Takao Urabe

Migration of enhanced green fluorescent protein expressing bone marrow-derived microglia/macrophage into the mouse brain following permanent focal ischemia

Brain ischemia induces a marked response of resident microglia and hematopoietic cells including monocytes/macrophages. The present study was designed to assess the distribution of microglia/macrophages in cerebral ischemia using bone marrow chimera mice known to express enhanced green fluorescent protein (EGFP). At 24 h after middle cerebral artery occlusion (MCAO), many round-shaped EGFP-positive cells migrated to the ischemic core and peri-infarct area. At 48-72 h after MCAO, irregular round- or oval-shaped EGFP/ionized calcium-binding adapter molecule 1 (Iba 1)-positive cells increased in the transition zone, while many amoeboid-shaped or large-cell-body EGFP/Iba 1-positive cells were increased in number in the innermost area of ischemia. At 7 days after MCAO, many process-bearing ramified shaped EGFP/Iba 1-positive cells were detected in the transition to the peri-infarct area, while phagocytic cells were distributed in the transition to the core area of the infarction. The distribution of these morphologically variable EGFP/Iba 1-positive cells was similar up to 14 days from MCAO. The present study directly showed the migration and distribution of bone marrow-derived monocytes/macrophages and the relationship between resident microglia and infiltrated hematogenous element in ischemic mouse brain. It is important to study the distribution of intrinsic and extrinsic microglia/macrophage in ischemic brain, since such findings may allow the design of appropriate gene-delivery system using exogenous microglia/macrophages to the ischemic brain area.

Exendin-4, a glucagon-like peptide-1 receptor agonist, provides neuroprotection in mice transient focal cerebral ischemia

Glucagon-like peptide-1 (GLP-1) is an incretin hormone known to stimulate glucose-dependent insulin secretion. The GLP-1 receptor agonist, exendin-4, has similar properties to GLP-1 and is currently in clinical use for type 2 diabetes mellitus. As GLP-1 and exendin-4 confer cardioprotection after myocardial infarction, this study was designed to assess the neuroprotective effects of exendin-4 against cerebral ischemia–reperfusion injury. Mice received a transvenous injection of exendin-4, after a 60-minute focal cerebral ischemia. Exendin-4-treated vehicle and sham groups were evaluated for infarct volume, neurologic deficit score, various physiologic parameters, and immunohistochemical analyses at several time points after ischemia. Exendin-4 treatment significantly reduced infarct volume and improved functional deficit. It also significantly suppressed oxidative stress, inflammatory response, and cell death after reperfusion. Furthermore, intracellular cyclic AMP (cAMP) levels were slightly higher in the exendin-4 group than in the vehicle group. No serial changes were noted in insulin and glucose levels in both groups. This study suggested that exendin-4 provides neuroprotection against ischemic injury and that this action is probably mediated through increased intracellular cAMP levels. Exendin-4 is potentially useful in the treatment of acute ischemic stroke.

Neurogenesis After Transient Global Ischemia in the Adult Hippocampus Visualized by Improved Retroviral Vector

Background and Purpose— Neurogenesis has been observed in the dentate gyrus of the adult hippocampus; however, the mechanisms involved in this process are still only partly understood. In this study, we visualized the proliferation, migration, and differentiation of neuronal progenitor cells in the dentate gyrus induced by ischemic stress using improved retroviral vector. Methods— Improved retroviral vector expressing enhanced green fluorescent protein (EGFP) as a transgene was injected into the dentate gyrus of adult Mongolian gerbils. After 48 hours, transient global ischemia (TGI) was induced by bilateral common carotid artery occlusion for 5 minutes using aneurysm clips. The morphological and immunohistological features of newly-generated cells in the dentate gyrus were analyzed at various times thereafter. Results— At 48 hours after viral injection, almost all EGFP-positive dividing cells were found in the subgranule layer (SGL). These cells proliferated and migrated to the granule cell layer (GCL), expressing the developing neuronal markers polysialic acid and doublecortin, and differentiated to neuronal nuclei–positive or calbindin-positive mature granule cells at 30 days after TGI or sham-operation. The number of GFP-positive cells in the GCL was significantly higher (P < 0.05) in the ischemic animals at 30 days than in sham-operated gerbils. Conclusions— We saw neurogenesis in the adult dentate gyrus. Furthermore, we showed that ischemic stress promoted the proliferation and normal development of neurons at this site.

