Temporal and regional changes during focal ischemia in rat brain studied by proton spectroscopic imaging and quantitative diffusion NMR imaging.

The early development of focal ischemia after permanent occlusion of the right middle cerebral artery (MCA) was studied in six rats using interleaved measurements by diffusion-weighted NMR imaging (DWI) of water and two variants of proton spectroscopic imaging (SI), multiecho SI (TE: 136, 272, 408 ms) and short TE SI (TE: 20 ms). Measurements on a 4.7-T NMR imaging system were performed between the control phase and approximately 6 h postocclusion. In the center of the ischemic lesion of all rats, the apparent diffusion coefficient (ADC) decreased rapidly to 84.4 +/- 4.2% (mean +/- SD) of the control values approximately 2 min postocclusion. Approximately 6 h postocclusion, the ADC was reduced to 67.1 +/- 5.9%. In contrast, large differences between the animals were observed for the temporal increase of lactate (Lac) in the ipsilateral hemisphere. The maximum Lac signal was reached in four rats after 0.5-1.5 h, and in two rats was not reached even after 6 h postocclusion. Six h postocclusion, SI spectra measured at a TE of 136 ms revealed a decrease in the CH3 signal of N-acetylaspartate (NAA) to 67 +/- 13% of the control values. Differences were observed between the spatial regions of decreased NAA and increased Lac. In the lesions, a T2 relaxation time of Lac of 292 +/- 40 ms, considering a J-coupling constant of 6.9 Hz, was measured. Furthermore, a prolongation of the T2 of the CH3 signal of creatine/phosphocreatine (Cr/PCr) was observed in the lesion, from 163 +/- 22 ms during control to 211 +/- 41 ms approximately 6 h postocclusion. The experiments proved that DWI and proton SI are valuable tools to provide complementary information on processes associated with brain infarcts.

Reperfusion after thrombolytic therapy of embolic stroke in the rat: magnetic resonance and biochemical imaging.

The effect of thrombolytic therapy was studied in rats submitted to thromboembolic stroke by intracarotid injection of autologous blood clots. Thrombolysis was initiated after 15 minutes with an intracarotid infusion of recombinant tissue-type activator (10 mg/kg body weight). Reperfusion was monitored for 3 hours using serial perfusion- and diffusion magnetic resonance imaging, and the outcome of treatment was quantified by pictorial measurements of ATP, tissue pH, and blood flow. In untreated animals, clot embolism resulted in an immediate decrease in blood flow and a sharp decrease in the apparent diffusion coefficient (ADC) that persisted throughout the observation period. Thrombolysis successfully recanalized the embolized middle cerebral artery origin and led to gradual improvement of blood flow and a slowly progressing reversal of ADC changes in the periphery of the ischemic territory, but only to transient and partial improvement in the center. Three hours after initiation of thrombolysis, the tissue volume with ADC values less than 80% of control was 39 +/- 22% as compared to 61 +/- 20% of ipsilateral hemisphere in untreated animals (means +/- SD, P = .03) and the volume of ATP-depleted brain tissue was 25 +/- 31% as compared to 46 +/- 29% in untreated animals. Recovery of ischemic brain injury after thromboembolism is incomplete even when therapy is started as early as 15 minutes after clot embolism. Possible explanations for our findings include downstream displacement of clot material, microembolism of the vascular periphery, and events associated with reperfusion injury.

Functional MRI of somatosensory activation in rat: effect of hypercapnic up-regulation on perfusion- and BOLD-imaging.

Functional activation of somatosensory cortex was studied in alpha-chloralose anesthetized rats by functional magnetic resonance imaging (fMRI), using both perfusion-weighted and T2*-weighted (blood oxygenation level dependent, BOLD) imaging. The sensitivity of functional activation was altered by ventilating animals for 3 minutes with 6% CO2. Before hypercapnic conditioning, electrical stimulation of the left forepaw at a frequency of 3 Hz led to an increase of signal intensity (relative to the unstimulated baseline condition) in the right somatosensory cortex by 6+/-2% (means+/-SD) in T2*-weighted images and by 45%+/-48% in perfusion-weighted images. After hypercapnic conditioning the signal intensity increase in perfusion-weighted images doubled to 91%+/-62% (P=0.034), whereas that of T2*-weighted images only marginally increased to 7+/-4% (not significant). This different behavior in both imaging modalities is interpreted as evidence for an increased flow response in combination with a higher oxygen extraction. Thus, the fMRI data reflect hypercapnia-induced resetting of the functional-metabolic coupling of the tissue during activation.

