Brain Redox Imaging

Abstract

Nitroxyl contrast agents (nitroxyl radicals, also known as nitroxide) are paramagnetic species, which can react with reactive oxygen species (ROS) to lose paramagnetism to be diamagnetic species. The paramagnetic nitroxyl radical forms can be detected by using electron paramagnetic resonance imaging (EPRI), Overhauser MRI (OMRI), or MRI. The time course of in vivo image intensity induced by paramagnetic redox-sensitive contrast agent can give tissue redox information, which is the so-called redox imaging technique. The redox imaging technique employing a blood–brain barrier permeable nitroxyl contrast agent can be applied to analyze the pathophysiological functions in the brain. A brief theory of redox imaging techniques is described, and applications of redox imaging techniques to brain are introduced.

1. Introduction

1.1. Redox-Sensitive Contrast Agent

Nitroxyl contrast agents (nitroxyl radicals, also known as nitroxide) are paramagnetic species, which have one unpaired electron. Structures of several commonly used nitroxyl radicals are shown in Fig. 20.1. Nitroxides belong to two main structural groups, i.e. five-member ring (pyrrolidine derivatives or so-called PROXYL derivatives) and six-member ring (piperidine derivatives or so-called TEMPO derivatives). Substitutional groups on the pyrrolidine ring of the PROXYL derivatives or the piperidine ring of the TEMPO derivatives determine the in vivo distribution of these agents as well as their sub-cellular distribution. Nitroxyl contrast agents with appropriate substitutional groups on the ring can be directed to intracellular locations, such as the mitochondria, or restricted to extracellular spaces or even penetrate the blood–brain barrier (BBB) and localize intracellularly. The membrane permeability of nitroxyl contrast agents can be relatively easily modified by chemical substituents on pyrrolidine or piperidine rings of the molecule. Various nitroxyl contrast agents which have distinct membrane permeabilities have been reported. For example, the carboxy-PROXYL can not go through the BBB; however, methoxy-carbonyl-PROXYL (also known as MC-PROXYL or CxP-M), which is synthesized by esterification of carboxy-PROXYL, can readily go through the BBB (1, 2).

Fig. 20.1.

Structures and common names of commercialized nitroxyl radicals. a PROXYL derivatives and b TEMPO derivatives.

The nitroxyl contrast agents are stable free radical species at room temperature in the solid form or even in solutions at physiological pH. However, the nitroxyl radical can be chemically and/or enzymatically reduced to the corresponding hydroxylamine form (Fig. 20.2). The nitroxyl radicals can also be oxidized to oxoammonium cation by reactive oxygen species (ROS), such as superoxide (·O₂⁻) and hydroxyl radical (·OH). The oxoammonium cation can be reduced by superoxide by a one-electron reduction back to the nitroxyl radical form or by two electrons to the corresponding hydroxylamine form by reduced glutathione (GSH), reduced nicotinamide adenine dinucleotide (NADH), and/or reduced nicotinamide adenine dinucleotide phosphate (NADPH) (3, 4). The hydroxylamines can be again re-oxidized to the corresponding nitroxyl radicals under oxidative conditions (5). When a nitroxyl radical is administrated to an experimental animal, the nitroxyl radical is mainly metabolized to the corresponding hydroxylamine form (6). This intrinsic in vivo one-electron reduction of nitroxyl radical can be modified (amplified or retarded) by abnormal tissue redox conditions, such as hypoxia, hyperoxia, and/or several oxidative stress conditions accompanying excess ROS generation. In addition to the redox sensitivity of nitroxyl radicals, BBB permeability with suitable substituents makes them suitable for imaging redox reactions in the brain.

Fig. 20.2.

Reactive oxygen species (ROS), such as superoxide (·O₂⁻) and hydroxyl radical (·OH), undergo one electron reduction of nitroxyl radicals in the presence of hydrogen donors (H-donors), such as reduced nicotinamide adenine dinucleotide (NADH), and/or reduced nicotinamide adenine dinucleotide phosphate (NADPH). The nitroxyl radicals were once oxidized to an oxoammonium cation by ROS and then reduced to hydroxylamine by receiving a hydrogen atom from H-donors. Reduced glutathione (GSH) and oxoammonium cations may make another complex irreversibly. Therefore, the generation of total ROS can be estimated by a reduction of the nitroxyl radical under coexisting GSH.

