MEK1 protein kinase inhibition protects against damage resulting from focal cerebral ischemia

The MEK1 (MAP kinase/ERK kinase)/ERK (extracellular-signal-responsive kinase) pathway has been implicated in cell growth and differentiation [Seger, R. & Krebs, E. G. (1995) FASEB J. 9, 726–735]. Here we show that the MEK/ERK pathway is activated during focal cerebral ischemia and may play a role in inducing damage. Treatment of mice 30 min before ischemia with the MEK1-specific inhibitor PD98059 [Alessi, D. R., Cuenda, A., Cohen, P., Dudley, D. T. & Saltiel, A. R. (1995) J. Biol. Chem. 270, 27489–27494] reduces focal infarct volume at 22 hr after ischemia by 55% after transient occlusion of the middle cerebral artery. This is accompanied by a reduction in phospho-ERK1/2 immunohistochemical staining. MEK1 inhibition also results in reduced brain damage 72 hr after ischemia, with focal infarct volume reduced by 36%. This study indicates that the MEK1/ERK pathway contributes to brain injury during focal cerebral ischemia and that PD98059, a MEK1-specific antagonist, is a potent neuroprotective agent.

Evidence for a protective role of metallothionein-1 in focal cerebral ischemia

Metallothioneins (MTs) are a family of metal binding proteins that have been proposed to participate in a cellular defense against zinc toxicity and free radicals. In the present study, we investigated whether increased expression of MT in MT-1 isoform-overexpressing transgenic mice (MT-TG) affords protection against mild focal cerebral ischemia and reperfusion. Transient focal ischemia was induced in control (wild type) and MT-TG mice by occluding the right middle cerebral artery for 45 min. Upon reperfusion, cerebral edema slowly developed and peaked at 24 hr as shown by T2-weighted MRI. The volume of affected tissue was on the average 42% smaller in MT-TG mice compared with control mice at 6, 9, 24, and 72 hr and 14 days postreperfusion (P < 0.01). In addition, functional studies showed that 3 weeks after reperfusion MT-TG mice showed a significantly better motor performance compared with control mice (P = 0.011). Although cortical baseline levels of MT-1 mRNA were similar in control and MT-TG mice, there was an increase in MT-1 mRNA levels in the ischemic cortex of MT-TG mice to 7.5 times baseline levels compared with an increase to 2.3 times baseline levels in control mice 24 hr after reperfusion. In addition, MT-TG mice showed an increased MT immunoreactivity in astrocytes, vascular endothelial cells, and neurons 24 hr after reperfusion whereas in control mice MT immunoreactivity was restricted mainly to astrocytes and decreased in the infarcted tissue. These results provide evidence that increased expression of MT-1 protects against focal cerebral ischemia and reperfusion.

Immunologic tolerance to myelin basic protein decreases stroke size after transient focal cerebral ischemia

Immune mechanisms contribute to cerebral ischemic injury. Therapeutic immunosuppressive options are limited due to systemic side effects. We attempted to achieve immunosuppression in the brain through oral tolerance to myelin basic protein (MBP). Lewis rats were fed low-dose bovine MBP or ovalbumin (1 mg, five times) before 3 h of middle cerebral artery occlusion (MCAO). A third group of animals was sensitized to MBP but did not survive the post-stroke period. Infarct size at 24 and 96 h after ischemia was significantly less in tolerized animals. Tolerance to MBP was confirmed in vivo by a decrease in delayed-type hypersensitivity to MBP. Systemic immune responses, characterized in vitro by spleen cell proliferation to Con A, lipopolysaccharide, and MBP, again confirmed antigen-specific immunologic tolerance. Immunohistochemistry revealed transforming growth factor β1 production by T cells in the brains of tolerized but not control animals. Systemic transforming growth factor β1 levels were equivalent in both groups. Corticosterone levels 24 h after surgery were elevated in all sham-operated animals and ischemic control animals but not in ischemic tolerized animals. These results demonstrate that antigen-specific modulation of the immune response decreases infarct size after focal cerebral ischemia and that sensitization to the same antigen may actually worsen outcome.

