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Biomedical subjects

M C Krishna

Publications and source records attributed to M C Krishna.

At least 19 recordsLinked to original sources
PubMed2004

Tailored sinc pulses for uniform excitation and artifact-free radio frequency time-domain EPR imaging.

A method to generate shaped radiofrequency pulses for uniform excitation of electron spins in time-domain radio frequency (RF) electron paramagnetic resonance (EPR) imaging is presented. A commercial waveform generator was integrated with the transmit arm of the existing time-domain RF-EPR spectrometer to generate tailored excitation pulses with sub-nano second resolution for excitation with a 90 degrees flip-angle. A truncated sinc [sin(x)/x] pulse, tailored to compensate for the Q-profile (RF frequency response) of the resonator, was shown to yield images from phantom objects as well as in vivo images, with minimal distortion. These studies point to the advantages in using shaped sinc pulses to achieve improved uniform excitation over a relatively wide bandwidth region in time-domain RF-EPR imaging (RF-FT-EPRI).

Animals↗
PubMed2002

Feasibility and assessment of non-invasive in vivo redox status using electron paramagnetic resonance imaging.

PURPOSE: To test the feasibility of electron paramagnetic resonance imaging (EPRI) to provide non-invasive images of tissue redox status using redox-sensitive paramagnetic contrast agents. MATERIAL AND METHODS: Nitroxide free radicals were used as paramagnetic agents and a custom-built 300 MHz EPR spectrometer/imager was used for all studies. A phantom was constructed consisting of four tubes containing equal concentrations of a nitroxide. Varying concentrations of hypoxanthine/xanthine oxidase were added to each tube and reduction of the nitroxide was monitored by EPR as a function of time. Tumor-bearing mice were intravenously infused with a nitroxide and the corresponding reduction rate was monitored on a pixel-by-pixel basis using 2D EPR of the tumor-bearing leg and normal leg serving as control. For animal studies, nitroxides were injected intravenously (1.25 mmol/kg) and EPR projections were collected every 3 min after injection using a magnetic field gradient of 2.5 G/cm. The reduction rates of signal intensity on a pixel-by-pixel basis were calculated and plotted as a redox map. Redox maps were also collected from the mice treated with diethylmaleate (DEM), which depletes tissue thiols and alters the global redox status. RESULTS: Redox maps obtained from the phantoms were in agreement with the intensity change in each of the tubes where the signals were decreasing as a function of the enzymatic activity, validating the ability of EPRI to accurately access changes in nitroxide reduction. Redox imaging capability of EPR was next evaluated in vivo. EPR images of the nitroxide distribution and reduction rates in tumor-bearing leg of mice exhibited more heterogeneity than in the normal tissue. Reduction rates were found to be significantly decreased in tumors of mice treated with DEM, consistent with the depletion of thiols and the consequent alteration of the redox status. CONCLUSION: Using redox-sensitive paramagnetic contrast agents, EPRI can non-invasively discriminate redox status differences between normal tissue and tumors.

Animals↗
PubMed2001

Cellular sites of H2O2-induced damage and their protection by nitroxides.

While the exact mechanism of H2O2-induced cytotoxicity is unknown, there is considerable evidence implicating DNA as a primary target. A recent study showed that a cell-impermeable nitroxide protected mammalian cells from H2O2-induced cell killing and suggested that the protection was mediated through cell membrane-bound or extracellular factors. To further define the protective properties of nitroxides, Chinese hamster V79 cells were exposed to H2O2 with or without cell-permeable and impermeable nitroxides and selected metal chelators. EPR spectroscopy and paramagnetic line broadening agents were used to distinguish between intra- and extracellular nitroxide distribution. To study the effectiveness of nitroxide protection, in the absence of a cell membrane, H2O2-mediated damage to supercoiled plasmid DNA was evaluated. Both deferrioxamine and Tempol cross the cell membrane, and inhibited H2O2-mediated cell killing, whereas the cell-impermeable DTPA and nitroxide, CAT-1, failed to protect. Similar protective effects of the chelators and nitroxides were observed when L-histidine, which enhances intracellular injury, was added to H2O2. In contrast, when damage to plasmid DNA was induced (in the absence of a cell membrane), both nitroxides were protective. Collectively, these results do not support a role for membrane-bound or extracellular factors in mediating H2O2 cytotoxicity in mammalian cells.

