Mary P. Stenzel-Poore

Abnormal adaptations to stress and impaired cardiovascular function in mice lacking corticotropin-releasing hormone receptor-2

The actions of corticotropin-releasing hormone (Crh), a mediator of endocrine and behavioural responses to stress, and the related hormone urocortin (Ucn) are coordinated by two receptors, Crhr1 (encoded by Crhr) and Crhr2 (refs 4,5). These receptors may exhibit distinct functions due to unique tissue distribution and pharmacology. Crhr-null mice have defined central functions for Crhr1 in anxiety and neuroendocrine stress responses. Here we generate Crhr2−/− mice and show that Crhr2 supplies regulatory features to the hypothalamic-pituitary-adrenal axis (HPA) stress response. Although initiation of the stress response appears to be normal, Crhr2−/− mice show early termination of adrenocorticotropic hormone (Acth) release, suggesting that Crhr2 is involved in maintaining HPA drive. Crhr2 also appears to modify the recovery phase of the HPA response, as corticosterone levels remain elevated 90 minutes after stress in Crhr2−/− mice. In addition, stress-coping behaviours associated with dearousal are reduced in Crhr2–/– mice. We also demonstrate that Crhr2 is essential for sustained feeding suppression (hypophagia) induced by Ucn. Feeding is initially suppressed in Crhr2−/− mice following Ucn, but Crhr2−/− mice recover more rapidly and completely than do wild-type mice. In addition to central nervous system effects, we found that, in contrast to wild-type mice, Crhr2−/− mice fail to show the enhanced cardiac performance or reduced blood pressure associated with systemic Ucn, suggesting that Crhr2 mediates these peripheral haemodynamic effects. Moreover, Crhr2−/− mice have elevated basal blood pressure, demonstrating that Crhr2 participates in cardiovascular homeostasis. Our results identify specific responses in the brain and periphery that involve Crhr2.

Mechanisms of ischemic brain damage

In the United States stroke is the third leading cause of death and the leading cause of disability. Brain injury following stroke results from the complex interplay of multiple pathways including excitotoxicity, acidotoxicity, ionic imbalance, peri-infarct depolarization, oxidative and nitrative stress, inflammation and apoptosis. There are very few treatments for stroke and the development of new treatments requires a comprehensive understanding of the diverse mechanisms of ischemic brain damage that are responsible for neuronal death. Here, we discuss the underlying pathophysiology of this devastating disease and reveal the intertwined pathways that are the target of therapeutic intervention.

Effect of ischaemic preconditioning on genomic response to cerebral ischaemia: similarity to neuroprotective strategies in hibernation and hypoxia-tolerant states.

BACKGROUND Molecular mechanisms of neuroprotection that lead to ischaemic tolerance are incompletely understood. Identification of genes involved in the process would provide insight into cell survival and therapeutic approaches for stroke. We developed a mouse model of neuroprotection in stroke and did gene expression profiling to identify potential neuroprotective genes and their associated pathways. METHODS Eight mice per condition were subjected to occlusion of the middle cerebral artery for 15 min (preconditioning), 60 min (injurious ischaemia), or preconditioning followed 72 h later by injurious ischaemia. RNA was extracted from the cortical regions of the ischaemic and non-ischaemic hemispheres. Three pools per condition were generated, and RNA was hybridised to oligonucleotide microarrays for comparison of ischaemic and non-ischaemic hemispheres. Real-time PCR and western blots were used to validate results. Follow-up experiments were done to address the biological relevance of findings. FINDINGS Microarray analysis revealed changes in gene expression with little overlap among the conditions of injurious ischaemia, ischaemic preconditioning, or both. Injurious ischaemia induced upregulation of gene expression; 49 (86%) of 57 genes regulated showed increased expression in the ischaemic hemisphere. By contrast, preconditioning followed by injurious ischaemia resulted in pronounced downregulation; 47 (77%) of 61 regulated genes showed lower expression. Preconditioning resulted in transcriptional changes involved in suppression of metabolic pathways and immune responses, reduction of ion-channel activity, and decreased blood coagulation. INTERPRETATION Preconditioning reprogrammes the response to ischaemic injury. Similar changes reported by others support an evolutionarily conserved endogenous response to decreased blood flow and oxygen limitation such as seen during hibernation.

Endotoxin Preconditioning Prevents Cellular Inflammatory Response During Ischemic Neuroprotection in Mice

Background and Purpose— Tolerance to ischemic brain injury is induced by several preconditioning stimuli, including lipopolysaccharide (LPS). A small dose of LPS given systemically confers ischemic protection in the brain, a process that appears to involve activation of an inflammatory response before ischemia. We postulated that LPS preconditioning modulates the cellular inflammatory response after cerebral ischemia, resulting in neuroprotection. Methods— Mice were treated with LPS (0.2 mg/kg) 48 hours before ischemia induced by transient middle cerebral artery occlusion (MCAO). The infarct was measured by 2,3,5-triphenyltetrazolium chloride staining. Microglia/macrophage responses after MCAO were assessed by immunofluorescence and flow cytometry. The effect of MCAO on white blood cells in the brain and peripheral circulation was measured by flow cytometry 48 hours after MCAO. Results— LPS preconditioning induced significant neuroprotection against MCAO. Administration of low-dose LPS before MCAO prevented the cellular inflammatory response in the brain and blood. Specifically, LPS preconditioning suppressed neutrophil infiltration into the brain and microglia/macrophage activation in the ischemic hemisphere, which was paralleled by suppressed monocyte activation in the peripheral blood. Conclusions— LPS preconditioning induces neuroprotection against ischemic brain injury in a mouse model of stroke. LPS preconditioning suppresses the cellular inflammatory response to ischemia in the brain and circulation. Diminished activation of cellular inflammatory responses that ordinarily exacerbate ischemic injury may contribute to neuroprotection induced by LPS preconditioning.

