In vivo neuropil density from anatomical MRI and machine learning.

Brain energy budgets specify metabolic costs emerging from underlying mechanisms of cellular and synaptic activities. While current bottom-up energy budgets use prototypical values of cellular density and synaptic density, predicting metabolism from a person's individualized neuropil density would be ideal. We hypothesize that in vivo neuropil density can be derived from magnetic resonance imaging (MRI) data, consisting of longitudinal relaxation (T1) MRI for gray/white matter distinction and diffusion MRI for tissue cellularity (apparent diffusion coefficient, ADC) and axon directionality (fractional anisotropy, FA). We present a machine learning algorithm that predicts neuropil density from in vivo MRI scans, where ex vivo Merker staining and in vivo synaptic vesicle glycoprotein 2A Positron Emission Tomography (SV2A-PET) images were reference standards for cellular and synaptic density, respectively. We used Gaussian-smoothed T1/ADC/FA data from 10 healthy subjects to train an artificial neural network, subsequently used to predict cellular and synaptic density for 54 test subjects. While excellent histogram overlaps were observed both for synaptic density (0.93) and cellular density (0.85) maps across all subjects, the lower spatial correlations both for synaptic density (0.89) and cellular density (0.58) maps are suggestive of individualized predictions. This proof-of-concept artificial neural network may pave the way for individualized energy atlas prediction, enabling microscopic interpretations of functional neuroimaging data.

Transmembrane pH gradient imaging in rodent glioma models.

A unique feature of the tumor microenvironment is extracellular acidosis in relation to intracellular milieu. Metabolic reprogramming in tumors results in overproduction of H+ ions (and lactate), which are extruded from the cells to support tumor survival and progression. As a result, the transmembrane pH gradient (ΔpH), representing the difference between intracellular pH (pHi) and extracellular pH (pHe), is posited to be larger in tumors compared with normal tissue. Controlling the transmembrane pH difference has promise as a potential therapeutic target in cancer as it plays an important role in regulating drug delivery into cells. The current study shows successful development of an MRI/MRSI-based technique that provides ΔpH imaging at submillimeter resolution. We applied this technique to image ΔpH in rat brains with RG2 and U87 gliomas, as well as in mouse brains with GL261 gliomas. pHi was measured with Amine and Amide Concentration-Independent Detection (AACID), while pHe was measured with Biosensor Imaging of Redundant Deviation in Shifts (BIRDS). The results indicate that pHi was slightly higher in tumors (7.40-7.43 in rats, 7.39-7.47 in mice) compared with normal brain (7.30-7.38 in rats, 7.32-7.36 in mice), while pHe was significantly lower in tumors (6.62-6.76 in rats, 6.74-6.84 in mice) compared with normal tissue (7.17-7.22 in rats, 7.20-7.21 in mice). As a result, ΔpH was higher in tumors (0.64-0.81 in rats, 0.62-0.65 in mice) compared with normal brain (0.13-0.16 in rats, 0.13-0.16 in mice). This work establishes an MRI/MRSI-based platform for ΔpH imaging at submillimeter resolution in gliomas.

High-resolution extracellular pH imaging of liver cancer with multiparametric MR using Deep Image Prior.

Noninvasive extracellular pH (pHe) mapping with Biosensor Imaging of Redundant Deviation in Shifts (BIRDS) using MR spectroscopic imaging (MRSI) has been demonstrated on 3T clinical MR scanners at 8 × 8 × 10 mm3 spatial resolution and applied to study various liver cancer treatments. Although pHe imaging at higher resolution can be achieved by extending the acquisition time, a postprocessing method to increase the resolution is preferable, to minimize the duration spent by the subject in the MR scanner. In this work, we propose to improve the spatial resolution of pHe mapping with BIRDS by incorporating anatomical information in the form of multiparametric MRI and using an unsupervised deep-learning technique, Deep Image Prior (DIP). Specifically, we used high-resolution T 1 , T 2 , and diffusion-weighted imaging (DWI) MR images of rabbits with VX2 liver tumors as inputs to a U-Net architecture to provide anatomical information. U-Net parameters were optimized to minimize the difference between the output super-resolution image and the experimentally acquired low-resolution pHe image using the mean-absolute error. In this way, the super-resolution pHe image would be consistent with both anatomical MR images and the low-resolution pHe measurement from the scanner. The method was developed based on data from 49 rabbits implanted with VX2 liver tumors. For evaluation, we also acquired high-resolution pHe images from two rabbits, which were used as ground truth. The results indicate a good match between the spatial characteristics of the super-resolution images and the high-resolution ground truth, supported by the low pixelwise absolute error.

