Costantino Iadecola

Pathobiology of ischaemic stroke: an integrated view

Brain injury following transient or permanent focal cerebral ischaemia (stroke) develops from a complex series of pathophysiological events that evolve in time and space. In this article, the relevance of excitotoxicity, peri-infarct depolarizations, inflammation and apoptosis to delayed mechanisms of damage within the peri-infarct zone or ischaemic penumbra are discussed. While focusing on potentially new avenues of treatment, the issue of why many clinical stroke trials have so far proved disappointing is addressed. This article provides a framework that can be used to generate testable hypotheses and treatment strategies that are linked to the appearance of specific pathophysiological events within the ischaemic brain.

Bright and dark sides of nitric oxide in ischemic brain injury

There is increasing evidence that nitric oxide (NO), a free radical that can act both as a signaling molecule and a neurotoxin, is involved in the mechanisms of cerebral ischemia. Although early investigations yielded conflicting results, the introduction of more-selective pharmacological tools and the use of molecular approaches for deletion of genes encoding for NO synthase have provided a better understanding of the role of NO in the mechanisms of ischemic brain damage. The evidence reviewed in this article suggests that NO is protective or destructive depending on the stage of evolution of the ischemic process and on the cellular source of NO. Defining the role of NO in cerebral ischemia provides the rationale for new neuroprotective strategies based on modulation of NO production in the post-ischemic brain.

Nitric Oxide Synthase Inhibition and Cerebrovascular Regulation

There is increasing evidence that nitric oxide (NO) is an important molecular messenger involved in a wide variety of biological processes. Recent data suggest that NO is also involved in the regulation of the cerebral circulation. Thus, NO participants in the maintenance of resting cerebrovascular tone and may play an important role in selected vasodilator responses of the cerebral circulation. Furthermore, evidence has been presented suggesting that NO participates in the mechanisms of cerebral ischemic damage. Despite the widespread attention that NO has captured in recent years and the large number of studies that have been published on the subject, there is considerable controversy regarding the role of this agent in cerebrovascular regulation and in ischemic damage. In this paper the results of investigations on NO and the cerebral circulation are reviewed and the evidence for and against a role of NO is critically examined.

Delayed Reduction of Ischemic Brain Injury and Neurological Deficits in Mice Lacking the Inducible Nitric Oxide Synthase Gene

Inducible nitric oxide synthase (iNOS), an enzyme that produces toxic amounts of nitric oxide, is expressed in a number of brain pathologies, including cerebral ischemia. We used mice with a null mutation of the iNOS gene to study the role of iNOS in ischemic brain damage. Focal cerebral ischemia was produced by occlusion of the middle cerebral artery (MCA). In wild-type mice, iNOS mRNA expression in the post-ischemic brain begun between 24 and 48 hr peaked at 96 hr and subsided 7 d after MCA occlusion. iNOS mRNA induction was associated with expression of iNOS protein and enzymatic activity. In contrast, mice lacking the iNOS gene did not express iNOS message or protein after MCA occlusion. The infarct and the motor deficits produced by MCA occlusion were smaller in iNOS knockouts than in wild-type mice (p < 0.05). Such reduction in ischemic damage and neurological deficits was observed 96 hr after ischemia but not at 24 hr, when iNOS is not yet expressed in wild-type mice. The decreased susceptibility to cerebral ischemia in iNOS knockouts could not be attributed to differences in the degree of ischemia or vascular reactivity between wild-type and knockout mice. These findings indicate that iNOS expression is one of the factors contributing to the expansion of the brain damage that occurs in the post-ischemic period. iNOS inhibition may provide a novel therapeutic strategy targeted specifically at the secondary progression of ischemic brain injury.