Phosphodiesterase III inhibition promotes differentiation and survival of oligodendrocyte progenitors and enhances regeneration of ischemic white matter lesions in the adult mammalian brain

Vascular dementia is caused by blockage of blood supply to the brain, which causes ischemia and subsequent lesions primarily in the white matter, a key characteristic of the disease. In this study, we used a chronic cerebral hypoperfusion rat model to show that the regeneration of white matter damaged by hypoperfusion is enhanced by inhibiting phosphodiesterase III. A rat model of chronic cerebral hypoperfusion was prepared by bilateral common carotid artery ligation. Performance at the Morris water-maze task, immunohistochemistry for bromodeoxyuridine, as well as serial neuronal and glial markers were analyzed until 28 days after hypoperfusion. There was a significant increase in the number of oligodendrocyte progenitor cells in the brains of patients with vascular dementia as well as in rats with cerebral hypoperfusion. The oligodendrocyte progenitor cells subsequently underwent cell death and the number of oligodendrocytes decreased. In the rat model, treatment with a phosphodiesterase III inhibitor prevented cell death, markedly increased the mature oligodendrocytes, and promoted restoration of white matter and recovery of cognitive decline. These effects were cancelled by using protein kinase A/C inhibitor in the phosphodiesterase III inhibitor group. The results of our study indicate that the mammalian brain white matter tissue has the capacity to regenerate after ischemic injury.

Edaravone attenuates white matter lesions through endothelial protection in a rat chronic hypoperfusion model

A multicenter randomized clinical trial demonstrated that acute ischemic stroke patients treated with edaravone, a scavenger of hydroxyl radicals, had significant functional improvement. We tested the hypothesis that edaravone has protective effects against white matter lesions (WML) and endothelial injury, using a rat chronic hypoperfusion model. Adult Wistar rats underwent ligation of bilateral common carotid artery (LBCCA) and were divided into the edaravone group (injected once only immediately after LBCCA [n=39, ED(1)]; and injected on three consecutive days [n=39, ED(3)]), the vehicle group (n=39), and the sham group (n=15). Cerebral blood flow, Morris water maze performance, footprint test for locomotor function, immunohistochemical analyses and Western blot analysis were performed before and after LBCCA. The ED(3) group upregulated endothelial nitric oxide synthase and attenuated Evans Blue extravasation at day 3 after LBCCA (P<0.05). Edaravone markedly suppressed accumulation of 4-hydroxy-2-nonenal-modified protein and 8-hydroxy-deoxyguanosine (P<0.01), and loss of oligodendrocytes (P<0.05) in the cerebral white matter at days 3, 7, 14, 21 and 28 after LBCCA. These results were more evident in the ED(3) group. Moreover, at day 21 after LBCCA, spatial memory but not motor function, and axonal damage were significantly improved by three-time treatment of edaravone (P<0.05). Our results indicated that 3-day treatment with edaravone provides protection against WML through endothelial protection and free radical scavenging and suggested that edaravone is potentially useful for the treatment of cognitive impairment.

Expression of glucose transporters in rat brain following transient focal ischemic injury.

Abstract: We have investigated the serial changes in the transcription and translation of the rat glucose transporter (GLUT) 1 and 3 genes after 3 h of middle cerebral artery (MCA) occlusion followed by reperfusion. Northern blot analysis and in situ hybridization study were performed to determine the chronological change and regional expression. In the ipsilateral anterior cerebral artery (ACA) cortex, GLUT1 mRNA expression was increased at 12 h (11.6‐fold) of reperfusion, and its expression was detected not only in vascular endothelial cells but also in neurons. At 48 h of reperfusion, GLUT3 mRNA expression was increased in the ipsilateral ACA (8.6‐fold) and in the contralateral MCA cortex (9.1‐fold). Immunohistochemical study failed to show GLUT1 protein synthesis in neurons in the ipsilateral ACA cortex. The immunoreactivity of GLUT3 protein was increased in neurons in ipsilateral ACA cortex and contralateral MCA cortex. Our results suggest that the expression of GLUT1 and GLUT3 is controlled differently after transient focal ischemic conditions. Furthermore, the postischemic localizations of both GLUT1 and GLUT3 expressions may be altered from the normal physiological expression pattern, which may be of importance in investigating postischemic cell function.

Postischemic accumulation of lipid peroxidation products in the rat brain: immunohistochemical detection of 4-hydroxy-2-nonenal modified proteins

We report an immunohistochemical study on the distribution and alterations of 4-hydroxy-2-nonenal (HNE)-modified proteins, an indicator of lipid peroxidation, in the rat brain after 3 h of middle cerebral artery (MCA) occlusion followed by reperfusion. HNE immunoreactivity was not observed in intact neurons, but it appeared in some shrunken neurons within the infarcted zone at 3 h after reperfusion. The number of HNE-positive neurons increased with the spread of the infarcted area. The pyramidal neurons in the third layer of the frontoparietal cortex were HNE-positive and the intensity of their HNE immunoreactivity was highest at 24 h after reperfusion. At 48 h, HNE-positive neurons were observed in the medial part of the striatum, the lateral side of the frontoparietal cortex, and at the boundary between the infarcted and noninfarcted zones. In addition, strong HNE immunoreactivity was seen in microglia (identified by OX-42 immunostaining). This method seems to be useful to follow the progress of lipid peroxidation at the cellular level after ischemic injury.

Accumulation of 4-hydroxynonenal-modified proteins in hippocampal CA1 pyramidal neurons precedes delayed neuronal damage in the gerbil brain.

