Robin A. de Graaf

Changes in the Diffusion of Water and Intracellular Metabolites After Excitotoxic Injury and Global Ischemia in Neonatal Rat Brain

The reduction of the apparent diffusion coefficient (ADC) of brain tissue water in acute cerebral ischemia, as measured by diffusion-weighted magnetic resonance imaging, is generally associated with the development of cytotoxic edema. However, the underlying mechanism is still unknown. Our aim was to elucidate diffusion changes in the intracellular environment in cytotoxic edematous tissue. The ADC of intracellular metabolites was measured by use of diffusion-weighted 1H-magnetic resonance spectroscopy after (1) unilateral N-methyl-D-aspartate (NMDA) injection and (2) cardiac arrest-induced global ischemia in neonatal rat brain. The distinct water ADC drop early after global ischemia was accompanied by a significant reduction of the ADC of all measured metabolites (P < 0.01, n = 8). In the first hours after excitotoxic injury, the ADC of water and the metabolites taurine and N-acetylaspartate dropped significantly (P < 0.05, n = 8). At 24 and 72 hours after NMDA injection brain metabolite levels were diminished and metabolite ADC approached contralateral values. Administration of the NMDA-antagonist MK-801 1.5 hours after NMDA injection completely normalized the water ADC but not the metabolite ADC after 1 to 2 hours (n = 8). No damage was detected 72 hours later and, water and metabolite ADC had normal values (n = 8). The contribution of brain temperature changes (calculated from the chemical shift between the water and N-acetylaspartate signals) and tissue deoxygenation to ischemia-induced intracellular ADC changes was minor. These data lend support to previous suggestions that the ischemia-induced brain water ADC drop may partly be caused by reduced diffusional displacement of intracellular water, possibly involving early alterations in intracellular tortuosity, cytoplasmic streaming, or intracellular molecular interactions.

In vivo 31P-NMR diffusion spectroscopy of ATP and phosphocreatine in rat skeletal muscle

The aim of this study was to measure the diffusion of ATP and phosphocreatine (PCr) in intact rat skeletal muscle, using (31)P-NMR. The acquisition of the diffusion-sensitized spectra was optimized in terms of the signal-to-noise ratio for ATP by using a frequency-selective stimulated echo sequence in combination with adiabatic radio-frequency pulses and surface coil signal excitation and reception. Diffusion restriction was studied by measuring the apparent diffusion coefficients of ATP and PCr as a function of the diffusion time. Orientation effects were eliminated by determining the trace of the diffusion tensor. The data were fitted to a cylindrical restriction model to estimate the unbounded diffusion coefficient and the radial dimensions of the restricting compartment. The unbounded diffusion coefficients of ATP and PCr were approximately 90% of their in vitro values at 37 degrees C. The diameters of the cylindrical restriction compartment were approximately 16 and approximately 22 microm for ATP and PCr, respectively. The diameters of rat skeletal muscle fibers are known to range from 60 to 80 microm. The modelling therefore suggests that the in vivo restriction of ATP and PCr diffusion is not imposed by the sarcolemma but by other, intracellular structures with an overall cylindrical orientation.

Cerebral ischemia and white matter edema in experimental hydrocephalus. A combined in vivo MRI and MRS study

T2 and diffusion weighted MRI, as well as 31P and 1H MRS were performed in kaolin-induced hydrocephalic rats. Extracellular white matter edema was detected in the early stages of progressive hydrocephalus. Phosphocreatine (PCr)/inorganic phosphate (Pi) ratios in hydrocephalic animals were decreased compared to controls, and lactate was detected during the acute and chronic stages of hydrocephalus. These MR spectroscopic results are indicative of a compromised energy metabolism and suggest the occurrence of cerebral ischemia in experimental hydrocephalus.

Off-resonance metabolite magnetization transfer measurements on rat brain in situ.

