Adaptation is one of physiology's fundamental tenets, operating not only at the level of species, as Darwin proposed, but also at the level of tissues, cells, molecules and, perhaps, genes. During recent years, stroke neurobiologists have advanced a considerable body of evidence supporting the hypothesis that, with experimental coaxing, the mammalian brain can adapt to injurious insults such as cerebral ischaemia to promote cell survival in the face of subsequent injury. Establishing this protective phenotype in response to stress depends on a coordinated response at the genomic, molecular, cellular and tissue levels. Here, I summarize our current understanding of how 'preconditioning' stimuli trigger a cerebroprotective state known as cerebral 'ischaemic tolerance'.

Programmed cell death (apoptosis) is a normal process in the developing nervous system. Recent data suggest that certain features seen in the process of programmed cell death may be favored in the developing versus the adult brain in response to different brain injuries. In a well characterized model of neonatal hypoxia-ischemia, we demonstrate marked but delayed cell death in which there is prominent DNA laddering, TUNEL-labeling, and nuclei with condensed chromatin. Caspase activation, which is required in many cases of apoptotic cell death, also followed a delayed time course after hypoxia-ischemia. Administration of boc-aspartyl(OMe)-fluoromethylketone, a pan-caspase inhibitor, was significantly neuroprotective when given by intracerebroventricular injection 3 h after cerebral hypoxia-ischemia. In addition, systemic injections of boc-aspartyl(OMe)-fluoromethylketone also given in a delayed fashion, resulted in significant neuroprotection. These findings suggest that caspase inhibitors may be able to provide benefit over a prolonged therapeutic window after hypoxic-ischemic events in the developing brain, a major contributor to static encephalopathy and cerebral palsy.

Results of recent studies reveal vascular and neuroprotective effects of matrix metalloproteinase-9 (MMP-9) inhibition and MMP-9 gene deletion in experimental stroke. However, the cellular source of MMP-9 produced in the ischemic brain and the mechanistic basis of MMP-9-mediated brain injury require elucidation. In the present study, we used MMP-9-/- mice and chimeric knockouts lacking either MMP-9 in leukocytes or in resident brain cells to test the hypothesis that MMP-9 released from leukocytes recruited to the brain during postischemic reperfusion contributes to this injury phenotype. We also tested the hypothesis that MMP-9 promotes leukocyte recruitment to the ischemic brain and thus is proinflammatory. The extent of blood-brain barrier (BBB) breakdown, the neurological deficit, and the volume of infarction resulting from transient focal stroke were abrogated to a similar extent in MMP-9-/- mice and in chimeras lacking leukocytic MMP-9 but not in chimeras with MMP-9-containing leukocytes. Zymography and Western blot analysis from these chimeras confirmed that the elevated MMP-9 expression in the brain at 24 h of reperfusion is derived largely from leukocytes. MMP-9-/- mice exhibited a reduction in leukocyte-endothelial adherence and a reduction in the number of neutrophils plugging capillaries and infiltrating the ischemic brain during reperfusion; microvessel immunopositivity for collagen IV was also preserved in these animals. These latter results document proinflammatory actions of MMP-9 in the ischemic brain. Overall, our findings implicate leukocytes, most likely neutrophils, as a key cellular source of MMP-9, which, in turn, promotes leukocyte recruitment, causes BBB breakdown secondary to microvascular basal lamina proteolysis, and ultimately contributes to neuronal injury after transient focal stroke.

Hypoxia‐inducible factor‐1 (HIF‐1) is a heterodimer composed of HIF‐1α and HIF‐1β protein subunits. This transcription factor is essential for the activation of hypoxia‐inducible genes like erythropoietin, some glucose transporters, the glycolytic enzymes, and vascular endothelial growth factor. Because HIF‐1 activation may promote cell survival in hypoxic tissues, we studied the effect of hypoxic preconditioning on HIF‐1 expression in neonatal rat brain. Hypoxic preconditioning (8% O2 for 3 hours), a treatment known to protect the newborn rat brain against hypoxic‐ischemic injury, markedly increased HIF‐1α and HIF‐1β expression. To support the role of HIF‐1 in protective preconditioning, we also studied the effect of two other known HIF‐1 inducers, cobalt chloride (CoCl2) and desferrioxamine (DFX), on HIF‐1 expression and neuroprotection in newborn brain. HIF‐1α and HIF‐1β protein levels were markedly increased after intraperitoneal injection of CoCl2 (60 mg/kg) and moderately increased after intraperitoneal injection of DFX (200 mg/kg) 1 to 3 hours after the injections. Preconditioning with CoCl2 or DFX 24 hours before hypoxia‐ischemia afforded 75 and 56% brain protection, respectively, compared with that in vehicle‐injected littermate controls. Thus, HIF‐1 activation could contribute to protective brain preconditioning, which could be used in high‐risk deliveries and other clinical situations. Ann Neurol 2000;48:285–296

