Electron Paramagnetic Resonance Imaging Research Articles

A 750-MHz Electronically Tunable Resonator Using Microstrip Line Couplers for Electron Paramagnetic Resonance Imaging of a Mouse Tumor-Bearing Leg.

The purpose of this work was to develop an electronically tunable resonator operating at 750 MHz for continuous-wave electron paramagnetic resonance (CW-EPR) imaging of a mouse tumor-bearing leg. The resonator had a multi-coil parallel-gap structure with a sample space of 16 mm in diameter and 20 mm in length. Microstrip line couplers were used in conjunction with varactor diodes to enable resonance frequency adjustment and to reduce the nonlinear effects of the varactor diodes. The resonator was modeled by the finite-element method and a microwave circuit simulation was performed to clarify its radiofrequency characteristics. A tunable resonator was evaluated in terms of its resonance frequency, tunable frequency band, and conversion efficiency of the RF magnetic field. The developed resonator provided a tunable frequency band of 4 MHz at a central frequency of 747 MHz and a conversion efficiency of 34 μT/W1/2. To demonstrate the application of this tunable resonator to EPR imaging, three-dimensional EPR images of a sample solution and a mouse tumor-bearing leg were obtained. The developed tunable resonator satisfied our initial requirements for in vivo EPR imaging and may be able to be further improved using the present finite-element and circuit models if any problems arise during future practical applications. This work may help to promote EPR imaging of tumor-bearing mice in cancer-related studies.

Investigating Pigment Radicals in Black Rice Using HPLC and Multi-EPR.

We investigated the location and distribution of paramagnetic species in black and white rice using electron paramagnetic resonance (EPR), X-band (9 GHz) EPR imaging (EPRI), and HPLC. EPR primarily detected two paramagnetic species in black rice, which were identified as a stable radical and Mn2+ species, based on the g values and hyperfine components of the EPR signals. The signal from the stable radical appeared at g ≈ 2.00 and was relatively strong and stable. Subsequent noninvasive two-dimensional (2D) EPRI revealed that this stable radical was primarily located in the pigmented region of black rice, while very few radicals were observed in the rice interior. Pigments extracted from black rice were analyzed using HPLC; the major compound was found to be cyanidin-3-glucoside. EPR and HPLC results indicate that the stable radical was only found within the pigmented region of the rice, and that it could either be cyanidin-3-glucoside, or one of its oxidative decomposition products.

In vivo electron paramagnetic resonance oximetry and applications in the brain.

Molecular oxygen (O2) is essential to brain function and mechanisms necessary to regulate variations in delivery or utilization of O2 are crucial to support normal brain homeostasis, physiology and energy metabolism. Any imbalance in cerebral tissue partial pressure of O2 (pO2) levels may lead to pathophysiological complications including increased reactive O2 species generation leading to oxidative stress when tissue O2 level is too high or too low. Accordingly, the need for oximetry methods, which assess cerebral pO2 in vivo and in real time, is imperative to understand the role of O2 in various metabolic and disease states, including the effects of treatment and therapy options. In this review, we provide a brief overview of the common in vivo oximetry methodologies for measuring cerebral pO2. We discuss the advantages and limitations of oximetry methodologies to measure cerebral pO2 in vivo followed by a more in-depth review of electron paramagnetic resonance oximetry spectroscopy and imaging using several examples of current electron paramagnetic resonance oximetry applications in the brain.

Electron Paramagnetic Resonance pO2 Image Tumor Oxygen-Guided Radiation Therapy Optimization.

Modern standards for radiation treatment do not take into account tumor oxygenation for radiation treatment planning. Strong correlation between tumor oxygenation and radiation treatment success suggests that oxygen-guided radiation therapy (OGRT) may be a promising enhancement of cancer radiation treatment. We have developed an OGRT protocol for rodents. Electron paramagnetic resonance (EPR) imaging is used for recording oxygen maps with high spatial resolution and excellent accuracy better than 1 torr. Radiation is delivered with an animal intensity modulated radiation therapy (IMRT) XRAD225Cx micro-CT/ therapy system. The radiation plan is delivered in two steps. First, a uniform 15% tumor control dose (TCD15) is delivered to the whole tumor. In the second step, an additional booster dose amounting to the difference between TCD98 and TCD15 is delivered to radio-resistant, hypoxic tumor regions. Delivery of the booster dose is performed using a multiport conformal beam protocol. For radiation beam shaping we used individual radiation blocks 3D-printed from tungsten infused ABS polymer. Calculation of beam geometry and the production of blocks is performed next to the EPR imager, immediately after oxygen imaging. Preliminary results demonstrate the sub-millimeter precision of the radiation delivery and high dose accuracy. The efficacy of the radiation treatment is currently being tested on syngeneic FSa fibrosarcoma tumors grown in the legs of C3H mice.