Off‐resonance metabolite magnetization transfer (MT) experiments were performed on rat brain in vivo and post mortem, with short (18 msec) and long (144 msec) echo‐time 1H nuclear magnetic resonance (NMR) spectroscopy. In vivo and post mortem, the methyl protons of total creatine and all protons from glutamate/glutamine showed a strong MT effect on off‐resonance saturation, as well as the methyl protons from lactate post mortem. Other resonances, like that of N‐acetyl aspartate, showed a much smaller, but detectable, MT effect. The results obtained were confirmed by combining off‐resonance saturation with two‐dimensional correlation spectroscopy. Three water suppression techniques, i.e., presaturation, chemical shift‐selective (CHESS), and selective water eliminated Fourier transform (WEFT) were evaluated for their ability to generate an MT effect, to assess their possible influence on metabolite quantification. Presaturation and selective WEFT led to alterations of the total creatine, lactate, and N‐acetyl aspartate resonance intensities, while CHESS had no effect. Finally, it was shown that water protons play an important role in the generation of the observed metabolite MT effects. Magn Reson Med 41:1136–1144, 1999.

Single-shot diffusion trace 1H NMR spectroscopy

Ignoring diffusion anisotropy can severely hamper the quantitative determination of water and metabolite diffusion in complex tissues. The measurement of the trace of the diffusion tensor provides unambiguous and rotationally invariant ADC values, but usually requires three separate experiments. A single‐shot technique developed earlier, originally designed for diffusion trace MR imaging (Mori and van Zijl, Magn Reson Med 1995;33:41–52), was improved and adapted for diffusion trace MR spectroscopy. A double spin‐echo pulse sequence was incorporated with four pairs of bipolar gradients with specific predetermined relative signs in each of the three orthogonal directions. The combination of gradient directions leads to cancellation of all off‐diagonal tensor elements while constructively adding the diagonal elements. Furthermore, the pulse scheme provides complete compensation for cross‐terms between static magnetic field gradients and the applied diffusion gradients, while simultaneously avoiding cross‐terms with localization gradients. The sequence was tested at 4.7 T in vivo on rat brain for MRI and on rat skeletal muscle and brain for MRS. It is shown that the average ADC as determined from the measurement of the ADCs in the three orthogonal directions is in close agreement with the ADC obtained along the trace of the diffusion tensor in a single acquisition, for both water and metabolite diffusion. The large differences in water and metabolite diffusion coefficients as measured in the individual orthogonal directions illustrate the need for diffusion trace measurements when accurate and rotationally invariant diffusion quantitation is required. The pulse scheme presented here may be applied for such purposes in MRS and MRI studies. Magn Reson Med 45:741–748, 2001.

Magnetic coupling of creatine/phosphocreatine protons in rat skeletal muscle, as studied by 1H‐magnetization transfer MRS

Off‐resonance saturation caused a reduction of the 3.04 ppm NMR signal from the methyl protons of creatine in rat hindleg skeletal muscle. 1H‐NMR spectra were recorded over a 200 kHz range of off‐resonance saturation frequencies. The span of frequencies over which the creatine signal was reduced greatly exceeded that expected for direct saturation by the off‐resonance RF‐field. This suggests that there is a motionally restricted proton pool which exchanges magnetization with the free creatine pool. The experimental data were fitted to characterize the immobilized proton pool and the exchange kinetics, using a two‐pool exchange model. The immobile pool was estimated to amount to ca. 2.5% of the mobile pool of free creatine, while the rate of exchange between the mobile and immobile configurations is ca. 2.3 sec‐1. After depletion of phosphocreatine by termination of the animal, the MT effect on the creatine methyl protons remained unchanged. This indicates that phosphocreatine and creatine both contribute to the MT phenomenon. Selective saturation of the mobile water pool also led to a reduction in the intensity of the total creatine methyl signal, suggesting that water and creatine are magnetically coupled via a macromolecular interface. The precise mechanism responsible for and the biological significance of the pronounced creatine magnetization transfer effect in rat skeletal muscle remains to be established. Magn Reson Med 42:665–672, 1999.

Effects of hypoxia-ischemia and inhibition of nitric oxide synthase on cerebral energy metabolism in newborn piglets.

