Masayasu Matsumoto

Neurogenesis by Progenitor Cells in the Ischemic Adult Rat Hippocampus

Background and Purpose— Recently, there has been great interest in adult neurogenesis. We investigated whether transient forebrain ischemia could influence the proliferation of neuronal progenitor in the subgranular zone (SGZ) of the rat hippocampus and whether aging could influence the neurogenesis after ischemia. Methods— Male Wistar rats were subjected to 4-vessel occlusion model. We used a bromodeoxyuridine (BrdU) labeling method to identify the postproliferation cells and double-immunostaining with confocal microscopy to determine the cell phenotype. Results— The number of BrdU-positive cells in the SGZ increased ≈5.7-fold 8 days after ischemia, compared with the control. BrdU-positive cells formed clusters, which suggested that these cells had divided from an original progenitor cell, and expressed Musashi1 (Msi1), a marker of neural stem/progenitor cells. Although astrocytes also expressed Msi1 in the adult brain, Msi1-positive cells that formed clusters in the SGZ did not express glial fibrillary acidic protein, an astrocyte marker. About 70% of all BrdU-positive cells in the SGZ represented the neuronal phenotype 4 weeks after the BrdU injection. Although proliferation of progenitor cells was stimulated in both young and older animals, aging accelerated the reduction in newborn cells after ischemia. Conclusions— Our results indicate that ischemic stress stimulated the proliferation of neuronal progenitor cells in the SGZ of both young and old rats but resulted in increased neurogenesis only in young animals. Our findings will be important in developing therapeutic intervention to enhance endogenous neurogenesis after brain injury.

Microtubule-associated protein 2 as a sensitive marker for cerebral ischemic damage--immunohistochemical investigation of dendritic damage.

We investigated the neuronal distribution of microtubule-associated protein 2 in gerbil brain and monitored the progression of ischemic damage immunohistochemically by using this protein as a dendritic marker. The reaction for microtubule-associated protein 2 in normal gerbil brain clearly visualized neuronal soma and dendrites but other structures such as axonal bundles, glia and endothelial cells exhibited little immunoreactivity. In a reproducible gerbil model of unilateral cerebral ischemia, we could detect the ischemic lesions as early as 3 min after right common carotid occlusion at the subiculum-CA1 region of the ipsilateral hippocampus as faint loss of the reaction in the dendrites. After ischemia for 30 min, the ischemic lesions were clearly detected as loss of the reaction in the nerve cell bodies, dendrites and the neuropil in the hippocampus, cerebral cortex, thalamus and the caudoputamen. Although the mechanism for prompt disappearance of the immunohistochemical reaction for microtubule-associated protein 2 is not clear, the present investigation suggests that dendrites in the vulnerable regions may be quite susceptible to ischemic stress and that the immunohistochemical procedure for microtubule-associated protein 2 may be very useful for demonstration of dendritic damage in various pathophysiological states of the central nervous system.

Rage: A Novel Cellular Receptor for Advanced Glycation End Products

Exposure of proteins to reducing sugars results in nonenzymatic glycation with the ultimate formation of advanced glycation end products (AGEs). One means through which AGEs modulate cellular functions is through binding to specific cell surface acceptor molecules. The receptor for AGEs (RAGE) is such a receptor and is a newly identified member of the immunoglobulin superfamily expressed on endothelial cells (ECs), mononuclear phagocytes (MPs), and vascular smooth muscle cells (SMCs) in both vivo and in vitro. Binding of AGEs to RAGE results in induction of cellular oxidant stress, as exemplified by the generation of thiobarbituric acid-reactive substances, expression of heme oxygenase type I, and activation of the transcription factor NF-kB, with consequences for a range of cellular functions. AGEs on the surface of diabetic red cells enhance binding to endothelial RAGE and result in enhanced oxidant stress in the vessel wall. By using reagents to selectively block access to RAGE, the role of this receptor in AGE-mediated perturbation of cellular properties can be dissected in detail.

Contribution of Microglia/Macrophages to Expansion of Infarction and Response of Oligodendrocytes After Focal Cerebral Ischemia in Rats