Animals↗
PubMed2001

Multifunctional antioxidant activity of HBED iron chelator.

The use of N,N'-bis (2-hydroxybenzyl) ethylenediamine-N,N'-diacetic acid (HBED) for iron chelation therapy is currently being tested. Besides its affinity for iron, bioavailability, and efficacy in relieving iron overload, it is important to assess its anti- and/or pro-oxidant activity. To address these questions, the antioxidant/pro-oxidant effects of HBED in a cell-free solution and on cultured Chinese hamster V79 cells were studied using UV-VIS spectrophotometry, oximetry, spin trapping, and electron paramagnetic resonance (EPR) spectrometry. The results indicate that HBED facilitates Fe(II) oxidation but blocks O2(.-)-induced reduction of Fe(III) and consequently pre-empts production of .OH or hypervalent iron through the Haber-Weiss reaction cycle. The efficacy of HBED as a 1-electron donor (H-donation) was demonstrated by reduction of the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate)-derived nitrogen-centered radical cation (ABTS(.+)), accompanied by formation of a short-lived phenoxyl radical. HBED also provided cytoprotection against toxicity of H2O2 and t-BuOOH. Our results show that HBED can act both as a H-donating antioxidant and as an effective chelator lacking pro-oxidant capacity, thus substantiating its promising use in iron chelation therapy.

Animals↗
PubMed2001

Magnetic resonance imaging for in vivo assessment of tissue oxygen concentration.

Magnetic resonance imaging (MRI) provides high-resolution morphological images useful in diagnostic radiology to differentiate normal from abnormal/pathological states. More recently, emerging developments in MRI seek to add a functional/physiological dimension to the anatomic images to provide better understanding of the physiology of pathological conditions. Three MRI methods offer the promise of providing important physiologic information, such as oxygen status and redox capability of tissues, and these are discussed in the context of their potential usefulness to radiation oncology. The techniques include blood oxygen level-dependent (BOLD) MRI, Overhauser enhanced MRI (OMRI), and electron paramagnetic resonance imaging (EPRI). BOLD MRI provides information of tumor oxygen status by using the differences in MRI images from tumors obtained when breathing air or carbogen. Deoxyhemoglobin serves as an endogenous BOLD MRI contrast agent. OMRI utilizes the enhancement of proton MRI images by a nontoxic free radical contrast agent. The advantages of this technique are the very low magnetic fields used and its capability to provide quantitative information of tissue oxygen concentration. EPRI also uses free radical contrast agents and can provide redox and oxygen status differences between tumor and normal tissues. Some of the contrast agents used in EPRI have been identified as radiation protectors. The images obtained from each of the technologies may ultimately be used to overlay their respective views (containing spatial tissue physiology information) onto detailed anatomic maps.

Humans↗
PubMed2001

Electron paramagnetic resonance for small animal imaging applications.