A tetracycline derivative, minocycline, reduces inflammation and protects against focal cerebral ischemia with a wide therapeutic window

The only treatment of patients with acute ischemic stroke is thrombolytic therapy, which benefits only a fraction of stroke patients. Both human and experimental studies indicate that ischemic stroke involves secondary inflammation that significantly contributes to the outcome after ischemic insult. Minocycline is a semisynthetic second-generation tetracycline that exerts antiinflammatory effects that are completely separate from its antimicrobial action. Because tetracycline treatment is clinically well tolerated, we investigated whether minocycline protects against focal brain ischemia with a wide therapeutic window. Using a rat model of transient middle cerebral artery occlusion, we show that daily treatment with minocycline reduces cortical infarction volume by 76 ± 22% when the treatment is started 12 h before ischemia and by 63 ± 35% when started even 4 h after the onset of ischemia. The treatment inhibits morphological activation of microglia in the area adjacent to the infarction, inhibits induction of IL-1β-converting enzyme, and reduces cyclooxygenase-2 expression and prostaglandin E2 production. Minocycline had no effect on astrogliosis or spreading depression, a wave of ionic transients thought to contribute to enlargement of cortical infarction. Treatment with minocycline may act directly on brain cells, because cultured primary neurons were also salvaged from glutamate toxicity. Minocycline may represent a prototype of an antiinflammatory compound that provides protection against ischemic stroke and has a clinically relevant therapeutic window.

Interaction between inducible nitric oxide synthase and cyclooxygenase-2 after cerebral ischemia

Focal cerebral ischemia is associated with expression of both inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), enzymes whose reaction products contribute to the evolution of ischemic brain injury. We tested the hypothesis that, after cerebral ischemia, nitric oxide (NO) produced by iNOS enhances COX-2 activity, thereby increasing the toxic potential of this enzyme. Cerebral ischemia was produced by middle cerebral artery occlusion in rats or mice. Twenty-four hours after ischemia in rats, iNOS-immunoreactive neutrophils were observed in close proximity (<20 μm) to COX-2-positive cells at the periphery of the infarct. In the olfactory bulb, only COX-2 positive cells were observed. Cerebral ischemia increased the concentration of the COX-2 reaction product prostaglandin E2 (PGE2) in the ischemic area and in the ipsilateral olfactory bulb. The iNOS inhibitor aminoguanidine reduced PGE2 concentration in the infarct, where both iNOS and COX-2 were expressed, but not in the olfactory bulb, where only COX-2 was expressed. Postischemic PGE2 accumulation was reduced significantly in iNOS null mice compared with wild-type controls (C57BL/6 or SV129). The data provide evidence that NO produced by iNOS influences COX-2 activity after focal cerebral ischemia. Pro-inflammatory prostanoids and reactive oxygen species produced by COX-2 may be a previously unrecognized factor by which NO contributes to ischemic brain injury. The pathogenic effect of the interaction between NO, or a derived specie, and COX-2 is likely to play a role also in other brain diseases associated with inflammation.

Erythropoietin prevents neuronal apoptosis after cerebral ischemia and metabolic stress

Erythropoietin (EPO) promotes neuronal survival after hypoxia and other metabolic insults by largely unknown mechanisms. Apoptosis and necrosis have been proposed as mechanisms of cellular demise, and either could be the target of actions of EPO. This study evaluates whether antiapoptotic mechanisms can account for the neuroprotective actions of EPO. Systemic administration of EPO (5,000 units/kg of body weight, i.p.) after middle-cerebral artery occlusion in rats dramatically reduces the volume of infarction 24 h later, in concert with an almost complete reduction in the number of terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling of neurons within the ischemic penumbra. In both pure and mixed neuronal cultures, EPO (0.1–10 units/ml) also inhibits apoptosis induced by serum deprivation or kainic acid exposure. Protection requires pretreatment, consistent with the induction of a gene expression program, and is sustained for 3 days without the continued presence of EPO. EPO (0.3 units/ml) also protects hippocampal neurons against hypoxia-induced neuronal death through activation of extracellular signal-regulated kinases and protein kinase Akt-1/protein kinase B. The action of EPO is not limited to directly promoting cell survival, as EPO is trophic but not mitogenic in cultured neuronal cells. These data suggest that inhibition of neuronal apoptosis underlies short latency protective effects of EPO after cerebral ischemia and other brain injuries. The neurotrophic actions suggest there may be longer-latency effects as well. Evaluation of EPO, a compound established as clinically safe, as neuroprotective therapy in acute brain injury is further supported.