It has been proposed that reactive oxygen species and lipid peroxidation have a role in the delayed neuronal death of pyramidal cells in the CA1 region. To explore the in situ localization and serial changes of 4-hydroxy-2-nonenal-modified proteins, which are major products of membrane peroxidation, we used immunohistochemistry of the gerbil hippocampus after transient forebrain ischemia with or without preconditioning ischemia. The normal gerbil hippocampus showed weak immunoreactivity for 4-hydroxy-2-nonenal-modified proteins in the cytoplasm of CA1 pyramidal cells. 4-hydroxy-2-nonenal immunoreactivity showed no marked changes after preconditioning ischemia. In the early period after ischemia and reperfusion, there was a transient increase of nuclear 4-hydroxy-2-nonenal immunoreactivity in CA1 pyramidal neurons. In contrast, cytoplasmic immunoreactivity transiently disappeared during same period and then increased markedly from 8h to seven days. One week after ischemia, 4-hydroxy-2-nonenal immunoreactivity was observed within reactive astrocytes in the CA1 region. Early nuclear accumulation of 4-hydroxy-2-nonenal in CA1 neurons may indicate a possible role in signal transduction between the nucleus and cytoplasm/mitochondria, while delayed accumulation of 4-hydroxy-2-nonenal-modified proteins in the cytoplasm may be related to mitochondrial damage. We conclude that 4-hydroxy-2-nonenal may be a key mediator of the oxidative stress-induced neuronal signaling pathway and may have an important role in modifying delayed neuronal death.

Prevalence of Abnormal Glucose Metabolism and Insulin Resistance Among Subtypes of Ischemic Stroke in Japanese Patients

Background and Purpose— The purpose was to assess the prevalence of disorders of glucose metabolism and insulin resistance in Japanese ischemic stroke patients with no history of diabetes by performing 75-gram oral glucose tolerance test (OGTT). Methods— We recruited 427 ischemic stroke patients (atherothrombotic infarction, n=220; lacunar infarction, n=125; cardioembolic infarction, n=82). OGTT was used to evaluate disorders of glucose metabolism in stroke patients without previously known diabetes (n=113). We investigated the relationships among the prevalence of abnormal glucose metabolism, ischemic stroke subtypes, and the prevalence of insulin resistance using homeostasis model assessment for insulin resistance and immunoreactive insulin at 120 minutes after glucose loading (IRI120). Results— OGTT identified the presence of disorders of glucose metabolism in 62.8% of ischemic stroke patients without previously known diabetes, including diabetes (24.8%) and impaired glucose tolerance (lone impaired glucose tolerance and impaired fasting glucose plus impaired glucose tolerance, 34.5%). The prevalence of newly diagnosed diabetes and impaired glucose tolerance was the highest in the atherothrombotic infarction group (68.9%). The highest values of homeostasis model assessment for insulin resistance and immunoreactive insulin at 120 minutes after glucose loading were found in atherothrombotic infarction patients with abnormal glucose tolerance. Conclusions— In this study, a significantly large percentage of Japanese patients with ischemic stroke and no history of diabetes were found to have disorders of glucose metabolism by OGTT. Impaired glucose tolerance and insulin resistance could play an important pathogenic role in the development of atherothrombotic infarction.

Mobile Aortic Plaques Are a Cause of Multiple Brain Infarcts Seen on Diffusion-Weighted Imaging

Background and Purpose— Multiple brain infarcts are often seen on diffusion-weighted images in cardioembolic stroke patients. Recently, mobile aortic plaques (MAPs) have been proposed as embolic sources. However, the clinical characteristics of patients with MAPs are unclear. Methods— We prospectively studied patients with acute ischemic stroke who underwent transesophageal echocardiography. The patients were classified into 3 groups based on transesophageal echocardiography findings: atheromatous aortic plaques <4 mm, atheromatous aortic plaques ≥4 mm without mobility, and MAPs. Based on their diffusion-weighted image findings, the patients were divided into 3 subgroups: (1) single lesion; (2) multiple lesions in a single vascular territory; and (3) multiple lesions in multiple vascular territories. We assessed the clinical characteristics and the diffusion-weighted image findings of stroke patients with MAPs. Results— One hundred sixty-seven patients (age, 70±12 years; 98 males) were enrolled; 128 (77%) had atheromatous aortic plaques <4 mm, 27 (16%) had atheromatous aortic plaques ≥4 mm, and 12 (7%) had MAPs. Older age, male gender, coronary artery disease, and cerebral arterial stenotic lesions were seen most frequently in patients with MAPs. On diffusion-weighted image findings, patients with MAPs were most frequent in the multiple lesions in multiple vascular territories group (P=0.001). On multiple logistic regression analysis, the National Institutes of Health Stroke Scale score (OR: 1.11; 95% CI: 1.01 to 1.22; P=0.039), arterial stenotic lesions (OR: 4.71; 95% CI: 1.35 to 16.41; P=0.015), and mobile aortic plaques (OR: 14.44; 95% CI: 2.87 to 72.66; P=0.001) were significantly associated with the multiple lesions in multiple vascular territories group. Conclusions— MAPs were not uncommonly observed in patients with acute ischemic stroke. MAPs could cause multiple brain infarcts on diffusion-weighted images.