Magnetic resonance imaging (MRI) provides high-resolution morphological images useful in diagnostic radiology to differentiate between normal and abnormal/pathological states in tissues. More recently, emerging developments in MRI have added a functional/physiological dimension to anatomical images. Electron paramagnetic resonance (EPR), a magnetic resonance technique similar to nuclear magnetic resonance, detects paramagnetic species such as free radicals. Like MRI, EPR can be implemented as an imaging technique for small animals and potentially human applications. Because of the low abundance of naturally occurring paramagnetic species, exogenous paramagnetic species are needed for in vivo EPR imaging (EPRI). The image data from EPRI contain both spatial distribution of paramagnetic species and spectral information. Hence, spatially encoded functional information such as tissue oxygen status and redox status can be extracted and coregistered with the spatial distribution of the spin probe, to the anatomy, or both by suitable means. Ultimately, the images obtained from EPRI may be used to overlay the functional information (containing spatial tissue physiology information) onto detailed anatomical maps. With its ability to enable whole animal imaging in mice, EPRI will be a useful imaging technique that complements other techniques such as MRI and positron emission tomography in obtaining valuable functional/physiological images.

Anatomy, Cross-Sectional↗
PubMed2000

Evaluation of the hydroxylamine Tempol-H as an in vivo radioprotector.

Nitroxides are stable free radical compounds that protect against the toxicity of reactive oxygen species in vitro and in vivo. Tempol (Aldrich, Milwaukee, WI, USA) is a cell-permeable hydrophilic nitroxide and has been shown to be an in vitro and in vivo radioprotector. The limitations of Tempol as a systemic radioprotector are that it causes substantial reductions in arterial blood pressure when administered intravenously and is associated with seizure activity. Furthermore, Tempol is rapidly reduced to its hydroxylamine form, Tempol-H, which limits the period of time the active form of the nitroxide is available for radioprotection. Based on initial pharmacological and blood pressure experiments performed in mice, we hypothesized that the systemic administration of Tempol-H in vivo would lead to an equilibration between Tempol and Tempol-H that would limit the toxicity of the nitroxide and provide in vivo radioprotection. Tempol-H was administered in increasing doses via an intraperitoneal route to C3H mice. The maximally tolerated dose was found to be 325 mg/kg. The whole-blood pharmacology of Tempol-H was investigated with electron paramagnetic resonance spectroscopy. These studies demonstrated the appearance of Tempol in whole blood immediately after intraperitoneal injection, suggesting that rapid oxidation of Tempol-H to Tempol takes place in vivo. Although the peak concentration of Tempol in whole blood after administration of Tempol-H did not reach the same levels as those observed when Tempol is administered, the whole-blood levels of Tempol were similar by 10 min after injection. Tempol-H provided protection against the lethality of whole-body radiation in C3H mice at 30 d with a dose modification factor of 1.3, which is similar to the results obtained with Tempol. Hemodynamic measurements in C3H mice after intravenous injection showed that Tempol-H produced little effect on blood pressure or pulse compared with Tempol. Tempol-H is a systemic in vivo radioprotector of C3H mice and is associated with less hemodynamic toxicity than Tempol.

Animals↗
PubMed2000

Three-dimensional whole body imaging of spin probes in mice by time-domain radiofrequency electron paramagnetic resonance.

Imaging of stable paramagnetic spin probes in phantom objects and in vivo was evaluated using a RF time domain EPR spectrometer/imager operating at 300 MHz. Projections were collected using static magnetic field gradients and images were reconstructed using filtered back-projection techniques. Results from phantom objects containing approximately 10(17) spins of stable paramagnetic probes with single narrow EPR spectra provide three-dimensional spatial images with resolution better than 2 mm. When the spin probe was administered to mice, the spin probe accumulation was temporally observed in the thoracic, abdominal, and pelvic regions. A three-dimensional image (from 144 projections) from a live mouse was collected in 5 min. Using fiducial markers, the spin probe accumulation in organs such as liver, kidney, and bladder could be observed. Differences in the oxygen status between liver and kidney were observed from the EPR images from mice administered with spin probe, by treating the time-domain responses with convolution difference approach, prior to image reconstruction. The results from these studies suggest that, with the use of stable paramagnetic spin probes and time-domain RF EPR, it is possible to perform in vivo imaging on animals and also obtain important spatially resolved physiologic information.

Animals↗
PubMed2000

Parallel coil resonators for time-domain radiofrequency electron paramagnetic resonance imaging of biological objects.

