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A substantial and constitutive expression of translocator protein (TSPO) in cerebral blood vessels hampers the sensitive detection of neuroinflammation characterized by greatly induced TSPO expression in activated glia. Here, we conducted in vivo positron emission tomography (PET) and in vitro autoradiographic imaging of normal and TSPO-deficient mouse brains to compare the binding properties of 18F-FEBMP, a relatively novel TSPO radioligand developed for human studies based on its insensitivity to a common polymorphism, with 11C-PK11195, as well as other commonly used TSPO radioligands including 11C-PBR28, 11C-Ac5216 and 18F-FEDAA1106. TSPO in cerebral vessels of normal mice was found to provide a major binding site for 11C-PK11195, 11C-PBR28 and 18F-FEDAA1106, in contrast to no overt specific binding of 18F-FEBMP and 11C-Ac5216 to this vascular component. In addition, 18F-FEBMP yielded PET images of microglial TSPO with a higher contrast than 11C-PK11195 in a tau transgenic mouse modeling Alzheimer’s disease (AD) and allied neurodegenerative tauopathies. Moreover, TSPO expression examined by immunoblotting was significantly increased in AD brains compared with healthy controls, and was well correlated with the autoradiographic binding of 18F-FEBMP but not 11C-PK11195. Our findings support the potential advantage of comparatively glial TSPO-selective radioligands such as 18F-FEBMP for PET imaging of inflammatory glial cells.

Total redox capacity (TRC) and oxidative stress (OxiStress) of biological objects (such as cells, tissues, body fluids) are one of the most frequently analyzed parameters in the life science. Development of highly sensitive molecular probes and analytical methods for detection of these parameters is a rapidly growing sector of BioTech R&D industry. The aim of the present study was to develop quantum sensors for tracking the TRC and OxiStress in living biological objects using EPR, MRI, and optical imaging. We describe a new two-set sensor system: (i) TRC sensor – QD@CD-TEMPO; and (ii) OxiStress sensor – QD@CDTEMPOH. Both redox-sensors are composed of small-size quantum dots (QDs), coated with multi-nitroxide-functionalized cyclodextrin (paramagnetic CD-TEMPO or diamagnetic CD-TEMPOH) conjugated with Triphenylphosphonium (TPP) groups. The TPP-groups were added to achieve intracellular delivery and mitochondrial localization. Nitroxide residues interact simultaneously with various oxidizers and reducers, and the sensors are transformed from paramagnetic radical form (QD@CD-TEMPO) into diamagnetic hydroxylamine form (QD@CD-TEMPOH) and vice-versa, due to nitroxide redox-cycling. These chemical transformations are accompanied by a characteristic dynamics of their contrast features due to quenching of QD fluorescence by nitroxide radical. The sensors were applied for EPR/fluorescent analysis of TRC and OxiStress in vitro on isolated cells with different proliferative index, as well as for non-invasive magnetic resonance imaging of redox imbalance and severe oxidative stress in vivo on mice with renal dysfunction.

The purpose of this study was to compare parameter estimates for the 2-compartment and diffusion kurtosis imaging models obtained from diffusion-weighted imaging (DWI) of aquaporin-4 (AQP4) expression-controlled cells, and to look for biomarkers that indicate differences in the cell membrane water permeability. DWI was performed on AQP4-expressing and non-expressing cells and the signal was analyzed with the 2-compartment and diffusion kurtosis imaging models. For the 2-compartment model, the diffusion coefficients (Df, Ds) and volume fractions (Ff, Fs, Ff = 1-Fs) of the fast and slow compartments were estimated. For the diffusion kurtosis imaging model, estimates of the diffusion kurtosis (K) and corrected diffusion coefficient (D) were obtained. For the 2-compartment model, Ds and Fs showed clear differences between AQP4-expressing and non-expressing cells. Fs was also sensitive to cell density. There was no clear relationship with the cell type for the diffusion kurtosis imaging model parameters. Changes to cell membrane water permeability due to AQP4 expression affected DWI of cell suspensions. For the 2-compartment and diffusion kurtosis imaging models, Ds was the parameter most sensitive to differences in AQP4 expression.

Background/Aim: The menadione/ascorbate (M/A) combination has attracted attention due to the unusual ability of pro-vitamin/vitamin combination to kill cancer cells without affecting the viability of normal cells. The aim of this study was to elucidate the role of M/A in targeting cancerous mitochondria. Materials and Methods: Several cancer and normal cell lines of the same origin were used. Cells were treated with different concentrations of M/A for 24 h. The cell viability, mitochondrial superoxide, mitochondrial membrane potential, and succinate were analyzed using conventional analytical tests. Results: M/A exhibited a highly specific suppression on cancer cell growth and viability, without adversely affecting the viability of normal cells at concentrations attainable by oral or parenteral administration in vivo. This effect was accompanied by: (i) an extremely high production of mitochondrial superoxide in cancer cells, but not in normal cells; (ii) a significant dose-dependent depolarization of mitochondrial membrane and depletion of oncometabolite succinate in cancer cells. Conclusion: The anticancer effect of M/A is related to the induction of severe mitochondrial oxidative stress in cancer cells only. Thus, M/A has a potential to increase the sensitivity and vulnerability of cancer cells to conventional anticancer therapy and immune system.

