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Global ischemia promotes neurogenesis in the dentate gyrus of the adult mouse hippocampus. Cyclooxygenase (COX)-2, the principal isoenzyme in the brain, modulates inflammation, glutamate-mediated cytotoxicity, and synaptic plasticity. We demonstrated that delayed treatment with different classes of COX inhibitor significantly blunted enhancement of dentate gyrus proliferation of neural progenitor cells after ischemia. COX-2 immunoreactivity was observed in both neurons and astrocytes in the dentate gyrus, but not in neural progenitor cells in the subgranular zone. Moreover, in the postischemic dentate gyrus of heterozygous and homozygous COX-2 knockout mice, proliferating bromodeoxyuridine-positive cells were significantly fewer than in wild-type littermates. These results demonstrate that COX-2 is an important modulator in enhancement of proliferation of neural progenitor cells after ischemia., (Copyright 2003 Wiley-Liss, Inc.)

Objective: In vitro experiments are performed on a daily basis to study the molecular biology of tumors. For some benign tumors, however, established cell lines are not always available, or it may not be feasible to establish new ones. In such cases, primary cultures are used to perform in vitro experiments. Gene expression profiles in vitro differ from those in vivo, but information as to which genes have significantly altered levels is limited. In this study, gene expression profiles of meningiomas in primary cultures and frozen tumors were compared., Methods: Affymetrix U95A chips were applied to three sets of meningiomas. For each tumor, the gene expression profiles in frozen specimens (Fr) and primary cultures from the same tumor at Pass 5 (P5) and Pass 10 (P10) were compared. A paired t test (P < 0.025) was applied between Fr and P5, and then between Fr and P10. Genes that demonstrated significantly different expression levels in both comparisons were then identified. The expression levels for a subset of these genes were confirmed by quantitative real-time reverse transcription-polymerase chain reaction., Results: Among 12,000 genes examined, up-regulation of 51 genes by fivefold or more and down-regulation of 19 genes by twofold or more was found in primary cultures (P5 and P10) compared with the corresponding Fr. Up-regulation of genes encoding for extracellular matrix, cytoskeleton, and cell surface receptors was particularly notable., Conclusion: Gene expression of tissue-cultured meningiomas and in situ meningiomas is significantly different for a large number of genes. Therefore, gene expression and therapeutic studies on cultured meningiomas need to be interpreted with caution.

Akt kinase is involved in growth factor-mediated neuronal protection. In the present study, we found in cultured neurons exposed to glutamate, that phosphorylation at Ser473 was transiently induced, but the level of phosphorylation at Thr308 and Akt activity were unchanged. Inhibition of phosphoinositide 3-kinase with LY294002 decreased phosphorylation and Akt activity, however, pretreatment with LY294002 did not affect glutamate toxicity. Our findings suggested that the endogenous Akt pathway does not play a crucial role in cell survival after exposure to glutamate., (Copyright 2002 Elsevier Science Ireland Ltd.)

Bone morphogenetic proteins (BMPs) delivered on scaffolds can induce ectopic bone formation after subcutaneous injection. Adenoviral vectors (Ad) carrying BMP2, BMP7, and BMP9 cDNAs have been shown to produce bone through endochondral ossification. The present study was performed to elucidate the histological events leading to ectopic ossification for two novel first-generation adenoviral constructs encoding BMPs, AdBMP4 and AdBMP6. In vitro, the viral constructs produced and secreted the mature BMP4 and BMP6 proteins. In vivo, the calf muscles of athymic nude rats were injected with AdBMP4, AdBMP6, AdBMP2, or AdlacZ. Rats were sacrificed 3, 6, 9, 16, 21, 60, and 90 days postinjection. Whereas AdBMP4 produced ectopic bone through mechanisms similar to endochondral ossification, AdBMP6 seemed to induce bone by way of mechanisms similar to both intramembranous and endochondral ossification pathways. At the relatively low vector dose used in this study, AdBMP2 caused an initial recruitment of primitive mesenchymal cells, without further development to bone. From computed tomographic analysis, AdBMP6 produced the most rapid tissue calcification. The ultimate density of ectopic bone formed by AdBMP4 and AdBMP6 was comparable. The current study demonstrates that AdBMP4 and AdBMP6 are more potent than the prototypical osteogenic adenoviral vector AdBMP2 and seem to induce ectopic bone by different mechanisms.