Resonators suitable for time-domain electron paramagnetic resonance spectroscopy and imaging at a radiofrequency capable of accommodating experimental animals such as mice are described. Design considerations included B(1) field homogeneity, optimal Q, spectral bandwidth, resonator ring-down, and sensitivity. Typically, a resonator with 25-mm diameter and 25-mm length was constructed by coupling 11 single loops in parallel with a separation of 2.5 mm. To minimize the resonator ring-down time and provide the necessary spectral bandwidth for in vivo imaging experiments, the Q was reduced predominantly by overcoupling. Capacitative coupling was utilized to minimize microphonic effects. The B(1) field in the resonator was mapped both radially and axially and found to be uniform and adequate for imaging studies. Imaging studies with phantom objects containing a narrow-line spin probe as well as in vivo objects administered with the spin probe show the suitability of these resonators for valid reproduction of the spin probe distribution in three dimensions. The fabrication of such resonators is simple and can be scaled up with relative ease to accommodate larger objects as well.

Animals↗
PubMed2000

Radiation, radicals, and images.

Nitroxide stable free radicals exhibit varied chemical and biological properties. Their biological applications have been greatly expanded over the past few years. Not only have they been shown to exhibit potent antioxidant and radioprotective properties, but also they can serve as in vivo functional imaging probes that non-invasively report on the oxygen status and redox properties of tissue, which may have utility in clinical biomedical research.

Animals↗
PubMed1999

Cytotoxicity, redox cycling and photodynamic action of two naturally occurring quinones.

Two naturally occurring anthraquinones, barleriaquinone-I (BQ-I) and barleriaquinone-II (BQ-II), extracted from Barleria buxifolia, are tested for their cytotoxic action by aerobic incubation with human breast adenocarcinoma cells (MCF7). Cytotoxicities, measured as LD(50) (50% inhibition of colony formation) values, show BQ-II to be more active than BQ-I. Electron paramagnetic resonance studies confirm that BQ-II is reductively activated by NADH:cytochrome c reductase to superoxide anion radical. Cyclic voltammetric studies show one quasi-reversible redox couple for both BQ-I and BQ-II. Also, aerobic solutions of both BQ-I and BQ-II on visible illumination generate reactive oxygen species. Formation of O*-2 is studied by both EPR spin trapping and SOD-inhibitable cytochrome c reduction techniques. BQ-I generates more singlet oxygen as evidenced from the photobleaching of N,N-dimethyl-4-nitrosoaniline.

Adenocarcinoma↗
PubMed1999

High-speed data acquisition system and receiver configurations for time-domain radiofrequency electron paramagnetic resonance spectroscopy and imaging.

Design strategies, system configuration, and operation of a dual-channel data acquisition system for a radiofrequency (RF) time-domain electron paramagnetic resonance (EPR) spectrometer/imager operating at 300 MHz are described. This system wasconfigured to incorporate high-speed analog-to-digital conversion (ADC) and summation capabilities with both internal and external triggering via GPIB interface. The sampling rate of the ADC is programmable up to a maximum of 1 GS/s when operating in a dual-channel mode or 2 GS/s when the EPR data are collected in a single-channel mode. By using high-speed flash ADCs, a pipelined 8-bit adder, and a 24-bit accumulator, a repetition rate of 230 kHz is realized to sum FIDs of 4096 points. The record length is programmable up to a maximum of 8K points and a large number of FIDs (2(24)) can be summed without overflow before the data can be transferred to a host computer via GPIB interface for further processing. The data acquisition system can operate in a two-channel (quadrature) receiver mode for the conventional mixing to baseband. For detection using the single-channel mode, the resonance signals around the center frequency of 300 MHz were mixed with a synchronized local oscillator of appropriate frequency leading to an intermediate frequency (IF) which is sampled at a rate of 2 GS/s. Comparison of quadrature-mode and an IF-mode operation for EPR detection is presented by studying the FID signal intensity across a bandwidth of 10 MHz and as a function of transmit RF power. Imaging of large-sized phantoms accommodated in appropriately sized resonators indicates that IF-mode operation can be used to obtain distortion-free images in resonators of size 50 mm diameter and 50 mm length.