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Sublethal periods of hypoxia or ischemia can induce adaptive mechanisms to protect against subsequent lethal ischemic insults in a process known as ischemic preconditioning. In the present study, we developed a murine model of cerebral preconditioning using several common strains of adult mice. Animals were exposed to sublethal hypoxia (11% oxygen for 2 h) 48 h prior to a 90 min period of transient focal middle cerebral artery occlusion, induced by an intraluminal filament; injury was assessed 24 h later by TTC staining. Infarct volume in hypoxia-preconditioned animals was reduced 46%, 58%, and 64% in C57Bl/6, 129SvEv, and Swiss-Webster ND4 mice relative to their respective untreated controls. This non-invasive murine model of ischemic tolerance should be useful for elucidating the molecular basis of this protection using transgenic and knockout mice.

Background and Purpose-Genetically engineered mice are used to study the role of single genes in cerebral ischemia, but inherent, strain-dependent differences in neuronal vulnerability may affect experimental end points. To examine this possibility, tissue injury resulting from focal ischemia and its relationship to cerebral hemodynamics were determined in 3 common mutant mouse strains. Methods-Permanent middle cerebral artery ligation was performed in male C57BL/6J, Balb/C, and 129X1/SvJ mice.Mean arterial blood pressure, blood gases, basal and postischemic cortical blood flow ([ 14 C]iodoantipyrine autoradiography and laser-Doppler flowmetry), posterior communicating artery patency, and infarct size were determined. Results-Basal cortical blood flow did not differ among strains. Ten minutes after middle cerebral artery ligation, relative red cell flow in the ischemic cortex was 6% to 7% of preischemic flow in every strain. Despite similar hemodynamics, cortical infarcts in Balb/C mice were 3-fold larger than those in 129X1/SvJ and C57BL/6J mice; infarct size in the latter 2 strains was not significantly different. The posterior communicating artery was either poorly developed or absent in Ͼ90% of the Balb/C and C57BL/6J but in Ͻ50% of the 129X1/SvJ mice. Conclusions-The extent of ischemic injury differed markedly between the 3 strains. The presence and patency of posterior communicating arteries, although variable among strains, did not affect preischemic or postischemic cortical blood flow or bear any relationship to ischemic injury. Therefore, intrinsic factors, other than hemodynamic variability, may contribute to the differences in ischemic vulnerability among strains. These findings underscore the importance of selecting genetically matched wild-type controls. (Stroke. 2000;31:2707-2714.)

OBJECTIVE Brief systemic hypoxia protects the rodent brain from subsequent ischemic injury, although the protection wanes within days. We hypothesized that the duration of ischemic tolerance could be extended from days to months by repeated intermittent hypoxia of varying magnitude and duration. METHODS Infarction volumes following a 60-min transient middle cerebral artery occlusion were determined in adult male mice 2 days through 8 wks after completion of a 2-week repetitive hypoxic preconditioning (RHP) protocol. Separate cohorts were studied for the protective effects of RHP on postischemic and cytokine-induced cerebrovascular inflammation, and for potential deleterious effects of the RHP stimulus itself. RESULTS RHP protection against transient focal stroke persisted for 8 weeks. Leukocyte adherence to cortical venules was attenuated in response to stroke, as well as following TNF-α administration, indicating that reductions in postischemic inflammation were not secondary to smaller infarct volumes. RHP reduced post-stroke leukocyte diapedesis concomitant with a long-lasting downregulation of endothelial adhesion molecule mRNAs, and also reduced postischemic blood-brain barrier permeability to endogenous IgG. RHP was without effect on hippocampal CA1 pyramidal cell viability, only transiently elevated hematocrit, and did not affect the magnitude of CBF during and after ischemia. INTERPRETATION Taken together, our findings reveal a novel form of epigenetic neurovascular plasticity characterized by a prominent anti-inflammatory phenotype that provides protection against stroke many weeks longer than previously established windows of preconditioning-induced tolerance. Translating these endogenous protective mechanisms into therapeutics could afford sustained periods of cerebroprotection in subpopulations of individuals at identified risk for stroke.