1.2. Redox Properties of Nitroxyl Contrast Agent

The reactivity of nitroxyl radicals with ROS is, in other words, that nitroxyl radicals can have antioxidant properties. In fact, several anti-oxidative properties of nitroxyl radicals have been reported (7–11). Leading such studies, the radio-protective activity of TEMPOL also has been studied prosperously (12–16). The radio-protective mechanism of TEMPOL has been considered that is based on its anti-oxidative property, i.e., the reaction of TEMPOL with ROS generated during irradiation of ionizing radiation, such as X-ray and/or γ-ray. In addition, the superoxide dismutase (SOD)-like activity of TEMPOL and its reaction mechanism has been reported (17–19). The currently known reaction mechanisms of nitroxyl radicals and ROS are summarized in Fig. 20.2. As described above, the process of one-electron reduction of nitroxyl radicals to corresponding hydroxylamines goes along with two steps, at first one-electron oxidation, then two-electron reduction. A recent study showed that the reaction of oxoammonium cation and GSH mainly gives a stable complex rather than hydroxyl radicals, although the structure of the complex has not been identified yet (20). However, the main in vivo reaction of nitroxyl radicals is the one-electron reduction to the hydroxylamme (6). Hydroxylamme can be re-oxidized to nitroxyl radical by some oxidants, such as ·OH and/or Fe³⁺, in vivo. This redox cycle reactions support SOD mimetic catalytic activity, which may underlie its radio-protective effect of TEMPOL.

The hydroxylamine form of TEMPOL, i.e., TEMPOL-H, does not have radio-protective activity for cell (12, 21). However, TEMPOL-H has a radio-protective effect, when TEMPOL-H was administered to a mouse (22). This is because of in vivo re-oxidation of hydroxylamine to the radio-protective nitroxyl radical. The re-oxidation of hydroxylamine to the nitroxyl radical, which is an oxygen-dependent reaction, cannot occur in a hypoxic tumor tissue. Therefore no radioprotective effects of nitroxyl radicals (23, 24) in hypoxic tumors can be expected because of their facile reduction to hydroxylamines. In contrast, slight nitroxyl radical can be continuously available in normoxic normal tissues after dynamic equilibrium between reduction of nitroxyl radical and oxidation of hydroxylamme. Recently, a significantly fast decay of TEMPOL in tumor tissue compared with other normal tissues was reported using an MR-based redox imaging technique (24). It is suggested that nitroxyl radicals can be proposed as a normal tissue-selective radio-protector.

The redox properties of nitroxyl radicals can give us double benefits which are (i) as redox-sensitive contrast agents and (ii) as a normal tissue-selective radio-protector. Both benefits are useful for a first diagnosis of a tumor and radiation therapy planning. These will give a radio-protective treatment for normal tissues in subsequent radiotherapy. Therefore, the magnetic resonance-based redox imaging techniques using nitroxyl contrast agents can have wide possibility.

1.3. Applying Nitroxyl Contrast Agent to Brain Redox Imaging

Redox imaging techniques using electron paramagnetic resonance imaging (EPRI), Overhauser MRI (OMRI), or MRI (25, 26) can visualize and estimate the redox status in living experimental animals. The basis of redox imaging is to obtain a time course of image intensity, which is induced by paramagnetic redox-sensitive contrast agent.

One suitable approach of redox imaging techniques is a brain image (27). The relation between ROS and several brain disorders, such as stroke, Parkinson’s disease, Alzheimer’s disease, is well established (28–30). Likewise, the relation between memory loss and radiation-induced oxidative injuries in brain/neuronal system has also been reported (31–33). Therefore, the redox imaging techniques employing a BBB permeable nitroxyl contrast agent can be another approach to analyze the pathophysiological function in the brain. A brief theory of redox imaging techniques is described, and applications of redox imaging techniques to the brain are introduced.

