Yoshifumi Kagamiishi

Involvement of Cathepsin E in Exogenous Antigen Processing in Primary Cultured Murine Microglia

We have attempted to elucidate an involvement of cathepsin E (CE) in major histocompatibility complex class II-mediated antigen presentation by microglia. In primary cultured murine microglia, CE was localized mainly in early endosomes and its expression level was markedly increased upon stimulation with interferon-γ. Pepstatin A, a specific inhibitor of aspartic proteases, significantly inhibited interleukin-2 production from an OVA-(266–281)-specific T helper cell hybridomas upon stimulation with native OVA presented by interferon-γ-treated microglia. However, pepstatin A failed to inhibit the presentation of OVA-(266–281) peptide. The possible involvement of CE in the processing of native OVA into antigenic peptide was further substantiated by that digested fragments of native OVA by CE could be recognized by OVA-specific Th cells. Cathepsin D also degraded native OVA into antigenic peptide, whereas microglia prepared from cathepsin D-deficient mice retained an ability for antigen presentation. On the other hand, the requirement for cysteine proteases such as cathepsins S and B in the processing of invariant chain (Ii) was confirmed by immunoblot analyses in the presence of their specific inhibitors. In conclusion, CE is required for the generation of an antigenic epitope from OVA but not for the processing of Ii in microglia.

The intra-arterial injection of microglia protects hippocampal CA1 neurons against global ischemia-induced functional deficits in rats

In the present study, we have attempted to elucidate the effects of the intra-arterial injection of microglia on the global ischemia-induced functional and morphological deficits of hippocampal CA1 neurons. When PKH26-labeled immortalized microglial cells, GMIR1, were injected into the subclavian artery, these exogenous microglia were found to accumulate in the hippocampus at 24 h after ischemia. In hippocampal slices prepared from medium-injected rats subjected to ischemia 48 h earlier, synaptic dysfunctions including a significant reduction of synaptic responses and a marked reduction of long-term potentiation (LTP) of the CA3-CA1 Schaffer collateral synapses were observed. At this stage, however, neither significant neuronal degeneration nor gliosis was observed in the hippocampus. At 96 h after ischemia, there was a total loss of the synaptic activity and a marked neuronal death in the CA1 subfield. In contrast, the basal synaptic transmission and LTP of the CA3-CA1 synapses were well preserved after ischemia in the slices prepared from the microglia-injected animals. We also found the microglial-conditioned medium (MCM) to significantly increase the frequency of the spontaneous postsynaptic currents of CA1 neurons without affecting the amplitude, thus indicating that MCM increased the provability of the neurotransmitter release. The protective effect of the intra-arterial injected microglia against the ischemia-induced neuronal degeneration in the hippocampus was substantiated by immunohistochemical and immunoblot analyses. Furthermore, the arterial-injected microglia prevented the ischemia-induced decline of the brain-derived neurotrophic factor (BDNF) levels in CA1 neurons. These observations strongly suggest that the arterial-injection of microglia protected CA1 neurons against the ischemia-induced neuronal degeneration. The restoration of the ischemia-induced synaptic deficits and the resultant reduction of the BDNF levels in CA1 neurons, possibly by the release of diffusible factor(s), might thus contribute to the protective effect of the arterial-injection of microglia against ischemia-induced neuronal degeneration.

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.

Hippocampal serotonergic system is involved in anxiety-like behavior induced by corticotropin-releasing factor

To clarify the interaction between anxiety-like behavior produced by corticotropin-releasing factor (CRF) and the 5-HT system, we investigated the effects of intracerebroventricular (i.c.v.) administration of CRF on an elevated plus-maze performance as indices of anxiety, measuring extracellular levels of 5-HT in the ventral hippocampus using an in vivo brain dialysis method in rats. The time spent in the open arms of the maze and the number of open arm entries were decreased in a dose-dependent manner by the administration of CRF (0.3-1.0 microg/rat). These effects of CRF were prevented by pretreatment with a 5-HT(1A) receptor agonist, 8-OH-DPAT (0.5 mg/kg, s.c.). In biochemical studies, CRF increased 5-HT release about 150-250% above baseline in the ventral hippocampus and this elevation was significantly inhibited by a CRF receptor antagonist, alpha-Helical CRF(9-41) (50 mug/rat), and 5-HT(1A) receptor agonist, 8-OH-DPAT (0.5 mg/kg, s.c.). These results suggested that the anxiety-like effect produced by CRF may have involved increased 5-HT transmission in the ventral hippocampus. Taken with the evidence for hypersecretion of CRF in patients with depression and anxiety-related disorders, our findings lead to the intriguing hypothesis that interaction between CRF and 5-HT, especially in the ventral hippocampus, plays a role in the etiology of affective and anxiety disorders.