Effect of amifostine, a radiation-protecting drug, on oxygen concentration in tissue measured by EPR oximetry and imaging.

Effect of amifostine, a radiation-protecting drug, on muscle tissue partial pressure of oxygen was investigated by electron paramagnetic resonance spectroscopy and imaging. When amifostine was administered intraperitoneally or intravenously to mice, the linewidth of the electron paramagnetic resonance spectra of the lithium octa-n-butoxy-substituted naphthalocyanine implanted in the mouse leg muscle decreased. Electron paramagnetic resonance oximetry using a lithium octa-n-butoxy-substituted naphthalocyanine probe and electron paramagnetic resonance oxygen mapping using a triarylmethyl radical paramagnetic probe was useful to quantify pressure of oxygen in the tissues of living mice. The result of electron paramagnetic resonance oximetric imaging showed that administration of amifostine could decrease pressure of oxygen in the muscle and also tumor tissues. This finding suggests that lowering pressure of oxygen in tissues might contribute in part to the radioprotection of amifostine.

Electron spin resonance and fluorescence imaging assisted electrochemical approach for accurate and comprehensive monitoring of cellular hydrogen peroxide dynamics.

Dynamic alteration in the levels of cellular hydrogen peroxide (H2O2) is closely related to a variety of human diseases, as well as signal transduction pathways that regulate cell survival and death. Although qualitative or quantitative methods are available for measuring either intra- or extra-cellular H2O2 levels, accurate and comprehensive in situ detection of the real-time H2O2 dynamics of living cells remains a significant challenge. To solve this problem, a novel multi-dimensional in situ cell assay platform combining electrochemistry, electron spin resonance (ESR) and optical imaging is designed. In this platform, the real-time concentration of extracellular H2O2 released from stimulated cells can be accurately detected by ESR assisted chronoamperometry, while the level of intracellular H2O2 is simultaneously monitored via the incorporated fluorescence imaging. Accurately and simultaneously analyzing the level variations of extra- and intra-cellular reactive oxygen species based on our assay platform can complement each other for further precise and in-depth investigation of their membrane transport and related cellular signaling, which will benefit disease diagnosis and treatment.

Synthesis and Characterization of PEGylated Trityl Radicals: Effect of PEGylation on Physicochemical Properties.

Tetrathiatriarylmethyl (TAM, trityl) radicals have attracted considerable attention as spin probes for biological electron paramagnetic resonance (EPR) spectroscopy and imaging owing to their sharp EPR singlet signals and high biostability. However, their in vivo applications were limited by the short blood circulation lifetimes and strong binding with albumins. Our previous results showed that PEGylation is a feasible method to overcome the issues facing in vivo applications of TAM radicals. In the present study, we synthesized a series of new PEGylated TAM radicals (TTP1, TPP2, TNP1, TNP2, d-TNP1, and d-TNP3) containing various lengths and numbers of mPEG chains. Our results found that the pattern of PEGylation exerts an important effect on physicochemical properties of the resulting TAM radicals. Dendritic PEGylated TAM radicals, TNP1 and TNP2, have higher water solubility and lower susceptibility for self-aggregation than their linear analogues TPP1 and TPP2. Furthermore, dendritic PEGylated TAM radicals exhibit extremely high stability toward various biological oxidoreductants as well as in rat whole blood, liver homogenate, and following in vivo intravenous administration in mice. Importantly, the deuterated derivatives, especially d-TNP3, exhibit excellent properties including the sharp and O2-sensitive EPR singlet signal, good biocompatibility, and prolonged kinetics with half-life time of ≥10 h in mice. These PEGylated TAM radicals should be suitable for a wide range of applications in in vivo EPR spectroscopy and imaging.

