Rationale: The elderly experience profound systemic responses after stroke, which contribute to higher mortality and more severe long-term disability. Recent studies have revealed that stroke outcomes can be influenced by the composition of gut microbiome. However, the potential benefits of manipulating the gut microbiome after injury is unknown. Objective: To determine if restoring youthful gut microbiota after stroke aids in recovery in aged subjects, we altered the gut microbiome through young fecal transplant gavage in aged mice after experimental stroke. Further, the effect of direct enrichment of selective bacteria producing short-chain fatty acids (SCFAs) was tested as a more targeted and refined microbiome therapy. Methods and Results: Aged male mice (18–20 months) were subjected to ischemic stroke by middle cerebral artery occlusion. We performed fecal transplant gavage 3 days after middle cerebral artery occlusion using young donor biome (2–3 months) or aged biome (18–20 months). At day 14 after stroke, aged stroke mice receiving young fecal transplant gavage had less behavioral impairment, and reduced brain and gut inflammation. Based on data from microbial sequencing and metabolomics analysis demonstrating that young fecal transplants contained much higher SCFA levels and related bacterial strains, we selected 4 SCFA-producers ( Bifidobacterium longum , Clostridium symbiosum , Faecalibacterium prausnitzii , and Lactobacillus fermentum ) for transplantation. These SCFA-producers alleviated poststroke neurological deficits and inflammation, and elevated gut, brain and plasma SCFA concentrations in aged stroke mice. Conclusions: This is the first study suggesting that the poor stroke recovery in aged mice can be reversed via poststroke bacteriotherapy following the replenishment of youthful gut microbiome via modulation of immunologic, microbial, and metabolomic profiles in the host.

The peripheral immune system plays a critical role in aging and in the response to brain injury. Emerging data suggest inflammatory responses are exacerbated in older animals following ischemic stroke; however, our understanding of these age-related changes is poor. In this work, we demonstrate marked differences in the composition of circulating and infiltrating leukocytes recruited to the ischemic brain of old male mice after stroke compared to young male mice. Blood neutrophilia and neutrophil invasion into the brain were increased in aged animals. Relative to infiltrating monocyte populations, brain-invading neutrophils had reduced phagocytic potential, and produced higher levels of reactive oxygen species and extracellular matrix-degrading enzymes (i.e., MMP-9), which were further exacerbated with age. Hemorrhagic transformation was more pronounced in aged versus young mice relative to infarct size. High numbers of myeloperoxidase-positive neutrophils were found in postmortem human brain samples of old (> 71 years) acute ischemic stroke subjects compared to non-ischemic controls. Many of these neutrophils were found in the brain parenchyma. A large proportion of these neutrophils expressed MMP-9 and positively correlated with hemorrhage and hyperemia. MMP-9 expression and hemorrhagic transformation after stroke increased with age. These changes in the myeloid response to stroke with age led us to hypothesize that the bone marrow response to stroke is altered with age, which could be important for the development of effective therapies targeting the immune response. We generated heterochronic bone marrow chimeras as a tool to determine the contribution of peripheral immune senescence to age- and stroke-induced inflammation. Old hosts that received young bone marrow (i.e., Young → Old) had attenuation of age-related reductions in bFGF and VEGF and showed improved locomotor activity and gait dynamics compared to isochronic (Old → Old) controls. Microglia in young heterochronic mice (Old → Young) developed a senescent-like phenotype. After stroke, aged animals reconstituted with young marrow had reduced behavioral deficits compared to isochronic controls, and had significantly fewer brain-infiltrating neutrophils. Increased rates of hemorrhagic transformation were seen in young mice reconstituted with aged bone marrow. This work suggests that age alters the immunological response to stroke, and that this can be reversed by manipulation of the peripheral immune cells in the bone marrow.

Recent studies have highlighted the crucial role of microglia (MG) and their interactions with the gut microbiome in post-stroke neuroinflammation. The activation of immunoregulatory pathways, including the aryl hydrocarbon receptor (AHR) pathway, is influenced by a dynamic balance of ligands derived from both the host and microbiota. This study aimed to investigate the association between stroke-induced dysbiosis and the resultant imbalance in AHR ligand sources (loss of microbiota-derived [indole-based] and increase of host-derived [kynurenine-based]) after stroke. Microbiota-derived AHR ligands decreased in human plasma and remained low for days following an ischemic stroke highlighting the translational significance. Transient-middle-cerebral-artery-occlusion was performed in aged wild-type and germ-free male mice. MG-AHR expression and activity increased in both in vivo and ex vivo stroke models. Germ-free mice showed altered neuroinflammation and antigen presentation while aged mice showed reduced infarct volume and neurological deficits following treatment with microbiota-derived AHR ligands after stroke. Restoring a balanced pool of host- and microbiota-derived AHR ligands may be beneficial after stroke and may represent a therapeutic target.

