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.

Bcl-xL is a well characterized death-suppressing molecule of the Bcl-2 family. Bcl-xL is expressed in embryonic and adult neurons of the CNS and may play a critical role in preventing neuronal apoptosis that occurs during brain development or results from diverse pathologic stimuli, including cerebral ischemia. In this study, we used a novel approach to study the potential neuroprotective effect of Bcl-xL as a therapeutic agent in the murine model of focal ischemia/reperfusion. We created a Bcl-xL fusion protein, designated as PTD-HA-Bcl-xL, which contains the protein transduction domain (PTD) derived from the human immunodeficiency TAT protein. We demonstrated that this fusion protein is highly efficient in transducing into primary neurons in cultures and potently inhibited staurosporin-induced neuronal apoptosis. Furthermore, intraperitoneal injection of PTD-HA-Bcl-xL into mice resulted in robust protein transduction in neurons in various brain regions within 1-2 hr, and decreased cerebral infarction (up to approximately 40%) in a dose-dependent manner, as determined at 3 d after 90 min of focal ischemia. PTD-HA-Bcl-xL was effective even when it was administered after the completion of ischemia (up to 45 min), and the protective effect was independent of the changes in cerebral blood flow or other physiological parameters. Finally, as shown by immunohistochemistry, Western blotting, and substrate-cleavage assays, PTD-HA-Bcl-xL attenuated ischemia-induced caspase-3 activation in ischemic neurons. These results thus confirm the neuroprotective effect of Bcl-xL against ischemic brain injury and provide the first evidence that the PTD can be used to efficiently transduce a biologically active neuroprotectant in experimental cerebral ischemia.

The major heat shock protein, Hsp70, can protect against cell death by directly interfering with mitochondrial apoptosis pathways. However, Hsp70 also sensitizes cells to certain apoptotic stimuli like TNF. Little is known about how Hsp70 enhances apoptosis. We demonstrate here that Hsp70 promotes TNF killing by specifically binding the coiled-coil domain of IB kinase ␥ (IKK␥) to inhibit IKK activity and consequently inhibit NF-B-dependent antiapoptotic gene induction. An IKK␥ mutant, which interacts with Hsp70, competitively inhibits the Hsp70-IKK␥ interaction and relieves heat-mediated NF-B suppression. Depletion of Hsp70 expression with RNA interference rescues TNF-mediated cell death. Although TNF may or may not be sufficient to trigger apoptosis on its own, TNF-triggered apoptosis was initiated or made worse when Hsp70 expression increased to high levels to disrupt NF-B signaling. These results provide significant novel insights into the molecular mechanism for the pro-apoptotic behavior of Hsp70 in death-receptor-mediated cell death.

Understanding transcriptional changes in brain after ischemia may provide therapeutic targets for treating stroke and promoting recovery. To study these changes on a genomic scale, oligonucleotide arrays were used to assess RNA samples from periinfarction cortex of adult Sprague-Dawley rats 24 h after permanent middle cerebral artery occlusions. Of the 328 regulated transcripts in ischemia compared with sham-operated animals, 264 were upregulated, 64 were downregulated, and 163 (49.7%) had not been reported in stroke. Of the functional groups modulated by ischemia: G-protein-related genes were the least reported; and cytokines, chemokines, stress proteins, and cell adhesion and immune molecules were the most highly expressed. Quantitative reverse transcription polymerase chain reaction of 20 selected genes at 2, 4, and 24 h after ischemia showed early upregulated genes (2 h) including Narp, Rad, G33A, HYCP2, Pim-3, Cpg21, JAK2, CELF, Tenascin, and DAF. Late upregulated genes (24 h) included Cathepsin C, Cip-26, Cystatin B, PHAS-I, TBFII, Spr, PRG1, and LPS-binding protein. Glycerol 3-phosphate dehydrogenase, which is involved in mitochondrial reoxidation of glycolysis derived NADH, was regulated more than 60-fold. Plasticity-related transcripts were regulated, including Narp, agrin, and Cpg21. A newly reported lung pathway was also regulated in ischemic brain: C/EBP induction of Egr-1 (NGFI-A) with downstream induction of PAI-1, VEGF, ICAM, IL1, and MIP1. Genes regulated acutely after stroke may modulate cell survival and death; also, late regulated genes may be related to tissue repair and functional recovery.

Geldanamycin (GA), a benzoquinone ansamycin, binds Hsp90 in vitro, releases heat shock factor (HSF1) and induces heat shock proteins (Hsps). Because viral and transgenic overexpression of Hsps protects cells against ischemia in vitro, we hypothesized that GA would protect brain from focal ischemia by inducing Hsps in vivo. Adult male SpragueDawley rats were subjected to 2-hour middle cerebral artery occlusions (MCAO) using the suture technique followed by 22-h reperfusions. GA or vehicle was injected into the lateral cerebral ventricles (i.c.v) 24 h before ischemia. Geldanamycin at 1 lg/kg decreased infarct volumes by 55.7% (p < 0.01) and TUNEL-positive cells by 30% in cerebral cortex. GA also improved behavioral outcomes (p < 0.01) and reduced brain edema (p < 0.05). Western blots showed that the 1 lg/kg GA dose induced Hsp70 and Hsp25 protein 8.2-fold and 2.7-fold, respectively, by 48 h following administration. Immunocytochemistry showed that GA induced Hsp70 in neurons and Hsp25 in glia and arteries in cortex, hippocampus, hypothalamus, and other brain regions. GA reduced co-immunoprecipitation of HSF1 with Hsp90 in brain tissue homogenates, promoted HSE-binding of HSF in brain nuclear extracts using gel shift assays, and increased luciferase reporter gene transcription for the Hsp70 promoter in PC12 cells. The data show that geldanamycin protects brain from focal ischemia and that this may be due, at least in part, to geldanamycin stimulation of heat shock gene transcription.