Concentration dependence of nitroxyl spin probes in liposomal solution: electron spin resonance and overhauser-enhanced magnetic resonance studies

In this work, the detailed studies of electron spin resonance (ESR) and overhauser-enhanced magnetic resonance imaging (OMRI) were carried out for permeable nitroxyl spin probe, MC-PROXYL as a function of agent concentration in liposomal solution. In order to compare the impermeable nature of nitroxyl radical, the study was also carried out only at 2 mM concentration of carboxy-PROXYL. The ESR parameters were estimated using L-band and 300 MHz ESR spectrometers. The line width broadening was measured as a function of agent concentration in liposomal solution. The estimated rotational correlation time is proportional to the agent concentration, which indicates that less mobile nature of nitroxyl spin probe in liposomal solution. The partition parameter and permeability values indicate that the diffusion of nitroxyl spin probe distribution into the lipid phase is maximum at 2 mM concentration of MC-PROXYL. The dynamic nuclear polarization (DNP) parameters such as DNP factor, longitudinal relaxivity, saturation parameter, leakage factor and coupling factor were estimated for 2 mM MC-PROXYL in 400 mM liposomal dispersion. The spin lattice relaxation time was shortened in liposomal solution, which leads to the high relaxivity. Reduction in coupling factor is due to less interaction between the electron and nuclear spins, which causes the reduction in enhancement. The leakage factor increases with increasing agent concentration. The increase in DNP enhancement was significant up to 2 mM in liposomal solution. These results paves the way for choosing optimum agent concentration and OMRI scan parameters used in intra and extra membrane water by loading the liposome vesicles with a lipid permeable nitroxyl spin probes in OMRI experiments.

C-Ion- and X-ray-Induced Sucrose Radicals Investigated by CW EPR and 9 GHz EPR Imaging

Abstract We investigated stable radical distribution and particle tracks in sucrose irradiated by C-ion irradiation with continuous wave (CW) electron paramagnetic resonance (EPR) and 9 GHz EPR imaging. Both EPR results were compared with X-ray irradiation at a similar dose. Radical distribution in sucrose crystals induced by C-ion and X-ray irradiation were completely different. The 2D EPR imaging results suggested that radical species were mostly located inside the sucrose crystal. Fewer radicals were found on the surface region of the sucrose crystal. The high radical intensities in relation to the C-ion energy deposition are clearly observed at Bragg peak region. No trace of the stable radicals was found after the peak region. The stable radicals of sucrose were distributed as a result of recombination of radicals induced by particle interaction. For the first time, the present EPR images showed the stable radical distribution and particle tracks in the crystal in association with particle-sucrose interaction.

Investigation of scavenging activities and distribution of paramagnetic species in Zanthoxylum limonella seeds

We investigated the scavenging activities of methanol-extracted oil and the distribution of paramagnetic species in Zanthoxylum limonella (ZL) seeds using noninvasive 9 GHz electron paramagnetic resonance (EPR) imaging and continuous wave EPR. EPR detected three different stable paramagnetic species that were assigned to stable organic radicals, Mn2+, and other paramagnetic metal complexes. Two-dimensional EPR imaging showed that the stable paramagnetic species were located in the pigmented seed region with a strong intensity. Gas chromatography–mass spectrophotometric (GC–MS) analyses were then performed to identify the compound possibly related to the scavenging activity. The DPPH scavenging activities of ZL were slightly higher than those of Piper nigrum and Coriandrum sativum. Based on the results of EPR, GC–MS, and other methods, limonene in ZL is one of the major compounds that can be related to the scavenging activities.

Multimodality Imaging Identifies Distinct Metabolic Profiles In Vitro and In Vivo