Inter-α inhibitor proteins (IAIPs) are a family of endogenous plasma and extracellular matrix molecules. IAIPs suppress proinflammatory cytokines, limit excess complement activation, and bind extracellular histones to form IAIP-histone complexes, leading to neutralization of histone-associated cytotoxicity in models of sepsis. Many of these detrimental processes also play critical roles in the pathophysiology of ischemic stroke. In this study, we first assessed the clinical relevance of IAIPs in stroke and then tested the therapeutic efficacy of exogenous IAIPs in several experimental stroke models. IAIP levels were reduced in both ischemic stroke patients and in mice subjected to experimental ischemic stroke when compared with controls. Post-stroke administration of IAIP significantly improved stroke outcomes across multiple stroke models, even when given 6 hours after stroke onset. Importantly, the beneficial effects of delayed IAIP treatment were observed in both young and aged mice. Using targeted gene expression analysis, we identified a receptor for complement activation, C5aR1, that was highly suppressed in both the blood and brain of IAIP-treated animals. Subsequent experiments using C5aR1-knockout mice demonstrated that the beneficial effects of IAIPs are mediated in part by C5aR1. These results indicate that IAIP is a potential therapeutic candidate for the treatment of ischemic stroke.

The gut is a major source of bacteria and antigens that contribute to neuroinflammation after brain injury. Colonic epithelial cells (ECs) are responsible for secreting major cellular components of the innate defense system, including antimicrobial proteins (AMP) and mucins. These cells serve as a critical regulator of gut barrier function and maintain host-microbe homeostasis. In this study, we determined post-stroke host defense responses at the colonic epithelial surface in mice. We then tested if the enhancement of these epithelial protective mechanisms is beneficial in young and aged mice after stroke. AMPs were significantly increased in the colonic ECs of young males, but not in young females after experimental stroke. In contrast, mucin-related genes were enhanced in young females and contributed to mucus formation that maintains the distance between the host and gut bacteria. Bacterial community profiling was done using universal amplification of 16S rRNA gene sequences. The sex-specific colonic epithelial defense responses after stroke in young females were reversed with ovariectomy and led to a shift from a predominately mucin response to the enhanced AMP expression seen in males after stroke. Estradiol (E₂) replacement prior to stroke in aged females increased mucin gene expression in the colonic ECs. Interestingly, we found that E₂ treatment reduced stroke-associated neuronal hyperactivity in the insular cortex, a brain region that interacts with visceral organs such as the gut, in parallel to an increase in the composition of Lactobacillus and Bifidobacterium in the gut microbiota. This is the first study demonstrating sex differences in host defense mechanisms in the gut after brain injury.

Background and Purpose: Stroke induces the expression of several long noncoding RNAs in the brain. However, their functional significance in poststroke outcome is poorly understood. We recently observed that a brain-specific long noncoding RNA called Fos downstream transcript (FosDT) is induced rapidly in the rodent brain following focal ischemia. Using FosDT knockout rats, we presently evaluated the role of FosDT in poststroke brain damage. Methods: FosDT knockout rats were generated using CRISPR-Cas9 genome editing on a Sprague-Dawley background. Male and female FosDT ^−/− and FosDT ^+/+ cohorts were subjected to transient middle cerebral artery occlusion. Postischemic sensorimotor deficits were evaluated between days 1 and 7 and lesion volume on day 7 of reperfusion. The developmental expression profile of FosDT was determined with real-time polymerase chain reaction and mechanistic implications of FosDT in the ischemic brain were conducted with RNA-sequencing analysis and immunostaining of pathological markers. Results: FosDT expression is developmentally regulated, with the adult cerebral cortex showing significantly higher FosDT expression than neonates. FosDT ^−/− rats did not show any anomalies in growth and development, fertility, brain cytoarchitecture, and cerebral vasculature. However, when subjected to transient focal ischemia, FosDT ^−/− rats of both sexes showed enhanced sensorimotor recovery and reduced brain damage. RNA-sequencing analysis showed that improved poststroke functional outcome in FosDT ^−/− rats is partially associated with curtailed induction of inflammatory genes, reduced apoptosis, mitochondrial dysfunction, and oxidative stress. Conclusions: Our study shows that FosDT is developmentally dispensable, mechanistically important, and a functionally promising target to reduce ischemic brain damage and facilitate neurological recovery.