Depletion of CD4 T cells provides therapeutic benefits in aged mice after ischemic stroke.

T-lymphocytes have a multifaceted role in ischemic stroke, but the majority of studies have been conducted in young mice, which may limit the translational value of these findings. Previous studies have shown that aging results in T cell dysfunction, leading to enhanced production of pro-inflammatory cytokines and chemokines, including interferon gamma (IFN-γ) and interferon-gamma-inducible protein (IP-10). This study assessed the role of T cells and pro-inflammatory factors on histologic and functional outcomes in an aged mouse model. Levels of IP-10 were measured in the brain and serum of young and aged male mice following middle cerebral artery occlusion (MCAo) or sham surgery. Additionally, IP-10 levels were evaluated in stroke patients. To directly determine the role of brain-infiltrating T cells after stroke, a separate cohort of aged male and female animals received either an anti-CD4 depletion antibody or IgG isotype control at 72 and 96 h following experimental stroke. Behavioral assessments were performed on day 7 post-MCAo. CD4 T cell depletion resulted in improved behavioral outcomes, despite the lack of differences in infarct size between the isotype control and anti-CD4 antibody treated stroke groups. Circulating IP-10 levels were increased in both humans and mice with age and stroke, and depletion of CD4 T cells led to a reduction in IFN-γ and IP-10 levels in mice. Since anti-CD4 treatment was administered three days after stroke onset, targeting this inflammatory pathway may be beneficial to aged stroke patients who present outside of the current time window for thrombolysis and thrombectomy.

Inhibition of miR-141-3p Ameliorates the Negative Effects of Poststroke Social Isolation in Aged Mice.

BACKGROUND AND PURPOSE: Social isolation increases mortality and impairs recovery after stroke in clinical populations. These detrimental effects have been recapitulated in animal models, although the exact mechanism mediating these effects remains unclear. Dysregulation of microRNAs (miRNAs) occurs in both strokes as well as after social isolation, which trigger changes in many downstream genes. We hypothesized that miRNA regulation is involved in the detrimental effects of poststroke social isolation in aged animals. METHODS: We pair-housed 18-month-old C57BL/6 male mice for 2 weeks before a 60-minute right middle cerebral artery occlusion or sham surgery and then randomly assigned mice to isolation or continued pair housing immediately after surgery. We euthanized mice either at 3, 7, or 15 days after surgery and isolated the perilesional frontal cortex for whole microRNAome analysis. In an additional cohort, we treated mice 1 day after stroke onset with an in vivo-ready antagomiR-141 for 3 days. RESULTS: Using whole microRNAome analysis of 752 miRNAs, we identified miR-141-3p as a unique miRNA that was significantly upregulated in isolated mice in a time-dependent manner up to 2 weeks after stroke. Posttreatment with an antagomiR-141-3p reduced the postisolation-induced increase in miR-141-3p to levels almost equal to those of pair-housed stroke controls. This treatment significantly reduced mortality (by 21%) and normalized infarct volume and neurological scores in poststroke-isolated mice. Quantitative PCR analysis revealed a significant upregulation of Tgfßr1 (transforming growth factor beta receptor 1, a direct target of miR-141-3p) and Igf-1 (insulin-like growth factor 1) mRNA after treatment with antagomiR. Treatment also increased the expression of other pleiotropic cytokines such as Il-6 (interleukin 6) and Tnf-α (tumor necrosis factor-α), an indirect or secondary target) in brain tissue. CONCLUSIONS: miR-141-3p is increased with poststroke isolation. Inhibition of miR-141-3p improved mortality, neurological deficits, and decreased infarct volumes. Importantly, these therapeutic effects occurred in aged animals, the population most at risk for stroke and poststroke isolation.

Nano-particle delivery of brain derived neurotrophic factor after focal cerebral ischemia reduces tissue injury and enhances behavioral recovery.