Aspm knockout ferret reveals an evolutionary mechanism governing cerebral cortical size.

The human cerebral cortex is distinguished by its large size and abundant gyrification, or folding. However, the evolutionary mechanisms that drive cortical size and structure are unknown. Although genes that are essential for cortical developmental expansion have been identified from the genetics of human primary microcephaly (a disorder associated with reduced brain size and intellectual disability) 1 , studies of these genes in mice, which have a smooth cortex that is one thousand times smaller than the cortex of humans, have provided limited insight. Mutations in abnormal spindle-like microcephaly-associated (ASPM), the most common recessive microcephaly gene, reduce cortical volume by at least 50% in humans2-4, but have little effect on the brains of mice5-9; this probably reflects evolutionarily divergent functions of ASPM10,11. Here we used genome editing to create a germline knockout of Aspm in the ferret (Mustela putorius furo), a species with a larger, gyrified cortex and greater neural progenitor cell diversity12-14 than mice, and closer protein sequence homology to the human ASPM protein. Aspm knockout ferrets exhibit severe microcephaly (25-40% decreases in brain weight), reflecting reduced cortical surface area without significant change in cortical thickness, as has been found in human patients3,4, suggesting that loss of 'cortical units' has occurred. The cortex of fetal Aspm knockout ferrets displays a very large premature displacement of ventricular radial glial cells to the outer subventricular zone, where many resemble outer radial glia, a subtype of neural progenitor cells that are essentially absent in mice and have been implicated in cerebral cortical expansion in primates12-16. These data suggest an evolutionary mechanism by which ASPM regulates cortical expansion by controlling the affinity of ventricular radial glial cells for the ventricular surface, thus modulating the ratio of ventricular radial glial cells, the most undifferentiated cell type, to outer radial glia, a more differentiated progenitor.

A 3D atlas of functional human brain energetic connectome based on neuropil distribution.

The human brain is energetically expensive, yet the key factors governing its heterogeneous energy distributions across cortical regions to support its diversity of functions remain unexplored. Here, we built up a 3D digital cortical energy atlas based on the energetic costs of all neuropil activities into a high-resolution stereological map of the human cortex with cellular and synaptic densities derived, respectively, from ex vivo histological staining and in vivo PET imaging. The atlas was validated with PET-measured glucose oxidation at the voxel level. A 3D cortical activity map was calculated to predict the heterogeneous activity rates across all cortical regions, which revealed that resting brain is indeed active with heterogeneous neuronal activity rates averaging around 1.2 Hz, comprising around 70% of the glucose oxidation of the cortex. Additionally, synaptic density dominates spatial patterns of energetics, suggesting that the cortical energetics rely heavily on the distribution of synaptic connections. Recent evidence from functional imaging studies suggests that some cortical areas act as hubs (i.e., interconnecting distinct and functionally active regions). An inverse allometric relationship was observed between hub metabolic rates versus hub volumes. Hubs with smaller volumes have higher synapse density, metabolic rate, and activity rates compared to nonhubs. The open-source BrainEnergyAtlas provides a granular framework for exploring revealing design principles in energy-constrained human cortical circuits across multiple spatial scales.

Neurovascular coupling is optimized to compensate for the increase in proton production from nonoxidative glycolysis and glycogenolysis during brain activation and maintain homeostasis of pH, pCO2 , and pO2.

During transient brain activation cerebral blood flow (CBF) increases substantially more than cerebral metabolic rate of oxygen consumption (CMRO2 ) resulting in blood hyperoxygenation, the basis of BOLD fMRI contrast. Explanations for the high CBF vs. CMRO2 slope, termed neurovascular coupling (NVC) constant, focused on maintainenance of tissue oxygenation to support mitochondrial ATP production. However, paradoxically the brain has a 3-fold lower oxygen extraction fraction (OEF) than other organs with high energy requirements, like heart and muscle during exercise. Here, we hypothesize that the NVC constant and the capillary oxygen mass transfer coefficient (which in combination determine OEF) are co-regulated during activation to maintain simultaneous homeostasis of pH and partial pressure of CO2 and O2 (pCO2 and pO2 ). To test our hypothesis, we developed an arteriovenous flux balance model for calculating blood and brain pH, pCO2 , and pO2 as a function of baseline OEF (OEF0 ), CBF, CMRO2 , and proton production by nonoxidative metabolism coupled to ATP hydrolysis. Our model was validated against published brain arteriovenous difference studies and then used to calculate pH, pCO2, and pO2 in activated human cortex from published calibrated fMRI and PET measurements. In agreement with our hypothesis, calculated pH, pCO2, and pO2 remained close to constant independently of CMRO2 in correspondence to experimental measurements of NVC and OEF0 . We also found that the optimum values of the NVC constant and OEF0 that ensure simultaneous homeostasis of pH, pCO2, and pO2 were remarkably similar to their experimental values. Thus, the high NVC constant is overall determined by proton removal by CBF due to increases in nonoxidative glycolysis and glycogenolysis. These findings resolve the paradox of the brain's high CBF yet low OEF during activation, and may contribute to explaining the vulnerability of brain function to reductions in blood flow and capillary density with aging and neurovascular disease.

