Ex vivo MR microimaging of neuronal damage after kainate-induced status epilepticus in rat: correlation with quantitative histology.

The present study was designed to investigate whether T(2)-weighted signal changes obtained by microimaging of paraformaldehyde-fixed brain correlate with the histologically quantified damage in a model of status epilepticus (SE) induced by kainic acid in the rat. Animals were killed at several time points up to 8 weeks after a single intraperitoneal kainate (KA) injection (9 mg/kg). Perfusion-fixed brains were embedded in gelatin for MR microimaging at 9.4T. After the MRI analysis, the gelatin was removed and the brains were cryoprotected and processed for quantitative histology. Severity of neuronal damage and gliosis were assessed from thionin-stained serial sections. Correlative analysis of microimaging and histology data was done in the hippocampus, amygdala, parietal rhinal cortex (PaRH), piriform cortex (Pir), and entorhinal cortex. The relative signal intensities in T(2)-weighted images correlate with the severity of neuronal damage in the matched histological sections (correlation coefficients of 0.752-0.826). Our data show that MR microimaging ex vivo detects the degree of neuronal damage and its anatomical distribution after KA-induced SE, thus providing a useful tool for detecting the dynamics of progressive neuronal damage after prolonged seizures.

Reduction in water and metabolite apparent diffusion coefficients during energy failure involves cation-dependent mechanisms. A proton nuclear magnetic resonance study of rat cortical brain slices.

Proton (1H) nuclear magnetic resonance (NMR) diffusion spectroscopy was used to assess apparent diffusion coefficients (ADCs) in rat brain slices. Aglycemic hypoxia caused reductions in the ADC of N-acetylaspartate (NAA) (0.15 to 0.09 x 10(-3) mm2/s) and "slow" diffusion coefficient (D2) of tissue water (0.51 to 0.37 x 10(-3) mm2/s), together with a 32+/-11% increase in tissue water volume, attributable to tissue swelling. The ADC and D2 reductions were diminished, however, by removing external Ca2+, and under 10 mmol/L Mg2+, normoxic diffusion coefficients persisted until 40 minutes of hypoxia. The data suggest that the shift of water into the intracellular space alone cannot satisfactorily explain the reduced cerebral diffusion upon energy failure and that external Mg2+ and Ca2+ play crucial modulatory roles.

Increased macromolecular resonances in the rat cerebral cortex during severe energy failure as detected by 1H nuclear magnetic resonance spectroscopy.

Changes in cerebral macromolecular 1H nuclear magnetic resonance (NMR) spectrum were studied in cortical brain slices in vitro. Aglycaemic hypoxia irreversibly increased various short T2 spectral components at 1.8-0.8 ppm in concordance with energy loss and independent of T1 and T2 relaxation effects. Removal of external calcium (Ca2+e) slightly attenuated the effect. The results suggest NMR-visible reorganisation of intracellular proteins due to hypoxic insult, and show that it may be possible to monitor early cytoplasmic changes due to brain energy depletion by NMR spectroscopy.

Glucose metabolism in the developing cerebral cortex as detected by 1H(13C) nuclear magnetic resonance spectroscopy ex vivo.

Metabolism of [1-13C]glucose was monitored in superfused cerebral cortex slice preparations from 1-, 2-, and 5-week-old rats using 1H-observed/13C-edited (1H(13C)) NMR spectroscopy. The rate of label incorporation into glutamate C-4 did not differ among the three age groups: 0.52-0.67% of total 1H NMR-detected glutamate/min. This was rather unexpected, as oxygen uptake proceeded at 1.1 +/- 0.1, 1.9 +/- 0.1, and 2.0 +/- 0.1 mumol/min/g wet weight in brain slices prepared from 1-, 2-, and 5-week-old animals, respectively. Steady-state glutamate C-4 fractional enrichments in the slice preparations were approximately 23% in all age groups. In the acid extracts of slices glutamate C-4 enrichments were smaller, however, in 1- and 2-week-old (17.8 +/- 1.7 and 16.8 +/- 0.8%, respectively) than in 5-week-old rats (22.7 +/- 0.7%) after 75 min of incubation with 5 mM [1-13C]glucose. We add a new assignment to the 1H(13C) NMR spectroscopy, as acetate C-2 was detected in slice preparations from 5-week-old animals. In the acid extracts of slice preparations acetate C-2 was labeled by approximately 30% in 5-week-old rats but by 15% in both 1- and 2-week-old animals, showing that the turnover rate was increased in 5-week-old animals. In the extracts 3-4% of the C-6 of N-acetyl-aspartate (NAA; CH3 of the acetyl group) contained label as determined by both NMR and mass spectrometry, which indicated that there was no significant labeling to other carbons in NAA.(ABSTRACT TRUNCATED AT 250 WORDS)

Lactate efflux and intracellular pH during severe hypoxia in rat cerebral cortex in vitro studied by nuclear magnetic resonance spectroscopy.