The study of alterations of tumor metabolism should allow the identification of new targets for innovative anticancer strategies. Metabolic alterations are generally established in vitro, and conclusions are often extrapolated to the in vivo situation without further tumor metabolic phenotyping. To highlight the key role of microenvironment on tumor metabolism, we studied the response of glycolytic and oxidative tumor models to metabolic modulations in vitro and in vivo. MDA-MB-231 and SiHa tumor models, characterized in vitro as glycolytic and oxidative, respectively, were studied. Theoretically, when passing from a hypoxic state to an oxygenated state, a Warburg phenotype should conserve a glycolytic metabolism, whereas an oxidative phenotype should switch from glycolytic to oxidative metabolism (Pasteur effect). This challenge was applied in vitro and in vivo to evaluate the impact of different oxic conditions on glucose metabolism. 18F-fluorodeoxyglucose uptake, lactate production, tumor oxygenation, and metabolic fluxes were monitored in vivo using positron emission tomography, microdialysis, electron paramagnetic resonance imaging, and 13C-hyperpolarizated magnetic resonance spectroscopy, respectively. In vitro, MDA-MB-231 cells were glycolytic under both hypoxic and oxygenated conditions, whereas SiHa cells underwent a metabolic shift after reoxygenation. On the contrary, in vivo, the increase in tumor oxygenation (induced by carbogen challenge) led to a similar metabolic shift in glucose metabolism in both tumor models. The major discordance in metabolic patterns observed in vitro and in vivo highlights that any extrapolation of in vitro metabolic profiling to the in vivo situation should be taken cautiously and that metabolic phenotyping using molecular imaging is mandatory in vivo.

EPR Imaging Spin Probe Trityl Radical OX063: A Method for Its Isolation from Animal Effluent, Redox Chemistry of Its Quinone Methide Oxidation Product, and in Vivo Application in a Mouse.

We report herein a method for the recovery, purification, and application of OX063, a costly, commercially available nontoxic spin probe widely used for electron paramagnetic resonance (EPR) imaging, as well as its corresponding quinone methide (QM) form. This precious probe can be successfully recovered after use in animal model experiments (25-47% recovery from crude lyophilizate with 98.5% purity), even from samples that are >2 years old. Significantly, the recovered trityl can be reused in further animal model EPR imaging experiments. The work also describes support for the observed formation of an air-sensitive radical derived from the QM under reducing conditions.

380 - In Vivo Brain Redox Status in Diethylmaleate-Treated Mice Using EPR Imaging

Under normal physiological conditions, oxidative damage is prevented by the regulation of ROS by the antioxidant network. Major antioxidants in brain are ascorbic acid (AsA) and glutathione (GSH), and the levels of these antioxidants change depending on the disease state. Nitroxides are redox-sensitive compounds and reduced in vivo in the presence of AsA and GSH. Therefore, EPR imaging with nitroxides has been used to visualize the redox status in oxidative brain diseases, but the role of GSH on the reduction reaction of nitroxides is not clear. In the present study, using the mouse model of GSH depletion with diethylmaleate (DEM), the role of GSH in the brain redox status was examined non-invasively by EPR imaging with blood brain barrier-permeable 3-methoxycarbonyl-PROXYL (MCP). The pixel-based reduction rates of MCP in control and DEM-treated mouse heads were calculated and plotted as a redox map. The obtained redox map clearly showed that reduction rates of MCP significantly decreased in DEM-treated mouse brain. The GSH assay showed a significant reduction of GSH levels in DEM-treated mouse brain compared with untreated one. Alteration of the GSH level correlates well with the change in redox status of mouse brain image.

Accelerated electron paramagnetic resonance imaging using partial Fourier compressed sensing reconstruction

PurposeElectron paramagnetic resonance (EPR) imaging has evolved as a promising tool to provide non-invasive assessment of tissue oxygenation levels. Due to the extremely short T2 relaxation time of electrons, single point imaging (SPI) is used in EPRI, limiting achievable spatial and temporal resolution. This presents a problem when attempting to measure changes in hypoxic state. In order to capture oxygen variation in hypoxic tissues and localize cycling hypoxia regions, an accelerated EPRI imaging method with minimal loss of information is needed. MethodsWe present an image acceleration technique, partial Fourier compressed sensing (PFCS), that combines compressed sensing (CS) and partial Fourier reconstruction. PFCS augments the original CS equation using conjugate symmetry information for missing measurements. To further improve image quality in order to reconstruct low-resolution EPRI images, a projection onto convex sets (POCS)-based phase map and a spherical-sampling mask are used in the reconstruction process. The PFCS technique was used in phantoms and in vivo SCC7 tumor mice to evaluate image quality and accuracy in estimating O2 concentration. ResultsIn both phantom and in vivo experiments, PFCS demonstrated the ability to reconstruct images more accurately with at least a 4-fold acceleration compared to traditional CS. Meanwhile, PFCS is able to better preserve the distinct spatial pattern in a phantom with a spatial resolution of 0.6mm. On phantoms containing Oxo63 solution with different oxygen concentrations, PFCS reconstructed linewidth maps that were discriminative of different O2 concentrations. Moreover, PFCS reconstruction of partially sampled data provided a better discrimination of hypoxic and oxygenated regions in a leg tumor compared to traditional CS reconstructed images. ConclusionsEPR images with an acceleration factor of four are feasible using PFCS with reasonable assessment of tissue oxygenation. The technique can greatly enhance EPR applications and improve our understanding cycling hypoxia. Moreover this technique can be easily extended to various MRI applications.