BACKGROUND AND PURPOSE The purpose of this study was (1) to examine the contribution of microglia and macrophages with their interleukin-1beta production and (2) to assess the vulnerability and response of oligodendrocytes in cerebral infarction. METHODS Male Wistar rats were subjected to permanent occlusion of the left middle cerebral artery. Expansion of ischemic infarction and response of oligodendrocytes were investigated together with accumulation of inflammatory cells, production of interleukin-1beta, and disruption of the blood-brain barrier. Apoptotic cell death was inferred from fragmented DNA and the expression of proapoptotic Bax protein. RESULTS During expansion of infarction, amoeboid microglia and extravasation of serum albumin were observed not only in the infarcted area but also in the adjacent surviving area, whereas macrophages accumulated along the boundary and granulocytes migrated into the center of the infarction. Both amoeboid microglia and macrophages produced interleukin-1beta, an inflammatory cytokine, during an early ischemic period. Furthermore, macrophages within the infarcted tissue expressed Bax protein and subsequently showed fragmented nuclear DNA. Oligodendrocytes were detected in the infarcted area even after 24 hours following middle cerebral artery occlusion, but they subsequently developed fragmented DNA. A week after onset of ischemia, oligodendrocytes were found to be accumulated in the intact area bordered with the infarct together with reactive astrocytes. CONCLUSIONS Our results suggest the importance of amoeboid microglia, macrophages, and their interleukin-1beta production in gradual expansion of cerebral infarction. Resident oligodendrocytes may be resistant to ischemic insults, and oligodendrocytes accumulated at the border of the infarction may participate in tissue repair after cerebral infarction.

Prevalence of carotid atherosclerosis in diabetic patients. Ultrasound high-resolution B-mode imaging on carotid arteries.

OBJECTIVE To quantitatively assess atherosclerosis of the carotid artery in subjects with and without diabetes. RESEARCH DESIGN AND METHODS Ultrasound high resolution B-mode imaging of carotid arteries was conducted on 71 nondiabetic subjects without hyperlipidemia or hypertension and 295 diabetic patients to determine IMT of the arterial wall. RESULTS IMT was linearly related with age in nondiabetic (IMT = [0.0087 × age] + 0.3318) and diabetic subjects (IMT = [0.0155 × age] + 0.32450). The regression coefficient for age was significantly greater in diabetic than nondiabetic subjects. IMT (mean ± SD) of diabetic subjects aged 20–29 was significantly > that of nondiabetic subjects aged 20–29 (0.73 ± 0.27 vs. 0.52 ± 0.07 mm, P < 0.01). Multivariate regression analysis of 275 NIDDM patients indicated smoking, hyperlipidemia, duration of diabetes, hypertension, and age were factors determining thickness of the carotid arterial wall. CONCLUSIONS Diabetes, along with age, hyperlipidemia, smoking, and hypertension, aggravates carotid atherosclerosis.

Cerebral Ischemia after Bilateral Carotid Artery Occlusion and Intraluminal Suture Occlusion in Mice: Evaluation of the Patency of the Posterior Communicating Artery:

Cerebral ischemia models using mice have drawn increasing attention, particularly because of the availability of transgenic animals. However, the variability of intracranial vasculature at the circle of Willis in mice can influence the degree of ischemia in both the bilateral common carotid artery (CCA) occlusion and intraluminal suture occlusion models. We have developed a method to predict the extent of the anastomosis between carotid and vertebrobasilar circulation in three mouse strains (C57BL/6, CBA, and DBA/2) by measuring cortical microperfusion with laser Doppler flowmetry during a 1-minute occlusion of both CCA. When animals showed residual cortical microperfusion of less than 12% during bilateral CCA occlusion, the mice showed absence of functional anastomosis, developed ATP depletion in the frontal cortex during occlusion, and had ischemic neuronal death in the hippocampus and caudoputamen after occlusion for 15 minutes and recirculation for 7 days. Furthermore, those mice exhibited decreased local cerebral blood flow and associated ischemic neuronal death in the hippocampus, within the territory supplied by the posterior cerebral artery, with the intraluminal suture occlusion model. The current study demonstrates the need for assessment of intracranial vasculature in each animal by measuring cortical microperfusion during temporary occlusion of both CCA, no matter whether cerebral ischemia is produced by bilateral CCA occlusion or intraluminal suture occlusion in transgenic mice.

C57BL/6 strain is most susceptible to cerebral ischemia following bilateral common carotid occlusion among seven mouse strains: selective neuronal death in the murine transient forebrain ischemia

Rats and gerbils have been used widely to investigate the molecular mechanism of selective neuronal death following transient global ischemia. Recently, the availability of transgenic mice has enabled us to examine the involvement of specific gene products in various pathophysiological conditions. However, there has been only limited information about the experimental model of cerebral ischemia in mice, particularly in regard to selective neuronal death. We examined whether bilateral carotid occlusion produced global forebrain ischemia in seven common mouse strains including C57BL/6, ICR, BALB/c, C3H, CBA, ddY and DBA/2, based on neurological signs, histological findings and cortical microcirculatory as well as India ink perfusion patterns. The C57BL/6 strain was found to be the most susceptible among seven strains. All C57BL/6 mice died within 6 h after permanent bilateral carotid occlusion. After transient bilateral carotid occlusion for 20 min, more than 90% of C57BL/6 mice showed typical neurological signs such as torsion of the neck and rolling fits, and developed selective neuronal death in the hippocampus and caudoputamen. Hypothermia prevented the neuronal death. Visualization of brain vasculature by India ink perfusion indicated that the susceptibility of the mice after bilateral carotid occlusion depended mainly on the degree of anastomosis between carotid and basilar arteries. Our results showed the feasibility of investigating selective neuronal death in transgenic mice with simple temporary occlusion of both common carotid arteries, when those from the C57BL/6 strain or inbred transgenic mice from other strains with the C57BL/6 strain in a back-cross manner are used.

