Dirk Megow

Increased Formation of Reactive Oxygen Species after Permanent and Reversible Middle Cerebral Artery Occlusion in the Rat

In barbiturate-anesthetized rats, we induced 3 hours of permanent middle cerebral artery occlusion (MCAO) by an intraluminal thread (n = 6), or 1 hour MCAO followed by 2 hours of reperfusion (n = 6). Through a closed cranial window over the parietal cortex, the production of reactive oxygen species (ROS) was measured in the infarct border using online in vivo chemiluminescence (CL) while monitoring the appearance of peri-infarct depolarizations (PID). The borderzone localization of the ROS and direct current (DC) potential measurements was confirmed in additional experiments using laser-Doppler scanning, mapping regional CBF changes through the cranial window after permanent (n = 5) or reversible (n = 5) MCAO. CL measurements revealed a short period (10 to 30 minutes) of reduced ROS formation after vessel occlusion, followed by a significant increase (to 162 ± 51%; baseline = 100%; P < .05) from 100 minutes of permanent MCAO onward. Reperfusion after a 1-hour period of MCAO led to a burst-like pattern of ROS production (peak: 489 ± 330%; P < .05). When the experiments were terminated 3 hours after induction of MCAO, CL was still significantly increased above baseline after permanent and reversible MCAO (to 190 ± 67% and 211 ± 64%, respectively; P < .05). Simultaneous DC potential recordings detected 6.4 ± 2.7 PID in the first, 4.7 ± 2.3 in the second, and 2.8 ± 2.0 in the third hour after permanent MCAO. In animals with reversible MCAO, PID were abolished from 15-minutes recirculation onward. There was no temporal relationship between ROS production and peri-infarct DC potential shifts. In conclusion, using a high temporal resolution ROS detection technique (CL), we found that permanent MCAO (after an initial decrease) was accompanied by a steady increase of ROS production during the 3-hour observation period, while reperfusion after 1 hour of MCAO produced a burst in ROS formation. Both patterns of ROS production were not related to the occurrence of PID.

Neuroprotective effects of atorvastatin against glutamate-induced excitotoxicity in primary cortical neurones

Statins [3‐hydroxy‐3‐methylglutaryl‐coenzyme A (HMG‐CoA) reductase inhibitors] exert cholesterol‐independent pleiotropic effects that include anti‐thrombotic, anti‐inflammatory, and anti‐oxidative properties. Here, we examined direct protective effects of atorvastatin on neurones in different cell damage models in vitro. Primary cortical neurones were pre‐treated with atorvastatin and then exposed to (i) glutamate, (ii) oxygen–glucose deprivation or (iii) several apoptosis‐inducing compounds. Atorvastatin significantly protected from glutamate‐induced excitotoxicity as evidenced by propidium iodide staining, nuclear morphology, release of lactate dehydrogenase, and mitochondrial tetrazolium metabolism, but not from oxygen–glucose deprivation or apoptotic cell death. This anti‐excitototoxic effect was evident with 2–4 days pre‐treatment but not with daily administration or shorter‐term pre‐treatment. The protective properties occurred independently of 3‐hydroxy‐3‐methylglutaryl‐CoA reductase inhibition because co‐treatment with mevalonate or other isoprenoids did not reverse or attenuate neuroprotection. Atorvastatin attenuated the glutamate‐induced increase of intracellular calcium, which was associated with a modulation of NMDA receptor function. Taken together, atorvastatin exerts specific anti‐excitotoxic effects independent of 3‐hydroxy‐3‐methylglutaryl‐CoA reductase inhibition, which has potential therapeutic implications.

Desferrioxamine Induces Delayed Tolerance against Cerebral Ischemia in Vivo and in Vitro

The widely prescribed drug desferrioxamine is a known activator of the hypoxia-inducible transcription factor 1 (HIF-1) and the subsequent transcription of erythropoietin. In the brain, HIF-1 is a master switch of the transcriptional response to hypoxia, whereas erythropoietin is a potent neuroprotectant. The authors show that desferrioxamine dose-dependently and time-dependently induces tolerance against focal cerebral ischemia in rats and mice, and against oxygen–glucose deprivation in purified cortical neurons. Desferrioxamine induced HIF-1 DNA binding and transcription of erythropoietin in vivo, the temporal kinetics of which were congruent with tolerance induction. Desferrioxamine is a promising drug for the induction of tolerance in humans when ischemia can be anticipated.

