Fudong Liu

TTC, fluoro-Jade B and NeuN staining confirm evolving phases of infarction induced by middle cerebral artery occlusion.

Considerable debate exists in the literature on how best to measure infarct damage and at what point after middle cerebral artery occlusion (MCAO) infarct is histologically complete. As many researchers are focusing on more chronic endpoints in neuroprotection studies it is important to evaluate histological damage at later time points to ensure that standard methods of tissue injury measurement are accurate. To compare tissue viability at both acute and sub-acute time points, we used 2,3,5-triphenyltetrazolium chloride (TTC), Fluoro-Jade B, and NeuN staining to examine the evolving phases of infarction induced by a 90-min MCAO in mice. Stroke outcomes were examined at 1.5h, 6h, 12h, 24h, 3d, and 7d after MCAO. There was a time-dependent increase in infarct volume from 1.5h to 24h in the cortex, followed by a plateau from 24h to 7d after stroke. Striatal infarcts were complete by 12h. Fluoro-Jade B staining peaked at 24h and was minimal by 7d. Our results indicated that histological damage as measured by TTC and Fluoro-Jade B reaches its peak by 24h after stroke in a reperfusion model of MCAO in mice. TTC staining can be accurately performed as late as 7d after stroke. Neurological deficits do not correlate with the structural lesion but rather transient impairment of function. As the infarct is complete by 24h and even earlier in the striatum, even the most efficacious neuroprotective therapies are unlikely to show any efficacy if given after this point.

Disruption of the Axon Initial Segment Cytoskeleton Is a New Mechanism for Neuronal Injury

Many factors contribute to nervous system dysfunction and failure to regenerate after injury or disease. Here, we describe a previously unrecognized mechanism for nervous system injury. We show that neuronal injury causes rapid, irreversible, and preferential proteolysis of the axon initial segment (AIS) cytoskeleton independently of cell death or axon degeneration, leading to loss of both ion channel clusters and neuronal polarity. Furthermore, we show this is caused by proteolysis of the AIS cytoskeletal proteins ankyrinG and βIV spectrin by the calcium-dependent cysteine protease calpain. Importantly, calpain inhibition is sufficient to preserve the molecular organization of the AIS both in vitro and in vivo. We conclude that loss of AIS ion channel clusters and neuronal polarity are important contributors to neuronal dysfunction after injury, and that strategies to facilitate recovery must preserve or repair the AIS cytoskeleton.

Changes in Experimental Stroke Outcome across the Life Span

Acute ischemic stroke is a leading cause of mortality and disability in the elderly. Age is the most important nonmodifiable risk factor for stroke, yet many preclinical models continue to examine only young male animals. It remains unclear how experimental stroke outcomes change with aging and with biologic sex. If sex differences are present, it is not known whether these reflect an intrinsic differing sensitivity to stroke or are secondary to the loss of estrogen with aging. We subjected both young and aging mice of both sexes to middle cerebral artery occlusion (MCAO). Young female mice had smaller strokes compared with age-matched males, an effect that was reversed by ovariectomy. Stroke damage increased with aging in female mice, whereas male mice had decreased damage after MCAO. Blood–brain barrier (BBB) permeability changes are correlated with infarct size. However, aging mice had significantly less edema formation, an effect that was independent of sex and histologic damage. Differences in the cellular response to stroke occur across the life span in both male and female mice. These differences need to be considered when developing relevant therapies for stroke patients, the majority of whom are elderly.

miR-23a regulation of X-linked inhibitor of apoptosis (XIAP) contributes to sex differences in the response to cerebral ischemia

