Laszlo Ligeti

Use of 19F NMR spectroscopy for measurement of cerebral blood flow: a comparative study using microspheres

19F NMR was used to determine washout curves of an inert, diffusible gas (CHF3) from the cat brain. The cerebral blood flow was estimated from a bi- or tri-phasic fit to the deconvoluted wash-out curve, using the Kety-Schmidt approach. Cerebral blood flow values determined by 19F NMR show the expected responsiveness to alterations in Paco2, but are approximately 28% lower than cerebral blood flow values determined simultaneously by radioactive microsphere techniques. High concentrations of CHF3 have little effect on intracranial pressure, mean arterial blood pressure or Paco2, but cause small changes in the blood flow to certain regions of the brain. We conclude that 19F NMR techniques utilizing low concentrations of CHF3 have potential for the noninvasive measurement of cerebral blood flow.

Simultaneous measurement of cerebral oxygen consumption and blood flow using 17O and 19F magnetic resonance imaging

17O and 19F magnetic resonance (MR) imaging were used to determine simultaneously the concentrations of H217O and CHF3 in 0.8-cc voxels in the cat brain during inhalation of a gas mixture containing both 17O2 and CHF3. The arterial time course of CHF3 was determined by “on-line” mass spectrometer detection of expired CHF3, and the arterial time course of H217O was determined by 17O MR analysis of arterial samples withdrawn during the inhalation period. The brain data and the arterial data for the two tracers were combined to calculate the cerebral oxygen consumption (CMRO2) and the CBF. The protocol was repeated on seven cats, using pentobarbital anesthesia. The average values of CMRO2 and CBF for a 0.8-cc voxel in the parietal cortex were 1.5 ± 0.5 mmol kg−1 min−1 and 38 ± 15 ml 100 g−1 min−1, respectively. In individual animals the average uncertainty in CMRO2 and CBF, calculated from Monte Carlo approaches, was ±9%.

19F magnetic resonance imaging of cerebral blood flow with 0.4-cc resolution

19F magnetic resonance imaging techniques were used to determine “wash-in” and “wash-out” curves of the inert, diffusible gas CHF3 from 0.4-cc voxels in the cat brain, and mass spectrometer gas detection was used to determine the CHF3 concentration in expired air. These two sets of data were used to calculate cerebral blood flow values in the 0.4-cc voxels, and the blood flow images were registered with high-resolution 1H magnetic resonance images. Data were collected both during the wash-in and wash-out phases of the experiment, but the two sets of data were analyzed separately to obtain independent estimates of the blood flow during the two phases, i.e., Qin and Qout. Repeated determinations of cerebral blood flow images were performed in individual animals, and the entire protocol was repeated on five different animals. The average values of Qin and Qout for a typical 0.4-cc voxel in the parietal cortex were 83 ml 100 g−1 min−1 and 72 ml 100 g−1 min−1, respectively. Monte Carlo calculations utilizing the noise in the 19F NMR signal from this voxel predict an average standard deviation for Qin and Qout of ± 10%. The average standard deviation for repeated measurements (in the same animal) of Qin and Qout in this voxel was ± 14%. We conclude that 19F magnetic resonance imaging approaches have the potential to image cerebral blood flow in humans.

Measurement of cerebral blood flow by volume-selective 19F NMR spectroscopy.

A stimulated echo sequence was used to obtain 19F NMR spectra from within a 4‐ml voxel in a cat brain. The time dependence of the 19F NMR signal from an inert gas (CHF3) was used to calculate the blood flow in the voxel. The position of the voxel was selected using a 1H MR image.

Can the Indo-1 fluorescence approach measure brain intracellular calcium in vivo? A multiparametric study of cerebrocortical anoxia and ischemia.