The present study was designed to examine the effects of inhibition of nitric oxide synthase on cerebral energy metabolism after hypoxia-ischemia in newborn piglets. Ten 1- to 3-d-old piglets received Nω-nitro-L-arginine (NNLA), an inhibitor of nitric oxide synthase (NNLA-hypoxia, n = 5), or normal saline (hypoxia, n = 5) 1 h before cerebral hypoxia-ischemia. After the infusion, hypoxia-ischemia was induced by bilateral occlusion of the carotid arteries and decreasing FiO2 to 0.07 and maintained for 60 min. Thereafter, animals were resuscitated and ventilated for another 3 h. Using 1H- and 31P-magnetic resonance spectroscopy, cerebral energy metabolism was measured in vivo at 15-min intervals throughout the experiment. Phosphocreatine to inorganic phosphate ratios decreased from 2.74 ± 0.14 to 0.74 ± 0.36 (hypoxia group) and 2.32 ± 0.17 to 0.18 ± 0.10 (NNLA-hypoxia group) during hypoxia-ischemia. Thereafter, phosphocreatine to inorganic phosphate ratios returned rapidly to baseline values in the hypoxia group, but remained below baseline values in the NNLA-hypoxia group. Intracellular pH decreased during hypoxia-ischemia and returned to baseline values on reperfusion in both groups. Intracellular pH values were lower in the NNLA-hypoxia group (p < 0.001, ANOVA). Lactate was not present during the baseline period. After hypoxia-ischemia, lactate to N-acetylaspartate ratios increased to 1.34 ± 0.28 (hypoxia group) and 2.22 ± 0.46 (NNLA-hypoxia group). Lactate had disappeared after 3 h of reperfusion in the hypoxia group, whereas lactate to N-acetylaspartate ratios were 1.37 ± 1.37 in the NNLA-hypoxia group. ANOVA demonstrated a significant effect of NNLA on lactate to N-acetylaspartate ratios (p < 0.001). Inhibition of nitric oxide synthase by NNLA tended to compromise cerebral energy status during and after cerebral hypoxia-ischemia in newborn piglets.

CEREBRAL METABOLISM OF NEWBORN PIGLETS FOLLOWING HYPOXIA- ISCHEMIA AND NITRIC OXIDE SYNTHASE INHIBITION, EXAMINED USING PROTON AND PHOSPHOROUS MAGNETIC RESONANCE SPECTROSCOPY. • 1729

Increased production of nitric oxide, a free radical with a potential for tissue damage, in cerebral tissue during hypoxia-ischemia (HI) and reperfusion has been demonstrated previously. The present study tests thehypothesis that inhibition of nitric oxide synthase (NOS) during cerebral HI reduces hypoxia-induced lactate production and changes in intracellular pH (pHi), phosphocreatine/ inorganic phosphate(PCr/Pi) ratios in cortical tissue of newborn piglets. Methods: Following anesthesia, 8 newborn piglets aged 1-3 days were instrumented, including placement of inflatable carotid occluders, and ventilated. The animals were placed in a 4.7 T magnet for examination of cerebral metabolism using 31P and 1H-MRS. Measurements were performed every 15 min throughout the experiment. One hour after the i.v. administration of 40 mg/kg Nω-nitro-L- arginine (n=4, NNLA-Hx group) or normal saline (n=4, Hx group), HI was induced by decreasing the FiO2 to 0.08 and carotid occlusion. After one hour, occluders were deflated, and resuscitation was started with an FiO2 sufficient to obtain PaO2 levels of 80-100 mmHg. pHi, PCr/Pi and lactate/N-acetylaspartate (Lac/NAA) ratios were calculated.Results: baseline pHi, PCr/Pi and Lac/NAA ratios(mean ± S.D.) were 7.22 ± 0.08, 2.80 ± 0.32, 0 in the Hx group and 7.12 ± 0.05, 2.46 ± 0.41, and 0 in the NNLA-Hx group, respectively (group differences not significant). After 1 hour of HI pHi, PCr/Pi and Lac/NAA were 6.79 ± 0.26, 1.13± 0.67, 1.10 ± 0.40 in the Hx group and 6.24 ± 0.31, 0.09± 0.06, and 2.07 ± 1.07 in the NNLA-Hx group. These differences are significant (P<0.05). Following 1 hour of resuscitation, pHi, PCr/Pi and Lac/NAA of the Hx group were 7.24 ± 0.18, 2.62 ± 0.36, and 0.34 ± 0.49, whereas in the NNLA-Hx group these values were 6.72 ± 0.44, 0.73 ± 0.93, and 1.11 ± 0.32, respectively. These differences are also significant (P<0.05).Conclusion: inhibition of NOS by NNLA during HI and reperfusion deteriorated cerebral energy metabolism in newborn piglets, which is in contrast with our hypothesis. We speculate that NO mediated effects on cerebral perfusion are important for cerebral energy metabolism following HI and reperfusion. Supported by the Wilhelmina Childrens Hospital Research Fund