It is increasingly recognized that the mechanisms underlying ischemic cell death are sexually dimorphic. Stroke-induced cell death in males is initiated by the mitochondrial release of apoptosis-inducing factor, resulting in caspase-independent cell death. In contrast, ischemic cell death in females is primarily triggered by mitochondrial cytochrome c release with subsequent caspase activation. Because X-linked inhibitor of apoptosis (XIAP) is the primary endogenous inhibitor of caspases, its regulation may play a unique role in the response to injury in females. XIAP mRNA levels were higher in females at baseline. Stroke induced a significant decrease in XIAP mRNA in females, whereas no changes were seen in the male brain. However, XIAP protein levels were decreased in both sexes after stroke. MicroRNAs (miRNAs) predominantly induce translational repression and are emerging as a major regulators of mRNA and subsequent protein expression after ischemia. The miRNA miR-23a was predicted to bind XIAP mRNA. miR-23a directly bound the 3′ UTR of XIAP, and miR-23a inhibition led to an increase in XIAP mRNA in vitro, demonstrating that XIAP is a previously uncharacterized target for miR-23a. miR-23a levels differed in male and female ischemic brains, providing evidence for sex-specific miRNA expression in stroke. Embelin, a small-molecule inhibitor of XIAP, decreased the interaction between XIAP and caspase-3 and led to enhanced caspase activity. Embelin treatment significantly exacerbated stroke-induced injury in females but had no effect in males, demonstrating that XIAP is an important mediator of sex-specific responses after stroke.

Middle Cerebral Artery Occlusion Model in Rodents: Methods and Potential Pitfalls

A variety of animal models have been developed for modeling ischemic stroke. The middle cerebral artery occlusion (MCAO) model has been utilized extensively, especially in rodents. While the MCAO model provides stroke researchers with an excellent platform to investigate the disease, controversial or even paradoxical results are occasionally seen in the literature utilizing this model. Various factors exert important effects on the outcome in this stroke model, including the age and sex of the animal examined. This paper discusses emerging information on the effects of age and sex on ischemic outcomes after MCAO, with an emphasis on mouse models of stroke.

Sex Differences in Caspase Activation After Stroke

BACKGROUND AND PURPOSE Over the past 5 years, experimental data have emerged that ischemia-induced cell death pathways may differ in males and females. Cell death in males is triggered by poly(ADP-ribose) polymerase activation and nuclear translocation of apoptosis-inducing factor. We have previously shown that interference with this pathway benefits males but not females after an experimental stroke. In contrast, caspase activation may be the major pathway activated after ischemic injury in females. The aim of this study is to examine whether sex differences exist in caspase activation in adult mice after stroke and to determine if interference with stroke-induced caspase activation preferentially protects females. METHODS Focal stroke was induced by reversible middle cerebral artery occlusion (90 minutes) in young and aging C57BL/6 mice of both sexes. The pan-caspase inhibitor, quinoline-Val-Asp(Ome)-CH2-O-phenoxy was administered at reperfusion. Histological outcomes were assessed 48 hours after middle cerebral artery occlusion. Separate cohorts were used for protein analysis of key cell death proteins, including caspase-3, caspase-8, cytochrome C, and apoptosis-inducing factor. RESULTS Drug-treated female mice had significantly decreased infarct volumes and improved neurological deficits after stroke compared to vehicle-treated mice. Quinoline-Val-Asp(Ome)-CH2-O-phenoxy administration had no effect in male mice. The expression of cytochrome C and nuclear caspase-8 levels were increased in females after stroke. CONCLUSIONS Female mice had an early release of cytochrome C and enhanced caspase activation after middle cerebral artery occlusion. Caspase inhibition benefited females but not males. Sex differences exist in both the response to ischemic injury and the efficacy of neuroprotective agents.

Sex Differences in the Response to Activation of the Poly (ADP-ribose) Polymerase Pathway after Experimental Stroke