Age-Related CNS Disorder and Early Death in Transgenic FVB/N Mice Overexpressing Alzheimer Amyloid Precursor Proteins

Transgenic FVB/N mice overexpressing human (Hu) or mouse (Mo) Alzheimer amyloid precursor protein (APP695) die early and develop a CNS disorder that includes neophobia and impaired spatial alternation, with diminished glucose utilization and astrogliosis mainly in the cerebrum. Age at onset of neophobia and age at death decrease with increasing levels of brain APP. HuAPP transgenes induce death much earlier than MoAPP transgenes expressed at similar levels. No extracellular amyloid was detected, indicating that some deleterious processes related to APP overexpression are dissociated from formation of amyloid. A similar clinical syndrome occurs spontaneously in approximately 20% of nontransgenic mice when they reach mid- to late-adult life, suggesting that APP overexpression may accelerate a naturally occurring age-related CNS disorder in FVB/N mice.

Regulation of the cerebral microcirculation during neural activity: is nitric oxide the missing link?

Although the mechanisms regulating the cerebral microcirculation during neural activity have been the subject of inquiry for a century or more, the mediators responsible for the changes in cerebral blood flow still remain to be clearly identified. The discovery that nitric oxide, a powerful cerebrovasodilator, is produced by active neurons has led to the hypothesis that this agent could be the long-sought mediator coupling brain activity to cerebral blood flow. This hypothesis is supported by recent experimental data suggesting that nitric oxide participates in the maintenance of resting cerebral blood flow and in the cerebrovasodilatation elicited by increased neural activity. In this article, this evidence is critically reviewed and discussed in the context of general principles of cerebrovascular regulation.

SOD1 rescues cerebral endothelial dysfunction in mice overexpressing amyloid precursor protein.

Peptides derived from proteolytic processing of the β–amyloid precursor protein (APP), including the amyloid–β peptide, are important for the pathogenesis of Alzheimers dementia. We found that transgenic mice overexpressing APP have a profound and selective impairment in endothelium–dependent regulation of the neocortical microcirculation. Such endothelial dysfunction was not found in transgenic mice expressing both APP and superoxide dismutase–1 (SOD1) or in APP transgenics in which SOD was topically applied to the cerebral cortex. These cerebrovascular effects of peptides derived from APP processing may contribute to the alterations in cerebral blood flow and to neuronal dysfunction in Alzheimers dementia.

Inducible Nitric Oxide Synthase Gene Expression in Vascular Cells After Transient Focal Cerebral Ischemia

BACKGROUND AND PURPOSEnWe investigated whether inducible nitric oxide synthase (iNOS) is expressed after transient cerebral ischemia and, if so, we sought to define the temporal profile and cellular localization of the expression and the role of iNOS in the mechanism of ischemic brain injury.nnnMETHODSnThe middle cerebral artery in rats was occluded for 2 hours by an intraluminal filament. The occurrence of transient ischemia and reperfusion was confirmed by laser-Doppler flowmetry (n = 5). iNOS message in the ischemic neocortex was determined by reverse-transcription polymerase chain reaction. iNOS enzymatic activity was assessed by citrulline assay. The cellular localization of iNOS expression was determined by immunohistochemistry.nnnRESULTSniNOS mRNA was maximally expressed in postischemic brain at 12 hours and was not present at 4 days (n = 3 per time point). iNOS mRNA was not observed in the contralateral cerebral cortex. iNOS enzymatic activity developed in the postischemic brain between 12 and 24 hours (P < .05) and subsided at 4 days (n = 4 to 8 per time point). iNOS immunoreactivity in the ischemic region was restricted to the wall of capillaries and of larger blood vessels at 12 to 24 hours. In regions of early necrosis, inflammatory cells were iNOS positive. Treatment with the iNOS inhibitor aminoguanidine (n = 5; 100 mg/kg IP, BID for 4 days), starting 6 hours after ischemia, reduced infarct size in neocortex by 36 +/- 7% in comparison with vehicle-treated controls (n = 5) (P < .05).nnnCONCLUSIONSnTransient focal ischemia leads to iNOS expression in postischemic brain. However, the spatial and temporal patterns of expression differ from those occurring in permanent ischemia: iNOS is induced earlier and predominantly in vascular cells rather than in neutrophils. Thus, the temporal profile and localization of postischemic iNOS expression depend on the nature of the ischemic insult. The finding that aminoguanidine reduces infarct size adds further support to the hypothesis that postischemic iNOS expression contributes to ischemic brain damage.

