Christoph Harms - Academia.edu (original) (raw)

Papers by Christoph Harms

Research paper thumbnail of Mild cerebral ischemia induces loss of cyclin-dependent kinase inhibitors and activation of cell cycle machinery before delayed neuronal cell death

The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 15, 2001

After mild ischemic insults, many neurons undergo delayed neuronal death. Aberrant activation of ... more After mild ischemic insults, many neurons undergo delayed neuronal death. Aberrant activation of the cell cycle machinery is thought to contribute to apoptosis in various conditions including ischemia. We demonstrate that loss of endogenous cyclin-dependent kinase (Cdk) inhibitor p16(INK4a) is an early and reliable indicator of delayed neuronal death in striatal neurons after mild cerebral ischemia in vivo. Loss of p27(Kip1), another Cdk inhibitor, precedes cell death in neocortical neurons subjected to oxygen-glucose deprivation in vitro. The loss of Cdk inhibitors is followed by upregulation of cyclin D1, activation of Cdk2, and subsequent cytoskeletal disintegration. Most neurons undergo cell death before entering S-phase, albeit a small number ( approximately 1%) do progress to the S-phase before their death. Treatment with Cdk inhibitors significantly reduces cell death in vitro. These results show that alteration of cell cycle regulatory mechanisms is a prelude to delayed neur...

Research paper thumbnail of Differential mechanisms of neuroprotection by 17 beta-estradiol in apoptotic versus necrotic neurodegeneration

The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 15, 2001

The major goal of this study was to compare mechanisms of the neuroprotective potential of 17 bet... more The major goal of this study was to compare mechanisms of the neuroprotective potential of 17 beta-estradiol in two models for oxidative stress-independent apoptotic neuronal cell death with that in necrotic neuronal cell death in primary neuronal cultures derived from rat hippocampus, septum, or cortex. Neuronal apoptosis was induced either by staurosporine or ethylcholine aziridinium (AF64A), as models for necrotic cell death glutamate exposure or oxygen-glucose deprivation (OGD) were applied. Long-term (20 hr) pretreatment (0.1 microm 17 beta-estradiol) was neuroprotective in apoptotic neuronal cell death induced by AF64A (40 microm) only in hippocampal and septal neuronal cultures and not in cortical cultures. The neuroprotective effect was blocked by the estrogen antagonists ICI 182,780 and tamoxifen and the phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002. In glutamate and OGD-induced neuronal damage, long-term pretreatment was not effective. In contrast, short-term (1...

Research paper thumbnail of TAT-apoptosis repressor with caspase recruitment domain protein transduction rescues mice from fulminant liver failure

Acute liver failure (ALF) is associated with massive hepatocyte cell death and high mortality rat... more Acute liver failure (ALF) is associated with massive hepatocyte cell death and high mortality rates. Therapeutic approaches targeting hepatocyte injury in ALF are hampered by the activation of distinct stimulus-dependent pathways, mechanism of cell death, and a limited therapeutic window. The apoptosis repressor with caspase recruitment domain (ARC) is a recently discovered death repressor that inhibits both death receptor and mitochondrial apoptotic signaling. Here, we investigated the in vivo effects of ARC fused with the transduction domain of human immunodeficiency virus 1 (HIV-1) (TAT-ARC) on Fas-and tumor necrosis factor (TNF)-mediated murine models of fulminant liver failure. Treatment with TAT-ARC protein completely abrogated otherwise lethal liver failure induced by Fasagonistic antibody (Jo2), concanavalin A (ConA), or D-galactosamine/lipopolysaccharide (GalN/LPS) administration. Importantly, survival of mice was even preserved when TAT-ARC therapy was initiated in a delayed manner after stimulation with Jo2, ConA, or GalN/LPS. ARC blocked hepatocyte apoptosis by directly interacting with members of the death-inducing signaling complex. TNF-mediated liver damage was inhibited by two independent mechanisms: inhibition of jun kinase (JNK)-mediated TNF-a expression and prevention of hepatocyte apoptosis by inhibition of both death receptor and mitochondrial death signaling. We identified JNK as a novel target of ARC. ARC's caspase recruitment domain (CARD) directly interacts with JNK1 and JNK2, which correlates with decreased JNK activation and JNK-dependent TNF-a production. Conclusion: This work suggests that ARC confers hepatoprotection upstream and at the hepatocyte level. The efficacy of TAT-ARC protein transduction in multiple murine models of ALF demonstrates its therapeutic potential for reversing liver failure. (HEPATOLOGY 2012;00:000-000)

Research paper thumbnail of Proteolysis of Oxidized Proteins After Oxygen???Glucose Deprivation in Rat Cortical Neurons Is Mediated by the Proteasome

Oxidative injury contributes to cellular damage during and after cerebral ischemia. However, the ... more Oxidative injury contributes to cellular damage during and after cerebral ischemia. However, the downstream catabolic pathways of damaged cellular components in neurons are largely unknown. In the current study, the authors examined the formation of oxidized proteins and their active degradation by the proteasome. In near-pure rat primary cortical neurons, it was found that protein-bound carbonyls as markers for oxidized proteins are increased after oxygen-glucose deprivation (OGD). During and after OGD, degradation of proteins metabolically radiolabeled before OGD increases two-to threefold compared with the normal protein turnover. Proteolysis after reoxygenation was attenuated by the presence of dimethylthiourea, a radical scavenger, and was blocked by lactacystin, a specific proteasome inhibitor. Lactacystin also increased the amount of protein carbonyls formed. In contrast, the activity of the proteasome complex itself after OGD was not different from sham-washed controls. The authors suggest that oxygenglucose deprivation increases free radicals, which, in turn, oxidize proteins that are recognized and actively degraded by the proteasome complex. This protease itself is relatively resistant against oxidative injury. The authors conclude that the proteasome may be an active part of the cellular defense system against oxidative stress after cerebral ischemia.

