Carolina M. Maier

Oxidative Stress in Ischemic Brain Damage: Mechanisms of Cell Death and Potential Molecular Targets for Neuroprotection

Significant amounts of oxygen free radicals (oxidants) are generated during cerebral ischemia/reperfusion, and oxidative stress plays an important role in brain damage after stroke. In addition to oxidizing macromolecules, leading to cell injury, oxidants are also involved in cell death/survival signal pathways and cause mitochondrial dysfunction. Experimental data from laboratory animals that either overexpress (transgenic) or are deficient in (knock-out) antioxidant proteins, mainly superoxide dismutase, have provided strong evidence of the role of oxidative stress in ischemic brain damage. In addition to mitochondria, recent reports demonstrate that NADPH oxidase (NOX), an important pro-oxidant enzyme, is also involved in the generation of oxidants in the brain after stroke. Inhibition of NOX is neuroprotective against cerebral ischemia. We propose that superoxide dismutase and NOX activity in the brain is a major determinant for ischemic damage/repair and that these major anti- and pro-oxidant enzymes are potential endogenous molecular targets for stroke therapy.

Optimal Depth and Duration of Mild Hypothermia in a Focal Model of Transient Cerebral Ischemia Effects on Neurologic Outcome, Infarct Size, Apoptosis, and Inflammation

BACKGROUND AND PURPOSE Mild hypothermia is possibly the single most effective method of cerebroprotection developed to date. However, many questions regarding mild hypothermia remain to be addressed before its potential implementation in the treatment of human stroke. Here we report the results of 2 studies designed to determine the optimal depth and duration of mild hypothermia in focal stroke and its effects on infarct size, neurological outcome, programmed cell death, and inflammation. METHODS Rats underwent a 2-hour occlusion of the left middle cerebral artery. In the first study (I) animals were kept (intraischemically) at either 37 degreesC (n=8), 33 degreesC (n=8), or 30 degreesC (n=8). Study II consisted of 4 groups: (1) controls (37 degreesC, n=10), (2) 30 minutes of hypothermia started at ischemic onset (33 degreesC, n=9), (3)1 hour (33 degreesC, n=8), and (4) 2 hours (33 degreesC, n=8). Brain temperature was measured by a thermocouple probe placed in the contralateral cortex. After suture removal, all animals were rewarmed and reperfused for 22 hours (I) or 70 hours (II). RESULTS Mild hypothermia to 33 degreesC or 30 degreesC was neuroprotective (17+/-7% and 27+/-6%, respectively) relative to controls (53+/-8%, P<0.02), but 33 degreesC was better tolerated and recovery from anesthesia was faster. The neurological score of hypothermic animals was significantly better than that of controls (I & II) at both 24 and 72 hours postischemia except for the 30-minute group (II), which showed no improvement. In Study II, 2 hours of hypothermia reduced injury by 59%, 1 hour reduced injury by 84% whereas 30 minutes did not reduce injury. Normalized for infarct size, 2 hours of mild hypothermia decreased neutrophil accumulation by 57% whereas both 1 hour and 30 minutes had no effect. At 72 hours, 1 and 2 hours of mild hypothermia decreased transferase dUTP nick-end labeling (TUNEL) staining by 78% and 99%, respectively, and 30 minutes of hypothermia had no effect. CONCLUSIONS Intraischemic mild hypothermia must be maintained for 1 to 2 hours to obtain optimal neuroprotection against ischemic cell death due to necrosis and apoptosis.

Neuroprotection by the α2-Adrenoreceptor Agonist Dexmedetomidine in a Focal Model of Cerebral Ischemia

BACKGROUND Dexmedetomidine, a highly selective alpha 2-adrenoreceptor agonist, decreases central sympathetic activity and reduces the anesthetic requirement of halothane. Preliminary studies show that dexmedetomidine improves the outcome from ischemic injury and, therefore, may have potential therapeutic value. METHODS The authors studied 14 rabbits that underwent a 2-h occlusion of the left internal carotid, anterior cerebral, and middle cerebral arteries, followed by 4 h of reperfusion. Ten minutes after occlusion, the animals were treated with either normal saline (n = 7) or dexmedetomidine (n = 7) using a computer-controlled infusion rate calculated to maintain a steady state plasma concentration. Halothane concentration was reduced by 50% for dexmedetomidine-treated animals to maintain a comparable level of anesthesia. Somatosensory evoked potentials were used to confirm adequate ischemia, and injury was assessed by histopathology. RESULTS There were significant differences in the area of ischemic neuronal damage between the groups in the cortex (halothane alone, 38.2 +/- 6.0% SEM vs. halothane plus dexmedetomidine, 20.0 +/- 2.7% SEM, P = 0.018), but not in the striatum (halothane alone, 68.7 +/- 12.6% SEM vs. halothane plus dexmedetomidine, 43.5 +/- 15.9% SEM, P = 0.24), nor in physiologic parameters. Dexmedetomidine plasma levels obtained every 90 min showed a mean of 4.0 +/- 0.15 ng/ml. CONCLUSIONS Results from this study indicate that postischemic administration of dexmedetomidine, in a dose that reduces the anesthetic requirements by 50%, has a neuroprotective effect in this model of focal cerebral ischemia.

