Edward Koellhoffer

The Effects of Estrogen in Ischemic Stroke

Stroke is a leading cause of death and the most common cause of long-term disability in the USA. Women have a lower incidence of stroke compared with men throughout most of the lifespan which has been ascribed to protective effects of gonadal steroids, most notably estrogen. Due to the lower stroke incidence observed in pre-menopausal women and robust preclinical evidence of neuroprotective and anti-inflammatory properties of estrogen, researchers have focused on the potential benefits of hormones to reduce ischemic brain injury. However, as women age, they are disproportionately affected by stroke, coincident with the loss of estrogen with menopause. The risk of stroke in elderly women exceeds that of men and it is clear that in some settings estrogen can have pro-inflammatory effects. This review will focus on estrogen and inflammation and its interaction with aging.

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.

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.

The G-quadruplex DNA stabilizing drug pyridostatin promotes DNA damage and downregulates transcription of Brca1 in neurons

The G-quadruplex is a non-canonical DNA secondary structure formed by four DNA strands containing multiple runs of guanines. G-quadruplexes play important roles in DNA recombination, replication, telomere maintenance, and regulation of transcription. Small molecules that stabilize the G-quadruplexes alter gene expression in cancer cells. Here, we hypothesized that the G-quadruplexes regulate transcription in neurons. We discovered that pyridostatin, a small molecule that specifically stabilizes G-quadruplex DNA complexes, induced neurotoxicity and promoted the formation of DNA double–strand breaks (DSBs) in cultured neurons. We also found that pyridostatin downregulated transcription of the Brca1 gene, a gene that is critical for DSB repair. Importantly, in an in vitro gel shift assay, we discovered that an antibody specific to the G-quadruplex structure binds to a synthetic oligonucleotide, which corresponds to the first putative G-quadruplex in the Brca1 gene promoter. Our results suggest that the G-quadruplex complexes regulate transcription in neurons. Studying the G-quadruplexes could represent a new avenue for neurodegeneration and brain aging research.

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

Age-related changes in intestinal permeability and gut microbiota after ischemic stroke

Modafinil is awakefulness promoting agent used in the treatment of narcolepsy. It is also beneficial in patients with multiple sclerosis and with Parkinsons disease who complain of fatigue and sleepiness. We and others have shown that patients with MS-related fatigue have a deficit of arousal that can be corrected by modafinil. The mechanisms of action of modafinil are not clearly elucidated, but it is known to target the dopamine transporter, and to enhance the availability of dopamine and catecholamine neurotransmitters. The wakefulness promoting effect is dependent on the presence of Dopamine receptor 1. Functional magnetic resonance studies in humans have implicated the thalamus, a gray matter component of the brain known to degenerate in MS, in the effects of modafinil. Behavioural and histological evidence suggests a neuroprotective effect of modafinil in experimental models of Parkinsons disease, and is conformed in other models of neurodegeneration. Based on this we first performed a retrospective review of MS patients on long term modafinil compared with matched MS controls without modafinil treatment and showed a slower progression of disability on modafinil, supporting a neuroprotective effect. In the present study, we investigated the effect of modafinil on MOG35-55 induced EAE in C57Bl/6 mice. Modafinil 33 mg/kg twice weekly intraperitoneally from the day of immunization showed a trend towards reducing EAE scores in the chronic phase (p = 0.06). In a subsequent set of experiments modafinil given in daily doses of 100 or 50 mg/kg from the development of EAEwith a score of 1 or higher, significantly reduced disease severity (p = 0.008 and 0.021 respectively). Gene expression profiling in the thalamus showed up-regulated expression of a number of genes associated with neuroprotection, in particular genes involved in the ubiquitin–proteasome pathway. Further potential immunomodulatory and neuroprotective mechanisms are investigated and discussed.