Indo-1 fluorescence was used to monitor intracellular calcium levels in the cat brain in vivo, using the approach proposed by Uematsu et al. [Uematsu D., Greenberg J. H., Reivich M., Karp A. In vivo measurement of cytosolic free calcium during cerebral ischemia and reperfusion. Ann Neurol 1988; 24: 420-428]. In addition, extracellular calcium and potassium levels, NADH redox state, electrocorticogram (ECoG), DC potential and relative cerebral blood flow were monitored simultaneously. Changes in the Indo-1 fluorescence ratio F400/F506 were monitored during anoxia, reversible ischemia and irreversible ischemia. Although these perturbations resulted in the expected changes in extracellular calcium and potassium levels, NADH redox state, ECoG and other physiological parameters, they did not result in significant increases in the F400/F506 ratio. The apparent insensitivity of the in vivo Indo-1 approach is due to the difficulty in obtaining accurate fluorescence signals from Indo-1 in the brain. Two reasons for this difficulty appear to be problems in loading Indo-1 into the brain, and problems in correcting Indo-1 fluorescence signals for changes in NADH fluorescence and changes in absorption of intrinsic chromophores. Under the conditions of our in vivo cat experiments, Indo-1 fluorescence is not a viable approach for measuring changes in cerebral intracellular calcium levels.

Effects of euthanasia on brain physiological activities monitored in real-time

Abstract Animal experimentation is terminated by the euthanasia procedure in order to avoid pain and minimize suffering. Very little is known about the real time physiological changes taking place in the brain of animals during the euthanasia. Since there is no way to evaluate the suffering of animals under euthanasia, it is assumed that objective physiological changes taking place could serve as a good way to compare various types of euthanasia procedures. In the present study we compared the effect of euthanasia induced by i.v. injection of concentrated KCL to that of Taxan T-61 (a standard mixture used by veterinarians). The responses of the cat brain were evaluated by monitoring the hemodynamic (CBF), metabolic (NADH redox state), electrical (EcoG) and extracellular ion levels, as an indicator to the ionic homeostasis.

Quantitative assessment of [Ca2+]ilevels in rat skeletal muscle in vivo

Intracellular free Ca2+ concentration ([Ca2+]i) plays an essential role in physiological regulatory processes and common pathological conditions. Better understanding of these phenomena is still hampered by problems encountered in the quantitative assessment of [Ca2+]i changes, especially in blood-perfused organs. This study demonstrates that the ratiometric fluorescence technique can be adapted for quantitative in vivo [Ca2+]i determinations. The rat spinotrapezius muscle was topically loaded with indo 1-AM and imaged by a cooled digital camera. Ratio images were calculated in small regions (100 micrometers x 100 micrometers) practically devoid of large vessels in the resting state, after 30 min of ischemia, 20 min of reperfusion, or ionomycin or manganate treatments. When we assumed an average [Ca2+]i of 100 nM in the resting blood-perfused muscle, ischemia increased [Ca2+]i to approximately 200 nM. During reperfusion [Ca2+]i decreased to approximately 140 nM. Ionomycin induced an increase in [Ca2+]i to well above 750 nM. Manganate reduced Ca2+-dependent fluorescence to virtually zero. Our main conclusion is that changes in [Ca2+]i can be monitored and quantitatively determined in vivo.Intracellular free Ca2+ concentration ([Ca2+]i) plays an essential role in physiological regulatory processes and common pathological conditions. Better understanding of these phenomena is still hampered by problems encountered in the quantitative assessment of [Ca2+]ichanges, especially in blood-perfused organs. This study demonstrates that the ratiometric fluorescence technique can be adapted for quantitative in vivo [Ca2+]ideterminations. The rat spinotrapezius muscle was topically loaded with indo 1-AM and imaged by a cooled digital camera. Ratio images were calculated in small regions (100 μm × 100 μm) practically devoid of large vessels in the resting state, after 30 min of ischemia, 20 min of reperfusion, or ionomycin or manganate treatments. When we assumed an average [Ca2+]iof 100 nM in the resting blood-perfused muscle, ischemia increased [Ca2+]ito ∼200 nM. During reperfusion [Ca2+]idecreased to ∼140 nM. Ionomycin induced an increase in [Ca2+]ito well above 750 nM. Manganate reduced Ca2+-dependent fluorescence to virtually zero. Our main conclusion is that changes in [Ca2+]ican be monitored and quantitatively determined in vivo.