Damaged mitochondria may be one of the earliest manifestations of Alzheimer`s disease (AD). Since oxidative phosphorylation is a primary source of neuronal energy, unlike glycolysis-dependent energy production in inflamed glia, mitochondrial respiration could provide a selective biomarker of neuronal deterioration in AD. Here we used a recently developed positron emission tomography (PET) probe targeting mitochondrial complex I (MC-I), 18F-BCPP-EF, to non-invasively visualize mitochondrial abnormalities in the brains of tau transgenic mice (rTg4510 TauTg). Tauopathy and neuroinflammation were visualized by PET using a tau probe 11C-PBB3 and a TSPO probe, 18F-FEBMP, respectively. A marked reduction in 18F-BCPP-EF uptake was observed in hippocampal and forebrain regions of TauTg mice, colocalizing with regions of tauopathy, neuronal damage and neuroinflammation. MC-I signals were highly correlated with atrophy assayed by MRI, but negatively associated with inflammatory signals measured by TSPO-PET, indicating that neuronal metabolic signals measured by MC-I PET were robust to inflammatory interference. MC-I may be a useful imaging biomarker to detect neuronal damage and metabolic changes with minimal interference from concomitant glial hypermetabolism.

Gadolinium(III) (Gd(III)) chelates are promising compounds for developing MRI‐guided neutron capture therapies (NCT). However, despite their success as MRI contrast agents (CAs), current Gd(III) chelates do not present appropriate accumulation in tumors for effective NCT. To overcome this limitation, we developed a series of biocompatible polymeric nanocarriers conjugating Gd(III)‐chelates, i. e. Gd‐DOTA, as NCT agents with high MRI sensitivity and tumor delivery. These polymers were based on poly(aspartic acid) (P(Asp)) and were modified with poly(ethylene glycol) (PEG) chains having different molecular weight (Mw) for controlling stability and pharmacokinetics. The T1 relaxivity of the Gd‐DOTA conjugated polymers increased 2‐fold compared to that of clinically used Gd(III) CAs. In vivo, the PEG‐modified polymers promoted the accumulation in tumor tissues, enhancing the MRI contrast of tumors. After neutron irradiation, the nanocarriers effectively suppressed the tumor growth, particularly the PEG‐P(Asp‐Gd‐DOTA) with shorter PEG chain, which promoted higher intracellular delivery, supporting their potential as NCT agents.

Synaptic dysfunction provoking dysregulated cortical neural circuits is currently hypothesized as a key pathophysiological process underlying clinical manifestations in Alzheimer's disease and related neurodegenerative tauopathies. Here, we conducted positron emission tomography (PET) along with postmortem assays to investigate time-course changes of excitatory and inhibitory synaptic constituents in an rTg4510 mouse model of tauopathy, which develops tau pathologies leading to noticeable brain atrophy at 5-6 months of age. Both male and female mice were analyzed in this study. We observed that radiosignals derived from [C]flumazenil, a tracer for benzodiazepine receptor, in rTg4510 mice were significantly lower than the levels in non-transgenic littermates at 2-3 months of age. In contrast, retentions of (E)-[C]ABP688, a tracer for mGluR5, were unaltered relative to controls at 2 months of age but then gradually declined with aging in parallel with progressive brain atrophy. Biochemical and immunohistochemical assessment of postmortem brain tissues demonstrated that inhibitory but not excitatory synaptic constituents selectively diminished without overt loss of somas of GABAergic interneurons in the neocortex and hippocampus of rTg4510 mice at 2 months of age, which was concurrent with enhanced immunoreactivity of cFos, a well-characterized immediate early gene, suggesting that impaired inhibitory neurotransmission may cause hyperexcitability of cortical circuits. Our findings indicate that tau-induced disruption of the inhibitory synapse may be a critical trigger of progressive neurodegeneration resulting in massive neuronal loss, and PET assessments of inhibitory versus excitatory synapses potentially offer indices for hyperexcitability and excitotoxicity early in the etiological pathway of neurodegenerative tauopathies.In this study, we examined the status of excitatory and inhibitory synapses in the brain of the rTg4510 tauopathy mouse model by PET imaging with (E)-[C]ABP688 and [C]flumazenil, respectively. We identified inhibitory synapse as being significantly dysregulated before brain atrophy at 2 months of age, while excitatory synapse stayed relatively intact at this stage. In line with this observation, postmortem assessment of brain tissues demonstrated selective attenuation of inhibitory synaptic constituents accompanied by the upregulation of cFos prior to the formation of tau pathology in the forebrain at young ages. Our findings indicate that selective degeneration of inhibitory synapse with hyperexcitability in the cortical circuit constitutes the critical early pathophysiology of tauopathy.

