Narito Tateishi

Astrocytic Activation and Delayed Infarct Expansion after Permanent Focal Ischemia in Rats. Part II: Suppression of Astrocytic Activation by a Novel Agent (R)-(−)-2-propyloctanoic acid (ONO-2506) Leads to Mitigation of Delayed Infarct Expansion and Early Improvement of Neurologic Deficits

A novel agent, (R)-(−)-2-propyloctanoic acid (ONO-2506), has a unique property in that it modulates functions of activated cultured astrocytes, including pronounced inhibition of S-100β synthesis. The present study examined whether administration of this agent would mitigate the delayed expansion of infarct volume and the neurologic deficits after permanent middle cerebral artery occlusion (pMCAO) in rats. Daily intravenous administration of ONO-2506 (10 mg/kg) abolished the delayed infarct expansion between 24 and 168 hours after pMCAO, whereas the acute infarct expansion until 24 hours was unaffected. The agent significantly reduced the expression of S-100β and glial fibrillary acidic protein in the activated astrocytes and the number of terminal deoxynucleotidyl transferase-mediated 2-deoxyuridine 5-triphosphate-biotin nick end labeling-positive cells in the periinfarct area. The neurologic deficits were significantly improved, compared with the vehicle-treated groups, as early as 24 hours after the initial administration of ONO-2506. The agent had a wide therapeutic time window of 0 to 48 hours after pMCAO. These results indicate that because of the pharmacologic modulation of astrocytic activation induced by ONO-2506, symptoms can regress whereas delayed expansion of the lesion is arrested. Pharmacologic modulation of astrocytic activation may confer a novel therapeutic strategy against stroke.

Astrocytic Activation and Delayed Infarct Expansion after Permanent Focal Ischemia in Rats. Part I: Enhanced Astrocytic Synthesis of S-100β in the Periinfarct Area Precedes Delayed Infarct Expansion

An astrocytic protein S-100β enhances the expression of inducible nitric oxide synthase in cultured astrocytes at micromolar concentrations, leading to nitric oxide-mediated death of cocultured neurons. The present study examined whether S-100β production by reactive astrocytes accumulating within the periinfarct area was related to delayed expansion of infarct volume after permanent middle cerebral artery occlusion in the rat. After rapid increases during the initial 24 hours, the increase of infarct volume then decelerated while maintaining the increasing tendency until 168 hours in this model, attaining a significant difference compared with that at 24 hours. In the periinfarct area, the number of reactive astrocytes expressing both S-100 and glial fibrillary acidic protein, the tissue level of S-100β as measured by the sandwich enzyme-linked immunosolvent assay method using anti-S-100β monoclonal antibody, and the number of terminal deoxynucleotidyl transferase-mediated 2-deoxyuridine 5-triphosphate-biotin nick end labeling-positive cells were significantly increased preceding the delayed expansion of infarct volume. The CSF concentration of S-100β showed a biphasic increase, presumably reflecting the immediate release from astrocytes within the ischemic core and the subsequent production in reactive astrocytes within the periinfarct area. These results show for the first time that the enhanced synthesis of S-100β by reactive astrocytes participates in the inflammatory responses within the periinfarct area, which may be related to the occurrence of delayed infarct expansion as a major component of the cytokine network.

Arundic Acid Ameliorates Cerebral Amyloidosis and Gliosis in Alzheimer Transgenic Mice