2. Redox Imaging in EPRI

Paramagnetic nitroxyl radicals can be directly detected by EPR, while the hydroxylamines which are the reduction products of nitroxyl radicals are diamagnetic and therefore EPR silent species. Therefore, detecting a decrease in EPR signal intensity from nitroxyl radicals due to their reduction in biological/chemical samples can be used to monitor redox reactions in vivo. Nitroxyl radicals have been utilized as in vivo molecular probes to estimate redox status in the tissue of experimental animals using in vivo EPR (34, 35). Using low frequency (300–1,200 MHz) EPR spectrometers (36–39) or X-band EPR spectrometers with special sampling techniques for in vivo measurements (40–43) the signal decay and/or the spectral change of nitroxyl spin probe in live animals can be monitored. Combining with EPR imaging techniques using field gradients, the distribution and time course of nitroxyl spin probes in tissues can be visualized (44–47).

Information related to tissue redox status using nitroxyl agents can be visualized by attaching some additional dimensions to an EPRI, such as time axis and/or spectral axis, to a simple image of spatial distribution of the probe molecule (Fig. 20.3). The time axis can be easily achieved by sequential measurement of several images of the spatial distribution of the spin probe. For example, when the image intensities decrease/increase in a time-dependent manner, a mapping of pixel-wise decay/increment rates of image intensities can be observed and the slopes of the image intensity change can be used to generate the “redox map” of the region of interest in an in vivo experiment

Fig. 20.3.

A schematic drawing of the concept of dynamic imaging. The additional time dimension to the spatial mapping of the paramagnetic contrast agent, functional information, such as pharmacokinetics of the contrast agent, can be tagged on to the EPR image. The time axis is by sequential measurement of several EPR images. Consequently, pixel-wise decay rates of EPR image intensity can be obtained.

Most in vivo EPR spectrometers operate in a continuous-wave (CW) modality, where the sample is irradiated continuously at low RF powers and the magnetic field is slowly swept to obtain an EPR spectrum. The temporal resolution of EPR spectroscopy and imaging is limited by the time of a relatively slow magnetic field scan. Recent developments in hardware and new data acquisition techniques achieved EPRI data acquisition capabilities in the range of seconds (48–50). Such a rapid data acquisition will improve the temporal resolution of EPRI in in vivo biological applications. However, the larger EPR linewidths of nitroxyl molecules and the available magnetic field gradients for imaging make the spatial resolution limited. In addition, no anatomical information is available by EPRI. To provide anatomic co-registration to EPR images of nitroxyl distribution in vivo, fusion techniques of EPRI and MRI have been developed by several groups (51–53).

3. Redox Imaging inOMRI

OMRI, which is also known as proton electron double resonance imaging (PEDRI), is a double-resonance technique involving both EPR and NMR (54–56). At a given magnetic field, by virtue of their stronger magnetic moments, the electrons’ spin levels are polarized to a greater extent than the corresponding spin levels of nuclei. When an aqueous solution containing a paramagnetic agent, such as a nitroxyl radical, is placed in a magnetic field within a resonator assembly which is tuned to the resonant frequencies of both the electron and the nucleus, a significant enhancement of the NMR signal results via the Overhauser effect (57) when the NMR signal of ¹H of H₂O is collected after a period of irradiation at the EPR frequency. Comparing the images obtained with the EPR-irradiation (OMRI signal) and without irradiation (native MRI signal), the paramagnetic contrast agents used can be quantified based on intensity enhancement patterns.

The imaging of nitroxyl contrast agents using OMRI was started by Lurie and colleagues in the late 1980s (54, 55). OMRI is a double resonance imaging technique. It relies on an EPR irradiation pulse followed by an MRI imaging sequence. In this technique, when a paramagnetic species, such as nitroxyls, are present in tissue, irradiating by an EPR pulse at the resonance frequency of the nitroxyl enhances the signal intensity of the surrounding water protons. This makes the image resolution dependent on MRI which is intrinsically a higher resolution technique compared to EPR imaging allowing the imaging of free radicals with ~ millimeter resolution. In addition, OMRI techniques can obtain not only distribution but also EPR spectral information of nitroxyl contrast agents (58). Nitroxyl radicals give a doublet or triplet EPR spectrum by hyper-fine-splitting due to ¹⁵N or ¹⁴N nuclear spin. Each of these can be separately obtained in the form of OMRI signal by using a different frequency for EPR excitation of either the ¹⁴N or the ¹⁵N nuclei. Utsumi et al. (58) proposed a novel imaging technique to separate the reduction and oxidation reactions in an identical sample simultaneously using a nitroxyl contrast agent and its hydroxylamine form labeled by different isotopes (i.e., ¹⁴N and ¹⁵N). They also showed that simultaneous reactions occurred separately inside and outside of liposomes using membrane permeable and impermeable nitroxyls. OMRI is an EPR-dependent spectroscopic imaging technique that displays a high temporal and spatial resolution. It hence affords the rapid imaging of in vivo metabolism of multiple nitroxyl radical probes simultaneously.