Intracellular pH (pHi) and lactate were monitored in a superfused brain slice preparation using NMR spectroscopy in order to study the role of lactate washout in maintenance of pHi during hypoxia. Data are consistence with a functioning lactate-H+ cotransport in the energetically intact cerebral cortex. This pathway is not, however, linked to regulation of pHi during energy failure with external pH of 6.8 and thus appears not to have physiological impact in H+ homeostasis during cerebral hypoxia.

Compartmentation of cerebral glutamate in situ as detected by 1H/13C n.m.r.

Incorporation of 13C label from either [1-13C]glucose to glutamate C-4 and lactate C-3 or from [2-13C]acetate to glutamate C-4 was monitored in situ in a superfused brain slice preparation by using 1H-detected/13C-edited (1H/13C) n.m.r. spectroscopy. The fractional enrichments of both metabolites were determined by this means in both brain slices and acid extracts of the preparations in order to assess their 1H-n.m.r. detectabilities. The 1H/13C satellite resonances from glutamate C-4 and lactate C-3 in brain tissue were followed from 4 min onwards in the presence of 5 mM [1-13C]glucose. Fractional enrichment of glutamate C-4 in the slice preparations was higher than in their acid extracts throughout the incubation of 100 min; at 30 min the enrichment was 15.9 +/- 0.6% in the slice preparations and 10.6 +/- 0.9% in extracts and at 100 min 24.5 +/- 1.7% compared with 19.7 +/- 0.4%, respectively. In contrast, lactate C-3 reached a steady-state fractional enrichment of approx. 43% by 15 min and there was no difference between the values determined in the slice preparations and the acid extracts. There was a significant difference between the glutamate C-4 fractional enrichments in the brain slices (7.4 +/- 0.6%) and extracts (5.1 +/- 0.3%) after 60 min of incubation with [2-13C]acetate. Thus 13C label from both glucose and exogenous acetate enters a pool of glutamate that is more amenable to 1H n.m.r. detection than total acid-extracted brain biochemical glutamate, whereas lactate is labelled with full 1H n.m.r. visibility. The results are discussed in the light of the biochemical factors that affect glutamate 1H-n.m.r. susceptibility and thus its n.m.r. visibility.

Regulation of intracellular pH in guinea pig cerebral cortex ex vivo studied by 31P and 1H nuclear magnetic resonance spectroscopy: role of extracellular bicarbonate and chloride.

The role of transmembrane processes that are dependent on external anions in the regulation of cerebral intracellular pH (pHi), high-energy metabolites, and lactate was investigated using 31P and 1H NMR spectroscopy in an ex vivo brain slice preparation. During oxygenated superfusion, removal of external HCO3-/CO2 in the presence of Na+ led to a sustained split of the inorganic phosphate (Pi) peak so that the pHi indicated by one part of the peak was 0.38 pH units more alkaline and by the other part 0.10 pH units more acidic at 5 min than in the presence of HCO3-. The pH in the compartment with a higher pHi value returned to 7.29 +/- 0.04 by 10.5 min of superfusion in a HCO3(-)-free medium, whereas the pHi in an acidic compartment was reduced to 7.02. In the presence of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid or the absence of external Cl-, removal of HCO3- caused alkalinization without split of the Pi peak. Both treatments reduced the rate of pHi normalization following alkalinization. Simultaneous omission of external HCO3- and Na+ did not inhibit alkalinization of the pHi following CO2 exit. All these data show that the acid loading mechanism at neutral pHi is mediated by an Na(+)-independent anion transport. During severe hypoxia, pHi dropped from 7.29 +/- 0.05 to 6.13 +/- 0.16 and from 7.33 +/- 0.03 to 6.67 +/- 0.05 in the absence and presence of HCO3-, respectively, in Na(+)-containing medium. Lactate accumulated to 18.7 +/- 2.8 and 19.6 +/- 1.5 mmol/kg under the respective conditions. In the HCO3(-)-free medium supplemented with 1 mM amiloride, the pHi fell only to 6.94 +/- 0.08 despite the lactate concentration of 18.9 +/- 2.4 mmol/kg.(ABSTRACT TRUNCATED AT 250 WORDS)

Extracellular pH and buffering power determine intracellular pH in cortical brain slices during and following hypoxia.