Neuronal Death/Survival Signaling Pathways in Cerebral Ischemia

SummaryCumulative evidence suggests that apoptosis plays a pivotal role in cell deathin vitro after hypoxia. Apoptotic cell death pathways have also been implicated in ischemic cerebral injury inin vivo ischemia models. Experimental ischemia and reperfusion models, such as transient focal/global ischemia in rodents, have been thoroughly studied and the numerous reports suggest the involvement of cell survival/death signaling pathways in the pathogenesis of apoptotic cell death in ischemic lesions. In these models, reoxygenation during reperfusion provides a substrate for numerous enzymatic oxidation reactions. Oxygen radicals damage cellular lipids, proteins and nucleic acids, and initiate cell signaling pathways after cerebral ischemia. Genetic manipulation of intrinsic antioxidants and factors in the signaling pathways has provided substantial understanding of the mechanisms involved in cell death/survival signaling pathways and the role of oxygen radicals in ischemic cerebral injury. Future studies of these pathways may provide novel therapeutic strategies in clinical stroke.

Book Review: Role of Superoxide Dismutases in Oxidative Damage and Neurodegenerative Disorders

In recent years, oxidative stress has been implicated in a variety of degenerative processes, diseases, and syndromes. Some of these include atherosclerosis, myocardial infarction, stroke, and ischemia/reperfusion injury; chronic and acute inflammatory conditions such as wound healing; central nervous system disorders such as forms of familial amyotrophic lateral sclerosis (ALS) and glutathione peroxidase-linked adolescent seizures; Parkinson’s disease and Alzheimer’s dementia; and a variety of other age-related disorders. Among the various biochemical events associated with these conditions, emerging evidence suggests the formation of superoxide anion and expression/activity of its endogenous scavenger, superoxide dismutase (SOD), as a common denominator. This review summarizes the function of SOD under normal physiological conditions as well as its role in the cellular and molecular mechanisms underlying oxidative tissue damage and neurological abnormalities. Experimental evidence from laboratory animals that either overexpress (transgenics) or are deficient (knockouts) in antioxidant enzyme/protein levels and the genetic SOD mutations observed in some familial cases of ALS are also discussed.

Oxidative stress and neuronal death/survival signaling in cerebral ischemia

It has been demonstrated by numerous studies that apoptotic cell death pathways are implicated in ischemic cerebral injury in ischemia models in vivo. Experimental ischemia and reperfusion models, such as transient focal/global ischemia in rodents, have been thoroughly studied and the numerous reports suggest the involvement of cell survival/death signaling pathways in the pathogenesis of apoptotic cell death in ischemic lesions. In these models, reoxygenation during reperfusion provides oxygen as a substrate for numerous enzymatic oxidation reactions and for mitochondrial oxidative phosphorylation to produce adenosine triphosphate. Oxygen radicals, the products of these biochemical and physiological reactions, are known to damage cellular lipids, proteins, and nucleic acids and to initiate cell signaling pathways after cerebral ischemia. Genetic manipulation of intrinsic antioxidants and factors in the signaling pathways has provided substantial understanding of the mechanisms involved in cell death/survival signaling pathways and the role of oxygen radicals in ischemic cerebral injury. Future studies of these pathways could provide novel therapeutic strategies in clinical stroke.

Reperfusion and Neurovascular Dysfunction in Stroke: from Basic Mechanisms to Potential Strategies for Neuroprotection

Effective stroke therapies require recanalization of occluded cerebral blood vessels. However, reperfusion can cause neurovascular injury, leading to cerebral edema, brain hemorrhage, and neuronal death by apoptosis/necrosis. These complications, which result from excess production of reactive oxygen species in mitochondria, significantly limit the benefits of stroke therapies. We have developed a focal stroke model using mice deficient in mitochondrial manganese-superoxide dismutase (SOD2−/+) to investigate neurovascular endothelial damage that occurs during reperfusion. Following focal stroke and reperfusion, SOD2−/+ mice had delayed blood-brain barrier breakdown, associated with activation of matrix metalloproteinase and high brain hemorrhage rates, whereas a decrease in apoptosis and hemorrhage was observed in SOD2 overexpressors. Thus, induction and activation of SOD2 is a novel strategy for neurovascular protection after ischemia/reperfusion. Our recent study identified the signal transducer and activator of transcription 3 (STAT3) as a transcription factor of the mouse SOD2 gene. During reperfusion, activation of STAT3 and its recruitment into the SOD2 gene were blocked, resulting in increased oxidative stress and neuronal apoptosis. In contrast, pharmacological activation of STAT3 induced SOD2 expression, which limits ischemic neuronal death. Our studies point to antioxidant-based neurovascular protective strategies as potential treatments to expand the therapeutic window of currently approved therapies.

