Ischemic brain and peripheral white blood cells release cytokines, chemokines and other molecules that activate the peripheral white blood cells after stroke. To assess gene expression in these peripheral white blood cells, whole blood was examined using oligonucleotide microarrays in 15 patients at 2.4 ± 0.5, 5 and 24 h after onset of ischemic stroke and compared with control blood samples. The 2.4 h blood samples were drawn before patients were treated either with tissue-type plasminogen activator (tPA) alone or with tPA plus Eptifibatide (the Combination approach to Lysis utilizing Eptifibatide And Recombinant tPA trial). Most genes induced in whole blood at 2 to 3 h were also induced at 5 and 24 h. Separate studies showed that the genes induced at 2 to 24 h after stroke were expressed mainly by polymorphonuclear leukocytes and to a lesser degree by monocytes. These genes included: matrix metalloproteinase 9; S100 calcium-binding proteins P, A12 and A9; coagulation factor V; arginase I; carbonic anhydrase IV; lymphocyte antigen 96 (cluster of differentiation (CD)96); monocarboxylic acid transporter (6); ets-2 (erythroblastosis virus E26 oncogene homolog 2); homeobox gene Hox 1.11; cytoskeleton-associated protein 4; N-formylpeptide receptor; ribonuclease-2; N-acetylneuraminate pyruvate lyase; BCL6; glycogen phosphorylase. The fold change of these genes varied from 1.6 to 6.8 and these 18 genes correctly classified 10/15 patients at 2.4 h, 13/15 patients at 5h and 15/15 patients at 24 h after stroke. These data provide insights into the inflammatory responses after stroke in humans, and should be helpful in diagnosis, understanding etiology and pathogenesis, and guiding acute treatment and development of new treatments for stroke.

Hypoxic preconditioning (8% O 2 , 3 h) produces tolerance 24 h after hypoxic-ischemic brain injury in neonatal rats. To better understand the ischemic tolerance mechanisms induced by hypoxia, we used oligonucleotide microarrays to examine genomic responses in neonatal rat brain following 3 h of hypoxia (8% O 2 ) and either 0, 6, 18, or 24 h of re-oxygenation. The results showed that hypoxia-inducible factor (HIF)-1-but not HIF-2-mediated gene expression may be involved in brain hypoxia-induced tolerance. Among the genes regulated by hypoxia, 12 genes were confirmed by real time reverse transcriptase-PCR as follows: VEGF, EPO, GLUT-1, adrenomedullin, propyl 4-hydroxylase ␣, MT-1, MKP-1, CELF, 12-lipoxygenase, t-PA, CAR-1, and an expressed sequence tag. Some genes, for example GLUT-1, MT-1, CELF, MKP-1, and t-PA did not show any hypoxic regulation in either astrocytes or neurons, suggesting that other cells are responsible for the up-regulation of these genes in the hypoxic brain. These genes were expressed in normal and hypoxic brain, heart, kidney, liver, and lung, with adrenomedullin, MT-1, and VEGF being prominently induced in brain by hypoxia. These results suggest that a number of endogenous molecular mechanisms may explain how hypoxic preconditioning protects against subsequent ischemia, and may provide novel therapeutic targets for treatment of cerebral ischemia.Impaired oxygen (hypoxia) or reduced blood flow (ischemia) to the brain is a major cause of morbidity and mortality in the perinatal period, often resulting in cognitive impairment, seizures, and other neurological disabilities. Although hypoxiaischemia animal models have increased our understanding of the processes leading to cell death, there are still no pharmacological treatments available to reduce cell death in ischemic neonatal brain.Interestingly, cells can be protected when a non-injurious hypoxic stress is performed several hours or days before a lethal hypoxic-ischemic stress (preconditioning). This phenomenon is called tolerance. Ischemic tolerance can be achieved in brain by several preconditioning sublethal stresses such as hypoxia (1-3), ischemia itself (4), hypothermia (5), hyperthermia (6), hyperbaric oxygenation (7), metabolic inhibitors (8), spreading depression (9), as well as cytokines (10, 11).As hypoxic preconditioning is non-invasive and reproducible, this model has been used to study the mechanisms protecting the brain against hypoxia-ischemia particularly in newborn rats (3,(12)(13)(14). In addition, hypoxic preconditioning also induces tolerance against focal transient (15) and permanent (16) cerebral ischemia in adult mice. These studies suggested that hypoxia-inducible factor-1 (HIF-1) 1 could be an important mediator of hypoxia-induced tolerance to ischemia (13,14,16). Indeed, hypoxic preconditioning induces expression of HIF-1␣ and its target genes in neonatal (14) and adult brain (16). In addition, desferrioxamine and cobalt chloride, two agents that activate HIF-1 (17), also induce tolerance against hypoxia-ischemia...