Research paper thumbnail of DEFICIENCIES IN FOLIC ACID AND UNG RESULT IN LEARNING DEFICITS, AND ALTERED MMP-9 LEVELS IN A MOUSE MODEL OF VASCULAR DEMENTIA

Research paper thumbnail of NECDIN MODULATES RESIDENT MICROGLIAL CELLS IN EXPERIMENTAL STROKE

Research paper thumbnail of P17. 25GLIOMA-INITIATING CELL INDUCED INTERLEUKIN-6 PRODUCTION IS MEDIATED BY TOLL-LIKE RECEPTOR 4 IN MICROGLIA

Research paper thumbnail of Neuronal gelsolin prevents apoptosis by enhancing actin depolymerization

Molecular and Cellular Neuroscience, 2004

Gelsolin (gsn), an actin-severing protein, protects neurons from excitotoxic cell death via inact... more Gelsolin (gsn), an actin-severing protein, protects neurons from excitotoxic cell death via inactivation of membranous Ca 2+ channels. Its role during apoptotic cell death, however, has remained unclear. Using several models of neuronal cell death, we demonstrate that endogenous gelsolin has anti-apoptotic properties that correlate to its dynamic actions on the cytoskeleton. We show that neurons lacking gelsolin (gsn À/À ) have enhanced apoptosis following exposure to staurosporine, thapsigargin, or the cholinergic toxin ethylcholine aziridinium (AF64A). AF64A-induced loss of mitochondrial membrane potential and activation of caspase-3 was specifically enhanced in gsn À/À neurons and could be reversed by pharmacological inhibition of mitochondrial permeability transition. Moreover, increased caspase-3 activation and cell death in AF64A-treated gsn À/À neurons were completely reversed by pharmacological depolymerization of actin filaments and further enhanced by their stabilization. In conclusion, actin remodeling by endogenous gelsolin or analogues protects neurons from apoptosis mediated by mitochondria and caspase-3. D

Research paper thumbnail of Elevated levels of plasma homocysteine, deficiencies in dietary folic acid and uracil–DNA glycosylase impair learning in a mouse model of vascular cognitive impairment

Behavioural Brain Research, 2015

• Impaired learning in Ung −/− mice on FADD with chronic hypoperfusion.

Research paper thumbnail of Folate Deficiency Induces Neurodegeneration and Brain Dysfunction in Mice Lacking Uracil DNA Glycosylase

Journal of Neuroscience, 2008

Folate deficiency and resultant increased homocysteine levels have been linked experimentally and... more Folate deficiency and resultant increased homocysteine levels have been linked experimentally and epidemiologically with neurodegenerative conditions like stroke and dementia. Moreover, folate deficiency has been implicated in the pathogenesis of psychiatric disorders, most notably depression. We hypothesized that the pathogenic mechanisms include uracil misincorporation and, therefore, analyzed the effects of folate deficiency in mice lacking uracil DNA glycosylase (UngϪ/Ϫ) versus wild-type controls. Folate depletion increased nuclear mutation rates in UngϪ/Ϫ embryonic fibroblasts, and conferred death of cultured UngϪ/Ϫ hippocampal neurons. Feeding animals a folate-deficient diet (FD) for 3 months induced degeneration of CA3 pyramidal neurons in UngϪ/Ϫ but not Ung؉/؉ mice along with decreased hippocampal expression of brain-derived neurotrophic factor protein and decreased brain levels of antioxidant glutathione. Furthermore, FD induced cognitive deficits and mood alterations such as anxious and despair-like behaviors that were aggravated in UngϪ/Ϫ mice. Independent of Ung genotype, FD increased plasma homocysteine levels, altered brain monoamine metabolism, and inhibited adult hippocampal neurogenesis. These results indicate that impaired uracil repair is involved in neurodegeneration and neuropsychiatric dysfunction induced by experimental folate deficiency.

Research paper thumbnail of Phosphatidylinositol 3-Akt-Kinase-Dependent Phosphorylation of p21Waf1/Cip1 as a Novel Mechanism of Neuroprotection by Glucocorticoids

Journal of Neuroscience, 2007

The role of glucocorticoids in the regulation of apoptosis remains incongruous. Here, we demonstr... more The role of glucocorticoids in the regulation of apoptosis remains incongruous. Here, we demonstrate that corticosterone protects neurons from apoptosis by a mechanism involving the cyclin-dependent kinase inhibitor p21 Waf1/Cip1 . In primary cortical neurons, corticosterone leads to a dose-and Akt-kinase-dependent upregulation with enhanced phosphorylation and cytoplasmic appearance of p21 Waf1/Cip1 at Thr 145. Exposure of neurons to the neurotoxin ethylcholine aziridinium (AF64A) results in activation of caspase-3 and a dramatic loss of p21 Waf1/Cip1 preceding apoptosis in neurons. These effects of AF64A are reversed by pretreatment with corticosterone. Corticosterone-mediated upregulation of p21 Waf1/Cip1 and neuroprotection are completely abolished by glucocorticoid and mineralocorticoid receptor antagonists as well as inhibitors of PI3-and Akt-kinase. Both germline and somatically induced p21 Waf1/Cip1 deficiency abrogate the neuroprotection by corticosterone, whereas overexpression of p21 Waf1/Cip1 suffices to protect neurons from apoptosis. We identify p21 Waf1/Cip1 as a novel antiapoptotic factor for postmitotic neurons and implicate p21 Waf1/Cip1 as the molecular target of neuroprotection by high-dose glucocorticoids.

Research paper thumbnail of Melatonin is protective in necrotic but not in caspase-dependent, free radical-independent apoptotic neuronal cell death in primary neuronal cultures

The FASEB Journal, 2000

To assess the neuroprotective potential of melatonin in apoptotic neuronal cell death, we investi... more To assess the neuroprotective potential of melatonin in apoptotic neuronal cell death, we investigated the efficacy of melatonin in serum-free primary neuronal cultures of rat cortex by using three different models of caspase-dependent apoptotic, excitotoxin-independent neurodegeneration and compared it to that in necrotic neuronal damage. Neuronal apoptosis was induced by either staurosporine or the neurotoxin ethylcholine aziridinium (AF64A) with a delayed occurrence of apoptotic cell death (within 72 h). The apoptotic component of oxygen-glucose deprivation (OGD) unmasked by glutamate antagonists served as a third model. As a model for necrotic cell death, OGD was applied. Neuronal injury was quantified by LDH release and loss of metabolic activity. Although melatonin (0.5 mM) partly protected cortical neurons from OGD-induced necrosis, as measured by a significant reduction in LDH release, it was not effective in all three models of apoptotic cell death. In contrast, exaggeration of neuronal damage by melatonin was observed in native cultures as well as after induction of apoptosis. The present data suggest that the neuroprotectiveness of melatonin strongly depends on the model of neuronal cell death applied. As demonstrated in three different models of neuronal apoptosis, the progression of the apoptotic type of neuronal cell death cannot be withhold or is even exaggerated by melatonin, in contrast to its beneficial effect in the necrotic type of cell death.-Harms, C., Lautenschlager, M., Bergk, A., Freyer, D., Weih, M., Dirnagl, U., Weber, J. R., Hörtnagl, H. Melatonin is protective in necrotic but not in caspase-dependent, free radical-independent apoptotic neuronal cell death in primary neuronal cultures.