Toll-like receptor signaling in endogenous neuroprotection and stroke

Stroke and other cerebral vascular diseases are a leading cause of morbidity and mortality in the United States. Despite intensive research to identify interventions that lessen cerebrovascular injury, no major therapies exist. Development of stroke prophylaxis involves an understanding of the mechanisms of damage following cerebral ischemia, and elucidation of the endogenous mechanisms that combat further brain injury. Toll-like receptors (TLRs) are critical components of the innate immune system that have been shown recently to mediate ischemic injury. Paradoxically, TLR ligands administered systemically induce a state of tolerance to subsequent ischemic injury. Herein we suggest that stimulation of TLRs prior to ischemia reprograms TLR signaling that occurs following ischemic injury. Such reprogramming leads to suppressed expression of pro-inflammatory molecules and enhanced expression of numerous anti-inflammatory mediators that collectively confer robust neuroprotection. Our findings indicate that numerous preconditioning stimuli lead to TLR activation, an event that occurs prior to ischemia and ultimately leads to TLR reprogramming. Thus genomic reprogramming of TLR signaling may be a unifying principle of tolerance to cerebral ischemia.

Preconditioning Reprograms the Response to Ischemic Injury and Primes the Emergence of Unique Endogenous Neuroprotective Phenotypes A Speculative Synthesis

Ischemic tolerance in the brain, in which sub-threshold insults increase resistance to subsequent injurious ischemia, is a powerful adaptive defense that involves an endogenous program of neuroprotection. Emerging evidence from genomic studies suggests diverse stimuli that trigger preconditioning achieve neuroprotection through a common process which depends on a fundamental reprogramming of the response to injury. Such reprogramming of the genomic response to injury leads to the induction of novel neuroprotective pathways not ordinarily found in the setting of ischemia. Genomic studies also indicate that the nature of the preconditioning stimulus (eg, brief ischemia or endotoxin [lipopolysaccharide]) dictates the phenotype of neuroprotection, a phenotype that parallels protective adaptations also found in certain physiological conditions where the preconditioning stimulus exists at levels that can induce injury. The idea that preconditioning leads to a fundamental reprogramming event that confers neuroprotection is a novel and important concept in the field of ischemic tolerance. Moreover, the view that distinct preconditioning stimuli confer neuroprotection via effectors that differ according to the nature of the preconditioning stimulus offers promise that multiple, nonoverlapping pathways may be discovered as novel neuroprotective therapies.

Effects of transgenic overproduction of CRH on anxiety-like behaviour

Central administration of corticotropin‐releasing hormone increases anxiety‐like behaviour and arousal in rodents, and increased anxiety‐like behaviour has been shown in mice overproducing corticotropin‐releasing hormone on an elevated plus maze and in a dark–light emergence task. However, evidence is accumulating that measures obtained from different anxiety tasks may reflect different aspects of anxiety‐like behaviour in animals. We therefore tested mice overproducing corticotropin‐releasing hormone in a battery of paradigms, studying spontaneous behaviour after a mild stressor, tasks of innate anxiety‐like behaviour (light–dark box), lick suppression (Vogel conflict), conditioned fear, and forced swimming. Exploratory behaviour was studied in a 16‐hole board task. Furthermore, pain threshold, water intake, locomotor activity and sensorimotor learning/co‐ordination were tested to control for confounding factors. In line with previous findings, increased anxiety‐like behaviour of transgenic mice was observed in the light–dark box paradigm. However, no differences were seen in the conflict paradigm. Conditioned fear was decreased 1 h but not 24 h after conditioning in transgenic mice, and immobility was increased in forced swimming in corticotropin‐releasing hormone overexpressors. Locomotor activity in a novel open field and on the hole board was reduced in transgenics. Exploratory behaviour (hole pokes) was reduced during initial exploration of an unfamiliar hole board. Moreover, sensorimotor performance on a rotorod was impaired, and water intake was reduced in corticotropin‐releasing hormone overproducing mice, while no changes were seen in nociception. No differences in locomotor activity were seen in a second group of mice, tested in a familiar open field. When these animals were challenged with diazepam, transgenic mice were less susceptible to the sedative effects of the drug on locomotor activity. These data suggest that corticotropin‐releasing hormone overproduction leads to specific effects in a subset of anxiety paradigms, and that these transgenic mice suffer from a motor deficit in addition to altered anxiety‐like behaviour/arousal.

