Variable MR and pathologic patterns of hemorrhage after iodinated contrast infusion in MCA occlusion/reperfusion model.

OBJECTIVE: To examine the hypothesis that IA reperfusion with iso-osmolar iodixanol, low-osmolar iopamidol, or saline causes different effects on MR signal changes and pathologic cut-brain section related to hemorrhagic transformation (HT) or iodinated radiographic contrast media (IRCM) deposition. METHODS: Infarct was induced in 30 rats by middle cerebral artery suture occlusion. Reperfusion was performed after 5 hours with iso-osmolar iodixanol (n=9), low-osmolar iopamidol (n=12) or saline (n=9). MR images were obtained immediately after reperfusion and rats were sacrificed at 24 hours. Hypointense areas within the infarction on T2-weighted (T2-WI) or gradient echo (GRE) images were recorded and compared with HT on pathology. Fisher's exact test was used for proportions, and receiver operator curve analysis to evaluate MRI discrimination of hemorrhage. RESULTS: Two types of HT were noted on pathology: confluent >0.2 mm petechial hemorrhage (PeH, 78%) or well-defined ≤0.2 mm hemorrhagic focus (HF, 22%). PeH was least common in the iodixanol subgroup (p<0.02). HF was more common in the IRCM group. Hypointense areas on T2-WI but not on GRE were significantly more common in the IRCM group (p<0.05). Hypointense areas on T2-WI and GRE discriminated HT (area under the curve: 0.714, p<0.002). CONCLUSIONS: IRCM and saline induced different MRI signal and pathologic patterns in our sample. We postulate that T2 hypointensity with no GRE hypointensity might be associated with IRCM deposition; and decreased frequency of PeH after iodixanol infusion and the presence of HF almost exclusively in the IRCM group might represent a direct/indirect effect of contrast infusion/deposition in the brain parenchyma after reperfusion. Our results support previous observations in IMS III and are hypothesis generating.

Hemorrhagic transformation after ischemic stroke in animals and humans.

Hemorrhagic transformation (HT) is a common complication of ischemic stroke that is exacerbated by thrombolytic therapy. Methods to better prevent, predict, and treat HT are needed. In this review, we summarize studies of HT in both animals and humans. We propose that early HT (<18 to 24 hours after stroke onset) relates to leukocyte-derived matrix metalloproteinase-9 (MMP-9) and brain-derived MMP-2 that damage the neurovascular unit and promote blood-brain barrier (BBB) disruption. This contrasts to delayed HT (>18 to 24 hours after stroke) that relates to ischemia activation of brain proteases (MMP-2, MMP-3, MMP-9, and endogenous tissue plasminogen activator), neuroinflammation, and factors that promote vascular remodeling (vascular endothelial growth factor and high-moblity-group-box-1). Processes that mediate BBB repair and reduce HT risk are discussed, including transforming growth factor beta signaling in monocytes, Src kinase signaling, MMP inhibitors, and inhibitors of reactive oxygen species. Finally, clinical features associated with HT in patients with stroke are reviewed, including approaches to predict HT by clinical factors, brain imaging, and blood biomarkers. Though remarkable advances in our understanding of HT have been made, additional efforts are needed to translate these discoveries to the clinic and reduce the impact of HT on patients with ischemic stroke.

Peroxynitrite decomposition catalyst prevents matrix metalloproteinase activation and neurovascular injury after prolonged cerebral ischemia in rats.

Matrix metalloproteinases (MMPs) play an important role in reperfusion-induced brain injury following ischemia. To define the effects of peroxynitrite decomposition catalyst on MMP activation and neurovascular reperfusion injury, 5,10,15,20-tetrakis (2,4,6-trimethyl-3,5-disulfonatophenyl)-porphyrin iron (III) (FeTMPyP) was administered intravenously 30 min prior to reperfusion following a middle cerebral artery occlusion. Activation of MMP was assessed by in situ and gel zymography. Neurovascular injury was assessed using endothelial barrier antigen, collagen IV immunohistochemistry and Cresyl violet staining. Results were compared with sham and ischemia alone groups. We found that administration of FeTMPyP just before reperfusion after ischemia inhibited MMP-9 activation and total MMP-2 increases in the cortex and decreased active MMP-9 along with the total amounts of active MMP-9 and active MMP-2 in the striatum. Reperfusion-induced injury to the basal lamina of collagen IV-immunopositive microvasculature and neural cells in cortex and striatum was ameliorated by FeTMPyP. Losses of blood vessel endothelium produced by ischemia or reperfusion were also decreased in the cortex. These results suggest that administration of FeTMPy prior to reperfusion decreases MMP activation and neurovascular injury after prolonged cerebral ischemia. This strategy may be useful for future therapies targeted at preventing breakdown of the blood-brain barrier and hemorrhagic transformation.

