Daisuke Uematsu

Combined therapy with MK-801 and nimodipine for protection of ischemic brain damage.

Calcium ion can enter ischemic neurons through both receptor-operated and voltage-sensitive Ca2+channels. To attenuate this Ca2+ entry and Ca2+-induced neuronal injury, we tried a combined treatment with the noncompetitive N-methyl-D-aspartate (NMDA) antagonist, MK-801, and the dihydropyridine calcium antagonist, nimodipine, in a cat middle cerebral artery occlusion (1 hour) and reperfusion (3 hours) model. We measured changes in cytosolic free calcium, nicotinamide adenine dinucleotide/reduced nicotinamide adenine dinucleotide redox state, and blood flow in the cat cortex using a newly developed fluorometric technique with indo-1, a fluorescent intracellular Ca2+ indicator. The combined treatment, starting 5 minutes into ischemia, was effective in reducing both Ca2+ entry and histologic damage and in enhancing recovery of the electroencephalogram following reperfusion. MK-801 alone was also effective, but to a lesser extent. These data suggest that the dual blockade of Ca2+ entry using MK-801 and nimodipine may be a useful tool for protection against ischemic brain damage.

Nimodipine attenuates both increase in cytosolic free calcium and histologic damage following focal cerebral ischemia and reperfusion in cats.

To clarify the mechanism of its effect on ischemic stroke, we investigated the effect of nimodipine, a dihydropyridine calcium antagonist, on changes in cytosolic free calcium, cortical blood flow, and histologic changes following focal cerebral ischemia and reperfusion in 14 cats. Using indo-1, a fluorescent intracellular Ca2+ indicator, we simultaneously measured changes in the Ca2+ signal ratio (400:506 nm), reduced nicotinamide adenine dinucleotide fluorescence (464 nm), and reflectance (340 nm) during an ultraviolet excitation (340 nm) directly from the cat cortex in vivo. In six cats treated with vehicle only, the calcium signal ratio increased from 5 minutes after middle cerebral artery occlusion to 30 minutes into reperfusion. The elevation of cytosolic free calcium was significantly attenuated by nimodipine, which was administered by intravenous infusion in eight cats starting 5 minutes after occlusion. Nimodipine had no effect on cortical blood flow during ischemia but induced a hyperperfused state following reperfusion. Nimodipine did not modify changes in the mitochondrial oxidation-reduction state. Nimodipine proved to have beneficial effects on recovery of the electroencephalogram following reperfusion as well as on the extent of focal histologic damage. Our results suggest that nimodipine, when administered during the early stage of focal ischemia, can favorably modify the outcome of stroke by reducing the Ca2+ entry during both the ischemic and reperfusion periods.

In vivo fluorometric measurement of changes in cytosolic free calcium from the cat cortex during anoxia.

A new approach to assess the mean changes in intracellular free calcium [Ca2+]i directly from the cortex in situ is described along with the [Ca2+]i changes during nitrogen anoxia. Following incision of the dura and part of the pia-arachnoid membrane, quin2 acetoxymethyl ester, 100 μM in artificial CSF, was superfused for 60 min onto the cat cortex. A small cortical area was irradiated with ultraviolet rays (350/30 nm) and the changes in the fluorescence and reflectance were recorded microfluorometrically at 506 and 366 nm, respectively. The net change in the quin2-Ca2+ fluorescence was calculated after correction for the hemodynamic artifact and subtraction of the basal NADH change. The quin2-Ca2+ fluorescence began to increase significantly (48.0 ± 13.4 units; p < 0.05) 20 s prior to the isoelectric electrocorticogram (ECoG) and remained elevated during nitrogen anoxia. It decreased steeply 7.3 ±1.7 s prior to the recovery of the ECoG activity after the animal was reoxygenated. Thus, the changes in the intracellular free calcium preceded those of the ECoG during a reversible anoxic insult, suggesting that the increase in the [Ca2+]i might be related to the electrical failure during anoxia.