Simultaneous cortex-wide fluorescence Ca2+ imaging and whole-brain fMRI.

Achieving a comprehensive understanding of brain function requires multiple imaging modalities with complementary strengths. We present an approach for concurrent widefield optical and functional magnetic resonance imaging. By merging these modalities, we can simultaneously acquire whole-brain blood-oxygen-level-dependent (BOLD) and whole-cortex calcium-sensitive fluorescent measures of brain activity. In a transgenic murine model, we show that calcium predicts the BOLD signal, using a model that optimizes a gamma-variant transfer function. We find consistent predictions across the cortex, which are best at low frequency (0.009-0.08 Hz). Furthermore, we show that the relationship between modality connectivity strengths varies by region. Our approach links cell-type-specific optical measurements of activity to the most widely used method for assessing human brain function.

MR Imaging-Based In Vivo Macrophage Imaging to Monitor Immune Response after Radiofrequency Ablation of the Liver.

PURPOSE: To establish molecular magnetic resonance (MR) imaging instruments for in vivo characterization of the immune response to hepatic radiofrequency (RF) ablation using cell-specific immunoprobes. MATERIALS AND METHODS: Seventy-two C57BL/6 wild-type mice underwent standardized hepatic RF ablation (70 °C for 5 minutes) to generate a coagulation area measuring 6-7 mm in diameter. CD68+ macrophage periablational infiltration was characterized with immunohistochemistry 24 hours, 72 hours, 7 days, and 14 days after ablation (n = 24). Twenty-one mice were subjected to a dose-escalation study with either 10, 15, 30, or 60 mg/kg of rhodamine-labeled superparamagnetic iron oxide nanoparticles (SPIONs) or 2.4, 1.2, or 0.6 mg/kg of gadolinium-160 (160Gd)-labeled CD68 antibody for assessment of the optimal in vivo dose of contrast agent. MR imaging experiments included 9 mice, each receiving 10-mg/kg SPIONs to visualize phagocytes using T2∗-weighted imaging in a horizontal-bore 9.4-T MR imaging scanner, 160Gd-CD68 for T1-weighted MR imaging of macrophages, or 0.1-mmol/kg intravenous gadoterate (control group). Radiological-pathological correlation included Prussian blue staining, rhodamine immunofluorescence, imaging mass cytometry, and immunohistochemistry. RESULTS: RF ablation-induced periablational infiltration (206.92 µm ± 12.2) of CD68+ macrophages peaked at 7 days after ablation (P < .01) compared with the untreated lobe. T2∗-weighted MR imaging with SPION contrast demonstrated curvilinear T2∗ signal in the transitional zone (TZ) (186 µm ± 16.9), corresponsing to Iron Prussian blue staining. T1-weighted MR imaging with 160Gd-CD68 antibody showed curvilinear signal in the TZ (164 µm ± 3.6) corresponding to imaging mass cytometry. CONCLUSIONS: Both SPION-enhanced T2∗-weighted and 160Gd-enhanced T1-weighted MR imaging allow for in vivo monitoring of macrophages after RF ablation, demonstrating the feasibility of this model to investigate local immune responses.

The Stroke Preclinical Assessment Network: Rationale, Design, Feasibility, and Stage 1 Results.

