Oxygenation of the cortex of the brain of cats during occlusion of the middle cerebral artery and reperfusion.
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Animals↗
Biomedical subjects
J H Greenberg
Publications and source records attributed to J H Greenberg.
Acute improvement in histological outcome by MK-801 following focal cerebral ischemia and reperfusion in the cat independent of blood flow changes.
The present study reports on the acute effects of MK-801 on the histopathological outcome and blood flow changes during focal cerebral ischemia and reperfusion. In addition, acute changes in the EEG and blood pressure are also reported. In 16 halothane-anesthetized cats, the left middle cerebral artery (MCA) was occluded for 2 h followed by 4 h of reperfusion. Thirty minutes after the onset of ischemia, eight animals were treated with 1 mg/kg of MK-801, while eight animals received saline. Blood flow from the peripheral MCA territory was measured with H2 clearance. There was a comparable reduction in blood flow (down to 20% of control) in the ischemic gyri of the two groups followed by a partial recovery after recirculation. There was a similar decrease in the EEG amplitude over the ischemic central MCA territory in the treated and the untreated group. Treatment with MK-801 induced a burst suppression in the EEG and a transient drop (11.4 +/- 6.5 mm Hg) in the mean arterial pressure. The volume of early ischemic damage decreased by one-third in the MK-801-treated group compared to the untreated one, both in the total hemisphere (from 29 +/- 10 to 20 +/- 5%) and in the hemispheric cortex (range 36 +/- 8 to 24 +/- 13%). A major fraction of this improvement was localized to the middle and posterior parietal (mainly perifocal) regions of the MCA territory. These results show that in our model, MK-801 improves histopathological outcome despite the lack of apparent effect on the cortical blood flow, and an adverse effect on the systemic blood pressure. This is the first report that describes data on a reproducible model of reperfusion after temporary occlusion of the MCA in a cat, extending the findings of the Glasgow group, who observed similar neuroprotection in models of permanent MCA occlusion.
Animals↗
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 less than 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 less than 0.01). The histologically damaged area in the SOD-treated group was significantly smaller than that in the vehicle-treated group (p less than 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.
Animals↗
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 less than 0.01) and in the hyperglycemic group (from 1.40 to 2.00 +/- 0.53, p less than 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 less than 0.01). These data suggest that hyperglycemia may be harmful to calcium recovery during the early recirculation period following focal cerebral ischemia.
Animals↗
Effect of ischemia and reperfusion on lambda of the lumped constant of the [14C]deoxyglucose technique.
We measured the parameter lambda, which is the ratio of the distribution spaces of 2-deoxy-D-glucose (DG) and glucose in the brain, in a model of focal cerebral ischemia in the cat. lambda is the parameter in the lumped constant of the [14C]DG technique most susceptible to changes in ischemia. Cats were subjected to occlusion of the middle cerebral artery for a period of 2 h. During the last 60 min of occlusion, [14C]DG was infused in a programmed fashion so as to obtain a stable arterial blood [14C]DG concentration. The brain was funnel-frozen to preserve tissue metabolites and the frozen brain was sampled regionally (4 to 7-mg samples) for local concentrations of glucose, ATP, phosphocreatine (PCr), and lactate. In a separate series of cats, the infusion of [14C]DG was started after 2 h of occlusion and 3 h of recirculation. In both series, lambda declined slightly for increased levels of tissue glucose and increased appreciably as tissue glucose decreased. A similar relationship was observed between lambda and ATP and PCr, although the correlation was not as clear. Since lambda, and hence the lumped constant, increases in ischemia as well as in postischemic tissue, it is important to measure tissue glucose concentration if quantitative values of local cerebral glucose metabolism are desired in this condition.
Adenosine Triphosphate↗
Effects of NG-nitro-L-arginine and L-arginine on regional cerebral blood flow in the cat.