Research paper thumbnail of In Vivo Imaging of the Inflammatory Receptor CD40 After Cerebral Ischemia Using a Fluorescent Antibody

Stroke, 2008

Background and Purpose-Brain inflammation is a hallmark of stroke, where it has been implicated i... more Background and Purpose-Brain inflammation is a hallmark of stroke, where it has been implicated in tissue damage as well as in repair. Imaging technologies that specifically visualize these processes are highly desirable. In this study, we explored whether the inflammatory receptor CD40 can be noninvasively and specifically visualized in mice after cerebral ischemia using a fluorescent monoclonal antibody, which we labeled with the near-infrared fluorescence dye Cy5.5 (Cy5.5-CD40MAb). Methods-Wild-type and CD40-deficient mice were subjected to transient middle cerebral artery occlusion. Mice were either intravenously injected with Cy5.5-CD40MAb or control Cy5.5-IgGMAb. Noninvasive and ex vivo near-infrared fluorescence imaging was performed after injection of the compounds. Probe distribution and specificity was further assessed with single-plane illumination microscopy, immunohistochemistry, and confocal microscopy. Results-Significantly higher fluorescence intensities over the stroke-affected hemisphere, compared to the contralateral side, were only detected noninvasively in wild-type mice that received Cy5.5-CD40MAb, but not in CD40-deficient mice injected with Cy5.5-CD40MAb or in wild-type mice that were injected with Cy5.5-IgGMAb. Ex vivo near-infrared fluorescence showed an intense fluorescence within the ischemic territory only in wild-type mice injected with Cy5.5-CD40MAb. In the brains of these mice, single-plane illumination microscopy demonstrated vascular and parenchymal distribution, and confocal microscopy revealed a partial colocalization of parenchymal fluorescence from the injected Cy5.5-CD40MAb with activated microglia and blood-derived cells in the ischemic region. Conclusions-The study demonstrates that a CD40-targeted fluorescent antibody enables specific noninvasive detection of the inflammatory receptor CD40 after cerebral ischemia using optical techniques. (Stroke. 2008;39:2845-2852.)

Research paper thumbnail of Histone Acetylation and CREB Binding Protein Are Required for Neuronal Resistance against Ischemic Injury

PLoS ONE, 2014

Epigenetic transcriptional regulation by histone acetylation depends on the balance between histo... more Epigenetic transcriptional regulation by histone acetylation depends on the balance between histone acetyltransferase (HAT) and deacetylase activities (HDAC). Inhibition of HDAC activity provides neuroprotection, indicating that the outcome of cerebral ischemia depends crucially on the acetylation status of histones. In the present study, we characterized the changes in histone acetylation levels in ischemia models of focal cerebral ischemia and identified cAMP-response element binding protein (CREB)-binding protein (CBP) as a crucial factor in the susceptibility of neurons to ischemic stress. Both neuron-specific RNA interference and neurons derived from CBP heterozygous knockout mice showed increased damage after oxygen-glucose deprivation (OGD) in vitro. Furthermore, we demonstrated that ischemic preconditioning by a short (5 min) subthreshold occlusion of the middle cerebral artery (MCA), followed 24 h afterwards by a 30 min occlusion of the MCA, increased histone acetylation levels in vivo. Ischemic preconditioning enhanced CBP recruitment and histone acetylation at the promoter of the neuroprotective gene gelsolin leading to increased gelsolin expression in neurons. Inhibition of CBP's HAT activity attenuated neuronal ischemic preconditioning. Taken together, our findings suggest that the levels of CBP and histone acetylation determine stroke outcome and are crucially associated with the induction of an ischemia-resistant state in neurons.

Research paper thumbnail of Roller Culture of Free-Floating Retinal Slices: A New System of Organotypic Cultures of Adult Rat Retina

Ophthalmic Research, 2006

of the retinal bodies was formed by cell and fi bre layers typical of mature retina with photorec... more of the retinal bodies was formed by cell and fi bre layers typical of mature retina with photoreceptors located on the outside. Initially, retinal bodies contained an inner cavity which later was completely obliterated and fi lled with glial cells, sprouting nerve fi bres, and vascular structures. This culture system was further developed into a robust model of glutamate-induced neurotoxicity. Using a novel culture method of adult rat retina, preservation of the three-dimensional organotypic retinal cytoarchitecture was achieved, including survival of neurons in the ganglion cell layer and sprouting of nerve fi bres of the axotomized retinal ganglion cells. This novel culture model promises to facilitate studies of retinal physiology and pathology.

Research paper thumbnail of Inhibition of Na+,K+-ATPase activity in cultured rat cerebellar granule cells prevents the onset of apoptosis induced by low potassium

Neuroscience Letters, 2000

In cerebellar granule cells in culture, lowering of extracellular [K 1 ] results in apoptotic dea... more In cerebellar granule cells in culture, lowering of extracellular [K 1 ] results in apoptotic death (D'Mello, S.R., Galli, C., Ciotti, T. and Calissano, P., Induction of apoptosis in cerebellar granule neurons by low potassium: inhibition of death by insulin-like growth factor I and cAMP, Proc. Natl. Acad. Sci. USA, 90 (1993) 10989-10993). In this model, we studied the in¯uence of Na 1 ,K 1 -ATPase inhibition on apoptosis. We demonstrate that cell death (93^2 vs. 46^1.6%) as well as fragmentation of nuclear DNA induced by low extracellular potassium were prevented by addition of ouabain (0.1 mM), a speci®c inhibitor of the Na 1 ,K 1 -ATPase. Blockade of glutamatergic N-methyl-d-aspartate and alpha-amino-3-hydroxy-5methyl-4-isoxazole propionic acid receptors by 5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohepten-5,10-imine hydrogen maleate (MK-801; 20 mM) and 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX; 50 mM) did not inhibit the protective effect of ouabain. 24 h treatment with ouabain also decreased cell death induced by Fe 21 /ascorbic acid (74^2% to 493 %). We speculate that ouabain pretreatment enhances the resistance against low [K 1 ]-induced apoptosis independent of glutamate-receptor activation. Since this effect can be mimicked by a free-radical generating system, we suggest an antioxidative effect underlying ouabain-induced neuroprotection. q