Systemic lipopolysaccharide protects the brain from ischemic injury by reprogramming the response of the brain to stroke: a critical role for IRF3.

Lipopolysaccharide (LPS) preconditioning provides neuroprotection against subsequent cerebral ischemic injury through activation of its receptor, Toll-like receptor 4 (TLR4). Paradoxically, TLR activation by endogenous ligands after ischemia worsens stroke damage. Here, we define a novel, protective role for TLRs after ischemia in the context of LPS preconditioning. Microarray analysis of brains collected 24 h after stroke revealed a unique set of upregulated genes in LPS-pretreated animals. Promoter analysis of the unique gene set identified an overrepresentation of type I interferon (IFN)-associated transcriptional regulatory elements. This finding suggested the presence of type I IFNs or interferon regulatory factors (IRFs), which upregulate interferon-stimulated genes. Upregulation of IFNβ was confirmed by real-time reverse transcription-PCR. Direct administration of IFNβ intracerebroventricularly at the time of stroke was sufficient for neuroprotection. TLR4 can induce both IFNβ and interferon-stimulated genes through its adapter molecule Toll/interleukin receptor domain-containing adaptor-inducing IFNβ (TRIF) and the IRF3 transcription factor. We show in oxygen glucose deprivation of cortical neurons, an in vitro model of stroke, that activation of TRIF after stroke reduces neuronal death. Furthermore, mice lacking IRF3 were not protected by LPS preconditioning in our in vivo model. Our studies constitute the first demonstration of the neuroprotective capacity of TRIF/IRF3 signaling and suggest that interferon-stimulated genes, whether induced by IFNβ or by enhanced TLR signaling to IRF3, are a potent means of protecting the brain against ischemic damage.

The use of flow cytometry to evaluate temporal changes in inflammatory cells following focal cerebral ischemia in mice.

Recent studies indicate that inflammation following cerebral ischemia contributes to neuronal damage. The local activation of resident cells and efficient recruitment of leukocytes into the central nervous system are critical steps in this inflammatory process. Here we describe studies using flow cytometry to examine the temporal pattern of inflammatory cell activation and infiltration following transient middle cerebral artery occlusion (MCAO) in mice. We found an increase in activated microglia/macrophages as early as 18 h post occlusion, which peaked at 48 h and remained abundant at 96 h post occlusion. Neutrophils were significantly increased by 48 h and remained elevated at 96 h post occlusion. T lymphocytes were increased relatively late (72 and 96 h) post occlusion. The flow cytometry data correlate well both quantitatively and qualitatively with immunohistochemistry analysis performed on the same mice. The present study demonstrates the power of flow cytometry in analyzing the inflammatory process following cerebral ischemia and offers temporal information on the cellular changes in mice following transient MCAO.

Lack of Interleukin-6 Expression Is Not Protective Against Focal Central Nervous System Ischemia

BACKGROUND AND PURPOSE Interleukin-6 (IL-6) appears to be involved in the inflammatory response associated with central nervous system (CNS) ischemia. Although IL-6 levels increase after stroke, it is not known whether IL-6 directly influences CNS ischemic injury. In this study, we used a focal reversible stroke model to investigate whether mice lacking IL-6 were protected against acute ischemic injury. METHODS We bred IL-6-deficient C57 black mice (I-129 IL-6 KO back-crossed with C57), including homozygous knockouts (IL-6 -/-), heterozygous littermates (IL-6 +/-), and normal littermates (IL-6 +/+). The status of all animals was confirmed by DNA sampling and polymerase chain reaction analysis. Reversible middle cerebral artery occlusion was produced by advancing a silicone-coated 8-0 filament into the internal carotid artery for 2 hours (experiment 1) or 45 minutes (experiment 2). At 24 hours, animals were evaluated on a 28-point clinical scale, blood and cerebrospinal fluid were obtained, and the brains were evaluated for infarct volume and IL-6 mRNA levels. RESULTS In experiment 1 (severe ischemia), no differences were seen in lesion size or neurological function between the groups: lesion volume was IL-6 -/- (n=15), 57+/-13 mm(3); IL-6 +/- (n=15), 58+/-23 mm(3); and IL-6 +/+ (n=15), 58+/-18 mm(3) (P=NS). ELISA testing confirmed very low to absent levels of IL-6 in the serum and cerebrospinal fluid of knockout animals. Brain mRNA levels of the other proinflammatory cytokines, including tumor necrosis factor-alpha, IL-1beta, and IL-1 receptor antagonist, were 50% lower in IL-6-deficient ischemic animals than in normal animals. In experiment 2 (mild ischemia), no differences were seen in lesion size or neurological function between the groups: lesion volume was IL-6 -/- (n=10), 16+/-8 mm(3); IL-6 +/- (n=10), 14+/-4 mm(3); and IL-6 +/+ (n=10), 19+/-12 mm(3) (P=NS). CONCLUSIONS In this study, infarct size and neurological function at 24 hours were not different in animals deficient in IL-6 after transient CNS ischemia. This suggests that IL-6 does not have a direct influence on acute ischemic injury. Further study investigating the role of IL-6 on long-term recovery after stroke is in progress.