4. Trial of EPR-Based Brain Redox Imaging

Using a suitable BBB-permeable nitroxyl contrast agent, the time course of nitroxyl reduction rates in the brain can be visualized. CxP-M (also known as MC-PROXYL: 3-methoxy-carbonyl-2,2,5,5-tetramethyl-pyrrolidine-l-oxyl) (2, 59, 60) and CxP-AM (acetoxymethyl-2,2,5,5-tetramethyl-pyrrolidine-l-oxyl-3-carboxylate) were designed as BBB-permeable spin probe for EPR imaging (2). CxP-AM can be metabolized to BBB-impermeable CxP by esterases in the brain and can also be retained in the brain (2). CxP-AM has been commercialized and is currently available from LABOTEC Co. (Tokyo, Japan).

Anzai et al. (59) reported the distribution of CxP-M in the rat brain using autoradiography with ¹⁴C-labeled CxP-M and 3D EPRI. Figure. 20.4a shows a scheme of chemical synthesizing ¹⁴C-labeled CxP-M. Figure 20.4b shows autoradiograms of coronal sections of rat head obtained after administration with ¹⁴C-CxP-M or ¹⁴C-carbamoyl-PROXYL. They suggested that CxP-M is a suitable paramagnetic probe for the study of free radical reactions in the brain using in vivo EPR imaging. Lee et al. (60) reported a faster nitroxyl decay in the brain of spontaneously hypertensive rats compared with Wistar rat using the in vivo EPRI technique with CxP-M. Miyake et al. (61) reported the development of other BBB-permeable nitroxyl radical probes designed for the purpose of oxygen mapping in the brain. Molecular oxygen with two unpaired electrons is paramagnetic and by collisional interaction with the paramagnetic nitroxyls, broadens their EPR spectra in a concentration-dependent manner via a T2 contrast process typical in MRI. The oxygen-dependent spectral broadening can be monitored by EPR imaging allowing the determination of tissue oxygen.

Fig. 20.4.

Autoradiograms of axial sections of rat head obtained after 3 min i.v. or 15 min after i.p. treatment with ¹⁴C-labeled MC-PROXYL or ¹⁴C-labeled carbamoyl-PROXYL. The black region shows high radioactivity and the white, no radioactivity, showing that MC-PROXYL, but not carbamoyl-PROXYL, can penetrate the BBB and is well distributed to brain tissue.

Figure 20.5 shows an example of EPR-based brain redox imaging in the mouse brain (2). From two small ROIs selected in the brain area, the result of EPR imaging showed that the different parts of the brain have different decay rates of the nitroxyl radical. However, anatomical information is not obtained from the EPR imaging experiment. The exact anatomical location of the ROIs in the brain cannot be estimated directly from the EPR image itself. Co-registering of EPR images of nitroxyl radical distribution with anatomical images from MRI is therefore necessary to reliably visualize the regions of interest where the redox reactions occur.

Fig. 20.5.

Time-resolved 2D EPR images in the head of a mouse. A 100 mL volume of CxP-AM (25 mM) in PBS with 10% (v/v) of ethanol was injected into the tail veins of mice and the data for images were collected at various time points. a Data collected 0.9–9.9 min after injection. b Data collected 10.4–19.3 min after injection, c Data collected 19.8–28.7 min after injection. d A semilogarithmic plot of the imaging intensity of two different regions (ROI 1 and ROI 2) in the brain after intravenous injection of CxP-AM.

