Rodney Ritzel

Sex, stroke, and inflammation: The potential for estrogen-mediated immunoprotection in stroke

Stroke is the third leading cause of death and the primary cause of disability in the developed world. Experimental and clinical data indicate that stroke is a sexually dimorphic disease, with males demonstrating an enhanced intrinsic sensitivity to ischemic damage throughout most of their lifespan. The neuroprotective role of estrogen in the female brain is well established, however, estrogen exposure can also be deleterious, especially in older women. The mechanisms for this remain unclear. Our current understanding is based on studies examining estrogen as it relates to neuronal injury, yet cerebral ischemia also induces a robust sterile inflammatory response involving local and systemic immune cells. Despite the potent anti-inflammatory effects of estrogen, few studies have investigated the contribution of estrogen to sex differences in the inflammatory response to stroke. This review examines the potential role for estrogen-mediated immunoprotection in ischemic injury.

Age- and location-related changes in microglial function

Inflammation in the central nervous system (CNS) is primarily regulated by microglia. No longer considered a homogenous population, microglia display a high degree of heterogeneity, immunological diversity and regional variability in function. Given their low rate of self-renewal, the microenvironment in which microglia reside may play an important role in microglial senescence. This study examines age-related changes in microglia in the brain and spinal cord. Using ex-vivo flow cytometry analyses, functional assays were performed to assess changes in microglial morphology, oxidative stress, cytokine production, and phagocytic activity with age in both the brain and spinal cord. The regional CNS environment had a significant effect on microglial activity with age. Blood-CNS barrier permeability was greater in the aging spinal cord compared with aging brain; this was associated with increased tissue cytokine levels. Aged microglia had deficits in phagocytosis at baseline and after stimulus-induced activation. The identification of age-specific, high scatter microglia together with the use of ex-vivo functional analyses provides the first functional characterization of senescent microglia. Age and regional-specificity of CNS disease should be taken into consideration when developing immune-modulatory treatments.

Age-Associated Resident Memory CD8 T Cells in the Central Nervous System Are Primed To Potentiate Inflammation after Ischemic Brain Injury

Aging is associated with an increase in basal inflammation in the CNS and an overall decline in cognitive function and poorer recovery following injury. Growing evidence suggests that leukocyte recruitment to the CNS is also increased with normal aging, but, to date, no systematic evaluation of these age-associated leukocytes has been performed. In this work, the effect of aging on CNS leukocyte recruitment was examined. Aging was associated with more CD45high leukocytes, primarily composed of conventional CD8+ T cells. These results were strain independent and seen in both sexes. Intravascular labeling and immunohistology revealed the presence of parenchymal CD8+ T cells in several regions of the brain, including the choroid plexus and meninges. These cells had effector memory (CD44+CD62L−) and tissue-resident phenotypes and expressed markers associated with TCR activation. Analysis of TCRvβ repertoire usage suggested that entry into the CNS is most likely stochastic rather than Ag driven. Correlational analyses revealed a positive association between CD8 T cell numbers and decreased proinflammatory function of microglia. However, the effects of cerebral ischemia and ex vivo stimulation of these cells dramatically increased production of TNF, IFN-γ, and MCP-1/CCL2. Taken together, we identified a novel population of resident memory, immunosurveillant CD8 T cells that represent a hallmark of CNS aging and appear to modify microglia homeostasis under normal conditions, but are primed to potentiate inflammation and leukocyte recruitment following ischemic injury.

Old Maids: Aging and Its Impact on Microglia Function

Microglia are highly active and vigilant housekeepers of the central nervous system that function to promote neuronal growth and activity. With advanced age, however, dysregulated inflammatory signaling and defects in phagocytosis impede their ability to perform the most essential of homeostatic functions, including immune surveillance and debris clearance. Microglial activation is one of the hallmarks of the aging brain and coincides with age-related neurodegeneration and cognitive decline. Age-associated microglial dysfunction leads to cellular senescence and can profoundly alter the response to sterile injuries and immune diseases, often resulting in maladaptive responses, chronic inflammation, and worsened outcomes after injury. Our knowledge of microglia aging and the factors that regulate age-related microglial dysfunction remain limited, as the majority of pre-clinical studies are performed in young animals, and human brain samples are difficult to obtain quickly post-mortem or in large numbers. This review outlines the impact of normal aging on microglial function, highlights the potential mechanisms underlying age-related changes in microglia, and discusses how aging can shape the recovery process following injury.