Both nestin and the neural RNA-binding protein Musashi1 (Msi1) are expressed in neural stem cells in the subventricular zone. Neurogenesis in the hippocampus has received much attention, so we evaluated the expression of Msi1 and nestin in the adult rat hippocampus after transient forebrain ischemia. Both Msi1 and nestin were induced in the reactive astrocytes after ischemia, especially in the CA1 region, until 35 days after ischemia. Induction of both molecules suggested that reactive astrocytes might have immature characteristics. In the subgranular zone (SGZ) of the hippocampal dentate gyrus, Msi1-positive cells formed clusters after ischemia. These cells were labeled by bromodeoxyuridine (BrdU) but did not express glial fibrillary acidic protein. In contrast, very few nestin-positive cells were labeled by BrdU. Our results suggest that neuronal progenitor cells in the SGZ expressed Msi1 but not nestin., (Copyright 2002 Wiley-Liss, Inc.)

Survivin is an inhibitor of apoptosis protein that blocks apoptosis by binding to caspases-3 and -7. It is highly expressed in less-differentiated embryonic cells and rapidly dividing tumors, but not in terminally differentiated adult tissues. Elevated survivin levels are found in malignant systemic tumors, and are associated with chemo-resistance, radiation resistance, and poor prognosis. However, expression of survivin in primary nervous system tumors has not been previously characterized. Immunohistochemistry using anti-human survivin antibody (SURV11-A) was performed on formalin-fixed, paraffin-embedded archival tissue from 112 primary central nervous system tumors. Survivin immunoreactivity was seen in most diffuse astrocytomas [WHO II (2/4), III (3/3), IV (9/10), giant-cell glioblastoma (1), and gliosarcoma (1)]. The intensity and degree of survivin expression showed trends with tumor grade, with glioblastomas having the highest positivity. Pilocytic astrocytomas (5) and pleomorphic xanthoastrocytoma (1) were positive to a lesser degree. In oligodendrogliomas (6) and mixed oligo-astrocytomas [grade II (5), II-III (3), and III (7)], oligodendroglial elements appear to be negative compared to positive mini-gemistocytic oligodendrocytes. Ependymomas [grade II (6) and grade III (1)] were positive. Medulloblastomas (5) and retinoblastoma (1/4) showed focal positivity. All meningiomas [grade I (12), II (9), III (4), and grade I (3) and II (5) with frank brain invasion] were intensely positive. All schwannomas (11) and neurofibromas (6) were intensely positive. Thus, survivin is expressed in the majority of the primary nervous system tumors, particularly in glioblastomas, meningiomas, schwannomas and neurofibromas. Overexpression of survivin in meningiomas and benign peripheral nerve sheath tumors contrasts with previous reports relating it to rapid division and poor prognosis.

Background and Purpose: Higher plasma total homocysteine (tHcy) levels have been associated with carotid atherosclerosis and cerebral infarction in whites. However, data regarding such associations are limited for Asians. This study examined associations between tHcy levels and severity of carotid atherosclerosis in Japanese subjects. Additionally, because lacunar infarction is the most prevalent type of ischemic stroke in Japan, we also investigated its associations with tHcy levels., Methods: The subjects were 152 Japanese patients (age, 66.2+/-11.0 years) at our hospital. Using ultrasound, we evaluated severity of carotid atherosclerosis by plaque score, which is defined by the sum of all plaque (intima-media thickness > or =1.1 mm) height in bilateral carotid arteries. In 112 of 152 patients, the existence of lacunar infarction was evaluated on brain MRI scans., Results: A moderate linear association was found between tHcy levels and plaque score (r=0.48, P<0.0001). Moreover, tHcy level was associated with plaque score (beta=0.26, P<0.001) independently of traditional atherosclerotic risk factors. In logistic regression analyses, each 1-micromol/L-higher tHcy level was associated with a 1.37-fold-higher [95% confidence interval (CI), 1.19 to 1.58] likelihood for lacunar infarction, increasing the likelihood by 1.22-fold (95% CI, 1.04 to 1.43) independently of traditional atherosclerotic risk factors., Conclusions: Higher tHcy levels appear to have associations with increased severity of carotid atherosclerotic plaques and prevalent lacunar infarction in the Japanese. Larger prospective studies are necessary to establish whether higher tHcy levels serve as a harbinger for insidious carotid and cerebrovascular diseases.