5. NMR-Based Redox Imaging

If one needs to monitor the levels of nitroxyl probes, EPR spectral information is not required. In such applications, larger linewidths and spectral multiplicity of the EPR spectra would be interference in attempts to obtain the spatial distribution of nitroxyl radicals in vivo. Paramagnetic nitroxyl radicals, which have proton T₁ shortening effect, can be detected using T₁-weighted MRI. The feasibility of nitroxyl radicals as T₁ contrast agents in MRI has been examined in the early 1980s, before their use in the field of EPR imaging. However, nitroxyl radicals were found to be not optimal as MR contrast agents in 1980s due to their low relaxivity and rapid in vivo bio-reduction. Now, recent MRI scanners operating at higher magnetic field with better signal to noise ratio and time-efficient T₁-weighted pulse sequences once again make it useful to reconsider nitroxyl radicals as potential T₁ contrast agents. Several problems (e.g., low spatial resolution, low temporal resolution, small sample volume) typical in EPR imaging experiments can be overcome using T1 contrast-based MRI. Two unique features distinguish nitroxyl radicals from Gd-based T₁ contrast agents. (i) Being organic molecules of molecular weight ~ 200 Da, they can be specifically directed to extra- or intracellular locations. (ii) Unlike Gd-complexes which do not undergo biological redox transformations, nitroxides not only undergo reversible biological redox transformations to diamagnetic species but also depend on cellular redox status (62, 63), oxygenation status (38, 64), focal inflammatory processes involving ROS (65).

Figure 20.6 shows a simulation of T₁-weighted gradient echo MR contrast as a function of concentration of a nitroxyl radical. The relaxivity of the nitroxyl radical used in this calculation was 0.13 mM⁻¹s⁻¹ at 37° C. At such a low relaxivity, the relationship between T₁ enhancement and concentration of a contrast agent is almost linear especially in the lower concentration region below 2 mM.

Fig. 20.6.

Relationship between a paramagnetic nitroxyl contrast agent and an intensity change of T₁-weighted SPGR-based MRI. a Simulated ΔM% increased with the concentration of paramagnetic nitroxyl contrast agent. b Low concentration region (<1.5 mM) in (a) (dotted rectangle area) was expanded. Fairly good linearity (R² = 0.9995) was obtained between simulated ΔM% and the concentration of the low concentration region. Values used for the simulation were as follows: r₁ = 0.13 mM⁻¹ s⁻¹, M₀ = 1,000, TR = 75 ms, TE = 3 ms, FA = 45°, T_1i = 2,350 ms, and T₂* = 50 ms.

Figure 20.7 is a comparison of EPR imaging and T₁-weighted MR imaging of nitroxyl radicals undergoing chemical reduction at a rate determined by the concentration of the reductant, ascorbic acid (AsA). Solutions of a nitroxyl radical and ascorbic acid were loaded in individual syringes and mixed rapidly to initiate the reaction in a cylindrical reactor placed within the resonator. Although MRI has a higher spatial and temporal resolution than EPR imaging, the decay rate was similar to that from EPR imaging (66). This decay rate obtained from MR T₁-contrast can be treated as a pseudo-decay rate (67).

Fig. 20.7.

Comparison of EPR and MR redox imaging demonstrated using a phantom. Upper cartoon is a representation of the experimental device.

In vivo nitroxyl decay rate imaging in SCC tumor implanted in a mouse leg was examined using MRI (66). Figure 20.8a indicates the location of a 2 mm axial slice, including the SCC tumor bearing leg and the normal leg. Figure 20.8b shows a time course of nitroxyl-induced T₁-weighted signal enhancement, ΔM%, after the injection of the nitroxyl contrast agent, carbamoyl-PROXYL, and the T₂ map of the corresponding slice. The T₁-weighted signal enhancement of a given pixel at (x, y) coordination, ΔM%_x,y is as follows:

$$\begin{gathered} \text{Δ}M{\text{\%}}_{x,y}=[\left(S_{x,y}\text{after CA injection}\right)/ \\ \left(S_{x,y}\text{before CA injection}\right)-1]\times 100 \end{gathered}$$ (1)

Fig. 20.8.