Simultaneous Blood Oxygenation Level-Dependent and Cerebral Blood Flow Functional Magnetic Resonance Imaging during Forepaw Stimulation in the Rat

The blood oxygenation level-dependent (BOLD) contrast mechanism can be modeled as a complex interplay between CBF, cerebral blood volume (CBV), and CMRO2. Positive BOLD signal changes are presumably caused by CBF changes in excess of increases in CMRO2. Because this uncoupling between CBF and CMRO2 may not always be present, the magnitude of BOLD changes may not be a good index of CBF changes. In this study, the relation between BOLD and CBF was investigated further. Continuous arterial spin labeling was combined with a single-shot, multislice echo-planar imaging to enable simultaneous measurements of BOLD and CBF changes in a well-established model of functional brain activation, the electrical forepaw stimulation of a-chloralose-anesthetized rats. The paradigm consisted of two 18- to 30-second stimulation periods separated by a 1-minute resting interval. Stimulation parameters were optimized by laser Doppler flowmetry. For the same cross-correlation threshold, the BOLD and CBF active maps were centered within the size of one pixel (470 µm). However, the BOLD map was significantly larger than the CBF map. Measurements taken from 15 rats at 9.4 T using a 10-millisecond echo-time showed 3.7 ± 1.7% BOLD and 125.67 ± 81.7% CBF increases in the contralateral somatosensory cortex during the first stimulation, and 2.6 ± 1.2% BOLD and 79.3 ± 43.6% CBF increases during the second stimulation. The correlation coefficient between BOLD and CBF changes was 0.89. The overall temporal correlation coefficient between BOLD and CBF time-courses was 0.97. These results show that under the experimental conditions of the current study, the BOLD signal changes follow the changes in CBF.

Nitric oxide donors increase blood flow and reduce brain damage in focal ischemia : evidence that nitric oxide is beneficial in the early stages of cerebral ischemia

We studied whether administration of nitric oxide (NO) donors reduces the ischemic damage resulting from middle cerebral artery (MCA) occlusion in spontaneously hypertensive rats (SHRs). In halothane-anesthetized and ventilated SHRs, the MCA was occluded. CBF was monitored using a laser-Doppler flowmeter. Three to five minutes after MCA occlusion, the NO donors sodium nitroprusside (SNP; 3 mg/kg/h) or 3-morpholino-sydnonimine (SIN 1; 1.5–6 mg/kg/h) were administered into the carotid artery for 60 min. As a control, the effect of papaverine (3.6 mg/kg/h), a vasodilator that acts independently of NO, was also studied. The hypotension evoked by these agents was counteracted by intravenous infusion of phenylephrine. At the end of the infusion, rats were allowed to recover. Stroke size was determined 24 h later in thionin-stained sections. In sham occluded rats, SNP (n = 5), SIN 1 (n = 5), and papaverine (n = 5) produced comparable increases in CBF (p > 0.05 from vehicle). After MCA occlusion, SNP (n = 5) and SIN 1 (n = 5), but not papaverine (n = 5), enhanced the recovery of CBF (p < 0.05 from vehicle) and reduced the size of the infarct by 28 ± 12 and 32 ± 7%, respectively (mean ± SD; p < 0.05 from vehicle). To determine whether NO donors could act by inhibiting platelet aggregation, we studied the effect of SNP on collagen-induced platelet aggregation. Intracarotid administration of SNP (3 mg/kg/h for 60 min) did not affect platelet aggregation to collagen, suggesting that the protective effect of NO donors was not due to inhibition of platelet function. We conclude that NO donors increase CBF to the ischemic territory and reduce the tissue damage resulting from focal ischemia. The protective effect may result from an increase in CBF to the ischemic territory, probably the ischemic penumbra. These findings suggest that NO donors may represent a new therapeutic strategy for the management of acute stroke.