Cerebral ischemia and reperfusion initiate cellular events in brain that lead to neurological disability. Investigating these cellular events provides ample targets for developing new treatments. Despite considerable work, no such therapy has translated into successful stroke treatment. Among other issues-such as incomplete mechanistic knowledge and faulty clinical trial design-a key contributor to prior translational failures may be insufficient scientific rigor during preclinical assessment: nonblinded outcome assessment; missing randomization; inappropriate sample sizes; and preclinical assessments in young male animals that ignore relevant biological variables, such as age, sex, and relevant comorbid diseases. Promising results are rarely replicated in multiple laboratories. We sought to address some of these issues with rigorous assessment of candidate treatments across 6 independent research laboratories. The Stroke Preclinical Assessment Network (SPAN) implements state-of-the-art experimental design to test the hypothesis that rigorous preclinical assessment can successfully reduce or eliminate common sources of bias in choosing treatments for evaluation in clinical studies. SPAN is a randomized, placebo-controlled, blinded, multilaboratory trial using a multi-arm multi-stage protocol to select one or more putative stroke treatments with an implied high likelihood of success in human clinical stroke trials. The first stage of SPAN implemented procedural standardization and experimental rigor. All participating research laboratories performed middle cerebral artery occlusion surgery adhering to a common protocol and rapidly enrolled 913 mice in the first of 4 planned stages with excellent protocol adherence, remarkable data completion and low rates of subject loss. SPAN stage 1 successfully implemented treatment masking, randomization, prerandomization inclusion/exclusion criteria, and blinded assessment to exclude bias. Our data suggest that a large, multilaboratory, preclinical assessment effort to reduce known sources of bias is feasible and practical. Subsequent SPAN stages will evaluate candidate treatments for potential success in future stroke clinical trials using aged animals and animals with comorbid conditions.

Comparison of Lanthanide Macrocyclic Complexes as 23Na NMR Sensors.

Nuclear magnetic resonance (NMR) agents, composed of paramagnetic lanthanide ions (Ln3+) complexed with negatively charged cyclic chelating agents (Che(n+3)-) forming polyanionic lanthanide complexes (LnChen-), perturb sodium-23 (23Na) signals, a phenomenon which depends sodium ions (Na+) exchanging with LnChen-. We analyzed 23Na shiftability and broadening due to hyperfine and bulk magnetic susceptibility (BMS) effects that arise from LnChen- designs using selective Ln3+ ions (i.e., thulium, Tm3+; gadolinium, Gd3+; and europium, Eu3+) and macrocyclics derived from 1,4,7,10-tetraazacyclododecane (cyclen) [i.e., with phosphonate (DOTP8-) and carboxylate (DOTMA4-) arms] and 1,4,7-triazacyclononane (TACN) [i.e., with phosphonate (NOTP6-) arms]. All LnChen- complexes showed downfield shifts, but Gd3+ and Tm3+ agents, respectively, were dominated by BMS and hyperfine effects, in good agreement with theory. While 23Na shiftability and broadening were minimally affected by pH and competing cations (K+, Ca2+, and Mg2+) within physiological ranges, the 23Na shiftability and broadening were most sensitive to LnChen- concentration in relation to the interstitial Na+ level in vivo. Greatest 23Na shiftability and broadening were obtained with Tm3+ and Gd3+ agents, respectively. While BMS contribution to shiftability was most impacted by the number of unpaired electrons on Ln3+, negative charge on LnChen- regulated Na+ exchange for line broadening. In brain tumor models, TmDOTP5- with 23Na-NMR has been used previously to separate Na+ in intracellular, blood, and interstitial pools, while evidence here shows that GdDOTP5- can distinguish Na+ within intracellular and extracellular (i.e., blood and interstitial) pools. Given the biological importance of Na+ in vivo, future macrocyclic designs of LnChen- should be sought for 23Na-NMR biomedical applications.

Engineering of human brain organoids with a functional vascular-like system.

Human cortical organoids (hCOs), derived from human embryonic stem cells (hESCs), provide a platform to study human brain development and diseases in complex three-dimensional tissue. However, current hCOs lack microvasculature, resulting in limited oxygen and nutrient delivery to the inner-most parts of hCOs. We engineered hESCs to ectopically express human ETS variant 2 (ETV2). ETV2-expressing cells in hCOs contributed to forming a complex vascular-like network in hCOs. Importantly, the presence of vasculature-like structures resulted in enhanced functional maturation of organoids. We found that vascularized hCOs (vhCOs) acquired several blood-brain barrier characteristics, including an increase in the expression of tight junctions, nutrient transporters and trans-endothelial electrical resistance. Finally, ETV2-induced endothelium supported the formation of perfused blood vessels in vivo. These vhCOs form vasculature-like structures that resemble the vasculature in early prenatal brain, and they present a robust model to study brain disease in vitro.

High-resolution pH imaging using ratiometric chemical exchange saturation transfer combined with biosensor imaging of redundant deviation in shifts featuring paramagnetic DOTA-tetraglycinate agents.