The authors have previously shown that bilirubin-oxidation products (BOXes) are present in CSF of subarachnoid hemorrhage patients with vasospasm, and that BOXes cause vasoconstriction in vitro. This study determined whether BOXes cause vasospasm in vivo. Identical volumes of either lysed blood or standardized amounts of BOXes were injected into the cisterna magna of adult rats. BOX injections caused 6 of 10 rats to die within 10 minutes, whereas 12 of 12 rats survived for 24 hours after blood injections. The mechanism for this significant (P < or = 0.01) increase in mortality was unclear. To directly test whether BOXes produced vasospasm, a cranial window technique was used. Application of 20 microL of 10-micromol/L bilirubin had little effect on the vessels. However, application of BOXes produced marked, dose-dependent small artery and arteriole vasospasm that approached a 90% decrease in diameter by 40 minutes after application in some vessels, and persisted for at least 24 hours. To determine if BOX-mediated vasospasm led to cortical injury, histology and immunocytochemistry were performed on animals that survived for 24 hours. There was a BOX-related stress protein response for HSP25 and HSP32 (HO-1) without evidence of infarction. The finding that the BOXes produce vasospasm of cerebral vessels in vivo, in conjunction with BOXes being found in CSF of vasospasm patients, supports our hypothesis that BOXes contribute to or cause cerebral vasospasm after subarachnoid hemorrhage.

Estradiol reduces brain injury from many diseases, including stroke and trauma. To investigate the molecular mechanisms of this protection, the effects of 17-beta-estradiol on heat shock protein (HSP) expression were studied in normal male and female rats and in male gerbils after global ischemia. 17-beta-estradiol was given intraperitoneally (46 or 460 ng/kg, or 4.6 microg/kg) and Western blots performed for HSPs. 17-beta-estradiol increased hemeoxygenase-1, HSP25/27, and HSP70 in the brain of male and female rats. Six hours after the administration of 17-beta-estradiol, hemeoxygenase-1 increased 3.9-fold (460 ng/kg) and 5.4-fold (4.6 microg/kg), HSP25/27 increased 2.1-fold (4.6 microg/kg), and Hsp70 increased 2.3-fold (460 ng/kg). Immunocytochemistry showed that hemeoxygenase-1, HSP25/27,and HSP70 induction was localized to cerebral arteries in male rats, possibly in vascular smooth muscle cells. 17-beta-estradiol was injected intraperitoneally 20 minutes before transient occlusion of both carotids in adult gerbils. Six hours after global cerebral ischemia, 17-beta-estradiol (460 ng/kg) increased levels of hemeoxygenase-1 protein 2.4-fold compared with ischemia alone, and HSP25/27 levels increased 1.8-fold compared with ischemia alone. Hemeoxygenase-1 was induced in striatal oligodendrocytes and hippocampal neurons, and HSP25/27 levels increased in striatal astrocytes and hippocampal neurons. Finally, Western blot analysis confirmed that estrogen induced heat shock factor-1, providing a possible mechanism by which estrogen induces HSPs in brain and other tissues. The induction of HSPs may be an important mechanism for estrogen protection against cerebral ischemia and other types of injury.

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Global ischemia causes an extensive cell death 3 days after the ischemia in the CA1 region of the hippocampus, which is preceded by induction of a spectrum of genes with both neuroprotective and detrimental properties. This delayed cell death has been suggested to be mainly caused by programmed cell death. Here we applied differential display to characterize transcripts induced by global ischemia after 1 day in Mongolian gerbils, when the cells in the CA1 region are still viable, but initiating the cell death pathway. One of the cloned transcripts turned out to be a repeat sequence termed SINE B2. We also cloned the other member of the SINE family, SINE B1, and found it also to be slightly induced by ischemia in the CA1 region. The SINE repeat regions are not translated and their role in ischemia may be related the neurons' attempt to cope with decreased translational levels and/or genomic reorganization. Together with the previous data demonstrating the inducibility of the SINE transcripts using in vitro stress models, the present study shows that SINE transcripts are stress-inducible factors in the central nervous system.