Ischemic stroke induces gut permeability and enhances bacterial translocation leading to sepsis in aged mice.

Aging is an important risk factor for post-stroke infection, which accounts for a large proportion of stroke-associated mortality. Despite this, studies evaluating post-stroke infection rates in aged animal models are limited. In addition, few studies have assessed gut microbes as a potential source of infection following stroke. Therefore we investigated the effects of age and the role of bacterial translocation from the gut in post-stroke infection in young (8-12 weeks) and aged (18-20 months) C57Bl/6 male mice following transient middle cerebral artery occlusion (MCAO) or sham surgery. Gut permeability was examined and peripheral organs were assessed for the presence of gut-derived bacteria following stroke. Furthermore, sickness parameters and components of innate and adaptive immunity were examined. We found that while stroke induced gut permeability and bacterial translocation in both young and aged mice, only young mice were able to resolve infection. Bacterial species seeding peripheral organs also differed between young (Escherichia) and aged (Enterobacter) mice. Consequently, aged mice developed a septic response marked by persistent and exacerbated hypothermia, weight loss, and immune dysfunction compared to young mice following stroke.

Stroke sensitivity in the aged: sex chromosome complement vs. gonadal hormones

Stroke is a sexually dimorphic disease. Elderly women not only have higher stroke incidence than age-matched men, but also have poorer recovery and higher morbidity and mortality after stroke. In older, post-menopausal women, gonadal hormone levels are similar to that of men. This suggests that tissue damage and functional outcomes are influenced by biologic sex (XX vs. XY) rather than the hormonal milieu at older ages. We employed the Four Core Genotype (FCG) mouse model to study the contribution of sex chromosome complement and gonadal hormones to stroke sensitivity in aged mice in which the testis determining gene (Sry) is removed from the Y chromosome, allowing for the generation of XX males and XY females. XXF, XXM, XYF, XYM and XYwt aged mice were subjected to middle cerebral artery occlusion (MCAO). XXF and XXM mice had significantly larger infarct volumes than XYF and XYM cohorts respectively. There was no significant difference in hormone levels among aged FCG mice. XXF/XXM mice also had more robust microglial activation and higher serum levels of pro-inflammatory cytokines than XYF/XYM cohort respectively. We concluded that the sex chromosome complement contributes to ischemic sensitivity in aged animals and leads to sex differences in innate immune responses.

Inhibition of mitogen-activated protein kinase phosphatase-1 (MKP-1) increases experimental stroke injury

BACKGROUND AND PURPOSE Activation of mitogen-activated protein kinases (MAPKs), particularly c-jun-N-terminal kinases (JNK) and p38 exacerbates stroke injury by provoking pro-apoptotic and pro-inflammatory cellular signaling. MAPK phosphatase-1 (MKP-1) restrains the over-activation of MAPKs via rapid de-phosphorylation of the MAPKs. We therefore examined the role of MKP-1 in stroke and studied its inhibitory effects on MAPKs after experimental stroke. METHODS Male mice were subjected to transient middle cerebral artery occlusion (MCAO). MKP-1 knockout (KO) mice and a MKP-1 pharmacological inhibitor were utilized. We utilized flow cytometry, immunohistochemistry (IHC), and Western blots analysis to explore MKP-1 signaling and its effects on apoptosis/inflammation in the brain and specifically in microglia after stroke. RESULTS MKP-1 was highly expressed in the nuclei of both neurons and microglia after stroke. MKP-1 genetic deletion exacerbated stroke outcome by increasing infarct, neurological deficits and hemorrhagic transformation. Additionally, delayed treatment of the MKP-1 pharmacological inhibitor worsened stroke outcome in wild type (WT) mice but had no effect in MKP-1 KO mice. Furthermore, MKP-1 deletion led to increased c-jun-N-terminal kinase (JNK) activation and microglial p38 activation after stroke. Finally, MKP-1 deletion or inhibition increased inflammatory and apoptotic response as evidenced by the increased levels of interleukin-6 (IL-6), tumor necrosis factor α (TNFα), ratio of p-c-jun/c-jun and cleaved caspase-3 following ischemia. CONCLUSIONS We have demonstrated that MKP-1 signaling is an endogenous protective mechanism in stroke. Our data imply that MKP-1 possesses anti-apoptotic and anti-inflammatory properties by simultaneously controlling the activities of JNK and microglial p38.