Chemical exchange saturation transfer (CEST) and biosensor imaging of redundant deviation in shifts (BIRDS) methods differ respectively by detecting exchangeable and nonexchangeable proton signals by magnetic resonance. Because CEST contrast depends on both temperature and pH, simultaneous CEST and BIRDS imaging can be employed to separate these contributions. Here, we test if high-resolution pH imaging in vivo is possible with ratiometric CEST calibrated for temperature variations measured by BIRDS. Thulium- and europium-based DOTA-tetraglycinate agents, TmDOTA-(gly)4- and EuDOTA-(gly)4- , were used for high-resolution pH mapping in vitro and in vivo, using BIRDS for temperature adjustments needed for a more accurate ratiometric CEST approach. Although neither agent showed pH dependence with BIRDS in vitro in the pH range 6 to 8, each one's temperature sensitivity was enhanced when mixed because of increased redundancy. By contrast, the CEST signal of each agent was affected by the presence of the other agent in vitro. However, pH could be measured more accurately when temperature from BIRDS was detected. These in vitro calibrations with TmDOTA-(gly)4- and EuDOTA-(gly)4- enabled high-resolution pH imaging of glioblastoma in rat brains. It was concluded that temperature mapping with BIRDS can calibrate the ratiometric CEST signal from a cocktail of TmDOTA-(gly)4- and EuDOTA-(gly)4- agents to provide temperature-independent absolute pH imaging in vivo.

Methylated tetra-amide derivatives of paramagnetic complexes for magnetic resonance biosensing with both BIRDS and CEST.

Paramagnetic agents that utilize two mechanisms to provide physiological information by magnetic resonance imaging (MRI) and magnetic resonance spectroscopic imaging (MRSI) are described. MRI with chemical exchange saturation transfer (CEST) takes advantage of the agent's exchangeable protons (e.g., -OH or -NHx , where 2 ≥ x ≥ 1) to create pH contrast. The agent's incorporation of non-exchangeable protons (e.g., -CHy , where 3 ≥ y ≥ 1) makes it possible to map tissue temperature and/or pH using an MRSI method called biosensor imaging of redundant deviation in shifts (BIRDS). Hybrid probes based upon 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate chelate (DOTA4- ) and its methylated analog (1,4,7,10-tetraazacyclododecane-α, α', α″, α‴-tetramethyl-1,4,7,10-tetraacetate, DOTMA4- ) were synthesized, and modified to create new tetra-amide chelates. Addition of several methyl groups per pendent arm of the symmetrical chelates, positioned proximally and distally to thulium ions (Tm3+ ), gave rise to favorable BIRDS properties (i.e., high signal-to-noise ratio (SNR) from non-exchangeable methyl proton peaks) and CEST responsiveness (i.e., from amide exchangeable protons). Structures of the Tm3+ probes elucidate the influence of methyl group placement on sensor performance. An eight-coordinate geometry with high symmetry was observed for the complexes: Tm-L1 was based on DOTA4- , whereas Tm-L2 and Tm-L3 were based on DOTMA4- , where the latter contained an additional carboxylate at the distal end of each arm. The distance of Tm3+ from terminal methyl carbons is a key determinant for sustaining BIRDS temperature sensitivity without compromising CEST pH contrast; however, water solubility was influenced by introduction of hydrophobic methyl groups and hydrophilic carboxylate. Combined BIRDS and CEST detection of Tm-L2, which features two high-SNR methyl peaks and a strong amide CEST peak, should enable simultaneous temperature and pH measurements for high-resolution molecular imaging in vivo.

Comparison of metabolic and immunologic responses to transarterial chemoembolization with different chemoembolic regimens in a rabbit VX2 liver tumor model.

OBJECTIVES: The goal of this study was to investigate the effects of TACE using Lipiodol, Oncozene™ drug-eluting embolics (DEEs), or LUMI™-DEEs alone, or combined with bicarbonate on the metabolic and immunological tumor microenvironment in a rabbit VX2 tumor model. METHODS: VX2 liver tumor-bearing rabbits were assigned to five groups. MRI and extracellular pH (pHe) mapping using Biosensor Imaging of Redundant Deviation in Shifts (BIRDS) were performed before and after intra-arterial therapy with conventional TACE (cTACE), DEE-TACE with Idarubicin-eluting Oncozene™-DEEs, or Doxorubicin-eluting LUMI™-DEEs, each with or without prior bicarbonate infusion, and in untreated rabbits or treated with intra-arterial bicarbonate only. Imaging results were validated with immunohistochemistry (IHC) staining of cell viability (PCNA, TUNEL) and immune response (HLA-DR, CD3). Statistical analysis was performed using Mann-Whitney U test. RESULTS: pHe mapping revealed that combining cTACE with prior bicarbonate infusion significantly increased tumor pHe compared to control (p = 0.0175) and cTACE alone (p = 0.0025). IHC staining revealed peritumoral accumulation of HLA-DR+ antigen-presenting cells and CD3 + T-lymphocytes in controls. cTACE-treated tumors showed reduced immune infiltration, which was restored through combination with bicarbonate. DEE-TACE with Oncozene™-DEEs induced moderate intratumoral and marked peritumoral infiltration, which was slightly reduced with bicarbonate. Addition of bicarbonate prior to LUMI™-beads enhanced peritumoral immune cell infiltration compared to LUMI™-beads alone and resulted in the strongest intratumoral immune cell infiltration across all treated groups. CONCLUSIONS: The choice of chemoembolic regimen for TACE strongly affects post-treatment TME pHe and the ability of immune cells to accumulate and infiltrate the tumor tissue. KEY POINTS: • Combining conventional transarterial chemotherapy with prior bicarbonate infusion increases the pHe towards a more physiological value (p = 0.0025). • Peritumoral infiltration and intratumoral accumulation patterns of antigen-presenting cells and T-lymphocytes after transarterial chemotherapy were dependent on the choice of the chemoembolic regimen. • Combination of intra-arterial treatment with Doxorubicin-eluting LUMI™-beads and bicarbonate infusion resulted in the strongest intratumoral presence of immune cells (positivity index of 0.47 for HLADR+-cells and 0.62 for CD3+-cells).