Inhibition of nonselective cation channels reduces focal ischemic injury of rat brain.

The effect of the novel inhibitor of receptor-activated and calcium store-operated nonselective cation channels, (RS)-(3,4-dihydro-6,7-dimethoxyisoquinoline-1-gamma 1)-2-phenyl-N, N-di-[2(2,3,4-trimethoxyphenyl) ethyl]acetamide (LOE 908 MS), on focal cerebral ischemia was studied in halothane-anesthetized rats submitted to permanent suture occlusion of the right middle cerebral artery (MCA). The treated group (n = 7) received subcutaneous injections of 30 mg/kg LOE 908 MS (in 1 ml saline) 10 min after vascular occlusion and again after 3 h. The untreated group (n = 11) was injected subcutaneously with 1 ml saline at the same times. Evolution of infarct was monitored by electrophysiological recording of EEG and cortical steady potential and by diffusion-weighted magnetic resonance imaging during the initial 6 h of vascular occlusion. The hemodynamic, biochemical, and morphological changes were studied after 6 h by combining autoradiographic measurement of blood flow with histological stainings and pictorial measurements of ATP, glucose, and tissue pH. In the untreated animals, the ischemic lesion volume [defined as the region in which the apparent diffusion coefficient (ADC) of water declined to below 80% of control] steadily increased by approximately 50% during the initial 6 h of vascular occlusion relative to the first set of data 10 min postocclusion. In the treated animals, in contrast, the ADC lesion volume declined by approximately 20% during the same interval. Treatment also led to a significant reduction in the number of periinfarct depolarizations. After 6 h of vascular occlusion, blood flow was significantly higher in the treated animals, and the volume of ATP-depleted and morphologically injured tissue representing the infarct core was 60-70% smaller. The volume of severely acidic tissue, in contrast, did not differ, indicating that LOE 908 MS does not reduce the size of ischemic penumbra. These findings demonstrate that postocclusion treatment of permanent focal ischemia with LOE 908 MS delays the expansion of the infarct core into the penumbra for a duration of at least 6 h and therefore substantially prolongs the window of opportunity for the reversal of the ischemic impact in the peripheral parts of the evolving infarct.

Potassium-induced cortical spreading depressions during focal cerebral ischemia in rats: contribution to lesion growth assessed by diffusion-weighted NMR and biochemical imaging.

In focal ischemia of rats, the volume of ischemic lesion correlates with the number of peri-infarct depolarizations. To test the hypothesis that depolarizations accelerate infarct growth, we combined focal ischemia with externally evoked spreading depression (SD) waves. Ischemic brain infarcts were produced in halothane-anaesthetized rats by intraluminal thread occlusion of the middle cerebral artery (MCA). In one group of animals, repeated SDs were evoked at 15-min intervals by microinjections of potassium acetate into the frontal cortex. In another group, the spread of the potassium-evoked depolarizations was prevented by application of the N-methyl-D-aspartate (NMDA) receptor antagonist dizocilpine (MK-801). The volume of ischemic lesion was monitored for 2 h by diffusion-weighted imaging (DWI) and correlated with electro-physiological recordings and biochemical imaging techniques. In untreated rats, each microinjection produced an SD wave and a stepwise rise of the volume and signal intensity of the DWI-visible cortical lesion. The volume of this lesion increased between 15 min and 2 h of MCA occlusion from 19 +/- 15% to 66 +/- 16% of ipsilateral cortex. In dizocilpine-treated animals, microinjections of potassium did not evoke SDs, nor did the volume and signal intensity of the DWI-visible cortical lesion change. At 15 min after MCA occlusion, the DWI-visible lesion was larger than in untreated animals-43 +/- 16% of the ipsilateral cortex; however, after 2 h, it increased only slightly further to 49 +/- 21%. Slower lesion growth in the absence of SDs was also reflected by the volume of ATP-depleted tissue, which, after 2 h of MCA occlusion, involved 26 +/- 12% of the ipsilateral cortex in treated and 49 +/- 9% in untreated animals (p < 0.01). These observations support the hypothesis that peri-infarct depolarizations accelerate cerebral infarct growth.

Variation of functional MRI signal in response to frequency of somatosensory stimulation in alpha-chloralose anesthetized rats.