In vivo MR redox imaging. a Position of the slice scanned. b A time course of T₁-weighted signal enhancement in the slice and a T₂-mapping of the identical slice as a scout image. c A comparison of the time course of T₁-weighted signal enhancement in the ROI-1 and ROI-2. d A redox map calculated based on the time course of nitroxyl-induced tissue T₁-weighted signal enhancement.

where S_x,y is an image intensity of pixel (x, y). Image intensity enhancement via T₁-contrast was enhanced in both normal and tumor tissues after the administration of carbamoyl-PROXYL and then gradually decreased with time. Figure 20.8c shows the time course of average ΔM% in ROI-1 and ROI-2. The tumor region showed faster decay rate than the normal region. The decay rate maps (Fig. 20.8d) showed clear difference between tumor region and normal tissues.

There is a question whether the in vivo decay of MR T₁-contrast is a reduction of nitroxyl radical to the corresponding hydroxylamine or just a simple clearance of nitroxyl radical from the tissue. Chemically, the hydroxylamine can be oxidized to the corresponding nitroxyl radical by adding mild oxidant, such as potassium ferricyanide (FeK₃(CN)₆) after which the total amount of the nitroxyl agent in the tissue samples (nitroxyl radical form plus hydroxylamine form) can be measured and quantified by EPR (Fig. 20.9). From a set of five animals, whose tissue (both normal and tumor) were analyzed for nitroxyl content after chemical oxidation, it was found that the time course of the total amount of the nitroxyl in both tumor and normal tissues was stable during the time period used in the imaging experiment (Fig. 20.10). These observations suggest that the in vivo disappearance of T₁-contrast induced by a nitroxyl contrast agent as a function of time is due to a reduction (67, 68).

Fig. 20.9.

Re-oxidation of a hydroxylamine to the corresponding nitroxyl radical by a chemical oxidant. Ferricyanide (FeK₃(CN)₆) has been usually used to oxidize hydroxylamine to the nitroxyl radical.

Fig. 20.10.

Time course of total amount (nitroxyl radical form + hydroxylamine form) of a contrast agent, carbamoyl-PROXYL, in normal and tumor tissues after i.v. injection.

6. Recent Trial of NMR-Based Brain Redox Imaging

Figure 20.11 shows nitroxyl-induced T₁ contrast in the head region of mice. These images show varying distributions of nitroxyl contrast agents in the brain depending on their membrane permeability. Highly membrane permeable TEMPOL and CxP-M showed significant T₁ contrast induction in whole brain area. Carbamoyl-PROXYL (CmP), which has a limited membrane permeability, showed modest T1 contrast in the brain, while the membrane impermeable CxP displayed no induction of T₁ contrast in the brain. These results show that a membrane permeable nitroxyl contrast agent, such as TEMPOL or CxP-M, in combination with their antioxidant properties and their ability to participate in intracellular redox reactions, can be useful with MRI to assess tissue redox status in vivo in various organs of interest, including the brain.

Fig. 20.11.

Nitroxyl-induced T₁ contrast at the head part of mice. a TEMPOL, b CxP-M, c carbamoyl-PROXYL, and d CxP showed difference of distribution in brain region.

Hyodo et al. (27) investigated the ability of CxP-M as a BBB-permeable MRI contrast agent in rats to monitor the production of reactive oxygen species (ROS) in the brain after reperfusion following defined periods of ischemia. In addition, they demonstrated fast T₁ mapping acquired by a Look–Locker (LL) sequence (69). T₁-weighted signal enhancement was readily noticed throughout the brain after administering CxP-M. Maximum T₁-weighted signal enhancement, occurred at about 3 min after injection of CxP-M and returned to baseline by 15 min (Fig. 20.12a, c). The BBB-impermeable CxP, however, showed no enhancement of the T₁-weighted signal in the brain region (Fig. 20.12b, d). Figure 20.12e shows the effect of cessation of blood flow on the reduction of CxP-M. When the blood flow was stopped by a KCl injection 40 s after administration of CxP-M, the respiration stopped, i.e., the rat died 15–20 s after KCl injection. MR signal intensity in the brain is still decreased after the death of a rat, except the reduction rate became slower than that found with normal cerebral perfusion. Disruption of blood perfusion and/or subsequent tissue injury, such as an ischemia-reperfusion (IR) model, can make abnormal redox status. For example, the reduction rate of CxP-M in the right hemisphere after reperfusion following ischemic periods was found to be significantly lower compared to that observed in the sham group (Fig. 20.12f) suggesting either a lower reduction rate of nitroxyls or a faster re-oxidation of the hydroxylamines to nitroxyls.