Data Collection↗
PubMed1999

Hemodynamic effect of the nitroxide superoxide dismutase mimics.

Reactive oxygen species play critical roles in a number of physiologic and pathologic processes. Nitroxides are stable free radical compounds that possess superoxide dismutase (SOD) mimetic activity and have been shown to protect against the toxicity of reactive oxygen species in vitro and in vivo. Tempol, a cell-permeable hydrophilic nitroxide, protects against oxidative stress and also is an in vitro and in vivo radioprotector. In the course of evaluating the pharmacology and toxicity of the nitroxides, Tempol and another nitroxide, 3-carbamoyl-PROXYL (3-CP), were administered intravenously in various concentrations to miniature swine. Tempol caused dose-related hypotension accompanied by reflex tachycardia and increased skin temperature. Invasive hemodynamic monitoring with Swan Ganz catheterization (SGC) confirmed the potent vasodilative effect of Tempol. However, 3-CP had no effect on porcine blood pressure. The hemodynamic effects of Tempol and 3-CP are discussed in the context of differential catalytic rate constants for superoxide disumation that may impact systemic nitric oxide (NO) levels and lead to vasodilation. These findings are consistent with a role for the superoxide ion in the modulation of blood pressure and have potential implications for the systemic use of nitroxides.

Animals↗
PubMed1998

Studies of structure-activity relationship of nitroxide free radicals and their precursors as modifiers against oxidative damage.

The protective effects of stable nitroxides, as well as their hydroxylamine and amine precursors, have been tested in Chinese hamster V79 cells subjected to H2O2 exposure at fixed concentration or exposure to ionizing radiation. Cytotoxicity was evaluated by monitoring the viability of the cells assessed by the clonogenic assay. The compounds tested at fixed concentration varied in terms of ring size, oxidation state, and ring substituents. Electrochemical studies were carried out to measure the redox midpoint potentials. The studies show that in the case of protection against H2O2 exposure, the protection was determined by the ring size, oxidation state, and redox midpoint potentials. In general the protection factors followed the order nitroxides > hydroxylamines > amines. Both the six-membered ring nitroxides and substituted five-membered ring nitroxides were efficient protectors. For six-membered ring nitroxides, the compounds exhibiting the lowest midpoint potentials exhibited maximal protection. In the case of X-radiation, nitroxides were the most protective though some hydroxylamines were also efficient. The amines were in some cases found to sensitize the toxicity of aerobic radiation exposure. The protection observed by the nitroxides was not dependent on the ring size. However, the ring substituents had significant influence on the protection. Compounds containing a basic side chain were found to provide enhanced protection. The results in this study suggest that these compounds are novel antioxidants which can provide cytoprotection in mammalian cells against diverse types of oxidative insult and identify structural determinants optimal for protection against individual types of damage.

Animals↗
PubMed1998

Preparation and EPR studies of lithium phthalocyanine radical as an oxymetric probe.