Neurogenesis in dentate subgranular zone and rostral subventricular zone after focal cerebral ischemia in the rat

Because neurogenesis persists in the adult mammalian brain and can be regulated by physiological and pathological events, we investigated its possible involvement in the brain's response to focal cerebral ischemia. Ischemia was induced by occlusion of the middle cerebral artery in the rat for 90 min, and proliferating cells were labeled with 5-bromo-2′-deoxyuridine-5′-monophosphate (BrdUrd) over 2-day periods before sacrificing animals 1, 2 or 3 weeks after ischemia. Ischemia increased the incorporation of BrdUrd into cells in two neuroproliferative regions—the subgranular zone of the dentate gyrus and the rostral subventricular zone. Both effects were bilateral, but that in the subgranular zone was more prominent on the ischemic side. Cells labeled with BrdUrd coexpressed the immature neuronal markers doublecortin and proliferating cell nuclear antigen but did not express the more mature cell markers NeuN and Hu, suggesting that they were nascent neurons. These results support a role for ischemia-induced neurogenesis in what may be adaptive processes that contribute to recovery after stroke.

Vascular endothelial growth factor: Direct neuroprotective effect in in vitro ischemia

Vascular endothelial growth factor (VEGF) is a hypoxia-inducible angiogenic peptide with recently identified neurotrophic effects. Because some neurotrophic factors can protect neurons from hypoxic or ischemic injury, we investigated the possibility that VEGF has similar neuroprotective properties. In HN33, an immortalized hippocampal neuronal cell line, VEGF reduced cell death associated with an in vitro model of cerebral ischemia: at a maximally effective concentration of 50 ng/ml, VEGF approximately doubled the number of cells surviving after 24 h of hypoxia and glucose deprivation. To investigate the mechanism of neuroprotection by VEGF, the expression of known target receptors for VEGF was measured by Western blotting, which showed that HN33 cells expressed VEGFR-2 receptors and neuropilin-1, but not VEGFR-1 receptors. The neuropilin-1 ligand placenta growth factor-2 failed to reproduce the protective effect of VEGF, pointing to VEGFR-2 as the site of VEGF's neuroprotective action. Two phosphatidylinositol 3′-kinase inhibitors, wortmannin and LY294002, reversed the neuroprotective effect of VEGF, implicating the phosphatidylinositol 3′-kinase/Akt signal transduction system in VEGF-mediated neuroprotection. VEGF also protected primary cultures of rat cerebral cortical neurons from hypoxia and glucose deprivation. We conclude that in addition to its known role as an angiogenic factor, VEGF may exert a direct neuroprotective effect in hypoxic-ischemic injury.

Neuroprotective activity of a new class of steroidal inhibitors of the N-methyl-d-aspartate receptor

Release of the excitatory neurotransmitter glutamate and the excessive stimulation of N-methyl-d-aspartate (NMDA)-type glutamate receptors is thought to be responsible for much of the neuronal death that occurs following focal hypoxia-ischemia in the central nervous system. Our laboratory has identified endogenous sulfated steroids that potentiate or inhibit NMDA-induced currents. Here we report that 3α-ol-5β-pregnan-20-one hemisuccinate (3α5βHS), a synthetic homologue of naturally occurring pregnanolone sulfate, inhibits NMDA-induced currents and cell death in primary cultures of rat hippocampal neurons. 3α5βHS exhibits sedative, anticonvulsant, and analgesic properties consistent with an action at NMDA-type glutamate receptors. Intravenous administration of 3α5βHS to rats (at a nonsedating dose) following focal cerebral ischemia induced by middle cerebral artery occlusion significantly reduces cortical and subcortical infarct size. The in vitro and in vivo neuroprotective effects of 3α5βHS demonstrate that this steroid represents a new class of potentially useful therapeutic agents for the treatment of stroke and certain neurodegenerative diseases that involve over activation of NMDA receptors.

