Turgay Dalkara

MECHANISMS, CHALLENGES AND OPPORTUNITIES IN STROKE

Over the past two decades, research has heavily emphasized basic mechanisms that irreversibly damage brain cells after stroke. Much attention has focused on what makes neurons die easily and what strategies render neurons resistant to ischaemic injury. In the past few years, clinical experience with clot-lysing drugs has confirmed expectations that early reperfusion improves clinical outcome. With recent research emphasizing ways to reduce tissue damage by both vascular and cell-based mechanisms, the spotlight is now shifting towards the study of how blood vessels and brain cells communicate with each other. This new research focus addresses an important need in stroke research, and provides challenges and opportunities that can be used to therapeutic advantage.

Neurological diseases: Mechanisms, challenges and opportunities in stroke

Over the past two decades, research has heavily emphasized basic mechanisms that irreversibly damage brain cells after stroke. Much attention has focused on what makes neurons die easily and what strategies render neurons resistant to ischaemic injury. In the past few years, clinical experience with clot-lysing drugs has confirmed expectations that early reperfusion improves clinical outcome. With recent research emphasizing ways to reduce tissue damage by both vascular and cell-based mechanisms, the spotlight is now shifting towards the study of how blood vessels and brain cells communicate with each other. This new research focus addresses an important need in stroke research, and provides challenges and opportunities that can be used to therapeutic advantage.

Suppression of cortical spreading depression in migraine prophylaxis.

Topiramate, valproate, propranolol, amitriptyline, and methysergide have been widely prescribed for migraine prophylaxis, but their mechanism or site of action is uncertain. Cortical spreading depression (CSD) has been implicated in migraine and as a headache trigger and can be evoked in experimental animals by electrical or chemical stimulation. We hypothesized that migraine prophylactic agents suppress CSD as a common mechanism of action.

Pericyte contraction induced by oxidative-nitrative stress impairs capillary reflow despite successful opening of an occluded cerebral artery

Here we show that ischemia induces sustained contraction of pericytes on microvessels in the intact mouse brain. Pericytes remain contracted despite successful reopening of the middle cerebral artery after 2 h of ischemia. Pericyte contraction causes capillary constriction and obstructs erythrocyte flow. Suppression of oxidative-nitrative stress relieves pericyte contraction, reduces erythrocyte entrapment and restores microvascular patency; hence, tissue survival improves. In contrast, peroxynitrite application causes pericyte contraction. We also show that the microvessel wall is the major source of oxygen and nitrogen radicals causing ischemia and reperfusion–induced microvascular dysfunction. These findings point to a major but previously not recognized pathophysiological mechanism; ischemia and reperfusion-induced injury to pericytes may impair microcirculatory reflow and negatively affect survival by limiting substrate and drug delivery to tissue already under metabolic stress, despite recanalization of an occluded artery. Agents that can restore pericyte dysfunction and microvascular patency may increase the success of thrombolytic and neuroprotective treatments.

L-arginine infusion promotes nitric oxide-dependent vasodilation, increases regional cerebral blood flow, and reduces infarction volume in the rat.

Background and Purpose We previously reported that ar-ginine infusion increased pial vessel diameter by nitric oxide-dependent mechanisms, improved regional cerebral blood flow (rCBF) distal to middle cerebral artery (MCA) occlusion, reduced infarction volume in spontaneously hypertensive when administered intraperitoneally before and after MCA occlusion. In this report we extend our findings (1) by exam-ining the time course of L-arginine on rCBF and pial vessel diameter under basal conditions and on rCBF after MCA occlusion and (2) by reproducing the protective effect L-arginine on infarct volume when given intravenously imme-diately after the onset of MCA occlusion in both normotensive and hypertensive models of focal cerebral ischemia. Methods Changes in pial vessel diameter (closed cranial window) and rCBF (laser-Doppler flowmetry) were measured over time after L-arginine infusion into anesthetized Sprague-Dawley rats. rCBF was also measured distal to MCA occlusion in a brain region showing rCBF reductions in the range of 80% of baseline. The effects of infusing L-arginine (300 mg/kg for 10 minutes beginning 5 minutes after occlu-sion) were assessed on infarction volume in Sprague-Dawley rats after proximal MCA occlusion and in spontaneously hypertensive rats after common carotid artery plus distal MCA occlusion.. Results Results L-Arginine (300 mg/kg IV) elevated rCBF by 20% when measured in the dorsolateral cortex of Sprague-Dawley rats and caused L-nitroarginine-methyl ester-inhibitable in-creases in pial vessel diameter. L-Arginine (& 30 mg/kg IV) increased blood flow distal to MCA occlusion by 50%. These effects were sustained throughout the observation period (70 to 105 minutes). Changes in mean arterial blood pressure were not observed. L-Arginine (300 mg/kg IV) reduced infarction volume by 35% and 28% in Sprague-Dawley and spontane-ously hypertensive rats, respectively, when examined 24 hours after vessel occlusion. Conclusions Conclusions These studies extend our previous findings by demonstrating that exogenous L-arginine induces sustained rCBF increases in normal brain as well as in a marginally perfused brain region distal to MCA occlusion. Our data in Sprague-Dawley rats support the conclusion that L-arginine-induced increases in rCBF can decrease infarction volume. We conclude that nitric oxide-mediated mechanisms increase rCBF and decrease infarction volume after MCA occlusion in both normotensive and hypertensive animals. (Stroke. 1994;25:429-435.)