Research paper thumbnail of Role of nitric oxide in the ethylcholine aziridinium model of delayed apoptotic neurodegeneration in vivo and in vitro

Neuroscience, 2000

The involvement of nitric oxide in neurodegenerative processes still remains incompletely charact... more The involvement of nitric oxide in neurodegenerative processes still remains incompletely characterized. Although nitric oxide has been reported to be an important mediator in neuronal degeneration in different models of cell death involving NMDA-receptor activation, increasing evidence for protective mechanisms has been obtained. In this study the role of nitric oxide was investigated in a model of NMDA-independent, delayed apoptotic cell death, induced by the neurotoxin ethylcholine aziridinium ethylcholine aziridinium both in vivo and in vitro. For the in vivo evaluation rats received bilateral intracerebroventricular injections of ethylcholine aziridinium (2nmol/ventricle) or vehicle. In the hippocampus a transient decrease in nitric oxide synthase activity occurred, reaching its lowest levels three days after ethylcholine aziridinium treatment (51.7^9.8% of controls). The decrease coincided with the maximal reduction in choline acetyltransferase activity as marker for the extent of cholinergic lesion. The effect of pharmacological inhibition of nitric oxide synthase was tested by application of various nitric oxide synthase inhibitors with different selectivity for the nitric oxide synthase-isoforms. Unspecific nitric oxide synthase inhibition resulted in a significant potentiation of the loss of choline acetyltransferase activity in the hippocampus measured seven days after ethylcholine aziridinium application, whereas the specific inhibition of neuronal or inducible nitric oxide synthase was ineffective. These pharmacological data are suggestive for a neuroprotective role of nitric oxide generated by endothelial nitric oxide synthase. In vitro experiments were performed using serum-free primary neuronal cell cultures from hippocampus, cortex and septum of E15-17 Wistar rat embryos. Ethylcholine aziridinium-application in a range of 5-80 mM resulted in delayed apoptotic neurodegeneration with a maximum after three days as confirmed by morphological criteria, life-death assays and DNA laddering. Nitric oxide synthase activity in harvested cells decreased in a dose-and time-dependent manner. Nitric oxide production as determined by measurement of the accumulated metabolite nitrite in the medium was equally low in controls and in ethylcholine aziridinium treated cells (range 0.77-1.86 mM nitrite). An expression of inducible nitric oxide synthase messenger RNA could not be detected by semiquantitative RT-PCR 13 h after ethylcholine aziridinium application.

Research paper thumbnail of Serotonin uptake and release mechanisms in developing cultures of rat embryonic raphe neurons: age- and region-specific differences

Neuroscience, 2000

The development of serotonergic neurons of the rat raphe was followed in primary neuronal cell cu... more The development of serotonergic neurons of the rat raphe was followed in primary neuronal cell cultures taken at embryonic days embryonic day 13 and embryonic day 14 from three different raphe sub-groups, topographically defined with respect to their position to the isthmus as rostral (R1), intermediate (R2) and caudal (R3). In neurons cultivated from embryonic day 13 raphe serotonin, immunoreactivity was detected after only two days in vitro in the rostral R1 and the intermediate R2 sub-groups. Within two weeks of cultivation the number of serotonergic neurons as well as the dendritic branching continuously increased in all three sub-groups. In cultures obtained from embryonic day 13 raphe a specific uptake of [ 3 H]serotonin could not be detected during the first days in vitro. Specific uptake as well as regulated serotonin release, however, was clearly discernible in these cultures after nine days in vitro, indicating developmental differentiation of the initially immature serotonergic neurons in culture. In contrast, serotonergic neurons obtained from the three raphe sub-groups at embryonic day 14 took up and released [ 3 H]serotonin, as early as after two days in culture. Basal as well as stimulated serotonin release was diminished when preincubating the cells with tetanus toxin, which cleaves synaptobrevin thereby blocking exocytosis.

Research paper thumbnail of Protein kinase CK2 links extracellular growth factor signaling with the control of p27Kip1 stability in the heart

Nature Medicine, 2008

p27 Kip1 (p27) blocks cell proliferation through the inhibition of cyclin-dependent kinase-2 (Cdk... more p27 Kip1 (p27) blocks cell proliferation through the inhibition of cyclin-dependent kinase-2 (Cdk2). Despite its robust expression in the heart, little is known about both the function and regulation of p27 in this and other nonproliferative tissues, in which the expression of its main target, cyclin E-Cdk2, is known to be very low. Here we show that angiotensin II, a major cardiac growth factor, induces the proteasomal degradation of p27 through protein kinase CK2-a¢-dependent phosphorylation. Conversely, unphosphorylated p27 potently inhibits CK2-a¢. Thus, the p27-CK2-a¢ interaction is regulated by hypertrophic signaling events and represents a regulatory feedback loop in differentiated cardiomyocytes analogous to, but distinct from, the feedback loop arising from the interaction of p27 with Cdk2 that controls cell proliferation. Our data show that extracellular growth factor signaling regulates p27 stability in postmitotic cells, and that inactivation of p27 by CK2-a¢ is crucial for agonist-and stressinduced cardiac hypertrophic growth.

Research paper thumbnail of Imaging Early Endothelial Inflammation Following Stroke by Core Shell Silica Superparamagnetic Glyconanoparticles That Target Selectin

Nano Letters, 2014

Activation of the endothelium is a pivotal first step for leukocyte migration into the diseased b... more Activation of the endothelium is a pivotal first step for leukocyte migration into the diseased brain. Consequently, imaging this activation process is highly desirable. We synthesized carbohydrate-functionalized magnetic nanoparticles that bind specifically to the endothelial transmembrane inflammatory proteins E and P selectin. Magnetic resonance imaging revealed that the targeted nanoparticles accumulated in the brain vasculature following acute administration into a clinically relevant animal model of stroke, though increases in selectin expression were observed in both brain hemispheres. Nonfunctionalized naked particles also appear to be a plausible agent to target the ischemic vasculature. The importance of these findings is discussed regarding the potential for translation into the clinic.