Detection of Redox Imbalance in Normal Lymphocytes with Induced Mitochondrial Dysfunction - EPR Study.

The present study describes a new approach for direct imaging of redox status in live cells using paramagnetic spin-probes, which allows evaluation of the level of oxidative stress due to overproduction of superoxide. The method is based on redox cycling of cell/mitochondria-penetrating nitroxide radicals (e.g. mito-TEMPO) and their electron-paramagnetic resonance (EPR) contrast, which makes them useful molecular sensors for analysis of redox status and oxidative stress in cells and tissues. Oxidative stress was induced in normal human lymphocytes by treatment with 2-methoxyestradiol and rotenone (ME/Rot) at different concentrations. This combination provokes mitochondrial dysfunction, which is accompanied by overproduction of superoxide. The EPR measurements were performed in dynamics on X-Band spectrometer after addition of mito-TEMPO to cell suspensions. The intensity of the EPR signal in untreated cells decreased significantly, which indicates a conversion of paramagnetic mito-TEMPO to its non-contrast diamagnetic form (hydroxylamine - mito-TEMPOH) due to reduction. In ME/Rot-treated cells, the signal decreased more slowly and to a lower level with increasing the concentration of ME/Rot. These data indicate an induction of oxidative stress in the cells in a concentration-dependent manner. A very good positive correlation between the intensity of EPR signal of mito-TEMPO and the intracellular level of superoxide was found, analyzed by conventional dihydroethidium test (R=0.9143, p<0.001). In conclusion, our study demonstrated that cell-penetrating paramagnetic spin-probes, such as mito-TEMPO, are valuable tools for EPR imaging of the superoxide level in live cells, as well as for EPR imaging of mitochondrial dysfunction and metabolic activity, accompanied by superoxide imbalance.

Accelerated dynamic EPR imaging using fast acquisition and compressive recovery

Electron paramagnetic resonance (EPR) allows quantitative imaging of tissue redox status, which provides important information about ischemic syndromes, cancer and other pathologies. For continuous wave EPR imaging, however, poor signal-to-noise ratio and low acquisition efficiency limit its ability to image dynamic processes in vivo including tissue redox, where conditions can change rapidly. Here, we present a data acquisition and processing framework that couples fast acquisition with compressive sensing-inspired image recovery to enable EPR-based redox imaging with high spatial and temporal resolutions. The fast acquisition (FA) allows collecting more, albeit noisier, projections in a given scan time. The composite regularization based processing method, called spatio-temporal adaptive recovery (STAR), not only exploits sparsity in multiple representations of the spatio-temporal image but also adaptively adjusts the regularization strength for each representation based on its inherent level of the sparsity. As a result, STAR adjusts to the disparity in the level of sparsity across multiple representations, without introducing any tuning parameter. Our simulation and phantom imaging studies indicate that a combination of fast acquisition and STAR (FASTAR) enables high-fidelity recovery of volumetric image series, with each volumetric image employing less than 10 s of scan. In addition to image fidelity, the time constants derived from FASTAR also match closely to the ground truth even when a small number of projections are used for recovery. This development will enhance the capability of EPR to study fast dynamic processes that cannot be investigated using existing EPR imaging techniques.

Triarylmethyl Radicals: EPR Study of 13C Hyperfine Coupling Constants.