Respiratory Chain Inhibition Induces Tolerance to Focal Cerebral Ischemia

The authors show that the inhibitor of the succinate dehydrogenase, 3-nitroproprionic acid (3-NPA), which in high doses and with chronic administration is a neurotoxin, can induce profound tolerance to focal cerebral ischemia in the rat when administered in a single dose (20 mg/kg) 3 days before ischemia. Infarcts were approximately 70% and 35% smaller in the 3-NPA preconditioned groups of permanent and transient focal cerebral ischemia, respectively. This regimen of 3-NPA preconditioning neither induced necrosis, apoptosis, or any other histologically detectable damage to the brain, nor did it affect behavior of the animals. 3-NPA led to an immediate (1-hour) and long-lasting (3-day) decrease in succinate dehydrogenase activity (30% reduction) throughout the brain, whereas only a short metabolic impairment occurred (ATP decrease of 35% within 30 minutes, recovery within 2 hours). The authors found that 3-NPA induces a burst of reactive oxygen species and the free radical scavenger dimethylthiourea, when administered shortly before the 3-NPA stimulus, completely blocked preconditioning. Inhibition of protein synthesis with cycloheximide given at the time of 3-NPA administration completely inhibited preconditioning. The authors were unsuccessful in showing upregulation of mRNA for the manganese superoxide dismutase, and did not detect increased activities of the copper-zinc and manganese superoxide dismutases, prototypical oxygen free radicals scavenging enzymes, after 3-NPA preconditioning. The authors conclude that it is possible to pharmacologically precondition the brain against focal cerebral ischemia, a strategy that may in principal have clinical relevance. The data show the relevance of protein synthesis for tolerance, and suggests that oxygen free radicals may be critical signals in preconditioning.

Preventive Antibacterial Treatment Improves the General Medical and Neurological Outcome in a Mouse Model of Stroke

Background and Purpose— Epidemiological studies have demonstrated a high incidence of infections after severe stroke and their prominent role in morbidity and mortality in stroke patients. In a mouse model, it has been shown recently that stroke is coupled with severe and long-lasting immunosuppression, which is responsible for the development of spontaneous systemic infections. Here, we investigated in the same model the effects of preventive antibiotic treatment on survival and functional outcome of experimental stroke. Methods— Mice were subjected to experimental stroke by occlusion of the middle cerebral artery (MCAO) for 60 minutes. A group of mice received moxifloxacin (6×100 mg/kg body weight every 2 hours over 12 hours) either immediately or 12 hours after MCAO. Control animals received the vector only. Behavior, neurological deficit, fever, survival, and body weight were monitored over 14 days. In a subgroup, infarct volume was measured 4 days after MCAO. Microbiological assessment was based on cultures of lung tissue, blood, and feces of animals 3 days after stroke. For a dose-response study, moxifloxacin was given immediately after MCAO in different doses and at different time points. Results— Microbiological analyses of blood and lung tissue demonstrated high bacterial burden, mainly Escherichia coli, 3 days after stroke. Accordingly, we observed clinical and histological signs of septicemia and pneumonia. Moxifloxacin prevented the development of infections and fever, significantly reduced mortality, and improved neurological outcome. Conclusions— Preventive antibiotic treatment may be an important new therapeutical approach to improve outcome in patients with severe stroke.

Improved reperfusion and neuroprotection by creatine in a mouse model of stroke

Stroke leads to energy failure and subsequent neuronal cell loss. Creatine and phosphocreatine constitute a cellular energy buffering and transport system, and dietary creatine supplementation was shown to protect neurons in several models of neurodegeneration. Although creatine has recently been found to reduce infarct size after cerebral ischemia in mice, the mechanisms of neuroprotection remained unclear. We provide evidence for augmented cerebral blood flow (CBF) after stroke in creatine-treated mice using a magnetic resonance imaging (MRI)-based technique of CBF measurement (flow-sensitive alternating inversion recovery-MRI). Moreover, improved vasodilatory responses were detected in isolated middle cerebral arteries obtained from creatine-treated animals. After 3 weeks of dietary creatine supplementation, minor changes in brain creatine, phosphocreatine, adenosine triphosphate, adenosine diphosphate and adenosine monophosphate levels were detected, which did not reach statistical significance. However, we found a 40% reduction in infarct volume after transient focal cerebral ischemia. Our data suggest that creatine-mediated neuroprotection can occur independent of changes in the bioenergetic status of brain tissue, but may involve improved cerebrovascular function.

Hyperbaric oxygenation induced tolerance against focal cerebral ischemia in mice is strain dependent

SV129 or C57BL/6 mice were exposed to hyperbaric oxygenation (HBO, 5 days, 1 h every day, 100% O(2) at 3 atm absolute). One day after the 5th HBO session focal cerebral ischemia was induced. In SV129 mice, HBO induced tolerance against permanent focal cerebral ischemia (n=42, mean infarct volume reduction 27%, P=0.001), but not against transient (30 or 60 min) focal cerebral ischemia. In the C57BL/6 strain of mice, HBO did not induce tolerance against focal cerebral ischemia, even when the duration of ischemia or the HBO protocol were modified. For the first time we demonstrate that HBO can induce tolerance to focal cerebral ischemia, but this effect is strain dependent.

HMG-CoA reductase inhibition causes neurite loss by interfering with geranylgeranylpyrophosphate synthesis.