The dependence of functional MRI contrast on the repetition rate (1.5-9 Hz) of a sensory stimulus was investigated with a T2*-weighted gradient echo method during forepaw stimulation of alpha-chloralose anesthetized rats (n = 5). An activation area was observed in the left or right somatosensory cortex dependent on the stimulation of the right or left forepaw, respectively. The activation intensity decreased for stimulation frequencies above 3 Hz, and was negligible at 9 Hz. The interpretation is that at low stimulation rates the neurons can respond to each stimulus, but at higher rates there is insufficient recovery time so that the response is progressively occluded.

Ultrafast perfusion-weighted MRI of functional brain activation in rats during forepaw stimulation: comparison with T2 -weighted MRI.

A fast version of the arterial spin tagging technique for the detection of cerebral perfusion is presented. Based on adiabatic spin inversion in combination with snapshot FLASH imaging, our technique allows the recording of perfusion changes with a temporal resolution of about 3 s. Differences of cerebral perfusion dependent on the choice of anesthesia were observed in rat brain. Furthermore, with this arterial spin tagging method we demonstrated perfusion increases in the somatosensory cortex of anaesthetized rats during forepaw stimulation. Comparison of the activated areas in the T2 -weighted BOLD images and the perfusion-weighted images showed good spatial correspondence, but the sensitivity to the functional activation was more than ten times higher in the perfusion technique.

Evolution of acute focal cerebral ischaemia in rats observed by localized 1H MRS, diffusion-weighted MRI, and electrophysiological monitoring.

Focal cerebral ischaemia was produced in 11 rats by permanent occlusion of the right middle cerebral artery (MCA) using a suture model modified to enable manipulation with the animals in situ in an NMR spectrometer. The development of the ischaemic insults and the resultant infarcts were observed for up to 6 h by localized 1H MRS and diffusion-weighted MRI while performing continuous monitoring of electroencephalogram and extracellular DC potential. The ischaemic areas were depicted as regions of hyperintensity in the diffusion-weighted images. Signals due to lactate became visible in the 1H spectra after MCA occlusion indicating the onset of anaerobic glycolysis. A depletion of N-acetylaspartate was seen in all animals post-occlusion. Transient or stepwise increases of lactate were observed to occur coincidentally with the events of spontaneous transient peri-infarct depolarization detected by the electrophysiological measurements. Expansion of the ischaemic area delineated in the diffusion-weighted images also accompanied peri-infarct depolarizations. These observations are consistent with transient peri-infarct depolarization playing a role in the growth of infarcts.

Simultaneous recording of EEG, DC potential and diffusion-weighted NMR imaging during potassium induced cortical spreading depression in rats.

Electrophysiological recording during in vivo NMR imaging for correlation of electrophysiological events with changes in NMR parameters has, until now, not been satisfactory. Here we present a method that uses specially prepared calomel electrodes for the continuous monitoring of both cortical steady potential and EEG during in vivo NMR imaging studies of experimental animal models. In order to demonstrate the reliability of this approach we elicited single episodes of cortical spreading depression in rats by intracortical potassium acetate injection, using an intracerebral micropipette. During the passage of DC potential shifts and associated reduction in EEG amplitude, hyperintense regions, reflecting the progression of cortical spreading depression over the cortex, were visible in diffusion-weighted images of the rat brains. No susceptibility artifacts due to the electrodes and micropipette were visible in the NMR data. Thus, we have demonstrated under controlled experimental conditions, that electrophysiological recording with calomel electrodes can be performed simultaneously with diffusion-weighted NMR imaging. The described method enables further NMR investigations of phenomena which are detectable by electrophysiology.

Transient cell depolarization after permanent middle cerebral artery occlusion: an observation by diffusion-weighted MRI and localized 1H-MRS.

Focal cerebral ischemia causes rapid intensity changes in diffusion-weighted images (DWI) and elevated lactate as detected by localized proton spectroscopy (1H-MRS). To investigate whether such changes can also be evoked by perischemic depolarizations, we combined DWI and 1H-MRS measurements with DC potential recordings. About 40 min after occlusion of the middle cerebral artery in a rat, a negative DC deflection was observed indicating transient cell depolarization. Coincidentally with the depolarization a transient increase of the DWI signal intensity and a partially reversible increase of lactate occurred in the periphery of the ischemic territory. These results show that peri-ischemic depolarization, known to contribute to the evolution of cerebral infarction, evokes disturbances that can be detected by DWI and 1H-MRS.

Proton MR spectroscopy of experimental brain tumors in vivo.