Transplantation of human‐induced pluripotent stem cell‐derived neural stem/progenitor cells (hiPSC‐NS/PCs) is a promising treatment for a variety of neuropathological conditions. Although previous reports have indicated the effectiveness of hiPSC‐NS/PCs transplantation into the injured spinal cord of rodents and nonhuman primates, long‐term observation of hiPSC‐NS/PCs post‐transplantation suggested some “unsafe” differentiation‐resistant properties, resulting in disordered overgrowth. These findings suggest that, even if “safe” NS/PCs are transplanted into the human central nervous system (CNS), the dynamics of cellular differentiation of stem cells should be noninvasively tracked to ensure safety. Positron emission tomography (PET) provides molecular‐functional information and helps to detect specific disease conditions. The current study was conducted to visualize Nestin (an NS/PC marker)‐positive undifferentiated neural cells in the CNS of immune‐deficient (nonobese diabetic‐severe combined immune‐deficient) mice after hiPSC‐NS/PCs transplantation with PET, using 18 kDa translocator protein (TSPO) ligands as labels. TSPO was recently found to be expressed in rodent NS/PCs, and its expression decreased with the progression of neuronal differentiation. We hypothesized that TSPO would also be present in hiPSC‐NS/PCs and expressed strongly in residual immature neural cells after transplantation. The results showed high levels of TSPO expression in immature hiPSC‐NS/PCs‐derived cells, and decreased TSPO expression as neural differentiation progressed in vitro. Furthermore, PET with [18F] FEDAC (a TSPO radioligand) was able to visualize the remnant undifferentiated hiPSC‐NS/PCs‐derived cells consisting of TSPO and Nestin+ cells in vivo. These findings suggest that PET with [18F] FEDAC could play a key role in the safe clinical application of CNS repair in regenerative medicine.

In vivo redox imaging using nitroxyl radicals as redox sensitive contrast agents was described. The cyclic redox mechanism of nitroxyl radical makes its possibility as a normal tissue selective radioprotector. In vivo assessment of paramagnetic and diamagnetic conversions based on such redox reactions can be an index of tissue redox status. Electron paramagnetic resonance imaging and/or T1-weighted MRI techniques can visualize this tissue redox status non-invasively. Using nitroxyl radical as a MR contrast agent has several advantages compared with that used in EPR imaging. Nitroxyl radicals as redox sensitive MR contrast agents can be a useful tool to detect abnormal tissue redox status, such as disordered oxidative stress, tumor hypoxia, and etc.

Time courses of the redox status in the brains of mice after X-ray or carbon-ion beam irradiation were observed by magnetic resonance redox imaging (MRRI). The relationship between radiation-induced oxidative stress on the cerebral nervous system and the redox status in the brain was discussed. The mice were irradiated by 8-Gy X-ray or carbon-ion beam (C-beam) on their head under anesthesia. C-beam irradiation was performed at HIMAC (Heavy-Ion Medical Accelerator in Chiba, NIRS/QST, Chiba, Japan). MRRI measurements using a blood-brain-barrier-permeable nitroxyl contrast agent, MCP or TEMPOL, were performed using 7-T scanner at several different times, i.e., 5-10 hours, 1, 2, 4, and 8 day(s) after irradiation. Decay rates of the nitroxyl-enhanced T1-weighted MR signals in the brains were estimated from MRRI data sets, and variation in the decay rates after irradiation was assessed. The variation in decay rates of MCP and TEMPOL after X-ray or C-beam irradiation was similar, but different variation patterns were observed between X-ray and C-beam. The apparent decay rate of both MCP and TEMPOL decreased due to the temporal reduction of blood flow in the brain several hours after X-ray and/or C-beam irradiation. After decreasing, the apparent decay rates of nitroxyl radicals in the brain gradually increased during the following days after X-ray irradiation or rapidly increased 1 day after C-beam irradiation. The sequential increase in nitroxyl decay rates may have been due to the oxidative atmosphere in the tissue due to ROS generation. X-ray and C-beam irradiation resulted in different redox responses, which may have been due to time-varying oxidative stress/injury, in the mouse brain. The C-beam irradiation effects were more acute and larger than those of X-ray irradiation.

