Real-time monitoring of drug-induced changes in the stomach acidity of living rats using improved pH-sensitive nitroxides and low-field EPR techniques.

New improved pH-sensitive nitroxides were applied for in vivo studies. An increased stability of the probes towards reduction was achieved by the introduction of the bulky ethyl groups in the vicinity of the paramagnetic NO fragment. In addition, the range of pH sensitivity of the approach was extended by the synthesis of probes with two ionizable groups, and, therefore, with two pKa values. Stability towards reduction and spectral characteristics of the three new probes were determined in vitro using 290 MHz radiofrequency (RF)- and X-band electron paramagnetic resonance (EPR), longitudinally detected EPR (LODEPR), and field-cycled dynamic nuclear polarization (FC-DNP) techniques. The newly synthesized probe, 4-[bis(2-hydroxyethyl)amino]-2-pyridine-4-yl-2,5,5-triethyl-2,5-dihydro-1H-imidazol-oxyl, was found to be the most appropriate for the application in the stomach due to both higher stability and convenient pH sensitivity range from pH 1.8 to 6. LODEPR, FC-DNP and proton-electron double resonance imaging (PEDRI) techniques were used to detect the nitroxide localization and acidity in the rat stomach. Improved probe characteristics allowed us to follow in vivo the drug-induced perturbation in the stomach acidity and its normalization afterwards during 1 h or longer period of time. The results show the applicability of the techniques for monitoring drug pharmacology and disease in the living animals.

Field-cycled PEDRI imaging of free radicals with detection at 450 mT.

This paper describes the design, construction and use of a field-cycled proton-electron double-resonance imaging (FC-PEDRI) system for the detection and imaging of free radicals. The unique feature of this imager is its use of a 450-mT detection magnetic field in order to achieve good image quality and sensitivity. The detection magnetic field is provided by a superconducting magnet, giving high stability and homogeneity. Field cycling is implemented by switching on and off the current in an internal, coaxial, resistive secondary magnet that partially cancels the superconducting magnet's field at the sample; the secondary magnet is actively shielded to avoid eddy currents. EPR irradiation takes place at approximately 5 mT, following which the field is switched to 450 mT in 40 ms for NMR signal detection. Full details of the imager's subsystems are given, and experiments to image the distribution of stable free radical contrast agents in phantoms and in anesthetized rats are described.

Evaluation of MRI for in vivo monitoring of retinal damage and detachment in experimental ocular inflammation.

Two quantitative methods were developed for investigation of the potential of MRI for in vivo monitoring of retinal damage and detachment in experimental autoimmune uveitis (EAU). Measurements of retinal thickness and detachment area were performed on matched MR and histologic (HIST) images of rat eyes at different stages of EAU. In vivo MR images of rat eyes were acquired at 4.7 T using a figure-of-eight surface coil and a spin echo pulse sequence. Ex vivo measurements were performed on HIST images acquired using a digital camera attached to a microscope. MR images mirrored the HIST appearance of inflamed eyes at each stage of disease. Retinal detachments as small as 0.1 mm(2) were measured in vivo by MRI and confirmed in the same eye ex vivo by histology. Measurements performed on corresponding MR and HIST images demonstrated a good agreement between the two techniques. The potential of MRI for in vivo visualization and for monitoring changes in the eye during development of EAU was demonstrated in this study.

In vitro and in vivo measurement of pH and thiols by EPR-based techniques.

In vitro and in vivo measurements of pH and thiols provide critical information on physiology and pathophysiology of living organisms, particularly related to oxidative stress. Stable nitroxides of imidazoline and imidazolidine types provide the unique possibility of measuring local values of pH and glutathione content in various biological systems, including in vivo studies. The basis for these applications is the observation of specific chemical reactions of these nitroxides with protons or thiols, followed by significant changes in the electron paramagnetic resonance (EPR) spectra of these probes, measured by low-frequency EPR techniques. The applications of some newly developed pH and SH probes in model systems of pharmacological interest, biological fluids, tissues, and cells as well as in vivo studies in isolated hearts and in the gut of living animals are discussed.

In vivo detection of a pH-sensitive nitroxide in the rat stomach by low-field ESR-based techniques.

A study was made of the in vivo detectability of a pH-sensitive, imidazolidine spin probe, and the efficacy of low-frequency electron spin resonance (ESR)-based techniques for pH measurement in vitro and in vivo in rats. The techniques used were longitudinally-detected ESR (LODESR) and field-cycled dynamic nuclear polarization (FC-DNP) for in vitro and in vivo measurements, and radiofrequency (RF)- and X-band ESR for comparisons in vitro. The spin probe was hexamethyl imidazolidine (HMI) with a pK of 4.6. All techniques detected HMI. Detection by FC-DNP implies coupling between the free radical and solvent water spins. Separations between the three spectral lines of the nitroxide radical, relative to measurement frequency, were consistent with theory. The overall spectrum width from unprotonated HMI (pH > pK) was greater than that from protonated agent (pH < pK). This was observed in vitro and in vivo. Longer-term studies showed that HMI is detectable and has the same spectral width (i.e., is at the same pH) up to 2 hr after gavage into the stomach, although the magnitude of the signal decreases rapidly during the first hour. These findings demonstrate the suitability of LODESR and FC-DNP for monitoring HMI and measuring pH in vivo. These techniques would be useful for monitoring disease and drug pharmacology in the living system.

Rapid imaging of free radicals in vivo using hybrid FISP field-cycled PEDRI.

A new pulse sequence for rapid imaging of free radicals is presented which combines snapshot imaging methods and conventional field-cycled proton electron double resonance imaging (FC-PEDRI). The new sequence allows the number of EPR irradiation periods to be optimized to obtain an acceptable SNR and spatial resolution of free radical distribution in the final image while reducing the RF power deposition and increasing the temporal resolution. Centric reordered phase encoding has been employed to counter the problem of rapid decay of the Overhauser-enhanced signal. A phase-correction scheme has also been used to correct problems arising from instability of the magnetic field following field-cycling. In vivo experiments were carried out using triaryl methyl free radical contrast agent, injected at a dose of 0.214 mmol kg(-1) body weight in anaesthetized adult male Sprague-Dawley rats. Transaxial images through the abdomen were collected using 1, 2, 4 and 8 EPR irradiation periods. Using 4 EPR irradiation periods it was possible to generate free radical distributions of acceptable SNR and resolution. The EPR power deposition is reduced by a factor of 16 and the acquisition time is reduced by a factor of 4 compared to an acquisition using the conventional FC-PEDRI pulse sequence.