Acute decrease in net glutamate uptake during energy deprivation

The extracellular glutamate concentration ([glu]o) rises during cerebral ischemia, reaching levels capable of inducing delayed neuronal death. The mechanisms underlying this glutamate accumulation remain controversial. We used N-methyl-d-aspartate receptors on CA3 pyramidal neurons as a real-time, on-site, glutamate sensor to identify the source of glutamate release in an in vitro model of ischemia. Using glutamate and l-trans-pyrrolidine-2,4-dicarboxylic acid (tPDC) as substrates and dl-threo-β-benzyloxyaspartate (TBOA) as an inhibitor of glutamate transporters, we demonstrate that energy deprivation decreases net glutamate uptake within 2–3 min and later promotes reverse glutamate transport. This process accounts for up to 50% of the glutamate accumulation during energy deprivation. Enhanced action potential-independent vesicular release also contributes to the increase in [glu]o, by ≈50%, but only once glutamate uptake is inhibited. These results indicate that a significant rise in [glu]o already occurs during the first minutes of energy deprivation and is the consequence of reduced uptake and increased vesicular and nonvesicular release of glutamate.

Neurotoxicity of advanced glycation endproducts during focal stroke and neuroprotective effects of aminoguanidine.

Cerebral infarction (stroke) is a potentially disastrous complication of diabetes mellitus, principally because the extent of cortical loss is greater in diabetic patients than in nondiabetic patients. The etiology of this enhanced neurotoxicity is poorly understood. We hypothesized that advanced glycation endproducts (AGEs), which have previously been implicated in the development of other diabetic complications, might contribute to neurotoxicity and brain damage during ischemic stroke. Using a rat model of focal cerebral ischemia, we show that systemically administered AGE-modified bovine serum albumin (AGE-BSA) significantly increased cerebral infarct size. The neurotoxic effects of AGE-BSA administration were dose- and time-related and associated with a paradoxical increase in cerebral blood flow. Aminoguanidine, an inhibitor of AGE cross-linking, attenuated infarct volume in AGE-treated animals. We conclude that AGEs may contribute to the increased severity of stroke associated with diabetes and other conditions characterized by AGE accumulation.

Aurintricarboxylic acid prevents GLUR2 mRNA down-regulation and delayed neurodegeneration in hippocampal CA1 neurons of gerbil after global ischemia

Aurintricarboxylic acid (ATA), an inhibitor of endonuclease activity and other protein–nucleic acid interactions, blocks apoptosis in several cell types and prevents delayed death of hippocampal pyramidal CA1 neurons induced by transient global ischemia. Global ischemia in rats and gerbils induces down-regulation of GluR2 mRNA and increased α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-induced Ca2+ influx in CA1 before neurodegeneration. This result and neuroprotection by antagonists of AMPA receptors suggests that formation of AMPA receptors lacking GluR2, and therefore Ca2+ permeable, leads to excessive Ca2+ influx in response to endogenous glutamate; the resulting delayed neuronal death in CA1 exhibits many characteristics of apoptosis. In this study, we examined the effects of ATA on expression of mRNAs encoding glutamate receptor subunits in gerbil hippocampus after global ischemia. Administration of ATA by injection into the right cerebral ventricle 1 h before (but not 6 h after) bilateral carotid occlusion prevented the ischemia-induced decrease in GluR2 mRNA expression and the delayed neurodegeneration. These findings suggest that ATA is neuroprotective in ischemia by blocking the transcriptional changes leading to down-regulation of GluR2, rather than by simply blocking endonucleases, which presumably act later after Ca2+ influx initiates apoptosis. Maintaining formation of Ca2+ impermeable, GluR2 containing AMPA receptors could prevent delayed death of CA1 neurons after transient global ischemia, and block of GluR2 down-regulation may provide a further strategy for neuroprotection.