The Effect of Hyperglycemia on Intracellular Calcium in Stroke

The effect of hyperglycemia on cytosolic free calcium ([Ca2+]i) during temporary focal cerebral ischemia was investigated in cats using a fluorometric technique. The middle cerebral artery (MCA) was occluded for a period of 1 h, after which the clip was removed. In seven animals, plasma glucose was raised to 500–700 mg/dl by infusion of a 50% glucose solution starting 30 min after MCA occlusion, while eight animals were kept normoglycemic during and following occlusion. MCA occlusion induced a significant, but identical, elevation of the [Ca2+]i signal ratio (400/506 nm) in both the normoglycemic group (from 1.40 to 1.97 ± 0.34, p < 0.01) and in the hyperglycemic group (from 1.40 to 2.00 ± 0.53, p < 0.01) at the end of the occlusion. Between 10 and 30 min after reopening, the [Ca2+]i signal ratio decreased to control levels in the normoglycemic group (1.40 ± 0.11 and 1.36 ± 0.08 at 10 and 30 min after reopening, respectively), but remained elevated in the hyperglycemic group (1.69 ± 0.18 and 1.65 ± 0.21 at 10 and 30 min after reopening, respectively). There was a statistically significant difference between the two groups (p < 0.01). These data suggest that hyperglycemia may be harmful to calcium recovery during the early recirculation period following focal cerebral ischemia.

Effect of superoxide dismutase on intracellular calcium in stroke.

To clarify the relationship between calcium metabolism and free radical damage during the reperfusion period following ischemia, we investigated the effect of superoxide dismutase (SOD) on changes in cytosolic free calcium, cortical blood flow, and histologic changes following focal cerebral ischemia and reperfusion in 12 cats. Using indo-1, a fluorescent intracellular Ca2+ indicator, we simultaneously measured changes in the Ca2 + signal ratio (400:500 nm), NADH signal (464 nm), and reflectance (340 nm) during ultraviolet excitation (340 nm) directly from the cortex in vivo. The middle cerebral artery (MCA) was occluded for 1 h; only cats in which the EEG amplitude was depressed to <10% of control during the occlusion were entered into the study. Starting 2 min prior to release of the occlusion and continuing for 4 min, SOD (10,000 U/kg) was slowly infused in six cats, while in six cats, the vehicle only was infused. During MCA occlusion, the Ca2+ signal ratio increased significantly in both groups with no significant difference between the groups. During reperfusion, the Ca2+ signal ratio remained at a high level in the vehicle-treated group, while in the SOD-treated group, the Ca2+ signal ratio decreased. There was a statistically significant difference between the two groups at 10, 20, and 30 min after reperfusion (p < 0.01). The histologically damaged area in the SOD-treated group was significantly smaller than that in the vehicle-treated group (p < 0.01). These data suggest that the histoprotective action of SOD may be due to its ability to attenuate increases in intracellular calcium during the recirculation period following focal cerebral ischemia.

Mechanism Underlying Protective Effect of MK-801 against NMDA-Induced Neuronal Injury in vivo

The effects of the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 and the dihydropyridine calcium antagonist nimodipine on NMDA-induced phenomena were investigated using an in vivo fluorometric technique with indo-1. Indo-1, a fluorescent cytosolic free calcium ([Ca2+]i) indicator, was loaded into the cat cortex approximately 500 μm in depth by super-fusion with the membrane-permeant indo-1 acetoxymethyl ester (indo-1-AM). Changes in [Ca2+]i signals (400 and 506 nm) and reduced nicotinamide adenine dinucleotide (NADH) fluorescence (464 nm) were simultaneously measured directly from the cortex during ultraviolet excitation (340 nm). Superfusion of 100 μM NMDA over the exposed cortex induced an elevation of the [Ca2+]i signal ratio (400/506 nm), biphasic changes in NAD/NADH redox state (initial oxidation followed by progressive reduction), and characteristic changes in the EEG (abrupt depression in amplitude followed by an excitatory pattern of 18–22 Hz poly spikes or sharp waves). These changes were completely blocked by treatment with MK-801 and reduced by nimodipine. The mechanism underlying the protective effects of systemically administered MK-801 on the NMDA-induced neuronal injury was verified in vivo.