A high-throughput imaging platform to characterize extracellular pH in organotypic three-dimensional in vitro models of liver cancer.

Given the extraordinary nature of tumor metabolism in hepatocellular carcinoma and its impact on oncologic treatment response, this study introduces a novel high-throughput extracellular pH (pHe ) mapping platform using magnetic resonance spectroscopic imaging in a three-dimensional (3D) in vitro model of liver cancer. pHe mapping was performed using biosensor imaging of redundant deviation in shifts (BIRDS) on 9.4 T and 11.7 T MR scanners for validation purposes. 3D cultures of four liver cancer (HepG2, Huh7, SNU475, VX2) and one hepatocyte (THLE2) cell line were simultaneously analyzed (a) without treatment, (b) supplemented with 4.5 g/L d-glucose, and (c) treated with anti-glycolytic 3-bromopyruvate (6.25, 25, 50, 75, and 100 µM). The MR results were correlated with immunohistochemistry (GLUT-1, LAMP-2) and luminescence-based viability assays. Statistics included the unpaired t-test and ANOVA test. High-throughput pHe imaging with BIRDS for in vitro 3D liver cancer models proved feasible. Compared with non-tumorous hepatocytes (pHe = 7.1 ± 0.1), acidic pHe was revealed in liver cancer (VX2, pHe = 6.7 ± 0.1; HuH7, pHe = 6.8 ± 0.1; HepG2, pHe = 6.9 ± 0.1; SNU475, pHe = 6.9 ± 0.1), in agreement with GLUT-1 upregulation. Glucose addition significantly further decreased pHe in hyperglycolytic cell lines (VX2, HepG2, and Huh7, by 0.28, 0.06, and 0.11, respectively, all p < 0.001), whereas 3-bromopyruvate normalized tumor pHe in a dose-dependent manner without affecting viability. In summary, this study introduces a non-invasive pHe imaging platform for high-yield screening using a translational 3D liver cancer model, which may help reveal and target mechanisms of therapy resistance and inform personalized treatment of patients with hepatocellular carcinoma.

Small loci of astroglial glutamine synthetase deficiency in the postnatal brain cause epileptic seizures and impaired functional connectivity.

OBJECTIVE: The astroglial enzyme glutamine synthetase (GS) is deficient in small loci in the brain in adult patients with different types of focal epilepsy; however, the role of this deficiency in the pathogenesis of epilepsy has been difficult to assess due to a lack of sufficiently sensitive and specific animal models. The aim of this study was to develop an in vivo approach for precise and specific deletions of the GS gene in the postnatal brain. METHODS: We stereotaxically injected various adeno-associated virus (AAV)-Cre recombinase constructs into the hippocampal formation and neocortex in 22-70-week-old GSflox/flox mice to knock out the GS gene in a specific and focal manner. The mice were subjected to seizure threshold determination, continuous video-electroencephalographic recordings, advanced in vivo neuroimaging, and immunocytochemistry for GS. RESULTS: The construct AAV8-glial fibrillary acidic protein-green fluorescent protein-Cre eliminated GS in >99% of astrocytes in the injection center with a gradual return to full GS expression toward the periphery. Such focal GS deletion reduced seizure threshold, caused spontaneous recurrent seizures, and diminished functional connectivity. SIGNIFICANCE: These results suggest that small loci of GS deficiency in the postnatal brain are sufficient to cause epilepsy and impaired functional connectivity. Additionally, given the high specificity and precise spatial resolution of our GS knockdown approach, we anticipate that this model will be extremely useful for rigorous in vivo and ex vivo studies of astroglial GS function at the brain-region and single-cell levels.

