Biomedical subjects

Frank R Sharp

Publications and source records attributed to Frank R Sharp.

46 records · Page 3Linked to original sources

PubMed2002

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.

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PubMed2002

Neurogenesis following brain ischemia.

Following 5 or 10 min of global ischemia in the adult gerbil there is a tenfold increase in the birth of new cells in the subgranular zone of dentate gyrus of the hippocampus as assessed using BrdU incorporation. This begins at 7 days, peaks at 11 days, and decreases thereafter. Over the next month approximately 25% of the newborn cells disappear. Of the remaining cells, 60% migrate into the granule cell layer where two-thirds become NeuN, calbindin and MAP-2 immunostained neurons. The remaining 40% of the cells migrate into the dentate hilus where 25% of these become GFAP labeled astrocytes. It is proposed that ischemia-induced neurogenesis contributes to the recovery of function, and specifically may serve to improve anterograde and retrograde recent memory function that is lost following global ischemia in animals and man.

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PubMed2002

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.

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PubMed2002

Genomic responses of the brain to ischemic stroke, intracerebral haemorrhage, kainate seizures, hypoglycemia, and hypoxia.

RNA expression profiles in rat brain were examined 24 h after ischemic stroke, intracerebral haemorrhage, kainate-induced seizures, insulin-induced hypoglycemia, and hypoxia and compared to sham- or untouched controls. Rat oligonucleotide microarrays were used to compare expression of over 8000 transcripts from three subjects in each group (n = 27). Of the somewhat less than 4000 transcripts called 'present' in normal or treated cortex, 5-10% of these were up-regulated 24 h after ischemia (415), haemorrhage (205), kainate (187), and hypoglycemia (302) with relatively few genes induced by 6 h of moderate (8% oxygen) hypoxia (15). Of the genes induced 24 h after ischemia, haemorrhage, and hypoglycemia, approximately half were unique for each condition suggesting unique components of the responses to each of the injuries. A significant component of the responses involved immune-process related genes likely to represent responses to dying neurons, glia and vessels in ischemia; to blood elements in haemorrhage; and to the selectively vulnerable neurons that die after hypoglycemia. All of the genes induced by kainate were also induced either by ischemia, haemorrhage or hypoglycemia. This strongly supports the concept that excitotoxicity not only plays an important role in ischemia, but is an important mechanism of brain injury after intracerebral haemorrhage and hypoglycemia. In contrast, there was only a single gene that was down-regulated by all of the injury conditions suggesting there is not a common gene down-regulation response to injury.

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PubMed2002

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.

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PubMed2002

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.

Animals↗

PubMed2002

Oxidation of bilirubin produces compounds that cause prolonged vasospasm of rat cerebral vessels: a contributor to subarachnoid hemorrhage-induced vasospasm.

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.

Animals↗

PubMed2002

Correlation between changes in apparent diffusion coefficient and induction of heat shock protein, cell-specific injury marker expression, and protein synthesis reduction on diffusion-weighted magnetic resonance images after temporary focal cerebral ischemia in rats.

OBJECT: The authors investigated the relationship between the time course of apparent diffusion coefficient (ADC) changes and stress protein induction, ischemic neuroglial damage, and cerebral protein synthesis (CPS) after temporary focal cerebral ischemia in rats. METHODS: In Group I, ADC changes were measured on magnetic resonance (MR) images obtained during the second half of a 1-hour middle cerebral artery (MCA) occlusion, during a 1-hour reperfusion, and after 23 hours of reperfusion in rats. Immunohistochemical studies for heat shock protein (hsp) 70, glial fibrillary acidic protein (GFAP), and neuronal nuclear (NeuN) protein were performed. In Group II, CPS was assessed using autoradiographic studies obtained after occlusion. At 36 minutes of occlusion, MR imaging demonstrated significantly less ADC reduction in the frontoparietal cortex (82 +/- 9% of the contralateral hemisphere) than in the striatum (64 +/- 11%; p < 0.05). After 1 hour of reperfusion, the lesion resolved and the difference between cortex and striatum was no longer evident. After 23 hours of reperfusion, the ADC lesion recurred in striatum (76 +/- 12%) compared with frontoparietal cortex (100 +/- 11%; p < 0.05). Immunohistochemical studies showed hsp 70 expression and an increased GFAP reactivity localized in the frontoparietal cortex of the ischemic hemisphere, along with a significant drop in striatal NeuN immunoreactivity. A trend toward greater reduction in striatal CPS (53 +/- 15%) than in frontoparietal cortex CPS (78 +/- 23%) was also observed. CONCLUSIONS: Sequential ADC maps correlate with the expression of neuroglial stress and injury markers after temporary focal ischemia in rats, distinguishing the striatum (infarct core) from the cortex (ischemic penumbra). A greater reduction in striatal CPS further supports the conclusion that the striatum is more susceptible to temporary MCA occlusion than the cortex.

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PubMedDate not supplied

Genomic abnormalities in patients with migraine and chronic migraine: preliminary blood gene expression suggests platelet abnormalities.

BACKGROUND: Migraine has strong genetic and environmental components and may also be a significant contributor to chronic migraine (CM). It is hypothesized that gene expression changes in peripheral blood cells can be used to detect the interaction of these influences. OBJECTIVE: Distinct genomic expression patterns for migraine and CM will be present. These genomic profiles will help clarify the interactions of inheritance and environment. This initial study begins to examine the feasibility of peripheral blood cell genomic analysis to assist in the understanding of the pathophysiology of migraine and CM. METHODS: Blood samples from patients were obtained either during an acute migraine or CM. Genomic expression patterns were analyzed using Affymetrix U95A microarrays. RESULTS: Expression patterns of 7 migraine and 15 CM patients were compared to four distinct control groups (total patients, n=56) including healthy subjects. A group of platelet genes were upregulated in both migraine and CM samples. Different gene expression patterns were also seen between migraine and CM. A group of immediate early genes including c-fos and cox-2 were expressed at higher levels in migraine, whereas specific mitochondrial genes were expressed at higher levels in CM. CONCLUSIONS: Increased expression of platelet genes in patients with migraine and CM suggests similar underlying pathophysiology. The differences seen between migraine and CM in other genes suggest an overlapping but not identical pathophysiology. Further genomic profiling studies will help define these relationships and provide further insights into headache pathogenesis.

Adolescent↗

PubMedDate not supplied

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.

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