To determine whether neurite outgrowth depends upon the mevalonate pathway, we blocked mevalonate synthesis in nerve growth factor‐treated PC12 cells or primary cortical neurones with atorvastatin, a 3‐hydroxymethylglutaryl coenzyme A (HMG‐CoA) reductase inhibitor, and substituted different intermediates of the mevalonate pathway. We show that HMG‐CoA reductase inhibition causes a profound reduction of neurite length, neurite loss and ultimatively cell death in undifferentiated and pre‐differentiated PC12 cells and also in rat primary cortical neurones. Geranylgeranylpyrophosphate, but not farnesylpyrophosphate, squalene or cholesterol, completely compensated for the lack of mevalonate. Our data indicate that, under HMG‐CoA reductase inhibition, geranylgeranylpyrophosphate rather than farnesylpyrophosphate or cholesterol is critical for neurite outgrowth and/or maintenance. Loss of neurites is an early manifestation of various neurodegenerative disorders, and dysfunction of isoprenylation might play a role in their pathogenesis.

Mitochondrial oxidation in rat hippocampus can be preconditioned by selective chemical inhibition of succinic dehydrogenase

Repeatedly it was reported that a short ischemic episode may ameliorate biochemical and morphological impairment upon succeeding severe ischemia. We investigated whether the pattern of respiratory enzyme activity (RA), adenine nucleotides, and membrane potential in hippocampal slices following low-dose in vivo (20 mg/kg) and high-dose in vitro (1 mM) application of 3-nitropropionic acid (3-np), a specific inhibitor of succinic dehydrogenase (SDH), indicates a similar tolerance phenomenon. One hour in vivo treatment decreased RA, spectrophotometrically quantitated by intensity of staining with 2,3,5-triphenyltetrazolium chloride (TTC), to 48 +/- 5% (mean +/- SE; P<0.01). Intermittent increase after 2 h (79 +/- 5%; P<0.05) was followed by gradual decline to 48 +/- 16% (P<0.01) after 8 h. The intermittent increase predominated in stratum pyramidale of hippocampal region CA1 (CA1sp) vs CA3 (CA3sp) (89 +/- 6% vs 57 +/- 6% of control; P <0.01). ATP levels paralleled the intensity of average (CA1sp, CA3sp, plus CA1 stratum radiatum) TTC staining (r=0.93). After pretreatment of 3-np in vivo for 1 h, no further decrease of RA upon 30-min in vitro treatment was seen in any region. At all other times, RA declined further upon in vitro treatment (P<0.01). Compared to 1-h in vivo treatment, hyperpolarization of CA1sp pyramidal cells upon in vitro application of 1 mM 3-np was reduced after 8-h pretreatment in vivo (P<0.04). At this time, depolarization upon glibenclamide (10 muM), an antagonist at KATP-channels, was reduced. We conclude that the severity of impairment of oxidative phosphorylation upon repeated inhibition of SDH in vivo and in vitro is not increased in an additive manner. At appropriate times, relative protection against further decrease of energy metabolism is observed-chemical preconditioning. Activation of KATP-channels is associated with chemical preconditioning.

Increased Extracellular K+ Concentration Reduces the Efficacy of N-methyl-d-aspartate Receptor Antagonists to Block Spreading Depression-Like Depolarizations and Spreading Ischemia

Background and Purpose— Spreading depression (SD)-like depolarizations may augment neuronal damage in neurovascular disorders such as stroke and traumatic brain injury. Spreading ischemia (SI), a particularly malignant variant of SD-like depolarization, is characterized by inverse coupling between the spreading depolarization wave and cerebral blood flow. SI has been implicated in particular in the pathophysiology of subarachnoid hemorrhage. Under physiological conditions, SD is blocked by N-methyl-d-aspartate receptor (NMDAR) antagonists. However, because both SD-like depolarizations and SI occur in presence of an increased extracellular K+ concentration ([K+]o), we tested whether this increase in baseline [K+]o would reduce the efficacy of NMDAR antagonists. Methods— Cranial window preparations, laser Doppler flowmetry, and K+-sensitive/reference microelectrodes were used to record SD, SD-like depolarizations, and SI in rats in vivo; microelectrodes and intrinsic optical signal measurements were used to record SD and SD-like depolarizations in human and rat brain slices. Results— In vivo, the noncompetitive NMDAR antagonist dizocilpine (MK-801) blocked SD propagation under physiological conditions, but did not block SD-like depolarizations or SI under high baseline [K+]o. Similar results were found in human and rat neocortical slices with both MK-801 and the competitive NMDAR antagonist D-2-amino-5-phosphonovaleric acid. Conclusions— Our data suggest that elevated baseline [K+]o reduces the efficacy of NMDAR antagonists on SD-like depolarizations and SI. In conditions of moderate energy depletion, as in the ischemic penumbra, or after subarachnoid hemorrhage, NMDAR inhibition may not be sufficient to block these depolarizations.