Sex differences in T cell immune responses, gut permeability and outcome after ischemic stroke in aged mice
Introduction
Stroke is a cerebrovascular disease that presents with sex differences in risk, prevalence and outcome (Benjamin et al., 2018, Bushnell et al., 2014). While the onset of stroke is earlier in men, the lifetime risk is higher in women due to the larger population of aged women (Ahnstedt et al., 2016, Towfighi et al., 2007). Women suffer from poor functional outcomes, are less likely to get discharged to home and account for higher number of stroke deaths (Ahnstedt et al., 2016, Glader et al., 2003, Go et al., 2014, Reeves et al., 2009). However, age, pre-stroke disability and stroke severity are major drivers of outcome and mortality after stroke in clinical populations (Lisabeth et al., 2015). This is evident in a recent meta-analysis study where sex differences in the mortality rate ratio were fully reversed after adjusting for age, pre-stroke function, stroke severity, as well as a history of atrial fibrillation (Phan et al., 2017). In unadjusted analysis, women were 35% more likely than men to be dead 1 year after the stroke, while in adjusted analysis mortality was higher in men (Phan et al., 2017). Sex differences in human stroke is complex; as the response to injury and stroke etiology vary with age. Differences cannot be fully explained by sex disparities in demographics, pre-stroke function and clinical factors (Lisabeth et al., 2015, Phan et al., 2019).
Sex differences in ischemic stroke are not limited to humans but also apparent in the experimental setting. Numerous studies have shown that young female animals are protected from ischemic stroke and demonstrate smaller brain injury than age-matched males (Alkayed et al., 1998, Hall et al., 1991, Manwani et al., 2013). These sex differences have mostly been attributed to sex hormones, as the protection is lost and brain injury is aggravated in ovariectomized and reproductively senescent middle-aged females (Alkayed et al., 2000, Dubal et al., 1998, Manwani et al., 2013, Simpkins et al., 1997). Aged animals of both sexes sustain smaller ischemic injury after stroke (young females still exhibit the smallest injury) yet aged animals have higher mortality and morbidity, a paradox that is also evident in humans (Agarwal et al., 2013, Liu et al., 2012, Manwani et al., 2013, Roussel et al., 2009). In prior studies, we found no significant differences in infarct volumes or mortality in aged males versus aged females (20–22 months), but this study was limited, as only acute endpoints were examined (24 h) (Manwani et al., 2013). Although stroke is a disease of the elderly, and despite these apparent sex differences, a limited number of studies on ischemic stroke have included aged animals of both sexes. The present study is the first that not only studies sex differences in aged animals, but incorporates chronic time-points of 15, 21 and 42 days after ischemic stroke, in addition to subacute time-points of 3 and 7 days.
Ischemic stroke induces a profound inflammatory response that is considered as an important pathological mechanism and contributor to stroke outcome (Chamorro et al., 2012). Neutrophils have been implicated in many detrimental processes in the acute phase of stroke, including capillary plugging, the no-reflow phenomenon, oxidative stress and hemorrhagic transformation (del Zoppo et al., 1991, Garcia et al., 1994, Jickling et al., 2015). Despite promising experimental data, early clinical studies failed to show benefit from neutrophil targeted therapies (Jickling et al., 2015). More recently, examination and targeting of other peripheral immune cells in ischemic stroke have been investigated (Jickling et al., 2015, Kleinschnitz et al., 2011, Liesz et al., 2011, Yilmaz et al., 2006). Yilmaz et al. demonstrated that CD4+ and CD8+ T cells contribute to the inflammatory response and brain injury associated with experimental stroke (Yilmaz et al., 2006). This was later supported by Liesz et al. showing infiltrating T cells were the principal mediators of delayed ischemic injury (Liesz et al., 2011). The delayed and prolonged dynamics of T cell brain infiltration provides a promising target for stroke therapy. It was recently demonstrated that aging is associated with an accumulation of resident effector memory CD8+ T cells in brain (Ritzel et al., 2016). However, T cell responses after stroke in aged animals have been less studied, especially in aged females (Ahnstedt and McCullough, 2019). Biological sex is an important factor to include in studies of immune responses as significant male-female differences exist in healthy and disease states throughout the lifespan (reviewed in Ahnstedt and McCullough (2019)). Therefore, the current study was aimed to investigate temporal and sex-specific T cell immune responses after ischemic stroke in mice of advanced age (>20 months) to more closely mimic an aging human population. In addition, neurological deficits, sensorimotor and cognitive function were evaluated in these animals. Ischemic stroke is increasingly recognized as a systemic disease with reported effects on spleen, heart and gut. As the gut hosts a large immune cell pool, in particular T cells, we determined peripheral effects on gut permeability, fecal microbiota diversity and short-chain fatty acid levels.