Down-regulation of interleukin 7 mRNA by hypoxia is calcium dependent.

OBJECTIVE: The discovery of IL-7R(alpha) polymorphisms implicated in the pathogenesis of multiple sclerosis has highlighted the importance of interleukin 7 (IL-7) in central nervous system diseases. Hypoxia affects neurological disease states in part by modulating expression of many early and late response genes. The present work used cultured PC12 cells to investigate the effect of hypoxia on IL-7 expression. METHOD: PC12 cells were cultured in Dulbecco's modified Eagle's medium (DMEM)/F12 medium. RNA was isolated and reverse transcriptase-polymerase chain reaction (RT-PCR) was run to quantify messenger RNA (mRNA) change. Western blots were used to assess IL-7 protein change in the medium. Extracellular free Ca(2+) was removed by using Ca(2+)-free DMEM/F12 with 1 mM ethylene glycol tetraacetic acid for 45 minutes before the start of hypoxia. RESULTS: Exposure of PC12 cells to 1% oxygen for 6 hours decreased IL-7 mRNA by 77% using RT-PCR (p<0.01). Exposure to 1% oxygen for 24 hours decreased IL-7 protein in the medium by 21% (p<0.05). As hypoxia duration increased (2, 4, 6 and 24 hours) or oxygen concentrations decreased (10%, 5% and 1%), IL-7 mRNA expression progressively decreased. Removal of extracellular free Ca(2+) completely prevented these hypoxia-induced decreases of IL-7 mRNA. DISCUSSION: Since IL-7 exhibits trophic properties in developing brain, down-regulation of IL-7 by hypoxia may contribute to hypoxia-induced injury to neural cells.

A novel 165-kDa Golgin protein induced by brain ischemia and phosphorylated by Akt protects against apoptosis.

A cDNA encoding a novel protein was cloned from ischemic rat brain and found to be homologous to testis Mea-2 Golgi-associated protein (Golga3). The sequence predicted a 165-kDa protein, and in vitro translated protein exhibited a molecular mass of 165-170 kDa. Because brain ischemia induced the mRNA, and the protein localized to the Golgi apparatus, this protein was designated Ischemia-Inducible Golgin Protein 165 (IIGP165). In HeLa cells, serum and glucose deprivation-induced caspase-dependent cleavage of the IIGP165 protein, after which the IIGP165 fragments translocated to the nucleus. The C-terminus of IIGP165, which contains a LXXLL motif, appears to function as a transcriptional co-regulator. Akt co-localizes with IIGP165 protein in the Golgi in vivo, and phosphorylates IIGP165 on serine residues 345 and 134. Though transfection of IIGP165 cDNA alone does not protect HeLa cells from serum deprivation or Brefeldin-A-triggered cell death, co-transfection of both Akt and IIGP165 cDNA or combined IIGP165-transfection with PDGF treatment significantly protects HeLa cells better than either treatment alone. These data show that Akt phosphorylation of IIGP165 protects against apoptotic cell death, and add to evidence that the Golgi apparatus also plays a role in regulating apoptosis.

Gene expression in blood changes rapidly in neutrophils and monocytes after ischemic stroke in humans: a microarray study.

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 5 h 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.

Hypoxia preconditioning in the brain.

Exposure to moderate hypoxia alone does not cause neuronal death as long as blood pressure and cerebral blood flow are maintained in mammals. In neonatal and adult mammals including rats and mice, carotid occlusion in combination with hypoxia produces neuronal death and brain infarction. However, preexposure to 8% oxygen for 3 h protects the brain and likely other organs of neonatal and adult rats against combined hypoxia-ischemia 24 h later. In this paper, the possible mechanisms of this so-called hypoxia-induced tolerance to ischemia is discussed. One mechanism likely involves hypoxia-inducible factor-1alpha (HIF-1alpha). HIF-1alpha is a transcription factor that - during hypoxia - binds with a second protein (HIF-1beta) in the nucleus to promoter elements in hypoxia-responsive target genes. This causes upregulation of HIF target genes including VEGF, erythropoietin, iNOS, glucose transporter-1, glycolytic enzymes, and many other genes to protect the brain against ischemia 24 h later. In addition, non-HIF pathways including MTF-1, Egr-1 and others act directly or indirectly on other target genes to also promote hypoxia-induced preconditioning. Hypoxia preconditioning can be mimicked by iron chelators like desferrioxamine and transition metals like cobalt chloride that inhibit prolyl hydroxylases, increase HIF-1alpha levels in the brain, and produce protection of the brain against combined hypoxia-ischemia 24 h later. This hypoxia preconditioning has potential clinical usefulness in protecting high-risk newborns or to provide protection prior to surgery.

Human blood genomics: distinct profiles for gender, age and neurofibromatosis type 1.

