Takao Asano

Magnetic resonance imaging shows delayed ischemic striatal neurodegeneration.

Brief focal ischemia leading to temporary neurological deficits induces delayed hyperintensity on T1‐weighted magnetic resonance imaging (MRI) in the striatum of humans and rats. The T1 hyperintensity may stem from biochemical alterations including manganese (Mn) accumulation after ischemia. To clarify the significance of this MRI modification, we investigated the changes in the dorsolateral striatum of rats from 4 hours through 16 weeks after a 15‐minute period of middle cerebral artery occlusion (MCAO), for MRI changes, Mn concentration, neuronal number, reactivities of astrocytes and microglia/macrophages, mitochondrial Mn‐superoxide dismutase (Mn‐SOD), glutamine synthetase (GS), and amyloid precursor protein. The cognitive and behavioral studies were performed in patients and rats and compared with striatal T1 hyperintensity to show whether alteration in brain function correlated with MRI and histological changes. The T1‐weighted MRI signal intensity of the dorsolateral striatum increased from 5 days to 4 weeks after 15‐minute MCAO, and subsequently decreased until 16 weeks. The Mn concentration of the dorsolateral striatum increased after ischemia in concert with induction of Mn‐SOD and GS in reactive astrocytes. The neuronal survival ratio in the dorsolateral striatum decreased significantly from 4 hours through 16 weeks, accompanied by extracellular amyloid precursor protein accumulation and chronic glial/inflammatory responses. The patients and rats with neuroradiological striatal degeneration had late‐onset cognitive and/or behavioral declines after brief focal ischemia. This study suggests that (1) the hyperintensity on T1‐weighted MRI after mild ischemia may involve tissue Mn accumulation accompanied by Mn‐SOD and GS induction in reactive astrocytes, (2) the MRI changes correspond to striatal neurodegeneration with a chronic inflammatory response and signs of oxidative stress, and (3) the subjects with these MRI changes are at risk for showing a late impairment of brain function even though the transient ischemia is followed by total neurological recovery. Ann Neurol 2003;54:732–747

Participation of Lipoxygenase Products from Arachidonic Acid in the Pathogenesis of Cerebral Vasospasm

Abstract: To examine the possible involvement of lipoxygenase products from arachidonic acid in the pathogenesis of delayed vasospasm after subarachnoid hemorrhage (SAH), we measured the contents of hydroxyeicosatetraenoic acids (HETEs) in the subarachnoid clot, the cerebrospinal fluid, and the basilar artery, using the canine “two‐hemorrhage” model. Lipoxygenase activity in the subarachnoid clot and the basilar artery was measured, ex vivo, using samples obtained 7 days after SAH. For a quantitative analysis of HETEs, each sample was homogenized with either ice‐cold saline or methanol. The lipid extract was then submitted to reverse‐phase HPLC. The identity of each HETE was further confirmed using straight‐phase HPLC and gas chromatography‐mass spectrometry. When the basilar artery was homogenized with ice‐cold saline, a significant increase in the 5‐HETE content was observed on SAH day 8. However, when the artery was homogenized with methanol, HETEs were not detected. In the case of incubation in the presence of arachidonic acid and calcium ionophore A23187, the 5‐lipoxygenase activity was remarkably increased in the basilar artery exposed to SAH, compared to that of normal dogs. The subarachnoid clot contained a significant amount of 12‐HETE (average 1.8 nmol/g wet weight) from day 2 to day 8. The administration of 1,2‐bis(nicotinamido)propane significantly ameliorated vasospasm in the two‐hemorrhage model, simultaneously inhibiting the 5‐lipoxygenase activity of the basilar artery. Our observations show that the activities of 12‐ and 5‐lipoxygenases are significantly increased after SAH in the subarachnoid clot and the basilar artery, respectively. The generated hydroperoxides and/or leukotrienes may play some pertinent role in the occurrence of delayed vasospasm.

Role of Nitric Oxide in Pathogenesis Underlying Ischemic Cerebral Damage

Abstract1. Based upon the intriguing report that nitric oxide synthase (NOS) inhibitor dose-dependently reverses N-methyl-D-aspartate (NMDA)-induced neurotoxicity observed in primary cortical cell cultures, many laboratories have investigated whether NOS inhibition is beneficial as a treatment for cerebral ischemia.2. Although the results are variable, it is likely thought that nitric oxide plays a key role in pathomechanism underlying ischemic brain damage.3. We review the experimental studies on effects of NOS inhibition on cerebral ischemia and measuring nitric oxide produced in the brain subjected to cerebral ischemia.4. Finally, the possibility of NOS inhibitors as a therapeutical tool is discussed.

Possible role of the superoxide anion in the development of neuronal tolerance following ischaemic preconditioning in rats.