1H and 31P nuclear magnetic resonance (NMR) spectroscopy were used to study the role of external pH (pHo) in regulation of intracellular pH (pHi) in the cerebral cortex in vitro. Lowering the pH of the oxygenated superfusion medium (pHb) from 7.5 to 7.0-6.8 decreased pHi from 7.33 +/- 0.08 to approximately 7.00 independently of the presence of HCO3- and medium buffering power. Reduction of medium (pHb 6.8) buffering power during hypoxia severely acidified pHi independently of the presence of HCO3-. Following hypoxia, recovery of pHi was not sensitive to pHb provided that medium buffering was maintained. Lactate removal proceeded independently of pHb, HCO3- and external buffering power. These results indicate that decrease in extracellular buffering capacity has a feed-back effect on pHi in the cerebral cortex.

1H nuclear magnetic resonance spectroscopy study of cerebral glutamate in an ex vivo brain preparation of guinea pig.

Cerebral glutamate was monitored in a superfused cerebral cortical preparation by 1H NMR spectroscopy using a semiselective spin-echo sequence N-acetyl aspartate (NAA) as an internal concentration reference. During controlled metabolic conditions, the cerebral 1H NMR-detected glutamate-to-NAA ratio was approximately 20-30% lower than expected from the ratio of neutralized perchloric acid extracts of the preparations. Inhibition of respiration in the presence of glucose did not change the 1H NMR glutamate-to-NAA ratio in brain slice preparation. In contrast, either complete depletion of ATP during cyanide poisoning together with 0 mM glucose, anoxia in the absence of glucose, or treatment with nigericin or with a protonophore, carbonyl cyanide-m-fluorophenylhydrazone, increased 1H NMR-detected glutamate/NAA in the cerebral preparations without a change in the relative and absolute concentration ratios determined from the tissue acid extracts. Spin-spin relaxation times of glutamate and NAA peaks in anoxic slices were 749 +/- 89 and 729 +/- 94 ms, respectively, and thus, the portion of glutamate that could not be detected by 1H NMR was quantified in absolute terms. It was calculated that an increase in the glutamate-to-NAA ratio from 0.55 +/- 0.02 to 0.67 +/- 0.02 during aglycemic anoxia corresponded to some 6 mmol/kg of tissue dry weight of glutamate from the total concentration of 28 mmol/kg dry weight. It is suggested that this 22% of total glutamate pool is present in a noncytoplasmic compartment during controlled metabolic state.

Delayed increase in intracellular Na+ in cerebral cortical slices during severe hypoxia as measured by double quantum filtered 23Na+ NMR.

We have used double quantum filtered (DQF) 23Na+ nuclear magnetic resonance (NMR) spectroscopy without shift reagents in order to monitor intracellular Na+ (Na+i) in a cortical brain slice preparation. The external Na+ (Na+o) signal was reduced by 95% by the DQF sequence compared with the directly observed 23Na+. The DQF 23Na+ signal is not exclusively due to Na+i, however, as 40% of this signal appears to arise from Na(+)-ions interacting with extracellular membrane proteins or proteins exposed at the cut surfaces of the slices. Veratridine increased instantly the DQF 23Na+ signal so that it reached 130.4 +/- 5.0% by 12 min. This shows that there was a significant contribution from Na+i in the DQF 23Na+ NMR spectra. Hypoxia of 30 min duration in the presence of 10 nM glucose did not influence intensity of the DQF 23Na+ signal. Aglycaemic hypoxia caused complete collapse of phosphocreatine (PCr) within 7 min whereas DQF 23Na+ first increased 15 min after the insult. This increase reached its maximal value of 125% after 25 min. There was an incomplete recovery of the DQF 23Na+ after aglycaemic hypoxia to 110% of the control value parallel to poor metabolic recovery. The presence of 10 mM extracellular Mg2+ had no apparent effect on the aglycaemic hypoxia-induced rise in Na+i indicating that it was linked to Ca2+ influx. Tetrodotoxin (TTx, 4.7 microM) did not influence the rise of Na+i caused by aglycaemic hypoxia. These results indicate that elevation of Na+i is a late consequence of energy failure in the cerebral cortex.