Application of gene expression profiling to human diseases will be limited by availability of tissue samples. It was postulated that germline genetic defects affect blood cells to produce unique expression patterns. This hypothesis was addressed by using a test neurological disease-neurofibromatosis type 1 (NF1), an autosomal dominant genetic disease caused by mutations of the NF1 gene at chromosome 17q11.2. Oligonucleotide arrays were used to survey the blood gene expression pattern of 12 NF1 patients compared to 96 controls. A group of genes related to tissue remodeling, bone development and tumor suppression were down-regulated in NF1 blood samples. In addition, there were blood genomic patterns for gender and age: Y chromosome genes showing higher expression in males, indicating a gene-dosage effect; and genes related to lymphocyte functions showing higher expression in children. The results suggest that genetic mutations can be manifested at the transcriptional level in peripheral blood cells and blood gene expression profiling may be useful for studying phenotypic differences of human genetic diseases and possibly providing diagnostic and prognostic markers.

Hsp70 mutant proteins modulate additional apoptotic pathways and improve cell survival.

Although wild-type Hsp70 (Hsp70WT) inhibits procaspase-3 processing by preventing apoptosome formation, Hsp70WT does not block active caspase-3. Because all caspase-3 inhibitors bear canonical DXXD caspase-3 recognition motifs, we determined whether mutated Hsp70s with caspase-binding motifs act as direct caspase-3 inhibitors. Based on Hsp70 molecular modeling, the DNQP, DEVQ, and EEVD regions localized on the surface of Hsp70WT were chosen, allowing us to design mutants while trying to avoid disrupting the global fold of the molecule and losing the possibility of protein-protein interactions. We replaced DNQP with DQMD, and DEVQ and EEVD with DEVD residues that should be optimal substrates for caspase-3. The resultant Hsp70 mutants directly interacted with active caspase-3 and blocked its proteolytic activity while retaining the ability to reverse protein denaturation and disrupt the interaction between Apaf-1 and procaspase-9. The Hsp70C-terminal mutants interacted with Apaf-1 and active caspase-3 significantly longer than Hsp70WT. The Hsp70 DXXD mutants protected neuron and teratocarcinoma (NT) cells against cell death much better than Hsp70WT whether given before or after serum withdrawal. Hsp70 mutants represent a possible approach to antiapoptotic biotherapeutics. Similar rational designs could be used to engineer inhibitors of additional caspase family members.

Hypoxia-ischemia induces DNA synthesis without cell proliferation in dying neurons in adult rodent brain.

Recent studies suggest that postmitotic neurons can reenter the cell cycle as a prelude to apoptosis after brain injury. However, most dying neurons do not pass the G1/S-phase checkpoint to resume DNA synthesis. The specific factors that trigger abortive DNA synthesis are not characterized. Here we show that the combination of hypoxia and ischemia induces adult rodent neurons to resume DNA synthesis as indicated by incorporation of bromodeoxyuridine (BrdU) and expression of G1/S-phase cell cycle transition markers. After hypoxia-ischemia, the majority of BrdU- and neuronal nuclei (NeuN)-immunoreactive cells are also terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL)-stained, suggesting that they undergo apoptosis. BrdU+ neurons, labeled shortly after hypoxia-ischemia, persist for >5 d but eventually disappear by 28 d. Before disappearing, these BrdU+/NeuN+/TUNEL+ neurons express the proliferating cell marker Ki67, lose the G1-phase cyclin-dependent kinase (CDK) inhibitors p16INK4 and p27Kip1 and show induction of the late G1/S-phase CDK2 activity and phosphorylation of the retinoblastoma protein. This contrasts to kainic acid excitotoxicity and traumatic brain injury, which produce TUNEL-positive neurons without evidence of DNA synthesis or G1/S-phase cell cycle transition. These findings suggest that hypoxia-ischemia triggers neurons to reenter the cell cycle and resume apoptosis-associated DNA synthesis in brain. Our data also suggest that the demonstration of neurogenesis after brain injury requires not only BrdU uptake and mature neuronal markers but also evidence showing absence of apoptotic markers. Manipulating the aberrant apoptosis-associated DNA synthesis that occurs with hypoxia-ischemia and perhaps neurodegenerative diseases could promote neuronal survival and neurogenesis.

Hsp70 promotes TNF-mediated apoptosis by binding IKK gamma and impairing NF-kappa B survival signaling.

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 I kappa B kinase gamma (IKK gamma) to inhibit IKK activity and consequently inhibit NF-kappa B-dependent antiapoptotic gene induction. An IKK gamma mutant, which interacts with Hsp70, competitively inhibits the Hsp70-IKK gamma interaction and relieves heat-mediated NF-kappa 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-kappa 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.

Genomics of the periinfarction cortex after focal cerebral ischemia.