Prolonged Therapeutic Window for Ischemic Brain Damage Caused by Delayed Caspase Activation

Apoptotic cell death is prominent in neurodegenerative disorders, such as Alzheimers disease and Huntingtons disease, and is found in cerebral ischemia. Using a murine model of delayed cell death, we determined that cleavage of zDEVD-amino-4-trifluoromethyl coumarin (zDEVD-afc) in brain homogenate, a measure of caspase activation, increased initially 9 hours after brief (30 minutes) middle cerebral artery occlusion along with caspase-3p20 immunoreactive cleavage product as determined by immunoblotting. zDEVD-afc cleavage activity was blocked by pretreatment or posttreatment with the caspase-inhibitor N-benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethyl-ketone (zDEVD-fmk), and ischemic damage was reduced when the drug was injected up to 9 hours after reperfusion. The protection was long lasting (21 days). Hence, the period before caspase activation defined the therapeutic opportunity for this neuroprotective agent after mild ischemic brain injury. Prolonged protection after caspase inhibition plus the extended treatment window may be especially relevant to the treatment of neurodegenerative disorders.

Reperfusion-Induced Oxidative/Nitrative Injury to Neurovascular Unit After Focal Cerebral Ischemia

Background and Purpose— Use of thrombolysis in stroke is limited by a short therapeutic window because delayed reperfusion may cause brain hemorrhage and edema. Available evidence suggests a role for superoxide, NO, and peroxynitrite in reperfusion-induced injury. However, depending on their cellular origin and interactions between them, these molecules may exert protective or deleterious actions, neither of which is characterized in the intact brain. Methods— Using fluorescent probes, we determined superoxide and peroxynitrite formation within neurons, astrocytes, and endothelium, and the association between oxidative/nitrative stress and vascular injury in mice brains subjected to 2-hour middle cerebral artery occlusion and 3 or 5 hours of reperfusion. Results— Both signals were colocalized, suggesting that the main source of peroxynitrite in the reperfused brain was a reaction between superoxide and NO. Superoxide and peroxynitrite formation was particularly intense in microvessels and astrocytic end-feet surrounding them, and overlapped with dense mitochondrial labeling. Sites of oxidative/nitrative stress on microvessels were colocalized with markers of vascular injury such as Evans blue (EB) leakage and matrix metalloproteinase-9 (MMP-9) expression, suggesting an association between peroxynitrite and microvascular injury. Supporting this idea, partial inhibition of endothelial NO synthesis at reperfusion with a low dose of L-nitroarginine (1 mg/kg IP) reduced 3-nitrotyrosine formation in microvessels and EB extravasation. Conclusion— During reperfusion, intense superoxide, NO, and peroxynitrite formation on microvessels and surrounding end-feet may lead to cerebral hemorrhage and edema by disrupting microvascular integrity. Combination of thrombolysis with agents diminishing oxidative/nitrative stress may reduce reperfusion-induced injury and extend the therapeutic window for thrombolysis.