The electron paramagnetic resonance (EPR) spectrum of the paramagnetic center in solid lithium phthalocyanine, LiPc, exhibits a pO2 (partial pressure of oxygen)-dependent line width. The compound is insoluble in water and is not easily biodegradable and, therefore, is a useful spin probe for quantitative in vivo oxymetry. Because EPR spectrometry is potentially a useful technique to quantitatively obtain in vivo tissue pO2, such probes can be used to obtain physiological information. In this paper, a simple experimental procedure for the preparation of LiPc using potentiostatic electrochemical methods is described. The setup was relatively inexpensive and easy to implement. A constant potential ranging from 0.05 to 0.75 V versus Ag+/AgCl(s) was used for obtaining LiPc. The EPR spectral studies were carried out using spectrometers operating at X-band and at radiofrequency (RF) at different pO2 values to characterize the spectral response of these crystals. The results indicate that, depending on the electrolysis conditions, the products contain mixtures of crystals exhibiting pO2-sensitive and pO2-insensitive line widths. Electrolysis conditions are reported whereby the pO2-sensitive LiPc crystals were the predominant product. The influence of the working surface of the electrode and the electrolysis time on the yield were also evaluated. The crystals of LiPc were also studied using a time-domain RF EPR spectrometer. In time-domain EPR, the signals that survive beyond the spectrometer dead time are mainly the narrow lines corresponding to the pO2-sensitive crystals, whereas the signals arising from the pO2-insensitive component of LiPc were found not to survive beyond the spectrometer dead time. This signal survival makes the time-domain EPR method more sensitive for pO2 measurements using LiPc because the line width becomes very narrow at very low pO2 and, concomitantly, the relaxation time T2 longer, with no modulation or power saturation artifacts that are encountered as in the continuous wave (cw) mode. Further, minimal contributions from object motion in the spectral data obtained using time-domain methods make it an advantage for in vivo applications.

Crystallization↗
PubMed1998

In vivo electron paramagnetic resonance imaging of tumor heterogeneity and oxygenation in a murine model.

Nitroxides are redox-sensitive probes, which are useful in noninvasively delineating tissue heterogeneity especially with respect to metabolic activity and tissue oxygenation. Recent studies have shown that nitroxides are in vitro and in vivo radioprotectors and selectively protect normal tissue compared to tumor tissue. It has been postulated that the basis for selective radioprotection of normal tissues is greater bioreduction of nitroxides in tumor tissue compared to normal tissue. The aim of the present study was to investigate the distribution and lifetime of nitroxides in tumor and normal tissues. Mice were implanted with tumor cells (RIF-1) in the thigh, and the tumor was allowed to grow to about 10-15 mm in diameter. After i.v. infusion of nitroxides, in vivo electron paramagnetic resonance spectroscopy and imaging of the tumor were performed using a specially built bridged-loop surface resonator. The pharmacokinetic and spatial distribution of the nitroxides in tumor tissue were followed and compared with those in normal tissue. Three-dimensional spatial images showed significant heterogeneity in the nitroxide distribution as well as reduction rates. The nitroxide reduction rates were significantly higher in tumors than in the normal tissue. Measurements using spin label oximetry showed a substantial difference in the level of oxygenation between normal tissue (muscle) and tumor tissue. Average pO2 levels in tumor tissue were found to be 3-fold lower than in a corresponding volume of normal tissue. The lower pO2 levels in tumor compared to normal tissue may explain the more rapid reduction of nitroxides in these tissues. This study demonstrates that electron paramagnetic resonance imaging can perform noninvasive anatomical as well as functional imaging and provide in vivo physiological information regarding cellular metabolism in tumor and normal tissues.

Animals↗
PubMed1998

Development of functional electron paramagnetic resonance imaging.

The potential use of electron paramagnetic resonance imaging (EPRI) to obtain physiological information noninvasively is reviewed. EPR, a spectroscopic technique similar to nuclear magnetic resonance (NMR), is useful in detecting and characterizing free radical species. The ability to obtain information about tissue redox and oxygen status using nontoxic free radical spin probes is presented. The capability to encode this information spatially using magnetic field gradients, similar to magnetic resonance imaging (MRI), gives this technique the ability to overlay functional information of tissue with anatomic information. The noninvasive and quantitative nature of EPRI makes it a potentially useful technique for obtaining physiological information from tumors. The requirements for the magnetic field strengths are approximately 600 times lower than that for proton MRI at an identical frequency, making this a low-cost diagnostic tool.

Journal Article↗