Research paper thumbnail of Mild cerebral ischemia induces loss of cyclin-dependent kinase inhibitors and activation of cell cycle machinery before delayed neuronal cell death

The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 15, 2001

After mild ischemic insults, many neurons undergo delayed neuronal death. Aberrant activation of ... more After mild ischemic insults, many neurons undergo delayed neuronal death. Aberrant activation of the cell cycle machinery is thought to contribute to apoptosis in various conditions including ischemia. We demonstrate that loss of endogenous cyclin-dependent kinase (Cdk) inhibitor p16(INK4a) is an early and reliable indicator of delayed neuronal death in striatal neurons after mild cerebral ischemia in vivo. Loss of p27(Kip1), another Cdk inhibitor, precedes cell death in neocortical neurons subjected to oxygen-glucose deprivation in vitro. The loss of Cdk inhibitors is followed by upregulation of cyclin D1, activation of Cdk2, and subsequent cytoskeletal disintegration. Most neurons undergo cell death before entering S-phase, albeit a small number ( approximately 1%) do progress to the S-phase before their death. Treatment with Cdk inhibitors significantly reduces cell death in vitro. These results show that alteration of cell cycle regulatory mechanisms is a prelude to delayed neur...

Research paper thumbnail of Differential mechanisms of neuroprotection by 17 beta-estradiol in apoptotic versus necrotic neurodegeneration

The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 15, 2001

The major goal of this study was to compare mechanisms of the neuroprotective potential of 17 bet... more The major goal of this study was to compare mechanisms of the neuroprotective potential of 17 beta-estradiol in two models for oxidative stress-independent apoptotic neuronal cell death with that in necrotic neuronal cell death in primary neuronal cultures derived from rat hippocampus, septum, or cortex. Neuronal apoptosis was induced either by staurosporine or ethylcholine aziridinium (AF64A), as models for necrotic cell death glutamate exposure or oxygen-glucose deprivation (OGD) were applied. Long-term (20 hr) pretreatment (0.1 microm 17 beta-estradiol) was neuroprotective in apoptotic neuronal cell death induced by AF64A (40 microm) only in hippocampal and septal neuronal cultures and not in cortical cultures. The neuroprotective effect was blocked by the estrogen antagonists ICI 182,780 and tamoxifen and the phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002. In glutamate and OGD-induced neuronal damage, long-term pretreatment was not effective. In contrast, short-term (1...

Research paper thumbnail of TAT-apoptosis repressor with caspase recruitment domain protein transduction rescues mice from fulminant liver failure

Acute liver failure (ALF) is associated with massive hepatocyte cell death and high mortality rat... more Acute liver failure (ALF) is associated with massive hepatocyte cell death and high mortality rates. Therapeutic approaches targeting hepatocyte injury in ALF are hampered by the activation of distinct stimulus-dependent pathways, mechanism of cell death, and a limited therapeutic window. The apoptosis repressor with caspase recruitment domain (ARC) is a recently discovered death repressor that inhibits both death receptor and mitochondrial apoptotic signaling. Here, we investigated the in vivo effects of ARC fused with the transduction domain of human immunodeficiency virus 1 (HIV-1) (TAT-ARC) on Fas-and tumor necrosis factor (TNF)-mediated murine models of fulminant liver failure. Treatment with TAT-ARC protein completely abrogated otherwise lethal liver failure induced by Fasagonistic antibody (Jo2), concanavalin A (ConA), or D-galactosamine/lipopolysaccharide (GalN/LPS) administration. Importantly, survival of mice was even preserved when TAT-ARC therapy was initiated in a delayed manner after stimulation with Jo2, ConA, or GalN/LPS. ARC blocked hepatocyte apoptosis by directly interacting with members of the death-inducing signaling complex. TNF-mediated liver damage was inhibited by two independent mechanisms: inhibition of jun kinase (JNK)-mediated TNF-a expression and prevention of hepatocyte apoptosis by inhibition of both death receptor and mitochondrial death signaling. We identified JNK as a novel target of ARC. ARC's caspase recruitment domain (CARD) directly interacts with JNK1 and JNK2, which correlates with decreased JNK activation and JNK-dependent TNF-a production. Conclusion: This work suggests that ARC confers hepatoprotection upstream and at the hepatocyte level. The efficacy of TAT-ARC protein transduction in multiple murine models of ALF demonstrates its therapeutic potential for reversing liver failure. (HEPATOLOGY 2012;00:000-000)

Research paper thumbnail of Proteolysis of Oxidized Proteins After Oxygen???Glucose Deprivation in Rat Cortical Neurons Is Mediated by the Proteasome

Oxidative injury contributes to cellular damage during and after cerebral ischemia. However, the ... more Oxidative injury contributes to cellular damage during and after cerebral ischemia. However, the downstream catabolic pathways of damaged cellular components in neurons are largely unknown. In the current study, the authors examined the formation of oxidized proteins and their active degradation by the proteasome. In near-pure rat primary cortical neurons, it was found that protein-bound carbonyls as markers for oxidized proteins are increased after oxygen-glucose deprivation (OGD). During and after OGD, degradation of proteins metabolically radiolabeled before OGD increases two-to threefold compared with the normal protein turnover. Proteolysis after reoxygenation was attenuated by the presence of dimethylthiourea, a radical scavenger, and was blocked by lactacystin, a specific proteasome inhibitor. Lactacystin also increased the amount of protein carbonyls formed. In contrast, the activity of the proteasome complex itself after OGD was not different from sham-washed controls. The authors suggest that oxygenglucose deprivation increases free radicals, which, in turn, oxidize proteins that are recognized and actively degraded by the proteasome complex. This protease itself is relatively resistant against oxidative injury. The authors conclude that the proteasome may be an active part of the cellular defense system against oxidative stress after cerebral ischemia.