Spreading Depression Triggers Headache by Activating Neuronal Panx1 Channels

How Migraine Develops Migraine is a common medical disorder. Unfortunately, how and why migraine headache is initiated is unclear. Karatas et al. (p. 1092) now describe a signaling pathway between stressed neurons and meningeal trigeminal afferents, which may explain how migraine headaches can be generated. Migraine results from a sequence of events starting from stressed cortical neurons and leading to the trigeminal nucleus. The initial phase in the development of a migraine is still poorly understood. Here, we describe a previously unknown signaling pathway between stressed neurons and trigeminal afferents during cortical spreading depression (CSD), the putative cause of migraine aura and headache. CSD caused neuronal Pannexin1 (Panx1) megachannel opening and caspase-1 activation followed by high-mobility group box 1 (HMGB1) release from neurons and nuclear factor κB activation in astrocytes. Suppression of this cascade abolished CSD-induced trigeminovascular activation, dural mast cell degranulation, and headache. CSD-induced neuronal megachannel opening may promote sustained activation of trigeminal afferents via parenchymal inflammatory cascades reaching glia limitans. This pathway may function to alarm an organism with headache when neurons are stressed.

Deciphering migraine mechanisms: Clues from familial hemiplegic migraine genotypes

The molecular and cellular origins of migraine headache are among the most enigmatic in clinical neuroscience. Most agree that susceptibility is inherited and that its clinical presentation is strongly modulated by both internal and external factors. Polymorphisms in genes regulating ion translocation have been implicated in two subtypes of familial hemiplegic migraine (FHM), a rare migraine disorder. Families with FHM type 1 express point mutations in the Cav2.1 channel, 1 whereas type 2 patients express mutations in the subunit of the Na ,K pump (Fig 1). Cav2.1 channels gate Ca , whereas the ATP-utilizing pumps distribute Na ,K ions across plasma membranes. Interestingly, the mutated 1A subunit of the P/Q calcium channel is found exclusively on neurons, whereas the 2 subunit of the pump is expressed primarily by astrocytes in adult brain. How then does a coherent migraine phenotype emerge as a consequence of point mutations expressed on distinctive cell types regulating monovalent or divalent cation fluxes? The simple answer is that we do not know. However, human studies strongly implicating cortical spreading depression (CSD) as the generator of migraine aura, together with evidence linking astrocytes and blood vessels to brain metabolism and synaptic activity, provide intriguing possibilities relevant to FHM, and perhaps by extrapolation, to more common forms of migraine headache.

Role of Endothelial Nitric Oxide Generation and Peroxynitrite Formation in Reperfusion Injury After Focal Cerebral Ischemia

BACKGROUND AND PURPOSE Reperfusion injury is one of the factors that unfavorably affects stroke outcome and shortens the window of opportunity for thrombolysis. Surges of nitric oxide (NO) and superoxide generation on reperfusion have been demonstrated. Concomitant generation of these radicals can lead to formation of the strong oxidant peroxynitrite during reperfusion. METHODS We have examined the role of NO generation and peroxynitrite formation on reperfusion injury in a mouse model of middle cerebral artery occlusion (2 hours) and reperfusion (22 hours). The infarct volume was assessed by 2,3,5-triphenyl tetrazolium chloride staining; blood-brain barrier permeability was evaluated by Evans blue extravasation. Nitrotyrosine formation and matrix metalloproteinase-9 expression were detected by immunohistochemistry. RESULTS Infarct volume was significantly decreased (47%) in animals treated with the nonselective nitric oxide synthase (NOS) inhibitor N(omega)-nitro-L-arginine (L-NA) at reperfusion. The specific inhibitor of neuronal NOS, 7-nitroindazole (7-NI), given at reperfusion, showed no protection, although preischemic treatment with 7-NI decreased infarct volume by 40%. Interestingly, prereperfusion administration of both NOS inhibitors decreased tyrosine nitration (a marker of peroxynitrite toxicity) in the ischemic area. L-NA treatment also significantly reduced vascular damage, as indicated by decreased Evans blue extravasation and matrix metalloproteinase-9 expression. CONCLUSIONS These data support the hypothesis that in addition to the detrimental action of NO formed by neuronal NOS during ischemia, NO generation at reperfusion plays a significant role in reperfusion injury, possibly through peroxynitrite formation. Contrary to L-NA, failure of 7-NI to protect against reperfusion injury suggests that the source of NO is the cerebrovascular compartment.