Cerebral cortical hyperactivation in response to mental stress in patients with coronary artery disease

The central nervous system (CNS) effects of mental stress in patients with coronary artery disease (CAD) are unexplored. The present study used positron emission tomography (PET) to measure brain correlates of mental stress induced by an arithmetic serial subtraction task in CAD and healthy subjects. Mental stress resulted in hyperactivation in CAD patients compared with healthy subjects in several brain areas including the left parietal cortex [angular gyrus/parallel sulcus (area 39)], left anterior cingulate (area 32), right visual association cortex (area 18), left fusiform gyrus, and cerebellum. These same regions were activated within the CAD patient group during mental stress versus control conditions. In the group of healthy subjects, activation was significant only in the left inferior frontal gyrus during mental stress compared with counting control. Decreases in blood flow also were produced by mental stress in CAD versus healthy subjects in right thalamus (lateral dorsal, lateral posterior), right superior frontal gyrus (areas 32, 24, and 10), and right middle temporal gyrus (area 21) (in the region of the auditory association cortex). Of particular interest, a subgroup of CAD patients that developed painless myocardial ischemia during mental stress had hyperactivation in the left hippocampus and inferior parietal lobule (area 40), left middle (area 10) and superior frontal gyrus (area 8), temporal pole, and visual association cortex (area 18), and a concomitant decrease in activation observed in the anterior cingulate bilaterally, right middle and superior frontal gyri, and right visual association cortex (area 18) compared with CAD patients without myocardial ischemia. These findings demonstrate an exaggerated cerebral cortical response and exaggerated asymmetry to mental stress in individuals with CAD.

N-tert-butyl-alpha-phenylnitrone improves recovery of brain energy state in rats following transient focal ischemia.

Recent results have demonstrated that the spin trapping agent N-tert-butyl-alpha-phenylnitrone (PBN) reduces infarct size due to middle cerebral artery occlusion (MCAO), even when given after ischemia. The objective of the present study was to explore whether PBN influences recovery of energy metabolism. MCAO of 2-hr duration was induced in rats by an intraluminal filament technique. Brains were frozen in situ at the end of ischemia and after 1, 2, and 4 hr of recirculation. PBN was given 1 hr after recirculation. Neocortical focal and perifocal ("penumbra") areas were sampled for analyses of phosphocreatine (PCr), creatine, ATP, ADP, AMP, glycogen, glucose, and lactate. The penumbra showed a moderate-to-marked decrease and the focus showed a marked decrease in PCr and ATP concentrations, a decline in the sum of adenine nucleotides, near-depletion of glycogen, and an increase in lactate concentration after 2 hr of ischemia. Recirculation for 1 hr led to only a partial recovery of energy state, with little further improvement after 2 hr and signs of secondary deterioration after 4 hr, particularly in the focus. After 4 hr of recirculation, PBN-treated animals showed pronounced recovery of energy state, with ATP and lactate contents in both focus and penumbra approaching normal values. Although an effect of PBN on mitochondria cannot be excluded, the results suggest that PBN acts by preventing a gradual compromise of microcirculation. The results justify a reevaluation of current views on the pathophysiology of focal ischemic damage and suggest that a therapeutic window of many hours exists in stroke.

Essential role of adenosine, adenosine A1 receptors, and ATP-sensitive K+ channels in cerebral ischemic preconditioning.

Preconditioning with sublethal ischemia protects against neuronal damage after subsequent lethal ischemic insults in hippocampal neurons. A pharmacological approach using agonists and antagonists at the adenosine A1 receptor as well as openers and blockers of ATP-sensitive K+ channels has been combined with an analysis of neuronal death and gene expression of subunits of glutamate and gamma-aminobutyric acid receptors, HSP70, c-fos, c-jun, and growth factors. It indicates that the mechanism of ischemic tolerance involves a cascade of events including liberation of adenosine, stimulation of adenosine A1 receptors, and, via these receptors, opening of sulfonylurea-sensitive ATP-sensitive K+ channels.