Research paper thumbnail of DEFICIENCIES IN FOLIC ACID AND UNG RESULT IN LEARNING DEFICITS, AND ALTERED MMP-9 LEVELS IN A MOUSE MODEL OF VASCULAR DEMENTIA

Research paper thumbnail of NECDIN MODULATES RESIDENT MICROGLIAL CELLS IN EXPERIMENTAL STROKE

Research paper thumbnail of P17. 25GLIOMA-INITIATING CELL INDUCED INTERLEUKIN-6 PRODUCTION IS MEDIATED BY TOLL-LIKE RECEPTOR 4 IN MICROGLIA

Research paper thumbnail of Neuronal gelsolin prevents apoptosis by enhancing actin depolymerization

Molecular and Cellular Neuroscience, 2004

Gelsolin (gsn), an actin-severing protein, protects neurons from excitotoxic cell death via inact... more Gelsolin (gsn), an actin-severing protein, protects neurons from excitotoxic cell death via inactivation of membranous Ca 2+ channels. Its role during apoptotic cell death, however, has remained unclear. Using several models of neuronal cell death, we demonstrate that endogenous gelsolin has anti-apoptotic properties that correlate to its dynamic actions on the cytoskeleton. We show that neurons lacking gelsolin (gsn À/À ) have enhanced apoptosis following exposure to staurosporine, thapsigargin, or the cholinergic toxin ethylcholine aziridinium (AF64A). AF64A-induced loss of mitochondrial membrane potential and activation of caspase-3 was specifically enhanced in gsn À/À neurons and could be reversed by pharmacological inhibition of mitochondrial permeability transition. Moreover, increased caspase-3 activation and cell death in AF64A-treated gsn À/À neurons were completely reversed by pharmacological depolymerization of actin filaments and further enhanced by their stabilization. In conclusion, actin remodeling by endogenous gelsolin or analogues protects neurons from apoptosis mediated by mitochondria and caspase-3. D

Research paper thumbnail of Elevated levels of plasma homocysteine, deficiencies in dietary folic acid and uracil–DNA glycosylase impair learning in a mouse model of vascular cognitive impairment

Behavioural Brain Research, 2015

• Impaired learning in Ung −/− mice on FADD with chronic hypoperfusion.

Research paper thumbnail of Folate Deficiency Induces Neurodegeneration and Brain Dysfunction in Mice Lacking Uracil DNA Glycosylase

Journal of Neuroscience, 2008

Folate deficiency and resultant increased homocysteine levels have been linked experimentally and... more Folate deficiency and resultant increased homocysteine levels have been linked experimentally and epidemiologically with neurodegenerative conditions like stroke and dementia. Moreover, folate deficiency has been implicated in the pathogenesis of psychiatric disorders, most notably depression. We hypothesized that the pathogenic mechanisms include uracil misincorporation and, therefore, analyzed the effects of folate deficiency in mice lacking uracil DNA glycosylase (UngϪ/Ϫ) versus wild-type controls. Folate depletion increased nuclear mutation rates in UngϪ/Ϫ embryonic fibroblasts, and conferred death of cultured UngϪ/Ϫ hippocampal neurons. Feeding animals a folate-deficient diet (FD) for 3 months induced degeneration of CA3 pyramidal neurons in UngϪ/Ϫ but not Ung؉/؉ mice along with decreased hippocampal expression of brain-derived neurotrophic factor protein and decreased brain levels of antioxidant glutathione. Furthermore, FD induced cognitive deficits and mood alterations such as anxious and despair-like behaviors that were aggravated in UngϪ/Ϫ mice. Independent of Ung genotype, FD increased plasma homocysteine levels, altered brain monoamine metabolism, and inhibited adult hippocampal neurogenesis. These results indicate that impaired uracil repair is involved in neurodegeneration and neuropsychiatric dysfunction induced by experimental folate deficiency.

Research paper thumbnail of Phosphatidylinositol 3-Akt-Kinase-Dependent Phosphorylation of p21Waf1/Cip1 as a Novel Mechanism of Neuroprotection by Glucocorticoids

Journal of Neuroscience, 2007

The role of glucocorticoids in the regulation of apoptosis remains incongruous. Here, we demonstr... more The role of glucocorticoids in the regulation of apoptosis remains incongruous. Here, we demonstrate that corticosterone protects neurons from apoptosis by a mechanism involving the cyclin-dependent kinase inhibitor p21 Waf1/Cip1 . In primary cortical neurons, corticosterone leads to a dose-and Akt-kinase-dependent upregulation with enhanced phosphorylation and cytoplasmic appearance of p21 Waf1/Cip1 at Thr 145. Exposure of neurons to the neurotoxin ethylcholine aziridinium (AF64A) results in activation of caspase-3 and a dramatic loss of p21 Waf1/Cip1 preceding apoptosis in neurons. These effects of AF64A are reversed by pretreatment with corticosterone. Corticosterone-mediated upregulation of p21 Waf1/Cip1 and neuroprotection are completely abolished by glucocorticoid and mineralocorticoid receptor antagonists as well as inhibitors of PI3-and Akt-kinase. Both germline and somatically induced p21 Waf1/Cip1 deficiency abrogate the neuroprotection by corticosterone, whereas overexpression of p21 Waf1/Cip1 suffices to protect neurons from apoptosis. We identify p21 Waf1/Cip1 as a novel antiapoptotic factor for postmitotic neurons and implicate p21 Waf1/Cip1 as the molecular target of neuroprotection by high-dose glucocorticoids.

Research paper thumbnail of Melatonin is protective in necrotic but not in caspase-dependent, free radical-independent apoptotic neuronal cell death in primary neuronal cultures

The FASEB Journal, 2000

To assess the neuroprotective potential of melatonin in apoptotic neuronal cell death, we investi... more To assess the neuroprotective potential of melatonin in apoptotic neuronal cell death, we investigated the efficacy of melatonin in serum-free primary neuronal cultures of rat cortex by using three different models of caspase-dependent apoptotic, excitotoxin-independent neurodegeneration and compared it to that in necrotic neuronal damage. Neuronal apoptosis was induced by either staurosporine or the neurotoxin ethylcholine aziridinium (AF64A) with a delayed occurrence of apoptotic cell death (within 72 h). The apoptotic component of oxygen-glucose deprivation (OGD) unmasked by glutamate antagonists served as a third model. As a model for necrotic cell death, OGD was applied. Neuronal injury was quantified by LDH release and loss of metabolic activity. Although melatonin (0.5 mM) partly protected cortical neurons from OGD-induced necrosis, as measured by a significant reduction in LDH release, it was not effective in all three models of apoptotic cell death. In contrast, exaggeration of neuronal damage by melatonin was observed in native cultures as well as after induction of apoptosis. The present data suggest that the neuroprotectiveness of melatonin strongly depends on the model of neuronal cell death applied. As demonstrated in three different models of neuronal apoptosis, the progression of the apoptotic type of neuronal cell death cannot be withhold or is even exaggerated by melatonin, in contrast to its beneficial effect in the necrotic type of cell death.-Harms, C., Lautenschlager, M., Bergk, A., Freyer, D., Weih, M., Dirnagl, U., Weber, J. R., Hörtnagl, H. Melatonin is protective in necrotic but not in caspase-dependent, free radical-independent apoptotic neuronal cell death in primary neuronal cultures.