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.

Heme and iron metabolism: role in cerebral hemorrhage.

Heme and iron metabolism are of considerable interest and importance in normal brain function as well as in neurodegeneration and neuropathologically following traumatic injury and hemorrhagic stroke. After a cerebral hemorrhage, large numbers of hemoglobin-containing red blood cells are released into the brain's parenchyma and/or subarachnoid space. After hemolysis and the subsequent release of heme from hemoglobin, several pathways are employed to transport and metabolize this heme and its iron moiety to protect the brain from potential oxidative stress. Required for these processes are various extracellular and intracellular transporters and storage proteins, the heme oxygenase isozymes and metabolic proteins with differing localizations in the various brain-cell types. In the past several years, additional new genes and proteins have been discovered that are involved in the transport and metabolism of heme and iron in brain and other tissues. These discoveries may provide new insights into neurodegenerative diseases like Alzheimer's, Parkinson's, and Friedrich's ataxia that are associated with accumulation of iron in specific brain regions or in specific organelles. The present review will examine the uptake and metabolism of heme and iron in the brain and will relate these processes to blood removal and to the potential mechanisms underlying brain injury following cerebral hemorrhage.

Blood genomic expression profile for neuronal injury.

This study determined whether stroke and other types of insults produced a gene expression profile in blood that correlated with the presence of neuronal injury. Adult rats were subjected to ischemic stroke, intracerebral hemorrhage, status epilepticus, and insulin-induced hypoglycemia and compared with untouched, sham surgery, and hypoxia animals that had no brain injury. One day later, microarray analyses showed that 117 genes were upregulated and 80 genes were downregulated in mononuclear blood cells of the "injury" (n = 12) compared with the "no injury" (n = 9) animals. A second experiment examined the whole blood genomic response of adult rats after global ischemia and kainate seizures. Animals with no brain injury were compared with those with brain injury documented by TUNEL and PANT staining. One day later, microarray analyses showed that 37 genes were upregulated and 67 genes were downregulated in whole blood of the injury (n = 4) animals compared with the no-injury (n = 4) animals. Quantitative reverse transcription-polymerase chain reaction confirmed that the vesicular monoamine transporter-2 increased 2.3- and 1.6-fold in animals with severe and mild brain injury, respectively, compared with no-injury animals. Vascular tyrosine phosphatase-1 increased 2.0-fold after severe injury compared with no injury. The data support the hypothesis that there is a peripheral blood genomic response to neuronal injury, and that this blood response is associated with a specific blood mRNA gene expression profile that can be used as a marker of the neuronal damage.

In Vivo Delivery of a Bcl-xL Fusion Protein Containing the TAT Protein Transduction Domain Protects against Ischemic Brain Injury and Neuronal Apoptosis.

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.

Geldanamycin induces heat shock proteins in brain and protects against focal cerebral ischemia.

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 Sprague-Dawley 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 microg/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 microg/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.

Suppressive effects of ansamycins on inducible nitric oxide synthase expression and the development of experimental autoimmune encephalomyelitis.

The production of nitric oxide by the inflammatory isoform of nitric oxide synthase (NOS2) in brain glial cells is thought to contribute to the causes and development of neurological diseases and trauma. We previously demonstrated that activation of a heat shock response (HSR) by hyperthermia reduced NOS2 expression in vitro, and in vivo attenuated the clinical and histological symptoms of the demyelinating disease experimental autoimmune encephalomyelitis (EAE; Heneka et al. [2001] J. Neurochem. 77:568-579). Benzoquinoid ansamycins are fungal-derived antibiotics with tyrosine kinase inhibitory properties, and which also induce a HSR by allowing activation of HS transcription factor HSF1. We now show that two members of this class of drugs (geldanamycin and 17-allylamino-17-demethoxygeldanamycin) also induce a HSR in primary rat astrocytes and rat C6 glioma cells. Both drugs dose-dependently reduced nitrite accumulation, NOS2 steady-state mRNA levels, and the cytokine-dependent activation of a rat 2.2-kB NOS2 promoter construct stably expressed in C6 cells. These inhibitory effects were partially reversed by quercetin, a bioflavonoid which prevents HSF1 binding to DNA and thus attenuates the HSR. Ansamycins increased mRNA levels of the inhibitory IkappaBalpha protein, suggesting that inhibition of NFkappaB activation could contribute to their suppressive effects. Finally, in C57BL/6 mice actively immunized to develop EAE, a single injection of geldanamycin at 3 days after immunization reduced disease onset by over 50%. These results indicate that ansamycins can exert potent anti-inflammatory effects on brain glial cells which may provide therapeutic benefit in neuroinflammatory diseases.

17-beta-estradiol induces heat shock proteins in brain arteries and potentiates ischemic heat shock protein induction in glia and neurons.

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.