F98 gliomas, E367 neuroblastomas, and RN6 Schwannomas in rat brain were studied non-invasively in vivo by localized proton MR spectroscopy (MRS). The spectra obtained from homotopic brain contralateral to the tumors were qualitatively indistinguishable from those of normal rat brain in vivo and showed resonance lines assigned to N-acetylaspartate, glutamate, total creatine (creatine and phosphocreatine), choline, glucose, and myo-inositol. The tumor spectra displayed marked differences compared to those obtained from contralateral brain. There were increases in choline, myo-inositol and lipids, which are presumably associated with increased membrane turnover. The presence of lactate indicated anaerobic glycolysis. Other differences included the absence of signals from NAA resulting from the destruction or displacement of neuronal tissue by the tumor. There was also a loss of total creatine. Although the spectra of all three tumor types were distinct from contralateral brain, there were no obvious differences between the different tumor types.

Localized proton NMR spectroscopy of experimental gliomas in rat brain in vivo.

Experimental brain tumors produced in rats (n = 10) by stereotactic implantation of cells from the F98 anaplastic glioma clone into the right caudate nucleus were studied in vivo using localized proton NMR and in vitro using high-resolution proton NMR, bioluminescent imaging of lactate, ATP and glucose distributions, and fluorescent imaging of regional pH. In vivo spectra from normal brain contralateral to the tumor regions showed resonances assignable to N-acetyl aspartate (NAA), creatines, choline-containing compounds, myo-inositol, glutamate and glucose in a pattern similar to those obtained from normal anaesthetized rats. In vivo tumor spectra were characterized by the almost complete absence of NAA, a substantial reduction of total creatine and glucose, and an increase of cholines. Based on the in vitro spectra the increase of the myo-inositol signal observed in vivo was mainly attributed to glycine. Histological examination as well as bioluminescent and fluorescent imaging indicated two stages of tumor development, i.e., solid vital tumors and tumors with necrosis. However, there was no consistent relationship between proton NMR observations and tumor development.

On the interpretation of proton NMR spectra from brain tumours in vivo and in vitro.

Localized proton NMR spectroscopy in vivo allows focal studies of cerebral metabolites in both man and laboratory animals from image-defined regions as small as 1 mL or 64 microL, respectively. Although brain tumours lead to remarkable spectral alterations relative to normal brain, a number of problems may compromise the interpretation of the results. Potential complications arise from the chosen experimental conditions (method, TE, size and location of volume of interest), from regional metabolic heterogeneity in and around tumours, from differences between human tumours and animal models, and from discrepancies between in vivo and in vitro findings. Strategies and pitfalls are illustrated with use of selected examples from primary brain tumours, a rat tumour model and perchloric acid extracts of resected specimens.

Diffusion imaging of the human brain in vivo using high-speed STEAM MRI.

This paper describes a new method for diffusion imaging of the human brain in vivo that is based on a combination of diffusion-encoding gradients with high-speed STEAM MR imaging. The single-shot sequence 90 degrees-TE/2-90 degrees-TM-(alpha-TE/2-STE)n generates n = 32-64 differently phase-encoded stimulated echoes STE yielding image acquisition times of 576 ms for a 48 x 128 data matrix. Diffusion encoding is performed during the first TE/2-interval as well as during each readout period. Phantom studies reveal a quantitative agreement of calculated diffusion coefficients with literature values. EKG triggering completely eliminates motion artifacts from diffusion-weighted single-shot STEAM images of human brain in vivo. While signal attenuation of the cerebrospinal fluid (CSF) is predominantly due to flow, that observed for gray and white matter results from diffusion. Evaluated diffusion coefficients yield (1.0 +/- 0.1) x 10(-5) cm2 s-1 for gray matter, (0.5 +/- 0.1) x 10(-5) cm2 s-1 for white matter with the diffusion encoding parallel to the main orientation of the myelin sheath of the neurofibrils, and (0.3 +/- 0.1) x 10(-5) cm2 s-1 for white matter and a perpendicular orientation. All studies were performed at 2.0 T using a conventional 10 mT m-1 gradient system.

High-speed STEAM MRI of the human heart.