Research paper thumbnail of In Vivo Imaging of the Inflammatory Receptor CD40 After Cerebral Ischemia Using a Fluorescent Antibody

Stroke, 2008

Background and Purpose-Brain inflammation is a hallmark of stroke, where it has been implicated i... more Background and Purpose-Brain inflammation is a hallmark of stroke, where it has been implicated in tissue damage as well as in repair. Imaging technologies that specifically visualize these processes are highly desirable. In this study, we explored whether the inflammatory receptor CD40 can be noninvasively and specifically visualized in mice after cerebral ischemia using a fluorescent monoclonal antibody, which we labeled with the near-infrared fluorescence dye Cy5.5 (Cy5.5-CD40MAb). Methods-Wild-type and CD40-deficient mice were subjected to transient middle cerebral artery occlusion. Mice were either intravenously injected with Cy5.5-CD40MAb or control Cy5.5-IgGMAb. Noninvasive and ex vivo near-infrared fluorescence imaging was performed after injection of the compounds. Probe distribution and specificity was further assessed with single-plane illumination microscopy, immunohistochemistry, and confocal microscopy. Results-Significantly higher fluorescence intensities over the stroke-affected hemisphere, compared to the contralateral side, were only detected noninvasively in wild-type mice that received Cy5.5-CD40MAb, but not in CD40-deficient mice injected with Cy5.5-CD40MAb or in wild-type mice that were injected with Cy5.5-IgGMAb. Ex vivo near-infrared fluorescence showed an intense fluorescence within the ischemic territory only in wild-type mice injected with Cy5.5-CD40MAb. In the brains of these mice, single-plane illumination microscopy demonstrated vascular and parenchymal distribution, and confocal microscopy revealed a partial colocalization of parenchymal fluorescence from the injected Cy5.5-CD40MAb with activated microglia and blood-derived cells in the ischemic region. Conclusions-The study demonstrates that a CD40-targeted fluorescent antibody enables specific noninvasive detection of the inflammatory receptor CD40 after cerebral ischemia using optical techniques. (Stroke. 2008;39:2845-2852.)

Research paper thumbnail of Histone Acetylation and CREB Binding Protein Are Required for Neuronal Resistance against Ischemic Injury

PLoS ONE, 2014

Epigenetic transcriptional regulation by histone acetylation depends on the balance between histo... more Epigenetic transcriptional regulation by histone acetylation depends on the balance between histone acetyltransferase (HAT) and deacetylase activities (HDAC). Inhibition of HDAC activity provides neuroprotection, indicating that the outcome of cerebral ischemia depends crucially on the acetylation status of histones. In the present study, we characterized the changes in histone acetylation levels in ischemia models of focal cerebral ischemia and identified cAMP-response element binding protein (CREB)-binding protein (CBP) as a crucial factor in the susceptibility of neurons to ischemic stress. Both neuron-specific RNA interference and neurons derived from CBP heterozygous knockout mice showed increased damage after oxygen-glucose deprivation (OGD) in vitro. Furthermore, we demonstrated that ischemic preconditioning by a short (5 min) subthreshold occlusion of the middle cerebral artery (MCA), followed 24 h afterwards by a 30 min occlusion of the MCA, increased histone acetylation levels in vivo. Ischemic preconditioning enhanced CBP recruitment and histone acetylation at the promoter of the neuroprotective gene gelsolin leading to increased gelsolin expression in neurons. Inhibition of CBP's HAT activity attenuated neuronal ischemic preconditioning. Taken together, our findings suggest that the levels of CBP and histone acetylation determine stroke outcome and are crucially associated with the induction of an ischemia-resistant state in neurons.

Research paper thumbnail of Roller Culture of Free-Floating Retinal Slices: A New System of Organotypic Cultures of Adult Rat Retina

Ophthalmic Research, 2006

of the retinal bodies was formed by cell and fi bre layers typical of mature retina with photorec... more of the retinal bodies was formed by cell and fi bre layers typical of mature retina with photoreceptors located on the outside. Initially, retinal bodies contained an inner cavity which later was completely obliterated and fi lled with glial cells, sprouting nerve fi bres, and vascular structures. This culture system was further developed into a robust model of glutamate-induced neurotoxicity. Using a novel culture method of adult rat retina, preservation of the three-dimensional organotypic retinal cytoarchitecture was achieved, including survival of neurons in the ganglion cell layer and sprouting of nerve fi bres of the axotomized retinal ganglion cells. This novel culture model promises to facilitate studies of retinal physiology and pathology.

Research paper thumbnail of Inhibition of Na+,K+-ATPase activity in cultured rat cerebellar granule cells prevents the onset of apoptosis induced by low potassium

Neuroscience Letters, 2000

In cerebellar granule cells in culture, lowering of extracellular [K 1 ] results in apoptotic dea... more In cerebellar granule cells in culture, lowering of extracellular [K 1 ] results in apoptotic death (D'Mello, S.R., Galli, C., Ciotti, T. and Calissano, P., Induction of apoptosis in cerebellar granule neurons by low potassium: inhibition of death by insulin-like growth factor I and cAMP, Proc. Natl. Acad. Sci. USA, 90 (1993) 10989-10993). In this model, we studied the in¯uence of Na 1 ,K 1 -ATPase inhibition on apoptosis. We demonstrate that cell death (93^2 vs. 46^1.6%) as well as fragmentation of nuclear DNA induced by low extracellular potassium were prevented by addition of ouabain (0.1 mM), a speci®c inhibitor of the Na 1 ,K 1 -ATPase. Blockade of glutamatergic N-methyl-d-aspartate and alpha-amino-3-hydroxy-5methyl-4-isoxazole propionic acid receptors by 5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohepten-5,10-imine hydrogen maleate (MK-801; 20 mM) and 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX; 50 mM) did not inhibit the protective effect of ouabain. 24 h treatment with ouabain also decreased cell death induced by Fe 21 /ascorbic acid (74^2% to 493 %). We speculate that ouabain pretreatment enhances the resistance against low [K 1 ]-induced apoptosis independent of glutamate-receptor activation. Since this effect can be mimicked by a free-radical generating system, we suggest an antioxidative effect underlying ouabain-induced neuroprotection. q