Triarylmethyl (TAM) radicals are widely used in Electron Paramagnetic Resonance (EPR) spectroscopy as spin labels and in EPR imaging as spin probes for in vivo oxymetry. One of the key advantages of TAMs is extremely narrow EPR line, especially in case of deuterated analogues (~5 μT). Another advantage is their slow spin relaxation even at physiological temperatures allowing, in particular, application of pulsed dipolar EPR methods for distance measurements in biomolecules. In this paper a large series of TAM radicals and their deuterated analogues is synthesized, and corresponding spectroscopic parameters including 13C hyperfine constants are obtained for the first time. The negligible dependence of 13C hyperfine constants on solvent, as well as on structure and number of substituents at para-C atoms of aromatic rings, has been found. In addition, we have demonstrated that 13C signals at natural abundance can be employed for successful room-temperature distance measurements using Pulsed Electron Double Resonance (PELDOR or DEER).

Novel blood–brain barrier-permeable spin probe for in vivo electron paramagnetic resonance imaging

A novel blood–brain barrier (BBB)-permeable compound 10 was discovered, wherein the nitroxide moiety was linked to a nicotine acetylcholine receptor ligand. It was applied as a probe for electron paramagnetic resonance (EPR) imaging of the mouse brain. The results demonstrated that the newly synthesized compound 10 exhibited BBB permeability. These findings provide an essential discovery for in vivo EPR imaging.

In Situ Monitoring of Diffusion of Guest Molecules in Porous Media Using Electron Paramagnetic Resonance Imaging.

A method is demonstrated to monitor macroscopic translational diffusion using electron paramagnetic resonance (EPR) imaging. A host-guest system with nitroxide spin probe 3-(2-Iodoacetamido)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (IPSL) as a guest inside the periodic mesoporous organosilica (PMO) aerogel UKON1-GEL as a host and ethanol as a solvent is used as an example to describe the protocol. Data is shown from a previous publication, where the protocol has been applied to both IPSL and Tris(8-carboxy-2,2,6,6-perdeutero-tetramethyl-benzo[1,2-d:4,5-d']bis(1,3)dithiole) methyl (Trityl) as guest molecules and UKON1-GEL and SILICA-GEL as host systems. A method is shown to prepare aerogel samples that cannot be synthesized directly in the sample tube for measurement due to a size change during synthesis. The aerogel is attached to sample tubes using heat shrink tubing and a pressure cooker to reach the necessary temperature without evaporating the solvent in the process. The method does not assume a clearly defined initial distribution of guest molecules at the start of the measurement. Instead, it requires a reservoir on top of the aerogel and experimentally determines the influx rate during data analysis. The diffusion is monitored continually over a period of 20 hr by recording the 1d spin density profile within the sample. The spectrometer settings for the imaging experiment are described quantitatively. Data analysis software is provided to take the resonator sensitivity profile into account and to numerically solve the diffusion equation. The software determines the macroscopic translational diffusion coefficient by least square minimization of the difference between the experiment and the numerical solution of the diffusion equation.

In vivo EPR oximetry using an isotopically-substituted nitroxide: Potential for quantitative measurement of tissue oxygen

Variations in brain oxygen (O2) concentration can have profound effects on brain physiology. Thus, the ability to quantitate local O2 concentrations noninvasively in vivo could significantly enhance understanding of several brain pathologies. However, quantitative O2 mapping in the brain has proven difficult. The electron paramagnetic resonance (EPR) spectra of nitroxides are sensitive to molecular O2 and can be used to estimate O2 concentrations in aqueous media. We recently synthesized labile-ester-containing nitroxides, such as 3-acetoxymethoxycarbonyl-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (nitroxide 4), which accumulate in cerebral tissue after in situ hydrolysis, and thus enable spatial mapping of O2 concentrations in the mouse brain by EPR imaging. In an effort to improve O2 quantitation, we prepared 3-acetoxymethoxycarbonyl-2,2,5,5-tetra(2H3)methyl-1-(3,4,4-2H3,1-15N)pyrrolidinyloxyl (nitroxide 2), which proved to be a more sensitive probe than its normo-isotopic version for quantifying O2 in aqueous solutions of various O2 concentrations. We now demonstrate that this isotopically substituted nitroxide is ∼2-fold more sensitive in vivo than the normo-isotopic nitroxide 4. Moreover, in vitro and in vivo EPR spectral-spatial imaging results with nitroxide 2 demonstrate significant improvement in resolution, reconstruction and spectral response to local O2 concentrations in cerebral tissue. Thus, isotopic-substituted nitroxides, such as 2, are excellent sensors for in vivo O2 quantitation in tissues, such as the brain.