High-speed STEAM MR images of the normal human heart were obtained from single cardiac cycles using a 2.0-T whole-body system equipped with conventional 10 mT m-1 gradients. The single-shot 90 degrees-TE/2-90 degrees-TM-(alpha-TE/2-Acq)n pulse sequence acquires n differently phase-encoded stimulated echoes. Measuring times of 127-254 ms were achieved using a "repetition time" of 3.96 ms in conjunction with data matrices of 32-64 x 128 pixels covering a field-of-view of 250-350 mm. The sequence provides easy access to anatomical short-axis and long-axis views of the heart by single and double oblique rotation of the image orientation. STEAM images resemble the features of spin-echo images with respect to chemical shifts, susceptibilities, and flow. Thus, no additional techniques are required for the suppression of blood signals. EKG-triggered acquisitions demonstrate that slice-selective STEAM sequences using short TM intervals allow an unambiguous delineation of those parts of the myocardium that remain stationary within the selected plane throughout the entire imaging process. Neither spins leaving nor entering the slice defined by the initial 90 degrees RF pulses give rise to a stimulated echo and therefore do not contribute to the resulting image.

[Localized proton MR spectroscopy. A non-invasive insight into brain metabolism]. / Lokalisierte Protonen-MR-Spektroskopie. Nichtinvasive Einblicke in den Hirnstoffwechsel.

Recent progress in image-controlled, localized proton MR spectroscopy offers a non-invasive means of gaining unique insights into brain metabolism in man. Combined studies with MR imaging can be performed within about 1 h. Results obtained in healthy subjects provide the basis for reliable identification and quantification of metabolite concentrations in the CNS and allow determination of their regional variability and age dependence. Clinical applications include infarcts, tumors, and neurodegenerative diseases, and also metabolic disturbances resulting from diseases of the internal organs, such as diabetes mellitus or liver cirrhosis.

Non-invasive 1H NMR spectroscopy of the rat brain in vivo using a short echo time STEAM localization sequence.

Fully localized proton NMR spectra were obtained from the brains of normal anaesthetized rats in vivo using stimulated echo (STEAM) spectroscopy sequences. Investigations were carried out at 2.35 T using a 40 cm bore magnet equipped with an actively shielded gradient system. Localized shimming resulted in water proton linewidths of 6.5-7.8 Hz permitting excellent water suppression. Thus, high-quality proton NMR spectra (TE = 20 ms) were acquired within measuring times of 1.5-6.4 min from 64 to 125 microL volumes-of-interest. The spectra show metabolite resonances due to N-acetyl aspartate, glutamate, creatine and phosphocreatine, cholines, taurine and inositols. The assignments of strongly spin-coupled resonances were confirmed by comparison with spectra from model solutions obtained under identical experimental conditions to those used in vivo. T1 relaxation times as well as relative metabolite concentrations were evaluated from spectra obtained for repetition times ranging from 900 to 6000 ms. Sequential acquisitions of 1.5 min spectra before, during and after killing the animals exhibited a rapid accumulation of lactate, but did not reveal significant changes in other metabolite levels for several hours post mortem.

Cerebral glucose is detectable by localized proton NMR spectroscopy in normal rat brain in vivo.

This contribution reports the first direct and noninvasive observation of cerebral glucose in normal anesthetized rats (n = 16) using short-echo-time localized proton NMR spectroscopy (2.35 T, STEAM, TR = 6000 ms, TE = 20 ms, 125 microliters). In addition to resonances from N-acetyl aspartate (NAA), glutamate, total creatine, cholines, taurine, and myoinositol, all spectra exhibit strongly coupled resonances from glucose (3.43, 3.80 ppm) that are readily identifiable using model solutions. The observed level of cerebral glucose in fasted rats covered a range of 15-40% of that of NAA giving absolute concentrations of 1.1-2.8 mM when NAA is taken to be 7 mM. The arterial blood glucose concentration was 7.7 +/- 0.8 mM in the same group of animals.

On the identification of cerebral metabolites in localized 1H NMR spectra of human brain in vivo.

Localized 1H NMR spectra of human brain in vivo are affected by signal overlap, strong spin-spin coupling, and complex J modulation, and therefore differ considerably from those obtained at higher magnetic fields. This paper deals with the assignment of 1H NMR resonances of cerebral metabolites under the experimental conditions used for human investigations. Conventional 7.0-T FID spectra and 2.0 T localized, short echo time STEAM spectra (TE = 20 ms) of aqueous metabolite solutions are compared to in vivo brain spectra of human volunteers and patients. In addition to singlet resonances from N-acetyl aspartate (NAA), creatines, and cholines, short echo time STEAM spectra exhibit multiplets due to the NAA aspartyl group, glutamate, taurine, and myo-inositol. Enhanced levels of cerebral glutamine are detected in patients with liver cirrhosis. For the first time elevated levels of brain glucose are observed in patients with diabetes mellitus.