1. We studied the effects of NG-nitro-L-arginine (NOLA), a potent inhibitor of the L-arginine-nitric oxide pathway, and L-arginine, the precursor of nitric oxide, on regional cerebral blood flow, electrocortical activity and ex vivo cerebrovascular reactivity in the cat. Flow was measured via radiolabelled microspheres, and vascular responses were studied by measuring isometric tension of isolated middle cerebral arterial rings. 2. NOLA (30 mg kg-1 bolus followed by 1 mg kg-1 min-1 infusion) caused an approximately 40 mmHg elevation in the mean arterial blood pressure, a regionally heterogenous increase of the regional cerebrovascular resistance and a decrease in the regional cerebral blood flow 15 and 40 min after the start of its administration. In contrast L-arginine (30 mg kg-1 bolus followed by 10 mg kg-1 min-1 infusion) did not alter blood pressure, cerebrovascular resistance nor regional cerebral blood flow 15 min after the start of its administration. The NOLA-induced changes in tissue flow were the most pronounced in the cerebellum, pituitary and medulla oblongata, whereas there was no decrease in the flow of the cortex and white matter. 3. NOLA caused characteristic changes in total fronto-occipital EEG power and in power spectra which were unlikely to have been due to cerebral ischaemia. In addition, the ex vivo reactivity of the middle cerebral arteries showed signs of impaired endothelial nitric oxide synthesis: there were enhanced noradrenaline-induced contractions and N-ethoxycarbonyl-3-morpholino-sydnonimine (SIN-1)-induced relaxations and markedly attenuated acetylcholine- and ATP-induced relaxations after NOLA treatment. 4. The present data indicate that resting cerebral blood flow and cerebrovascular resistance are regulated by nitric oxide derived from L-arginine in a regionally heterogenous way and that exogenous L-arginine availability is not a limiting factor in this nitric oxide generation. Possibly, both the vascular endothelium and the neurons contribute to this basal nitric oxide release.
Animals↗
Peripheral neuropathy after chronic endoneurial ischemia.
We have developed a method for producing chronic regional nerve ischemia in rats by creating proximal limb arteriovenous shunts. This procedure results in a 50 to 75% reduction in endoneurial blood flow within the distal sciatic nerve as measured by the iodoantipyrine method. Nerve conduction velocities in sciatic nerves ipsilateral to the shunt fell by 25 to 30% within 2 weeks after creation of the shunt and did not recover for up to 10 months after the procedure. Morphological studies of the ischemic nerves showed structural abnormalities at nodes of Ranvier and mild axonal atrophy. Neither segmental demyelination nor axonal degeneration were evident. These results indicate that reduced endoneurial blood flow, insufficient to cause infarction, may result in measurable functional and morphological abnormalities in peripheral nerves.
Action Potentials↗
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 microns in depth by superfusion with the membrane-permeant indo-1 acetoxy-methyl 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 microM 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 polyspikes 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.
Animals↗
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.
Animals↗
Local cerebral tissue glucose utilization in graded arterial hemorrhagic hypotension, studied by the 14C-2-deoxyglucose method in rats.
The effect of hemorrhagic arterial hypotension on local cerebral glucose metabolism was studied on 33 rats. The mean arterial pressure was set with the aid of a reservoir at 80, 60, 50 and 40 mmHg pressures. Local cerebral glucose utilization was measured with the 14C-2-D-deoxyglucose accumulation autoradiographic technique. Local glucose consumption decreased somewhat in cortical structures when mean arterial pressure was reduced to 60-50 mmHg. Further decrease in mean arterial pressure to 50-40 mmHg caused inhomogeneity of tissue metabolism. Columns and patches of high glucose consumption interchanged with areas of very low glucose consumption in most telencephalic and cerebellar gray matter structures. Brain stem and white matter structures seem to be less sensitive to decreased mean arterial pressure in the range studied. We found a decrease in glucose utilization rather than an increase with decreasing mean arterial pressure down to 60-50 mmHg (in the range of the autoregulation of cerebral circulation). This finding makes it improbable that autoregulation would be connected with elevated anaerobic metabolism of the tissue. Patchy areas and columns of high glucose consumption found at 50-40 mmHg in all probability reflect areas of increased anaerobic metabolism of glucose. Here, circulation was not enough to transport adequate quantity of oxygen to the tissue, but still it transported relative large amount of glucose. Columns and patches of very low glucose consumption should reflect areas, where circulation was inadequate to transport both enough glucose and oxygen.
Animals↗
Intracellular calcium and pathophysiological changes in cerebral ischemia.
The role of calcium as a mediator in neuronal death during ischemia is now quite strong. Evidence supporting this link included studies in cell cultures, measurements of calcium accumulation in the mitochondria during ischemia as well as direct measurements of shifts in extracellular calcium using microelectrodes. Since high concentrations of intracellular free calcium have been hypothesized to lead to neuronal damage, direct in vivo measurements of this parameter in ischemia are important. The studies outlined demonstrate that changes in intracellular free calcium occur in focal ischemia and describe the time course of these changes. They indicate that cellular damage can be attenuated by the use of agents that block calcium channels (both voltage-sensitive and receptor-operated) and support the concept that these agents owe their beneficial effects to their ability to reduce the accumulation of intracellular calcium.
Animals↗
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.
Animals↗
Xenon-enhanced computed tomography compared with [14C]iodoantipyrine for normal and low cerebral blood flow states in baboons.