Ischemia-induced impairment of 2-deoxyglucose uptake and CA1 field potentials in rat hippocampal slices: Protection by 5-HT1A receptor agonists and 5-HT2 receptor antagonists

Various in vitro models have been developed to study ischemia and/or hypoxia. In the present experiment, we examined whether hypoxia/hypoglycemia (ischemia) in rat hippocampal slices reduced the 2-deoxyglucose (2-DG) uptake and CA1 field potentials evoked by stimulation of Schaffer collaterals. Autoradiograms revealed that ischemia for 15 or 20 min reduced 2-DG uptake in the stratum radiatum of the CA1 and the dentate gyrus. Similarly, the CA1 field potentials of slices exposed to ischemia for 15 and 20 min decreased by about 70 and 90% after a 6-h washout. In the second experiment, we evaluated the neuroprotective effect of the 5-HT1A receptor agonists 8-OH-DPAT and buspirone, and the 5-HT2 receptor antagonists cyproheptadine, mianserin and ketanserin on deficits of 2-DG uptake and Schaffer-CA1 field potentials induced by ischemia. The 5-HT1A receptor agonists and 5-HT2 receptor antagonists exhibited significant neuroprotective actions against ischemia-induced deficits. Therefore, impairments of 2-DG uptake and CA1 field potentials induced by ischemia may be good markers of ischemia-induced functional deficits. The attenuating action of 5-HT1A receptor agonists and 5-HT2 receptor antagonists were assessed using this model of ischemia.

Neuroprotective effect of 5-HT3 receptor antagonist on ischemia-induced decrease in CA1 field potential in rat hippocampal slices

The effect of 5-HT3 receptor agonists and antagonists on the hypoxia/hypoglycemia (ischemia)-induced decrease in CA1 field potential elicited by stimulation of Schaffer collaterals was investigated using rat hippocampal slices. Treatment with the 5-HT3 receptor agonist, 2-methyl-5-HT (1-10 microM), exacerbated the ischemia-induced decreased in CA1 field potential, whereas treatment with the 5-HT3 receptor antagonist, Y-25130 (0.1-100 microM), or the 5-HT2 receptor antagonist, ketanserin (10, 100 microM), produced dose-dependent neuroprotection against the ischemia-induced decrease. However, in normal non-ischemic solution these treatments did not significantly change the CA1 field potential. The protective action of Y-25130 was blocked by co-treatment with 2-methyl-5-HT. The magnitude of protection in the Y-25130-treated group (EC50, 1.8 microM) was about 20 times greater than that in the ketanserin-treated group (EC50, 33 microM). The present study demonstrated that stimulation of 5-HT3 receptors plays a detrimental role in the development of ischemic damage, whereas blockade of the 5-HT3 receptor plays a neuroprotective role in ischemic damage, suggesting a facilitatory role of 5-HT neurons in ischemia-induced neuronal deficits.

Neuroprotective effect of protein kinase C inhibitors on oxygen/glucose free-induced decreases in 2-deoxyglucose uptake and CA1 field potentials in rat hippocampal slices

Staurosporine, a protein kinase C inhibitor, was found to produce a neuroprotective effect against an ischemic insult in both gerbils and rats in vivo. We have demonstrated that rat hippocampal slices exposed to oxygen/glucose-free medium showed decreases in 2-deoxyglucose (2-DG) uptake and CA1 field potentials elicited by the stimulation of Schaffer collaterals. Therefore we examined the effect of protein kinase C inhibitors on oxygen/glucose free-induced impairments of 2-DG uptake and CA1 field potentials. Pretreatment with staurosporine, K252a and H-7 attenuated decreases in 2-DG uptake and CA1 field potentials. Treatment with phorbol ester, a protein kinase C activator, for a long period (90 min) was found to induce a down-regulation of protein kinase C activity. Therefore we examined the effect of pretreatment with phorbol ester for 90 min on oxygen/glucose free-induced decreases in 2-DG uptake and CA1 field potentials. These decrements were not attenuated by 5-min treatment with phorbol ester but were attenuated by 90-min treatment. The present results suggest that the treatment which decreases protein kinase C activity shows a neuroprotective action against oxygen/glucose free-induced deficits of metabolic and synaptic activity in hippocampal slices.

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.

Protective effects of microglia-conditioned medium (MCM) against NMDA-induced dendritc injury through an inhibition of non-selective cation conductance

By the use of a transient 90-min middle cerebral artery occlusion (MCAO) model of rats, we have shown that highly proliferative macrophage-like cells derived from bone marrow, started to accumulate in 2 days post reperfusion (2 dpr), and their number peaked at 7 dpr. Such macrophage-like cells expressed Iba1 and NG2 proteoglycan, therefore they were termed BINCs (Brain Iba1+/NG2+ Cells). Since the cells elicited ameliorative effects on the ischemic brains, some methods that enhance the accumulation of BINCs in ischemic lesions may become an effective treatment. However, it is an open question what kinds of factors are responsible for the recruitment of BINCs or their progenitors from the bone marrow to the ischemic brain lesions. In this study, we focused on fractalkine and MCP-1 as candidate factors responsible for the recruitment. BINCs were isolated from the cerebral ischemic lesion at 7 dpr, and mRNAs encoding MCP-1 and fractalkine as well as their receptors CCR2 and CX3CR1 were quantified using real-time RT-PCR. The expression levels of mRNAs by BINCs were compared with those by primary astrocytes and microglia. BINCs expressed CCR2 and CX3CR1 at the highest levels among the three types of cells, whereas astrocytes their ligands fractalkine and MCP-1 at the highest levels. Kinetic study using in vivo samples revealed that ischemic brain tissue contained significant level of mRNAs of both chemokines by 2 dpr, but the expressed level of the mRNAs decreased in time by 5dpr. The chemokines induced directed chemotaxis of BINCs, as revealed by migration assay using Boyden chambers. Taken together the present data and our previous study showing that BINCs or BINC progenitors may invade into ischemic brain tissue by 2dpr, it is probable that the two chemokines are responsible for the recruitment of BINCs or BINC progenitor from bone marrow.