Research paper thumbnail of Role of nitric oxide in the ethylcholine aziridinium model of delayed apoptotic neurodegeneration in vivo and in vitro

Neuroscience, 2000

The involvement of nitric oxide in neurodegenerative processes still remains incompletely charact... more The involvement of nitric oxide in neurodegenerative processes still remains incompletely characterized. Although nitric oxide has been reported to be an important mediator in neuronal degeneration in different models of cell death involving NMDA-receptor activation, increasing evidence for protective mechanisms has been obtained. In this study the role of nitric oxide was investigated in a model of NMDA-independent, delayed apoptotic cell death, induced by the neurotoxin ethylcholine aziridinium ethylcholine aziridinium both in vivo and in vitro. For the in vivo evaluation rats received bilateral intracerebroventricular injections of ethylcholine aziridinium (2nmol/ventricle) or vehicle. In the hippocampus a transient decrease in nitric oxide synthase activity occurred, reaching its lowest levels three days after ethylcholine aziridinium treatment (51.7^9.8% of controls). The decrease coincided with the maximal reduction in choline acetyltransferase activity as marker for the extent of cholinergic lesion. The effect of pharmacological inhibition of nitric oxide synthase was tested by application of various nitric oxide synthase inhibitors with different selectivity for the nitric oxide synthase-isoforms. Unspecific nitric oxide synthase inhibition resulted in a significant potentiation of the loss of choline acetyltransferase activity in the hippocampus measured seven days after ethylcholine aziridinium application, whereas the specific inhibition of neuronal or inducible nitric oxide synthase was ineffective. These pharmacological data are suggestive for a neuroprotective role of nitric oxide generated by endothelial nitric oxide synthase. In vitro experiments were performed using serum-free primary neuronal cell cultures from hippocampus, cortex and septum of E15-17 Wistar rat embryos. Ethylcholine aziridinium-application in a range of 5-80 mM resulted in delayed apoptotic neurodegeneration with a maximum after three days as confirmed by morphological criteria, life-death assays and DNA laddering. Nitric oxide synthase activity in harvested cells decreased in a dose-and time-dependent manner. Nitric oxide production as determined by measurement of the accumulated metabolite nitrite in the medium was equally low in controls and in ethylcholine aziridinium treated cells (range 0.77-1.86 mM nitrite). An expression of inducible nitric oxide synthase messenger RNA could not be detected by semiquantitative RT-PCR 13 h after ethylcholine aziridinium application.

Research paper thumbnail of Serotonin uptake and release mechanisms in developing cultures of rat embryonic raphe neurons: age- and region-specific differences

Neuroscience, 2000

The development of serotonergic neurons of the rat raphe was followed in primary neuronal cell cu... more The development of serotonergic neurons of the rat raphe was followed in primary neuronal cell cultures taken at embryonic days embryonic day 13 and embryonic day 14 from three different raphe sub-groups, topographically defined with respect to their position to the isthmus as rostral (R1), intermediate (R2) and caudal (R3). In neurons cultivated from embryonic day 13 raphe serotonin, immunoreactivity was detected after only two days in vitro in the rostral R1 and the intermediate R2 sub-groups. Within two weeks of cultivation the number of serotonergic neurons as well as the dendritic branching continuously increased in all three sub-groups. In cultures obtained from embryonic day 13 raphe a specific uptake of [ 3 H]serotonin could not be detected during the first days in vitro. Specific uptake as well as regulated serotonin release, however, was clearly discernible in these cultures after nine days in vitro, indicating developmental differentiation of the initially immature serotonergic neurons in culture. In contrast, serotonergic neurons obtained from the three raphe sub-groups at embryonic day 14 took up and released [ 3 H]serotonin, as early as after two days in culture. Basal as well as stimulated serotonin release was diminished when preincubating the cells with tetanus toxin, which cleaves synaptobrevin thereby blocking exocytosis.

Research paper thumbnail of Protein kinase CK2 links extracellular growth factor signaling with the control of p27Kip1 stability in the heart

Nature Medicine, 2008

p27 Kip1 (p27) blocks cell proliferation through the inhibition of cyclin-dependent kinase-2 (Cdk... more p27 Kip1 (p27) blocks cell proliferation through the inhibition of cyclin-dependent kinase-2 (Cdk2). Despite its robust expression in the heart, little is known about both the function and regulation of p27 in this and other nonproliferative tissues, in which the expression of its main target, cyclin E-Cdk2, is known to be very low. Here we show that angiotensin II, a major cardiac growth factor, induces the proteasomal degradation of p27 through protein kinase CK2-a¢-dependent phosphorylation. Conversely, unphosphorylated p27 potently inhibits CK2-a¢. Thus, the p27-CK2-a¢ interaction is regulated by hypertrophic signaling events and represents a regulatory feedback loop in differentiated cardiomyocytes analogous to, but distinct from, the feedback loop arising from the interaction of p27 with Cdk2 that controls cell proliferation. Our data show that extracellular growth factor signaling regulates p27 stability in postmitotic cells, and that inactivation of p27 by CK2-a¢ is crucial for agonist-and stressinduced cardiac hypertrophic growth.

Research paper thumbnail of Imaging Early Endothelial Inflammation Following Stroke by Core Shell Silica Superparamagnetic Glyconanoparticles That Target Selectin

Nano Letters, 2014

Activation of the endothelium is a pivotal first step for leukocyte migration into the diseased b... more Activation of the endothelium is a pivotal first step for leukocyte migration into the diseased brain. Consequently, imaging this activation process is highly desirable. We synthesized carbohydrate-functionalized magnetic nanoparticles that bind specifically to the endothelial transmembrane inflammatory proteins E and P selectin. Magnetic resonance imaging revealed that the targeted nanoparticles accumulated in the brain vasculature following acute administration into a clinically relevant animal model of stroke, though increases in selectin expression were observed in both brain hemispheres. Nonfunctionalized naked particles also appear to be a plausible agent to target the ischemic vasculature. The importance of these findings is discussed regarding the potential for translation into the clinic.