BACKGROUND: Low levels of brain-derived neurotrophic factor (BDNF) are linked to delayed neurological recovery, depression, and cognitive impairment following stroke. Supplementation with BDNF reverses these effects. Unfortunately, systemically administered BDNF in its native form has minimal therapeutic value due to its poor blood brain barrier permeability and short serum half-life. In this study, a novel nano-particle polyion complex formulation of BDNF (nano-BDNF) was administered to mice after experimental ischemic stroke. METHODS: Male C57BL/6J (8-10weeks) mice were randomly assigned to receive nano-BDNF, native-BDNF, or saline treatment after being subjected to 60min of reversible middle cerebral artery occlusion (MCAo). Mice received the first dose at 3 (early treatment), 6 (intermediate treatment), or 12h (delayed treatment) following stroke onset; a second dose was given in all cohorts at 24h after stroke onset. Post-stroke outcome was evaluated by behavioral, histological, and molecular analysis for 15days after stroke. RESULTS: Early and intermediate nano-BDNF treatment led to a significant reduction in cerebral tissue loss. Delayed treatment led to improved memory/cognition, reduced post-stroke depressive phenotypes, and maintained myelin basic protein and brain BDNF levels, but had no effect on tissue atrophy. CONCLUSIONS: The results indicate that administration of a novel nano-particle formulation of BDNF leads to both neuroprotective and neuro-restorative effects after stroke.

Reversal of the Detrimental Effects of Post-Stroke Social Isolation by Pair-Housing is Mediated by Activation of BDNF-MAPK/ERK in Aged Mice.

Social isolation (SI) increases stroke-related mortality and morbidity in clinical populations. The detrimental effects of SI have been successfully modeled in the laboratory using young animals. Mechanistically, the negative effects of SI in young animals are primarily mediated by an enhanced inflammatory response to injury and a reduction in neurotrophic factors. However, the response to brain injury differs considerably in the aged. Given that SI is more prevalent in aged populations, we hypothesized that isolation, even when initiated after stroke, would delay recovery in aged mice. We found that aged isolated male mice had significantly increased infarct volume, neurological deficits, and serum IL-6 levels three days after stroke compared to pair housed (PH) mice. Using RT(2) Profiler PCR Array and real-time quantitative PCR we found several important synaptic plasticity genes were differentially expressed in post-stroke SI mice. Furthermore, paired mice showed improved memory and neurobehavioral recovery four weeks after injury. Mechanistic and histological studies showed that the beneficial effects of pair housing are partially mediated by BDNF via downstream MAPK/ERK signaling and restoration of axonal basic myelin protein levels.

Pair housing reverses post-stroke depressive behavior in mice.

Social isolation (SI) has been linked epidemiologically to high rates of morbidity and mortality following stroke. In contrast, strong social support enhances recovery and lowers stroke recurrence. However, the mechanism by which social support influences stroke recovery has not been adequately explored. The goal of this study was to examine the effect of post-stroke pair housing and SI on behavioral phenotypes and chronic functional recovery in mice. Young male mice were paired for 14 days before a 60 min transient middle cerebral artery occlusion (MCAO) or sham surgery and assigned to various housing environments immediately after stroke. Post-stroke mice paired with either a sham or stroke partner showed significantly higher (P<0.05) sociability after MCAO than isolated littermates. Sociability deficits worsened over time in isolated animals. Pair-housed mice showed restored sucrose consumption (P<0.05) and reduced immobility in the tail suspension test compared to isolated cohorts. Pair-housed stroked mice demonstrated significantly reduced cerebral atrophy after 6 weeks (17.5 ± 1.5% in PH versus 40.8 ± 1.3% in SI; P<0.001). Surprisingly, total brain arginase-1, a marker of a M2 "alternatively activated" myeloid cells was higher in isolated mice. However, a more detailed assessment of cellular expression showed a significant increase in the number of microglia that co-labeled with arginase-1 in the peri-infarct region in PH stroke mice compared to SI mice. Pair housing enhances sociability and reduces avolitional and anhedonic behavior. Pair housing reduced serum IL-6 and enhanced peri-infarct microglia arginase-1 expression. Social interaction reduces post-stroke depression and improves functional recovery.