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.

Multiparity improves outcomes after cerebral ischemia in female mice despite features of increased metabovascular risk.

Significance Stroke is an age-related disease that disproportionately affects women. Although experimental studies have identified several hormonal and genetic factors underlying these differences, little is known about how reproductive experience influences risk. This study examined the role of pregnancy and parturition on neurovascular function and behavior in both normal female mice and in females exposed to stroke. We found that reproductive experience increases systemic metabolic risk and results in significant behavioral deficits that are associated with CNS immunosuppression. After stroke, however, multiparous females exhibited smaller infarct volumes, attenuated inflammatory responses, enhanced angiogenesis, and improved behavioral recovery. Although the precise mechanisms underlying this paradoxical finding remain unknown, parity was associated with higher VEGF and improved postischemic vascular remodeling. Females show a varying degree of ischemic sensitivity throughout their lifespan, which is not fully explained by hormonal or genetic factors. Epidemiological data suggest that sex-specific life experiences such as pregnancy increase stroke risk. This work evaluated the role of parity on stroke outcome. Age-matched virgin (i.e., nulliparous) and multiparous mice were subjected to 60 min of reversible middle cerebral artery occlusion and evaluated for infarct volume, behavioral recovery, and inflammation. Using an established mating paradigm, fetal microchimeric cells present in maternal mice were also tracked after parturition and stroke. Parity was associated with sedentary behavior, weight gain, and higher triglyceride and cholesterol levels. The multiparous brain exhibited features of immune suppression, with dampened baseline microglial activity. After acute stroke, multiparous mice had smaller infarcts, less glial activation, and less behavioral impairment in the critical recovery window of 72 h. Behavioral recovery was significantly better in multiparous females compared with nulliparous mice 1 mo after stroke. This recovery was accompanied by an increase in poststroke angiogenesis that was correlated with improved performance on sensorimotor and cognitive tests. Multiparous mice had higher levels of VEGF, both at baseline and after stroke. GFP+ fetal cells were detected in the blood and migrated to areas of tissue injury where they adopted endothelial morphology 30 d after injury. Reproductive experience has profound and complex effects on neurovascular health and disease. Inclusion of female mice with reproductive experience in preclinical studies may better reflect the life-long patterning of ischemic stroke risk in women.

Tilting the balance of M1/M2 microglial phenotypes: Inhibition of Ezh2 leads to decreased M1 and enhanced M2 phenotype

Infection of Theilers murine encephalomyelitis virus (TMEV) in the central nervous system (CNS) induces an immune-mediated demyelinating disease in susceptible mouse strains and serves as a relevant infection model for human multiple sclerosis (MS). Alpha 4 integrins function as adhesive structures, providing mechanical support for cell adhesion andmigration and, in addition, as bona fide signaling receptors. HCA3551 is a newly synthesized, orally active small molecule alpha 4 integrin antagonist. In this study, we examined the therapeutic effect of HCA3551 in the development of TMEV-induced demyelinating disease (TMEV-IDD). HCA3551 is provided by Ajinomoto Pharmaceuticals Co., Ltd. Micewere orally administrated twice daily with vehicle or HCA3551 (100 mg/kg) every 12 hours. HCA3551 treatment significantly suppressed the disease development of TMEV-IDD both clinically and histologically. The number of infiltrating mononuclear cells (MNCs) in the CNS was significantly decreased in mice treated with HCA3551 (p b 0.05) compared to control mice orally administrated with vehicle. The significant increase in peripheral lymphocyte countwas observed on day 20 (p b 0.01), day 30 (p b 0.05) and day 40 (p b 0.01) post infection compared to vehicle treated control mice. Flow cytometric analysis of cytokine staining revealed that absolute cell numbers of TNF-producing CD4 and IFN-gamma-producing CD8 T cells were significantly decreased in the CNS ofmice treatedwith HCA3551 compared to vehicle treated control mice (p b 0.01). Taken together these data suggest that HCA3551 treatment may ameliorate TMEV-IDD by inhibiting alpha 4 integrin accompanied with the decreasing number of MNCs and proinflammatory cytokine producing cells in the CNS. Therefore, HCA3551 could be used as a novel therapeutic treatment of MS. doi:10.1016/j.jneuroim.2014.08.422