There is a large body of evidence that reactive oxygen species play a major role in the pathogenesis of ischaemic brain damage. On the other hand, it has recently been suggested that superoxide anions participate in the development of neuronal tolerance against lethal ischaemia following ischaemic preconditioning (PC). The present study aimed to examine whether or not the intravenous administration of human recombinant Cu/Zn superoxide dismutase (hr SOD) prior to PC would affect the subsequent development of neuronal tolerance. Animals were randomly assigned to the following three groups: group 1, sham PC treated with vehicle; group 2, PC treated with hr SOD and group 3, PC treated with vehicle. For PC, 10 min occlusion of the middle cerebral artery (MCA) by a modified intraluminal suture method was followed by 60 min recirculation and this procedure was successively repeated three times. The procedures were similar for sham PC except that the MCA was kept unoccluded. Just prior to PC or sham PC, a bolus of hr SOD (6 × 103 IU/2 ml/kg) was administered intravenously. Seventy‐two hours thereafter, rats were subjected to lethal ischaemia, i.e. MCA occlusion for 100 min followed by recirculation for 48 h. The infarct area and volume were assessed with the 2,3,5‐triphenyltetrazolium stain. A significant difference in the infarct volume was revealed between the sham PC/vehicle and the PC/vehicle groups (total and cortex P < 0.01; striatum P < 0.05), showing that PC induced a marked neuronal tolerance against lethal ischaemia. The infarct volume in the PC/SOD group was close to that in the sham PC/vehicle group, being significantly greater than that in the PC/vehicle group (total and cortex P < 0.01) and showing that the administration of hr SOD suppressed the development of neuronal tolerance induced by PC. In a parallel experiment, expression of 72‐kDa heat‐shock protein (hsp 72) at 72 h after PC was considerably reduced in rats treated with hr SOD compared with those treated with vehicle. These results suggest that superoxide anions intraluminally generated within cerebral microvessels participate in the development of neuronal tolerance as well as the induction of hsp 72 following PC.

Attenuation of a delayed increase in the extracellular glutamate level in the peri-infarct area following focal cerebral ischemia by a novel agent ONO-2506.

A novel agent, ONO-2506 [(R)-(-)-2-propyloctanoic acid, ONO Pharmaceutical Co. Ltd.] was previously shown to mitigate delayed infarct expansion through inhibition of the enhanced production of S-100beta, while inducing a prompt symptomatic improvement that attained a significant level as early as 24h after drug administration. To elucidate the mechanism underlying the prompt symptomatic improvement, the present study aimed to examine whether ONO-2506 modulates the level of extracellular glutamate ([Glu]e) in the rat subjected to transient middle cerebral artery occlusion (tMCAO). In this model, it had been shown that ONO-2506 reduces the infarct volume, improves the neurological deficits, and enhances the mRNA expression of glial glutamate transporters (GLT-1 and GLAST). The [Glu]e levels in the ischemic cortices were continuously measured using intracerebral microdialysis. The alterations in the [Glu]e levels in the sham-operated and tMCAO-operated groups with or without drug administration were compared. In the tMCAO groups, the [Glu]e level increased during tMCAO to a similar extent, returned to normal on reperfusion, and increased again around 5h. In the saline-treated group, however, the [Glu]e level further increased from 15 h on to reach about 280% of the normal level at 24h. This secondary increase in the [Glu]e level in the late phase of reperfusion was prevented by ONO-2506. The intracerebral infusion of glutamate transporter inhibitor, l-trans-pyrrolidine-2,4-dicarboxylic acid, at 24h after tMCAO induced an increase in the [Glu]e level, which was marked in both the sham-operated and ONO-2506-treated groups, but much less pronounced in the saline-treated group. The above results suggest that functional modulation of activated astrocytes by pharmacological agents like ONO-2506 may inhibit the secondary rise of [Glu]e level in the late phase of reperfusion, leading to amelioration of delayed infarct expansion and neurological deficits.

Effect of indomethacin and a free radical scavenger on cerebral blood flow and edema after cerebral artery occlusion in cats.

Using the middle cerebral artery occlusion model in cats, we evaluated the possible role of the cyclooxygenase pathway in alterations of local cerebral blood flow and the development of cortical edema following prolonged ischemia or recirculation. We divided 57 cats into three groups, and each cat received saline (control), indomethacin, or the free radical scavenger ONO-3144. Each group was subdivided into prolonged ischemia (4 hours of occlusion: PI) and recirculation (2 hours of occlusion followed by 2 hours of recirculation: RC) subgroups. We compared local cerebral blood flow and cortical specific gravity between the PI and RC subgroups of the control and drug-treated groups. In the PI subgroup, indomethacin did not influence the time course of local cerebral blood flow but significantly worsened the decrease in cortical specific gravity. On the other hand, indomethacin significantly improved postischemic hypoperfusion and ameliorated the decrease in cortical specific gravity in the RC subgroup. The effects of ONO-3144 were similar to those of indomethacin, except that ONO-3144 did not affect cortical specific gravity in the PI subgroup. Indomethacin inhibits cyclooxygenase activity, whereas ONO-3144 scavenges the oxygen-centered radical released in the conversion of prostaglandin G2 to prostaglandin H2. Thus, prostaglandins do not seem to play a major role in the occurrence of brain edema due to prolonged regional ischemia. By contrast, oxygen-centered radicals released from the cyclooxygenase pathway appear to be at least partially responsible for the occurrence of recirculation-induced edema and postischemic hypoperfusion.