Detection of thymosin beta 4 in situ in a guinea pig cerebral cortex preparation using 1H NMR spectroscopy.

In the present work we have investigated the macromolecules that contribute to the brain 1H NMR spectrum. The cerebral cortex showed distinct resonances at the uncrowded methyl- and methylene chemical shift scale of the spin-echo 1H NMR spectrum. The peaks at 1.22 and 1.40 ppm (relative to the methyl protons of N-acetyl aspartate at 2.02 ppm) arise from cerebral macromolecules without evidence for co-resonances from low molecular weight metabolites as shown by the spin-spin relaxation decays of these resonances. In addition to these NMR signals, peaks at 0.9 and 1.7 ppm from macromolecules were detected. These resonances are from proteins, and we have identified the polypeptides that contributed to the 1H NMR peaks. Two proteins that were present at concentrations of 250 and 350 micrograms/g of dryed tissue showed 1H NMR spectra that resembled the macromolecular pattern in the cerebral 1H NMR spectrum. They were identified as thymosin beta 4 and histone H1, respectively. Thymosin beta 4 was present in soluble high speed cytoplasmic fraction and in P2 pellet, whereas histone H1 was detected in nuclear enriched fraction. A chemical shift-correlated two-dimensional 1H NMR spectrum of thymosin beta 4 in vitro revealed a coupling pattern that matched the macromolecule in the cerebral cortex which we have previously noted (Kauppinen R. A., Kokko, H., and Williams, S. R. (1992) J. Neurochem. 58, 967-974). On the basis of both one- and two-dimensional NMR evidence, subcellular distribution and high concentration, we assign the 1H NMR signals at 0.9, 1.22, 1.40, and 1.7 ppm in the cerebral cortex to thymosin beta 4.

Recovery of intracellular pH in cortical brain slices following anoxia studied by nuclear magnetic resonance spectroscopy: role of lactate removal, extracellular sodium and sodium/hydrogen exchange.

[31P]- and [1H]nuclear magnetic resonances recorded in an interleaved fashion were used in order to quantify high-energy phosphates, intracellular pH and lactate in cortical brain slices of the guinea-pig superfused in a CO2/HCO3(-)-buffered medium during and after anoxic insults. The volume-averaged intracellular pH and energy status of the preparation following anoxia were determined. In the presence of external Na+, intracellular pH normalized in 3 min and was significantly more alkaline from 10 to 12 min of recovery, but lactate remained elevated for 12 min of reoxygenation following anoxia. The amount of lactate removed was only 40% of the quantity of acid extruded showing operation of H+ neutralizing transmembrane mechanisms other than transport of lactic acid. Amiloride (1 or 2 mM) did not prevent the recovery of intracellular pH, but it blocked the "overshoot" of the alkalinization at 10-12 min of recovery. In a medium containing 70 mM K+, 60 mM Na+ and 0.1 mM Ca2+, the recovery of pH, but not lactate washout, was significantly delayed. Removal of external Na+ caused severe energetic failure, decreases both in oxygen uptake and in N-acetyl aspartate concentration, indicating loss of viable tissue. In Na(+)-free superfusion, lactic acidosis caused a more severe drop in intracellular pH than in the presence of Na+. Complexing of extracellular Ca2+ in the Na(+)-free medium inhibited the acidification by 0.38 pH units during anoxia which is as much as the acidification caused by lactate accumulation in the absence of Na+. In Na(+)-free medium intracellular pH recovered, however, from an anoxic level to a normoxic value in 6 min. Metabolic damage of the slice preparation induced by anoxia in the absence of Na+ was as profound in the presence as in the absence of Ca2+ showing that accumulation of Ca2+ is not the only reason for the damage. It is concluded that recovery of intracellular pH from lactic-acidosis can occur independently of energetic recovery and involves acid extrusion mechanism(s) that is(are) dependent on external Na+ and sensitive to high K+.