This study reports a non-invasive magnetic resonance imaging (MRI) of kidney dysfunction in mice, based on the induction of redox-imbalance and oxidative stress in the renal tissues, using mito-TEMPO as redox-sensitive contrast probe. Kidney dysfunction was triggered by hypercholesterolemia. The mice were divided in three groups: (i) on normal diet (ND); (ii) on cholesterol diet (CD); (iii) on cholesterol plus cholestyramine diet (CC). After 15 weeks feeding, the mice were subjected to the following analyses: plasma cholesterol levels; serum test for renal functionality; nitroxide-enhanced MRI of tissue redox-status in vivo; histochemical staining of tissue section to visualize renal damage; evaluation of total antioxidant capacity and oxidative stress on isolated tissue specimens. MRI signal of mito-TEMPO in the kidney was characterized by: high intensity and long life-time in CD mice, indicating a high oxidative capacity of renal tissues; poor intensity and short life-time in ND mice, indicating a high reducing capacity; moderate intensity and relatively short life-time in CC mice, indicating a protective effect of lipid-lowering drug. The data were confirmed on isolated tissue specimens, using conventional tests. They suggest that hypercholesterolemia induces redox-imbalance in kidney and this process could be visualized using MRI and mito-TEMPO as a redox-sensitive contrast.

(Fe(Salen)) is an anti-cancer agent with intrinsic magnetic property. Here, we covalently linked Fe(Salen) to paclitaxel (PTX), a widely used anti-cancer drug, to obtain a magnetized paclitaxel conjugate (M-PTX), which exhibited magnetic characteristics for magnet-guided drug delivery and MRI visualization. M-PTX increased apoptosis and G2/M arrest of cultured human oral cancer cell lines in the same manner as PTX. Furthermore, marked contrast intensity was obtained in magnetic resonance imaging (MRI) of M-PTX. In a mouse oral cancer model, a permanent magnet placed on the body surface adjacent to the tumor resulted in distinct accumulation of M-PTX, and the anti-cancer effect was greater than that of M-PTX without the magnet. We believe that this strategy may improve future cancer chemotherapy by providing conventional anti-cancer drugs with novel functionalities such as magnet-guided drug delivery or MRI-based visualization/quantitation of drug distribution.

We used a mitochondria-penetrating nitroxide, mito-TEMPO, as a contrast probe for imaging of kidney dysfunction in mice, based on the redox-imbalance and oxidative stress in the renal tissues. Kidney dysfunction was triggered by hypercholesterolemia. The mice were divided in three groups: (i) on normal diet (ND; control); (ii) on cholesterol diet (CD); (iii) on cholesterol plus cholestyramine diet (CC). CD mice showed increased plasma levels of total cholesterol and non-HDL-cholesterol, as well as increased serum levels of blood urea nitrogen, uric acid and creatinine, compared to ND mice. CC mice showed slightly increased plasma levels of total cholesterol and HDL-cholesterol, but not non-HDL-cholesterol, compared to ND mice. The serum levels of blood urea nitrogen, uric acid and creatinine in CC mice were equal to those in ND mice. The MRI signal of mito-TEMPO in the kidneys was characterized by: high intensity and long life-time in CD mice, indicating a high oxidative capacity of renal tissues; poor intensity and short life-time in ND mice, indicating a high reducing capacity of renal tissues; moderate intensity and relatively short life-time in CC mice, which shows the protective effect of lipid-lowering agents against oxidative damage. The data suggest that hypercholesterolemia induces redox-imbalance and oxidative stress in kidneys and this process could be visualized using MRI and mito-TEMPO as a redox-sensitive contrast substance.

Glioblastoma (GBM) is the most common malignant brain tumor, and infiltrates into the surrounding normal brain tissue. Induction of a tumor-specific immune response is one of the best methods to obtain tumor-specific cytotoxicity. Photodynamic therapy (PDT) is known to effectively induce an antitumor immune response. We have developed a clinically translatable nanoparticle, liposomally formulated phospholipid-conjugated indocyanine green (LP-iDOPE), applicable for PDT. This nanoparticle accumulates in tumor tissues by the enhanced permeability and retention effect, and releases heat and singlet oxygen to injure cancer cells when activated by near infrared (NIR) light. We assessed the effectiveness of the LP-iDOPE system in brain using the rat 9L glioblastoma model. Treatment with LP-iDOPE and NIR irradiation resulted in significant tumor growth suppression and prolongation of survival. Histopathological examination showed induction of both apoptosis and necrosis and accumulation of CD8+ T-cells and macrophages/microglia accompanied by marked expressions of heat shock protein-70 (HSP70), which was not induced by mild hyperthermia alone at 45° C or an interleukin-2-mediated immune reaction. Notably, the efficacy was lost in immunocompromised nude rats. These results collectively show that the novel nanoparticle LP-iDOPE in combination with NIR irradiation can efficiently induce a tumor-specific immune reaction for malignant gliomas possibly by inducing HSP70 expression which is known to activate antigen-presenting cells through toll-like receptor signaling.