Oxygen extraction fraction mapping with multi-parametric quantitative BOLD MRI: Reduced transverse relaxation bias using 3D-GraSE imaging.

Magnetic resonance imaging (MRI)-based quantification of the blood-oxygenation-level-dependent (BOLD) effect allows oxygen extraction fraction (OEF) mapping. The multi-parametric quantitative BOLD (mq-BOLD) technique facilitates relative OEF (rOEF) measurements with whole brain coverage in clinically applicable scan times. Mq-BOLD requires three separate scans of cerebral blood volume and transverse relaxation rates measured by gradient-echo (1/T2∗) and spin-echo (1/T2). Although the current method is of clinical merit in patients with stroke, glioma and internal carotid artery stenosis (ICAS), there are relaxation measurement artefacts that impede the sensitivity of mq-BOLD and artificially elevate reported rOEF values. We posited that T2-related biases caused by slice refocusing imperfections during rapid 2D-GraSE (Gradient and Spin Echo) imaging can be reduced by applying 3D-GraSE imaging sequences, because the latter requires no slice selective pulses. The removal of T2-related biases would decrease overestimated rOEF values measured by mq-BOLD. We characterized effects of T2-related bias in mq-BOLD by comparing the initially employed 2D-GraSE and two proposed 3D-GraSE sequences to multiple single spin-echo reference measurements, both in vitro and in vivo. A phantom and 25 participants, including young and elderly healthy controls as well as ICAS-patients, were scanned. We additionally proposed a procedure to reliably identify and exclude artefact affected voxels. In the phantom, 3D-GraSE derived T2 values had 57% lower deviation from the reference. For in vivo scans, the formerly overestimated rOEF was reduced by -27% (p â€‹< â€‹0.001). We obtained rOEF â€‹= â€‹0.51, which is much closer to literature values from positron emission tomography (PET) measurements. Furthermore, increased sensitivity to a focal rOEF elevation in an ICAS-patient was demonstrated. In summary, the application of 3D-GraSE improves the mq-BOLD-based rOEF quantification while maintaining clinically feasible scan times. Thus, mq-BOLD with non-slice selective T2 imaging is highly promising to improve clinical diagnostics of cerebrovascular diseases such as ICAS.

Orthonasal versus retronasal glomerular activity in rat olfactory bulb by fMRI.

Odorants can reach olfactory receptor neurons (ORNs) by two routes: orthonasally, when volatiles enter the nasal cavity during inhalation/sniffing, and retronasally, when food volatiles released in the mouth pass into the nasal cavity during exhalation/eating. Previous work in humans has shown that both delivery routes of the same odorant can evoke distinct perceptions and patterns of neural responses in the brain. Each delivery route is known to influence specific responses across the dorsal region of the glomerular sheet in the olfactory bulb (OB), but spatial distributions across the entire glomerular sheet throughout the whole OB remain largely unexplored. We used functional MRI (fMRI) to measure and compare activations across the entire glomerular sheet in rat OB resulting from both orthonasal and retronasal stimulations of the same odors. We observed reproducible fMRI activation maps of the whole OB during both orthonasal and retronasal stimuli. However, retronasal stimuli required double the orthonasal odor concentration for similar response amplitudes. Regardless, both the magnitude and spatial extent of activity were larger during orthonasal versus retronasal stimuli for the same odor. Orthonasal and retronasal response patterns show overlap as well as some route-specific dominance. Orthonasal maps were dominant in dorsal-medial regions, whereas retronasal maps were dominant in caudal and lateral regions. These different whole OB encodings likely underlie differences in odor perception between these biologically important routes for odorants among mammals. These results establish the relationships between orthonasal and retronasal odor representations in the rat OB.

APOE genotype-dependent pharmacogenetic responses to rapamycin for preventing Alzheimer's disease.