Cytosolic free calcium, NAD/NADH redox state and hemodynamic changes in the cat cortex during severe hypoglycemia

Summary Using indo-1, a fluorescent Ca2+ indicator, in vivo fluorometric measurements were made of changes in cytosolic free Ca2+, NAD/NADH redox state, and hemodynamics directly from the cat cortex during and after severe insulin-induced hypoglycemia. Cytosolic free Ca2+ started to increase when the EEG became isoelectric, remained at a significantly high level (p < 0.05) during the period of isoelectric EEG (IEEG), and recovered to the control level 6 min following an intravenous infusion of glucose. The NAD/NADH redox state oxidized significantly during IEEG and then recovered rapidly to the control level after the glucose infusion. Local cortical blood volume (LCBV) increased gradually during the progression of hypoglycemia, reaching the maximal level (146 ± 7%) at the end of IEEG, and then started to recover. The mean transit time (MTT) through the cortical microcirculation was shortened during the IEEG (control: 3.84 ± 0.41 s versus IEEG: 2.73 ± 0.17 s, p < 0.05), whereas it was prolonged during the 30-min recovery period (5.68 ± 0.58 s, p < 0.05). Local cortical blood flow calculated from the LCBV and MTT showed a twofold increase 5 min into IEEG (201 ± 27% of control, p < 0.05), recovered 15 min into the recovery period, and then decreased to 77% of control (p < 0.05) by 30 min. The data support the hypothesis that hypoglycemic brain damage might be mediated by an elevation of cytosolic free calcium.

Alterations in cytosolic free calcium in the cat cortex during bicuculline-induced epilepsy

Influx of calcium ion (Ca++) into the neurons has recently been implicated in the generation of seizure activity. Utilizing indo-1, a fluorescent Ca++ indicator, changes in cytosolic free calcium ([Ca++]i), NAD/NADH redox state and hemodynamics were simultaneously measured in vivo from the cat cortex during bicuculline-induced seizure activity. A ratio of indo-1-Ca++ fluorescence at 400 and 506 nm during ultraviolet excitation (340 nm) was utilized as a measure of changes in [Ca++]i. Alterations in the NAD/NADH redox state and local cortical blood volume (1CBV) were assessed at 464 nm and 340 nm, respectively. Local cortical blood flow (1CBF) was calculated from 1CBV and mean transit time determined from cortical hemodilution curves. Electroencephalogram (EEG) was monitored from the same cortical region as the optical measurements. The [Ca++]i signal ratio started to increase 19 +/- 2 sec prior to the onset of seizure activity on the EEG and remained elevated until the activity was suppressed by an intravenous administration of diazepam (2 mg/kg). The early increase in [Ca++]i is presumably due to a synaptic Ca++ entry associated with facilitated excitatory neurotransmission. The NAD/NADH redox state became oxidized during the seizure activity and started to recover as the EEG activity was suppressed. The 1CBV and 1CBF increased by 17 +/- 8% and 68 +/- 16%, respectively, 10 min into the seizure activity. This study provides direct in vivo evidence suggesting a possible role of calcium entry into the neurons in the epileptogenesis.

Cytosolic free calcium and NAD/NADH redox state in the cat cortex during in vivo activation of NMDA receptors

Activation of the N-methyl-D-aspartate (NMDA) receptors and the concomitant Ca2+ entry have been implicated in neuronal injury in a variety of pathological states. The effects of extracellular Mg2+ concentrations and D,L-2-amino-5-phosphonovaleric acid (APV), a competitive NMDA receptor antagonist on the NMDA-induced responses were investigated in vivo. In vivo fluorometric measurements were made of changes in cytosolic free Ca2+ ([Ca2+]i) and NADH fluorescence directly from the cat cortex using indo-1, a fluorescent Ca2+ indicator. Changes in [Ca2+]i were assessed utilizing the ratio of indo-1 emission at two wavelengths (400 and 506 nm) during excitation with ultraviolet light (340 nm). Application of 100 microM NMDA to the cortex produced a significant increase in the [Ca2+]i signal ratio at physiological concentrations of Mg2+ (1.2 mM). This increase was enhanced in the absence of Mg2+ and was completely blocked either at 5 mM Mg2+ or in the presence of 50 microM APV. The NAD/NADH redox state was initially oxidized, which was also blocked by either high Mg2+ or APV. The application of NMDA elicited characteristic electroencephalogram (EEG) changes consisting of a marked reduction in amplitude and regular spikes (17-20 Hz). These EEG changes did not appear in the presence of APV. In addition to NMDA receptor antagonists, the level of extracellular Mg2+ is a potent physiological modulator of the NMDA response.

Randomized double-blind placebo-controlled multicenter trial of Yokukansan for neuropsychiatric symptoms in Alzheimer's disease.

Yokukansan (YKS), a traditional herbal medicine, has been used to treat behavioral and psychological symptoms of dementia (BPSD). The present study is the first double‐blind, randomized, placebo‐controlled trial to determine the efficacy and safety of YKS for the treatment of BPSD in Alzheimers disease (AD).