Recent studies have demonstrated that apolipoprotein E (APOE) deficiency worsened neuronal injuries after transient focal and global cerebral ischemia. However, the molecular mechanism underlying the protective effect of APOE remains uncertain, even though several mechanisms, including excitotoxicty, free radicals, and apoptosis, have been cited as causes of selective neuronal vulnerability in cerebral ischemia. In the present study, we first compared the vulnerability of cultured neurons prepared from APOE-knockout mice upon exposure to glutamate, hydrogen peroxide, and staurosporine. No significant difference in cell viability was observed after exposure to glutamate or staurosporine between APOE-deficient and wild-type mice. However, exposure to hydrogen peroxide significantly increased the level of cell death in APOE-deficient mice compared with that in wild-type mice. After transient forebrain ischemia for 12 min, APOE-deficient mice showed more neuronal death than wild-type mice. Pretreatment of APOE-deficient mice with vitamin E for 2 months markedly reduced neuronal death caused by ischemia. The results suggest that APOE exerted its neuroprotective effect against ischemia through its antioxidant action but not through mitigation of glutamate toxicity or blocking of apoptosis., (Copyright 2002 Wiley-Liss, Inc.)

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.

Background and Purpose: Although chronic-stage crossed cerebellar diaschisis (CCD) is reported to be associated with the neurologic state or clinical improvement after infarct, the prognostic value of early-stage CCD remains controversial. Our aim was to determine whether measurements of CCD in the acute and subacute stages obtained at single-photon emission CT (SPECT) facilitate the prediction of stroke outcome., Methods: The pattern of cerebral blood flow changes after the occurrence of acute middle cerebral artery ischemia with severe cortical symptoms was examined by using technetium 99m-hexamethylpropyleneamine oxime ((99m)Tc-HMPAO) SPECT. Fifteen patients (mean age, 73 years +/- 8 [SD]) with unilateral ischemia were examined in the early subacute stage (10 days +/- 5). In 11 patients, SPECT was performed in both the acute (16 hours +/- 10) and subacute stages. From the total counts obtained from each cerebellar hemisphere, the asymmetry index (AI) was calculated as follows: [(value in unaffected hemisphere--value in affected hemisphere)/value in unaffected hemisphere] x 100. Clinical outcome (at 60 days) was assessed by means of the Scandinavian Stroke Scale (SSS) and Barthel Index (BI)., Results: AIs in the acute stage and clinical outcome (ie, SSS and BI scores) showed no significant correlation, but the severity of AI in the early subacute stage correlated significantly with both the final SSS (r = -0.69; P <.01) and BI scores (r = -0.82; P <.01)., Conclusion: Cerebellar hypoperfusion detected at (99m)Tc-HMPAO SPECT in the early subacute stage in patients with supratentorial infarct indicates a worse clinical outcome.

Recent clinical evidence has indicated that the level of soluble ICAM-1 (sICAM-1) is correlated with the severity of atherosclerosis and can predict future cardiovascular events. Here, using apolipoprotein E (APOE)-deficient mice, we investigated the level of sICAM-1 in parallel with endothelial ICAM-1 expression and aortic atherosclerosis. We also examined the effect of ICAM-1 deficiency during the progression of atherosclerosis using double knockout mice. The level of sICAM-1 increased significantly in parallel with the progression of atherosclerosis in APOE-deficient mice, while the sICAM-1 level remained constant in wild-type mice from 3 to 10 months of age. ICAM-1 staining was detected in virtually all endothelial cells, however, ICAM-1 was expressed strongly in the endothelium overlying atheromatous palque in APOE-deficient mice. Deficiency of ICAM-1 in APOE-deficient mice significantly reduced lesions after 5 and 10 months. The present study supported the notion that the level of sICAM-1 is closely related with the severity of atherosclerosis and cardiovascular events, and also suggested that inhibition of ICAM-1 can delay the progression of atherosclerosis.

Lung resistance-related protein (LRP) was identified as the major vault protein (MVP), the main component of multimeric vault particles. It functions as a transport-associated protein that can be associated with multidrug resistance. In previous studies, expression of MVP/LRP has been documented in tumors of various types. In general, good correlations have been reported for expression of MVP/LRP and decreased sensitivity to chemotherapy and poor prognosis. MVP/LRP expression has been documented in glioblastomas, but its expression in nervous system tumors in general has not been well characterized. Immunohistochemistry using anti-human MVP/LRP antibody (LRP-56) was performed on formalin-fixed, paraffin-embedded archival tissue from 69 primary central nervous system tumors. Expression of MVP/LRP was observed in 81.2% (56/69) of primary nervous system tumors, including astrocytomas (11/13), oligodendrogliomas (1/2), oligoastrocytomas (5/5), ependymoma (1/1), meningiomas (35/45), schwannomas (2/2), and neurofibroma (1/1). Various degrees and distributions of immunoreactivity to MVP/ LRP were observed. Neither the presence nor the degree of immunoreactivity to MVP/LRP showed any correlation with either tumor grade or the presence of brain invasion.