results. CONCLUSION: Lock-in suppressed imaging introduces a conceptually new approach to MRI. Heightened sensitivity to underlying susceptibility variations and their relative contribution to total magnetization may thus be achieved to yield new and enhanced contrast.

Simple mobile single-sided NMR apparatus with a relatively homogeneous B0 distribution.

This work presents a simple design for a mobile single-sided nuclear magnetic resonance (NMR) apparatus with a relatively homogeneous static magnetic field (B(0)) distribution. In the proposed design, the B(0) magnetic field of the apparatus is synthesized using only two permanent magnet blocks, i.e., a cube (main) magnet and a small shim magnet placed above the main magnet. The magnetic flux of the shim magnet partially cancels out that of the main magnet, subsequently creating a smooth B(0) profile above the shim magnet where low-resolution NMR experiments are performed. Compared with many previously published designs, this straightforward design simplifies the construction of the apparatus and simultaneously generates a B(0) field parallel to the apparatus surface, allowing the use of a simple loop-type radiofrequency (RF) coil. Additionally, an apparatus prototype is constructed according to the proposed design. Weighing only 1.8 kg, the constructed apparatus has a compact structure and can be held in the palm of a hand. The apparatus generates a B(0) strength of about 0.0746 T. Within a B(0) field deviation of 3 mT, the region with a relatively homogeneous B(0) distribution extends to about 11 mm above the shim magnet. The proposed apparatus can detect a clear Hahn echo or Carr-Purcell-Meiboom-Gill (CPMG) echoes of a pencil eraser block or a bottle of oil placed on the apparatus in 5 s with signal averaging using an RF transmitter power of only 19 W; the detection range of the apparatus exceeds 6 mm. The strength of the residual static magnetic field gradient of the apparatus is roughly estimated at 0.58 T/m. Applying different CPMG echo spacings in this residual static gradient leads to various transverse relaxation time (T(2)) contrasts for liquids with distinct viscosities such as water and oil. Two nondestructive inspection applications of the apparatus, including correlating the concentrations of magnetic nanoparticle solutions with their measured transverse relaxation rates (R(2)) and monitoring the outgassing from an opened bottle of oxygen-supersaturated water by measuring its longitudinal relaxation rate (R(1)), are also demonstrated.

A small MRI contrast agent library of gadolinium(III)-encapsulated supramolecular nanoparticles for improved relaxivity and sensitivity.

We introduce a new category of nanoparticle-based T(1) MRI contrast agents (CAs) by encapsulating paramagnetic chelated gadolinium(III), i.e., Gd(3+)·DOTA, through supramolecular assembly of molecular building blocks that carry complementary molecular recognition motifs, including adamantane (Ad) and ß-cyclodextrin (CD). A small library of Gd(3+)·DOTA-encapsulated supramolecular nanoparticles (Gd(3+)·DOTA⊂SNPs) was produced by systematically altering the molecular building block mixing ratios. A broad spectrum of relaxation rates was correlated to the resulting Gd(3+)·DOTA⊂SNP library. Consequently, an optimal synthetic formulation of Gd(3+)·DOTA⊂SNPs with an r(1) of 17.3 s(-1) mM(-1) (ca. 4-fold higher than clinical Gd(3+) chelated complexes at high field strengths) was identified. T(1)-weighted imaging of Gd(3+)·DOTA⊂SNPs exhibits an enhanced sensitivity with a contrast-to-noise ratio (C/N ratio) ca. 3.6 times greater than that observed for free Gd(3+)·DTPA. A Gd(3+)·DOTA⊂SNPs solution was injected into foot pads of mice, and MRI was employed to monitor dynamic lymphatic drainage of the Gd(3+)·DOTA⊂SNPs-based CA. We observe an increase in signal intensity of the brachial lymph node in T(1)-weighted imaging after injecting Gd(3+)·DOTA⊂SNPs but not after injecting Gd(3+)·DTPA. The MRI results are supported by ICP-MS analysis ex vivo. These results show that Gd(3+)·DOTA⊂SNPs not only exhibits enhanced relaxivity and high sensitivity but also can serve as a potential tool for diagnosis of cancer metastasis.