Like microglia, reactive astrocytes produce a myriad of neurotoxic substances in various brain pathologies, such as Alzheimers disease (AD), trauma, and cerebral ischemia. Among the numerous products of reactive astrocytes, attention has recently been directed toward the possible detrimental role of S100B, because the protein has been shown to be highly expressed along with the progression of brain damage and to exert neurotoxic effects at high concentrations. The present study aimed to examine the possible role of astrocyte-derived S100B in the progression of cerebral amyloidosis and gliosis in transgenic mice overproducing mutant amyloid precursor protein (Tg APPsw mice, line 2576). For this purpose, arundic acid (Ono Pharmaceutical Co., Ltd., Mishima, Osaka, Japan), which is known to negatively regulate astrocyte synthesis of S100B, was orally administered to Tg APPsw mice for 6 months from 12 months of age, and the effects of the agent on the above parameters were examined. Here, we report that β-amyloid deposits along with amyloid-β peptide/S100B levels, as well as β-amyloid plaque-associated reactive gliosis (astrocytosis and microgliosis), were significantly ameliorated in arundic acid-treated Tg APPsw mice relative to vehicle-treated Tg APPsw mice at 19 months of age. Based on the above results, arundic acid is considered to deserve further exploration as a promising therapeutic agent for AD.

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.

Modulation of astrocytic activation by arundic acid (ONO-2506) mitigates detrimental effects of the apolipoprotein E4 isoform after permanent focal ischemia in apolipoprotein E knock-in mice

Using homozygous human apolipoprotein E2 (apoE2) (2/2)-, apoE3 (3/3)-, or apoE4 (4/4)-knock-in (KI) mice, we have shown that delayed infarct expansion and reactive astrocytosis after permanent middle cerebral artery occlusion (pMCAO) were markedly exacerbated in 4/4-KI mice as compared with 2/2- or 3/3-KI mice. Here, we probed the putative causal relationship between enhanced astrocytic activation and exacerbation of brain damage in 4/4-KI mice using arundic acid (ONO-2506, Ono Pharmaceutical Co. Ltd), which is known to oppose astrocytic activation through its inhibitory action on S100B synthesis. In all of the KI mice, administration of arundic acid (10u2009mg/kgu2009day, intraperitoneal, started immediately after pMCAO) induced significant amelioration of brain damage at 5 days after pMCAO in terms of infarct volumes (results expressed as the mean infarct volume (mm3) ±1s.d. in 2/2-, 3/3-, or 4/4-KI mice in the vehicle groups: 16±2, 15±2, or 22±2; in the arundic acid groups: 11±2 (P<0.001), 11±2 (P<0.001), or 12±2 (P<0.001), as compared with the vehicle groups), neurologic deficits, and S100/glial fibrillary acidic protein burden in the peri-infarct area. The beneficial effects of arundic acid were most pronounced in 4/4-KI mice, wherein delayed infarct expansion together with deterioration of neurologic deficits was almost completely mitigated. The above results support the notion that the apoE4 isoform exacerbates brain damage during the subacute phase of pMCAO through augmentation of astrocytic activation. Thus, pharmacological modulation of astrocytic activation may confer a novel therapeutic strategy for ischemic brain damage, particularly in APOE ɛ4 carriers.

Functional modulation of astrocytes by a novel agent ONO-2506 mitigates delayed infarct expansion with a wide therapeutic time window, inducing prompt neurological recovery through reduction of the extracellular level of glutamate

Abstract ONO-2506, a novel agent which modulates miscellaneous functions of cultured astrocytes was shown to abolish delayed infarct expansion, while improving the neurological deficits following permanent focal ischemia in rats. The present study aimed to clarify its therapeutic time window and the mechanism underlying its beneficial effects. Treatment started at 24 and 48 h after the onset of ischemia significantly decreased the infarct volume at 168 h, whereas that started at 72 h was ineffective. In the former experimental group, the neurological deficits were significantly improved since 72 h on. Next, using a transient focal ischemia model in rats, the effect of the agent on the extracellular glutamate level in the periinfarct area was examined by microdialysis. During the period between 22 and 26 h after ischemia, the glutamate level as measured by the OPA-HPLC method markedly increased in the saline-treated group, whereas it remained close to normal in the treatment group. The glial glutamate transporter inhibitor-induced increases in the glutamate level were much greater in the drug-treated group than in the saline-treated group. Our results show that ONO-2506 has a wide therapeutic time window and is capable to induce rapid symptomatic improvement through preservation of the activities of glial glutamate transporters.