It is increasingly recognized that histological and functional outcomes after stroke are shaped by biologic sex. Emerging data suggests that ischemic cell death pathways are sexually dimorphic (Hurn, P., Vannucci, S., Hagberg, H. (2005) Adult or perinatal brain injury: does sex matter?. Stroke 36, 193-195 ; Lang, J.T., McCullough, L.D. (2008) Pathways to ischemic neuronal cell death: are sex differences relevant?. J. Transl. Med. 6). Reducing neuronal nitric oxide (NO) or poly-ADP-ribose polymerase (PARP1) activation protects only the male brain (Hagberg, H., et al. PARP-1 gene disruption in mice preferentially protects males from perinatal brain injury. J. Neurochem. 90, 1068-1075 (2004)), and paradoxically enhances ischemic injury in females (McCullough, L.D., et al. Ischemic nitric oxide and poly (ADP-ribose) polymerase-1 in cerebral ischemia: male toxicity, female protection. J. Cereb. Blood Flow Metab. 25, 502-512 (2005)). In this study, we examined downstream mediators of NO/PARP activation to investigate possible mediators of ischemic sexual dimorphism. Nuclear translocation of Apoptosis Inducing Factor (AIF) was equivalent in wild type males and females after stroke and was unaffected by estrogen exposure. Deletion of PARP1 led to a dramatic reduction in stroke-induced poly (ADP-ribose) polymerase (PAR) formation and AIF translocation in both sexes, yet ischemic damage was reduced only in males. Subsequent examination of AIF-deficient Harlequin mice demonstrated that male Harlequin mice had less PAR formation, reduced AIF translocation and less ischemic damage than male wild type mice. In contrast, female Harlequin mice had no neuroprotective effect of gene deletion despite robust reductions in PAR formation and AIF translocation. Although equivalent activation of this cell death pathway occurs in both sexes after ischemia, detrimental effects are only present in males. AIF translocation and PAR formation do not mediate ischemic injury in the female brain, therefore agents designed to reduce PARP1 activation are unlikely to benefit females.

Functional recovery in aging mice after experimental stroke

Aging is a non-modifiable risk factor for stroke. Since not all strokes can be prevented, a major emerging area of research is the development of effective strategies to enhance functional recovery after stroke. However, in the vast majority of pre-clinical stroke studies, the behavioral tests used to assess functional recovery have only been validated for use in young animals, or are designed for rats. Mice are increasingly utilized in stroke models but well validated behavioral tests designed for rats are not necessarily reproducible in mice. We examined a battery of behavioral tests to evaluate functional recovery in an aging murine model of stroke. We found that the vertical pole, hanging wire and open field can accurately assess acute behavioral impairments after stroke in both young and aging male mice, but animals recover rapidly on these tasks. The corner test can accurately and repeatedly differentiate stroke from sham animals up to 30 days post stroke and can be performed reliably in aging mice. Aging male mice had significantly worse behavioral impairment compared to young male mice in the first two weeks after stroke but eventually recovered to the same degree as young mice. In contrast, chronic infarct size, as measured by ipsilateral cerebral atrophy, was significantly lower in aging male mice compared to young male mice. Reactive gliosis, formation of glial scar, and an enhanced innate immune response was seen in the aging brain and may contribute to the delayed behavioral recovery seen in the aging animals.

Differential effects of aging and sex on stroke induced inflammation across the lifespan

Aging and biological sex are critical determinants of stroke outcome. Post-ischemic inflammatory response strongly contributes to the extent of ischemic brain injury, but how this response changes with age and sex is unknown. We subjected young (5-6 months), middle aged (14-15 months) and aged (20-22 months), C57BL/6 male and female mice to transient middle cerebral artery occlusion (MCAO) and found that a significant age by sex interaction influenced histological stroke outcomes. Acute functional outcomes were worse with aging. Neutrophils, inflammatory macrophages, macrophages, dendritic cells (DCs) and microglia significantly increased in the brain post MCAO. Cycling females had higher Gr1(-) non-inflammatory macrophages and lower T cells in the brain after stroke and these correlated with serum estradiol levels. Estrogen loss in acyclic aged female mice exacerbated stroke induced splenic contraction. Advanced age increased T cells, DCs and microglia at the site of injury, which may be responsible for the exacerbated behavioral deficits in the aged. We conclude that aging and sex have differential effects on the post stroke inflammatory milieu. Putative immunomodulatory therapies need to account for this heterogeneity.

Inflammatory responses in hypoxic ischemic encephalopathy

Inflammation plays a critical role in mediating brain injury induced by neonatal hypoxic ischemic encephalopathy (HIE). The mechanisms underlying inflammatory responses to ischemia may be shared by neonatal and adult brains; however, HIE exhibits a unique inflammation phenotype that results from the immaturity of the neonatal immune system. This review will discuss the current knowledge concerning systemic and local inflammatory responses in the acute and subacute stages of HIE. The key components of inflammation, including immune cells, adhesion molecules, cytokines, chemokines and oxidative stress, will be reviewed, and the differences between neonatal and adult inflammatory responses to cerebral ischemic injury will also be discussed.