Fig. 20.12.

Time-course SPGR MR images of rat head region after injection of a CxP-M (cell-permeable) and b CxP (cell-impermeable). Contrast agents were injected 2 min after the MR scan was started. Sixty serial images were obtained in 20 min. The SPGR MR parameters were as follows: image resolution was 256 × 128 zero-filled to 256 × 256 (0.125 mm resolution), FOV = 3.2 × 3.2 cm, slice thickness = 2.0 and 0.2 mm gap. The number of slices was six. The green color shows % enhancement of MR signal intensity, ΔM%. The time courses of intensity change of c CxP-M and d CxP in the ROI of cerebral cortex (red color) and thalamus (blue color) are shown. e Intensity change of CxP-M without blood flow is shown. KCI (2 mL) was injected 40 s after CxP-M injection and rats died within 20 s.

While T₁ is a quantitative value, the T₁-weighted signal enhancement ratio is not always quantitative especially in a tissue of relatively short T₁. Figure 20.13a shows a time course of T₁ maps of the rat head region scanned before and after injection of CxP-M. T₁ maps were obtained every 20 s with 13 slices (1.5 mm slice thickness) by the EPI-LL sequence. The T₁ maps clearly showed the difference of T₁ relaxation time between cerebral cortex and thalamus (Fig. 20.13a). The T₁ relaxation times in cerebral cortex and thalamus before injection were 1,577 ± 19.9 and 1,315 ± 11.9 ms, respectively (Fig. 20.13b). The T₁ values of these regions rapidly decreased after injection of CxP-M, and the minimum T₁ values of cerebral cortex and thalamus regions were 1,034 ± 70.4 and 779 ± 70.2 ms, respectively. The recovery of T₁ in the cerebral cortex and thalamus showed similar trends. According to the T₁ changes before and after injection of CxP-M and in vitro relaxivity of CxP-M (0.16 mM⁻¹s⁻¹), the expected in vivo concentrations of CxP-M were calculated for all time points (Fig. 20.13c). The maximum concentrations in cerebral cortex and thalamus 30 s after injection were 1.9 ± 0.35 and 3.0 ± 0.50 mM, respectively. The CxP-M concentration calculated in the brain based on the T₁ maps was in agreement with the results directly measured by ex-vivo EPR.

Fig. 20.13.

MRI signal dynamic of a SLENU and b SLCNUgly in the brain and surrounding tissues after i.v. injection in mice (0.4 mmol/kg b.w.). Each image was obtained within 20-s intervals using a Gradient-echo T₁-weighted MRI. The red color in the images is the extraction of the signal between every single slide and the averaged baseline signal (first five slides – before injection). The red and black colors in the chart represent the MRI signal dynamic in the brain or entire area, respectively. A representative image from three independent experiments is shown in the figure.

7. MR Imaging of BBB-Permeable Drugs

A recent study demonstrated the feasibility of using a combination of nitroxyl radical linked with a therapeutic drug and the use of T₁-weighted MRI to examine the BBB permeability of a nitroxyl labeled drug (70). This study demonstrates a BBB permeability for two TEMPO-labeled analogs (SLENU and SLCNUgly) of the anticancer drug Lomustine [1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea] using MRI at 7 T. SLENU and SLCNUgly were rapidly transported and randomly distributed in the brain tissue, which indicated that the exchange of the cyclohexyl part of Lomustine with TEMPO radical did not suppress the BBB permeability of the anticancer drug. In drug delivery imaging, nitroxyl-labeled anti-tumor drugs can therefore work not only as a tracer but may also have a role as a diagnostic probe for tumor physiology.

8. Conclusion

Brain redox imaging using a nitroxyl contrast agent as described in this chapter has a great potential to visualize the redox status in brain tissue. Non-invasive detection and visualization of the redox status in the brain will be an important contribution to the study of the pathophysiology of neurodegenerative diseases, such as stroke, Parkinson’s disease, and Alzheimer’s disease. Future therapeutic applications in these diseases can also be considered. Developing brain redox imaging techniques will be crucial to analyze redox phenomena and their influence on brain functions.