Mild hypothermia reduces ICAM-1 expression, neutrophil infiltration and microglia/monocyte accumulation following experimental stroke

Mild hypothermia is an established neuroprotectant against cerebral ischemic injury. Studies have shown that inflammation potentiates cerebral ischemic injury, particularly in the setting of reperfusion. To further elucidate the mechanism by which mild hypothermia attenuates the inflammatory response, we assessed endothelial intercellular adhesion molecule-1 (ICAM-1) expression, neutrophil and monocyte infiltration, and microglial activation following 2 h of transient focal cerebral ischemia under normothermic and mildly hypothermic conditions. Ischemia was induced using the intraluminal suture method in Sprague-Dawley rats. Immunohistochemistry was used to detect endothelial ICAM-1, infiltrating neutrophils and monocytes, and microglia at 1, 3, and 7 days post-ischemia. Immunopositive cell and vessel densities were measured in the peri-infarct region. Mild hypothermia was associated with decreased neutrophils at 1 and 3 days post-ischemia, decreased ICAM-1-positive vessels at 1, 3, and 7 days, and decreased monocytes/activated microglia at 3 and 7 days, but not at 1 day. These data demonstrate that mild hypothermia significantly reduces endothelial adhesion molecule expression, acute (neutrophil) and subacute (monocyte) leukocyte infiltration, and microglial activation up to 7 days following insult in a rodent model of transient focal cerebral ischemia.

Oxidative Damage to the Endoplasmic Reticulum is Implicated in Ischemic Neuronal Cell Death

The endoplasmic reticulum (ER), which plays important roles in apoptosis, is susceptible to oxidative stress. Because reactive oxygen species (ROS) are robustly produced in the ischemic brain, ER damage by ROS may be implicated in ischemic neuronal cell death. We induced global brain ischemia on wild-type and copper/zinc superoxide dismutase (SOD1) transgenic rats and compared ER stress and neuronal damage. Phosphorylated forms of eukaryotic initiation factor 2α (eIF2α) and RNA-dependent protein kinase-like ER eIF2α kinase (PERK), both of which play active roles in apoptosis, were increased in hippocampal CA1 neurons after ischemia but to a lesser degree in the transgenic animals. This finding, together with the finding that the transgenic animals showed decreased neuronal degeneration, indicates that oxidative ER damage is involved in ischemic neuronal cell death. To elucidate the mechanisms of ER damage by ROS, we analyzed glucose-regulated protein 78 (GRP78) binding with PERK and oxidative ER protein modification. The proteins were oxidatively modified and stagnated in the ER lumen, and GRP78 was detached from PERK by ischemia, all of which were attenuated by SOD1 overexpression. We propose that ROS attack and modify ER proteins and elicit ER stress response, which results in neuronal cell death.

Neurodegeneration in Striatum Induced by the Mitochondrial Toxin 3-Nitropropionic Acid: Role of Matrix Metalloproteinase-9 in Early Blood-Brain Barrier Disruption?

Blood-brain barrier (BBB) dysfunction is a potential mechanism involved in progressive striatal damage induced by the mitochondrial excitotoxin, 3-nitropropionic acid (3-NP). After activation by proteases and free radicals, matrix metalloproteinases (MMPs), particularly MMP-9 and -2, can digest the endothelial basal lamina leading to BBB opening. Using CD-1 mice, we show that MMP-9 expression by zymography is increased in the injured striatum compared with the contralateral striatum 2 hr after 3-NP injection [133.50 ± 57.17 vs 50.25 ± 13.56; mean ± SD of optical densities in arbitrary units (A.U.); p < 0.005] and remains elevated until 24 hr (179.33 ± 78.24 A.U.). After 4 hr, MMP-9 expression and activation are accompanied by an increase in BBB permeability. MMP inhibition attenuates BBB disruption, swelling, and lesion volume compared with vehicle-treated controls. There is a clear spatial relationship between MMP-9 expression and oxidized hydroethidine, indicating reactive oxygen species (ROS) production. Furthermore, transgenic mice that overexpress copper/zinc-superoxide dismutase (SOD1) show decreased lesion size and edema along with decreased immunoreactivity for MMP-9, compared with wild-type littermates (lesion: 38.8 ± 15.1 and 53.3 ± 10.3, respectively, p ≤ 0.05; edema: 21.8 ± 11.2 and 35.28 ± 11, respectively, p ≤ 0.05; MMP-9-positive cells: 352 ± 57 and 510 ± 45, respectively, p ≤ 0.005), whereas knock-out mice deficient in SOD1 display significantly greater swelling (48.65 ± 17; p ≤ 0.05). We conclude that early expression and activation of MMP-9 by ROS may be involved in early BBB disruption and progressive striatal damage after 3-NP treatment.