The correlation between the acute, invasive diffusible [14C]iodoantipyrine technique for cerebral blood flow and the noninvasive xenon-enhanced computed tomographic method has been assessed by simultaneous measurements in the baboon. Blood flows in small tissue volumes (about 0.125 cm3) were directly compared in normal and low flow states. These studies demonstrate a statistically significant association between the two methods (p less than 0.001). Similar correlations were obtained by both the Kendall (tau) and the Spearman (r) methods (r = 0.67 to 0.92, n greater than or equal to 19 for each study). The problems and limitations of such correlations are discussed.
Animals↗
Local cerebral blood flow during and after bilateral carotid artery occlusion in unanesthetized gerbils.
Using [14C]iodoantipyrine autoradiography, we measured regional cerebral blood flow in unanesthetized gerbils subjected to 2 (n = 5) or 30 (n = 6) minutes of bilateral carotid artery occlusion or 5 (n = 6), 30 (n = 6), or 120 (n = 5) minutes of reflow after 30 minutes of occlusion. Blood pressure, respiratory rate, and blood gases were recorded, and these and other gerbils were evaluated with periodic neurologic examinations. Blood flow to structures above the level of the diencephalon ceased almost totally during occlusion. The lateral thalamus, the rostral three quarters of the hypothalamus, and the superior colliculi were also markedly ischemic. Blood flow to the brainstem and cerebellum was only slightly affected. After release of the occlusion, blood flow was restored in some of the affected areas but to levels somewhat below that in eight sham-operated gerbils. In several areas, principally column-shaped areas in the cortex as well as patchy areas in other structures, blood flow did not recover. This inhomogeneous blood flow distribution lasted at least 30 minutes after release of the occlusion. Thereafter, the inhomogeneity slowly disappeared in such a manner that blood flow to originally well reperfused areas appeared to decrease while that to poorly reperfused areas increased. During reflow, blood flow in the brainstem and cerebellum slowly and continuously decreased. We show that there is an early no-reflow phenomenon that is inhomogeneous and appears to be of vascular origin and lasts approximately 30 minutes after release of the occlusion.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Cytosolic free calcium during focal cerebral ischemia and the effects of nimodipine on calcium and histologic damage.
The role of calcium as a mediator in neuronal death during ischemia is now quite strong. Evidence supporting this link includes studies in cell cultures and measurements of calcium accumulation in the mitochondria during ischemia, as well as direct measurements of shifts in extracellular calcium using microelectrodes. Since it is dangerously high concentrations of the intracellular free calcium that have been hypothesized to lead to neuronal damage, direct in vivo measurements of this parameter in ischemia are important. A technique for the measurement of intracellular free calcium is described, along with data from studies that dramatically demonstrate the time course of changes in intracellular free calcium induced by focal ischemia. Additional data are also presented that indicate that cellular damage can be attenuated by the use of agents that block calcium channels (nimodipine, which blocks voltage-sensitive calcium channels, and MK-801, which blocks receptor-operated channels) and support the concept that these agents owe their beneficial effects to their ability to reduce the accumulation of intracellular calcium.
Animals↗
Validation studies of iodine-123-iodoamphetamine as a cerebral blood flow tracer using emission tomography.
We studied the radioisotope iodine-123-iodoamphetamine ([123I]IMP) and its performance in single-photon emission computed tomographic (SPECT) studies of cerebral blood flow (CBF). In seven normal volunteers, IMP/SPECT CBF measurements were calculated using a two-compartment model and were compared with the results of CBF measurements utilizing (O15)-H2O and positron emission tomography (PET). Calculated mean PET CBF was 57.6 ml/100 g/min while the SPECT CBF value was 47.3 ml/100 g/min. The response of IMP/SPECT CBF to alterations in arterial PaCO2 was studied in hypo-, eu- and hypercarbic subjects. SPECT CBF values showed a reactivity of 1.03 ml/100 g/min per mmHg PaCO2 change. These results show that the IMP/SPECT CBF technique may be used for quantitative imaging of CBF in man. They provide further support for IMP as a CBF tracer.
Amphetamines↗
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.
2-Amino-5-phosphonovalerate↗
Direct evidence for calcium-induced ischemic and reperfusion injury.
Changes in cytosolic free calcium [( Ca2+]i) in the cat cortex were measured in vivo by indo-1 fluorometry during cerebral ischemia and reperfusion and were correlated to the histopathological ischemic changes. These changes were most pronounced in stroke cases with an increase in [Ca2+]i throughout the ischemic and reperfusion periods. Cases without a [Ca2+]i increase showed no histopathological change in the cortical gyrus in which [Ca2+]i was measured. The data support the hypothesis that an increase in [Ca2+]i during cerebral ischemia and reperfusion leads to neuronal damage.
Animals↗