Activation of the arachidonate 5-lipoxygenase pathway in the canine basilar artery after experimental subarachnoidal hemorrhage.

Abstract: Severe cerebral vasospasm as confirmed by an‐giography was induced in dogs by injection of autologous blood into the cisterna magna, and the resultant leukotriene formation in the isolated basilar artery was examined. When stimulated with calcium ionophore (A 23187), the arteries of the treated animals produced a significant amount of leu‐kotrienes B4 (85 ± 12 pmol/mg protein, n = 3) and C4 (72 ± 14 pmol/mg), in addition to 5(S)‐hydroxy‐6,8,l 1,14‐ei‐cosatetraenoic acid. Structural elucidations of these metabolites were performed by radioimmunoassays or gas chro‐matography‐mass spectrometry, following purification with HPLC. The artery of the untreated dog produced none of these compounds from either exogenous or endogenous ar‐achidonic acid, under stimulation with the calcium ionophore. However, the homogenates from both animals converted exogenous leukotriene A4 to leukotrienes B4 and C4. These observations suggest that the normal basilar artery contains no detectable amount of 5‐lipoxygenase, and that a prominent activation of this enzyme occurred (2.1 nmol 5‐HETE/5 min/mg of protein) after subarachnoidal hemorrhage. The observation that fatty acid hydroperoxides stimulated the 5‐lipoxygenase activity indicates a possible role of lipid peroxides in the development of vasospasm.

Intraluminal increase of superoxide anion following transient focal cerebral ischemia in rats

Using a modification of Karnovskys Mn2+/diaminobenzizine (DAB) technique, we examined the production of superoxide anion (.O-2) in the vascular lumen following transient occlusion and reperfusion of the left middle cerebral artery (MCA) in Sprague-Dawley rats. The MCA was occluded for 2 h using an intraluminal suture method. Zero, 15, 30, and 60 min after reperfusion, animals were perfused transcardially with buffer containing Mn2+ and DAB, and brain samples were prepared for light and electron microscopic examination. The amber reaction deposits of.O-2 were observable to the naked eye along the major cerebral vessels of the ischemic hemisphere after each reperfusion period. Upon microscopic examination the deposits were revealed to be within arterial, capillary, and venular lumen. The amount of reaction deposits in the ischemic hemisphere corresponded to the duration of reperfusion. The formation of.O-2 was suppressed when the perfusate contained superoxide dismutase and when either Mn2+ or DAB was omitted, confirming that the reaction products produced are due to the enhanced production of.O-2. These results show that there is a progressive increase in intraluminal. O-2 during reperfusion following an ischemic insult which may participate in the aggravation of cerebral damage.

The role of free radicals and eicosanoids in the pathogenetic mechanism underlying ischemic brain edema

Results of our consecutive study on the pathogenic mechanism underlying ischemic brain edema are summarized in this paper. Pertinent findings are as follows: (1) there is a close correlation between the influxes of water and sodium following ischemia; (2) the edema fluid can be regarded as the ultrafiltrate of serum; (3) there is a significant increase in the brain content of HETEs following ischemia; (4) the lipoxygenase activity of brain microvessels is increased following ischemia; (5) the lipoxygenase activity as well as the Na+, K+-ATPase activity of brain microvessels are enhanced by a hydroperoxide, 15-HPETE; (6) inhibition of Na+, K+-ATPase of brain microvessels by intraarterial infusion of ouabain resulted in a significant decrease in edema formation; and (7) not the cyclooxygenase, but the lipoxygenase pathway seems to be involved in the enhancement of microvessel Na+, K+-ATPase. Lipoxygenase(s) and Na+-K+-ATPase of brain microvessels, the activities of which are enhanced by an increased level of free radicals and/or hydroperoxides, may play a significant role in the occurrence of ischemic brain edema.

Functional image of dynamic computed tomography for the evaluation of cerebral hemodynamics.

We report the usefulness of functional image of dynamic computed tomography as a technique for evaluating cerebral hemodynamics. Although the technique itself has been reported, the advance of computer technology has made it possible to obtain high-resolution functional images within only a few minutes. We conducted 70 examinations on 57 patients with cerebral ischemia and correlated the findings with clinical outcome. Those patients whose abnormalities were detected on all corrected mean transit time, time to peak, and peak value images developed massive cortical infarcts. On the other hand, patients with abnormalities detected only on the corrected mean transit time image had only partial low-density lesions on follow-up computed tomograms. Patients with abnormalities detected only on the time to peak image suffered repeated transient ischemic attacks, but follow-up computed tomograms showed no low-density lesion in most cases. Single-photon emission computed tomography with N-isopropyl-p-(123I)iodoamphetamine performed at the time of functional image of dynamic computed tomography showed a high concordance of the findings in many cases, especially those with hyperacute-stage cerebral ischemia, in which the concordance rate was 90.5% (19 of 21). Comparing images constructed from different parameters, functional image of dynamic computed tomography can delineate other than hemodynamic factors, such as extent of the vascular bed or the degree of collateral circulation. Thus, functional image of dynamic computed tomography is a potentially important and useful technique for the further analysis of cerebral hemodynamics.