Ischemic stroke events and carotid atherosclerosis : results of the Osaka follow-up study for ultrasonographic assessment of carotid atherosclerosis (the OSACA study)

BACKGROUND AND PURPOSE To clarify the clinical significance of carotid atherosclerosis for ischemic stroke events, a follow-up study was performed in Japanese patients. METHODS Two hundred fourteen patients were registered from nine hospitals in the Osaka community. All patients were checked for a prior history of stroke, and the risk factors for stroke and atherosclerosis were evaluated. Carotid atherosclerosis was assessed by 7.5-MHz duplex ultrasonography. We studied the relationship between the ischemic stroke event rate and the severity and appearance of the carotid atherosclerosis. We also studied the relationship between stroke events and various risk factors. RESULTS The average duration of follow-up was 16 months. Ten patients suffered new ischemic stroke episodes during this follow-up period. At the initial ultrasonographic study, 16 patients had high-grade stenosis and 21 had ulcerated plaque. Proportional hazard regression analysis showed that grade of stenosis and plaque ulceration were positively related to the event rate. Patients with ulcerated plaque had a sevenfold higher hazard ratio for stroke in comparison to those without (P < .01). The ipsilateral stroke recurrence rate was 11 times higher in patients with ulcerated high-grade stenotic carotid lesions. CONCLUSIONS The present findings demonstrate that the severity of carotid atherosclerosis as evaluated by ultrasonography is a useful indicator of the risk of ischemic stroke in symptomatic patients.

High frequency of open-angle glaucoma in Japanese patients with Alzheimer's disease

The clinical and genetic relationships between Alzheimers disease (AD) and glaucoma remain obscure. The aim of this study was to determine the prevalence of open-angle glaucoma (OAG) in patients with AD and whether the apolipoprotein E (APOE) 4 allele is associated with AD, with or without OAG, in Japanese. The groups consisted of 172 patients with the diagnostic criteria of AD and 176 age-matched controls. Ophthalmic examinations were conducted, and genomic analysis was performed by PCR and digestion of products with an enzyme. OAG was found in 41 (23.8%) of the AD patients, which was a significantly (p = 0.0002) higher prevalence than that in the controls (9.9%). Furthermore, there was no significant difference between intraocular pressures (IOPs) in AD patients with OAG and without OAG. The percentage of AD patients who carried an APOE epsilon4 allele (29.5%) was significantly (p = 0.0007) higher than that of the controls (9.1%). However, the percentage of AD patients with OAG who carried an APOE epsilon4 allele (35.7%) was not significantly different than that of AD patients without OAG (27.7%, p = 0.42). In summary, the prevalence of OAG is high in Japanese patients with AD, suggesting that common factors other than APOE may contribute to the two diseases.

Increased Proliferation of Neural Progenitor Cells but Reduced Survival of Newborn Cells in the Contralateral Hippocampus After Focal Cerebral Ischemia in Rats

Recent studies demonstrated that neurogenesis in the adult hippocampus increased after transient global ischemia; however, the molecular mechanism underlying increased neurogenesis after ischemia remains unclear. The finding that proliferation of progenitor cells occurred at least a week after ischemic insult suggests that the stimulus was not an ischemic insult to progenitor cells. To clarify whether focal ischemia increases the rate of neurogenesis in the remote area, the authors examined the contralateral hemisphere in rats subjected to permanent occlusion of the middle cerebral artery. In the subgranular zone of the hippocampal dentate gyrus, the numbers of bromodeoxyuridine (BrdU)-positive cells increased approximately sixfold 7 days after ischemia. In double immunofluorescence staining, more than 80% of newborn cells expressed Musashi1, a marker of neural stem/progenitor cells, but only approximately 10% of BrdU-positive cells expressed glial fibrillary acidic protein (GFAP), a marker of astrocytes. The number of BrdU-positive cells markedly decreased 28 days after BrdU administration after ischemia, but it was still elevated compared with that of sham-operated rats. In double immunofluorescence staining, 80% of newborn cells expressed NeuN, a marker of differentiated neurons, and 10% of BrdU-positive cells expressed GFAP. However, in the other areas of the contralateral hemisphere including the rostral subventricular zone, the number of BrdU-positive cells remained unchanged. These results showed that focal ischemia stimulated the proliferation of neuronal progenitor cells, but did not support survival of newborn cells in the contralateral hippocampus.