The ε4 allele of Apolipoprotein (APOE4) is the strongest genetic risk factor for Alzheimer's disease (AD), the most common form of dementia. Cognitively normal APOE4 carriers have developed amyloid beta (Aß) plaques and cerebrovascular, metabolic and structural deficits decades before showing the cognitive impairment. Interventions that can inhibit Aß retention and restore the brain functions to normal would be critical to prevent AD for the asymptomatic APOE4 carriers. A major goal of the study was to identify the potential usefulness of rapamycin (Rapa), a pharmacological intervention for extending longevity, for preventing AD in the mice that express human APOE4 gene and overexpress Aß (the E4FAD mice). Another goal of the study was to identify the potential pharmacogenetic differences in response to rapamycin between the E4FAD and E3FAD mice, the mice with human APOE ε3 allele. We used multi-modal MRI to measure in vivo cerebral blood flow (CBF), neurotransmitter levels, white matter integrity, water content, cerebrovascular reactivity (CVR) and somatosensory response; used behavioral assessments to determine cognitive function; used biochemistry assays to determine Aß retention and blood-brain barrier (BBB) functions; and used metabolomics to identify brain metabolic changes. We found that in the E4FAD mice, rapamycin normalized bodyweight, restored CBF (especially in female), BBB activity for Aß transport, neurotransmitter levels, neuronal integrity and free fatty acid level, and reduced Aß retention, which were not observe in the E3FAD-Rapa mice. In contrast, E3FAD-Rapa mice had lower CVR responses, lower anxiety and reduced glycolysis in the brain, which were not seen in the E4FAD-Rapa mice. Further, rapamycin appeared to normalize lipid-associated metabolism in the E4FAD mice, while slowed overall glucose-associated metabolism in the E3FAD mice. Finally, rapamycin enhanced overall water content, water diffusion in white matter, and spatial memory in both E3FAD and E4FAD mice, but did not impact the somatosensory responses under hindpaw stimulation. Our findings indicated that rapamycin was able to restore brain functions and reduce AD risk for young, asymptomatic E4FAD mice, and there were pharmacogenetic differences between the E3FAD and E4FAD mice. As the multi-modal MRI methods used in the study are readily to be used in humans and rapamycin is FDA-approved, our results may pave a way for future clinical testing of the pharmacogenetic responses in humans with different APOE alleles, and potentially using rapamycin to prevent AD for asymptomatic APOE4 carriers.

Molecular MRI of the Immuno-Metabolic Interplay in a Rabbit Liver Tumor Model: A Biomarker for Resistance Mechanisms in Tumor-targeted Therapy?

Background The immuno-metabolic interplay has gained interest for determining and targeting immunosuppressive tumor micro-environments that remain a barrier to current immuno-oncologic therapies in hepatocellular carcinoma. Purpose To develop molecular MRI tools to reveal resistance mechanisms to immuno-oncologic therapies caused by the immuno-metabolic interplay in a translational liver cancer model. Materials and Methods A total of 21 VX2 liver tumor-bearing New Zealand white rabbits were used between October 2018 and February 2020. Rabbits were divided into three groups. Group A (n = 3) underwent intra-arterial infusion of gadolinium 160 (160Gd)-labeled anti-human leukocyte antigen-DR isotope (HLA-DR) antibodies to detect antigen-presenting immune cells. Group B (n = 3) received rhodamine-conjugated superparamagnetic iron oxide nanoparticles (SPIONs) intravenously to detect macrophages. These six rabbits underwent 3-T MRI, including T1- and T2-weighted imaging, before and 24 hours after contrast material administration. Group C (n = 15) underwent extracellular pH mapping with use of MR spectroscopy. Of those 15 rabbits, six underwent conventional transarterial chemoembolization (TACE), four underwent conventional TACE with extracellular pH-buffering bicarbonate, and five served as untreated controls. MRI signal intensity distribution was validated by using immunohistochemistry staining of HLA-DR and CD11b, Prussian blue iron staining, fluorescence microscopy of rhodamine, and imaging mass cytometry (IMC) of gadolinium. Statistical analysis included Mann-Whitney U and Kruskal-Wallis tests. Results T1-weighted MRI with 160Gd-labeled antibodies revealed localized peritumoral ring enhancement, which corresponded to gadolinium distribution detected with IMC. T2-weighted MRI with SPIONs showed curvilinear signal intensity representing selective peritumoral deposition in macrophages. Extracellular pH-specific MR spectroscopy of untreated liver tumors showed acidosis (mean extracellular pH, 6.78 ± 0.09) compared with liver parenchyma (mean extracellular pH, 7.18 ± 0.03) (P = .008) and peritumoral immune cell exclusion. Normalization of tumor extracellular pH (mean, 6.96 ± 0.05; P = .02) using bicarbonate during TACE increased peri- and intratumoral immune cell infiltration (P = .002). Conclusion MRI in a rabbit liver tumor model was used to visualize resistance mechanisms mediated by the immuno-metabolic interplay that inform susceptibility and response to immuno-oncologic therapies, providing a therapeutic strategy to restore immune permissiveness in liver cancer. © RSNA, 2020 Online supplemental material is available for this article.