Section snippets
Animals
Aged (18–20 months) C57BL/6CR mice were purchased from the National Institute of Aging (NIA) and acclimatized in house for two months before use at 20–22 months of age. All animals had access to chow and water ad libitum. Animal procedures were performed in accordance with National Institutes of Health Guidelines for the care and use of laboratory animals and approved by the Animal Welfare Committee at the University of Texas Health Science Center at Houston, Texas (AWC-15-0140).
Middle cerebral artery occlusion model of ischemic stroke
Cerebral
Higher mortality and hemorrhagic transformation rates in aged male mice after ischemic stroke
Despite similar brain injury at day 3, a time-point when the infarct has fully evolved, (Fig. 1A), a significant sex-specific effect on survival probability was observed. Aged males had a significantly higher mortality compared to aged females up to 15 days post-stroke (Fig. 1B, Log-Rank Mantel Cox test p < 0.05). In 16 of the male mortality cases, 7 (44%) had hemorrhagic transformation with petechial bleedings (Fig. 1C), while none of the females that died had hemorrhagic transformation
Discussion
In this study, we report several novel findings after ischemic stroke in aged mice (Fig. 7). These include; (1) A higher mortality and significantly increased rates of hemorrhagic transformation in aged males compared to females despite equivalent infarct size at day 3 post-stroke; (2) Systemic effects of stroke were more pronounced in males, including enhanced gut permeability and long-lasting changes in fecal microbiota diversity; (3) Brain T cell immune responses were temporally distinct and
Conclusions
The present study reveals significant sex differences in central T cell responses as well as greater peripheral effects after ischemic stroke in aged mice. Our findings show that males have a more chronic neuroinflammatory response compared to females during the chronic phase of stroke, which may be associated with greater cognitive decline long-term after stroke.
Funding
This work was supported by the National Institute of Neurological Disorders and Stroke (NINDS) grant R01 NS108779, NS094543, NS103592, NS096493 (United States, to LDM), and the American Heart Association (AHA) Postdoctoral Fellowship #17POST33660010 (to HA).
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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2022, Neurobiology of DiseaseCitation Excerpt :Collectively the baseline microbiome diversity, immunity, and gut function in reproductively senescent females results in increased mortality and morbidity after stroke when compared to non-senescent females. In addition to susceptibility and incidence, there are sex differences in post stroke recovery, with aged males fairing worse than aged females as measured by mortality and hemorrhagic conversion two weeks after an induced stroke (Ahnstedt et al., 2020). These changes were associated with more persistent changes in gut integrity, microbiome composition, and inflammation in males.
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2022, Brain ResearchCitation Excerpt :Three studies analyzed post-stroke mortality (Spychala et al., 2018; El-Hakim et al., 2021; Ahnstedt et al., 2020), four the infarct volume (Park et al., 2020; Spychala et al., 2018; El-Hakim et al., 2021; Ahnstedt et al., 2020), five the neurological deficits following MCAO (Park et al., 2020; El-Hakim et al., 2021; Ahnstedt et al., 2020; Lee et al., 2020; Spychala et al., 2018). Gut permeability was assessed through dextran extravasation (El-Hakim et al., 2021; Ahnstedt et al., 2020; Crapser et al., 2016; Lee et al., 2020), histology and gene expression (El-Hakim et al., 2021; Blasco et al., 2020), and fecal Short-Chain Fatty Acids (SCFA) (Park et al., 2020; El-Hakim et al., 2021; Ahnstedt et al., 2020; Lee et al., 2020; Spychala et al., 2018). Most of the studies measured cytokines and other pro-inflammatory molecules in plasma (El-Hakim et al., 2021; Spychala et al., 2018; Jackson et al., 2019; Park et al., 2020; Blasco et al., 2020; Crapser et al., 2016), five evaluated changes in leukocytes population (Ahnstedt et al., 2020; Jackson et al., 2019; Blasco et al., 2020; Crapser et al., 2016; Lee et al., 2020).