Interdisciplinary Stroke research

Targeting eNOS for stroke protection

Focal cerebral ischemia is caused by a local loss of cerebral blood flow (CBF). In this situation collateral arteries respond via dilation to preserve CBF in the affected region. The degree of collateral blood flow determines the lesion core where cells undergo ischemic damage. This ischemic core is surrounded by the so called "penumbra" where cells are functionally silent but metabolically still intact. Therefore, one major aim in the treatment of ischemic stroke is the rapid restoration of CBF.

eNOS-derived NO may preserve collateral blood flow during ischemia thus reducing neuronal damage. HMG-CoA reductase-inhibitors (statins), drugs which lower elevated cholesterol levels, were shown to enhance eNOS expression and augment CBF which results in acute neuro-protection. Furthermore, studies like the Heart Protection Study and the Anglo-Scandinavian Cardiac Outcomes Trial provide strong support for statin therapy in reducing stroke incidence in patients with average or low LDL (low-density lipoprotein) cholesterol levels. In addition to the experimental evidence of neuroprotection (i.e. better outcome and reduced cerebral infarct size), this data supports a role for statins in stroke prevention (i.e. fewer strokes).

Moderately or highly active individuals show reduced stroke incidence or mortality relative to low-active individuals. In addition to statins, also physical activity may provide stroke-protective effects via eNOS-dependent mechanisms. Our group was able to correlate voluntary physical activity to eNOS mediated CBF augmentation and neuroprotection in experimental stroke studies. Physical activity was shown to improve endothelial function, which enhances vasodilation and vasomotor function. The exact mechanism of eNOS regulation in this paradigm is still unclear, but it is known that physical activity acts on the endothelium via shear stress, which is the main physiological stimulus for the activation of flow-mediated dilation.

Taken together, augmentation of eNOS appears as a preventive and therapeutic target for stroke treatment.

EPCAS - Endothelial Function and Progenitor Cells in Acute Ischemic Stroke (NCT01289795)

Endothelial dysfunction is a key component of atherosclerosis which contributes to the development of cardio- and cerebrovascular diseases. However, endothelial dysfunction (ED) is not established as a risk factor for ischemic stroke. As a novelty the proposed trial investigates the following variety of indirect markers of endothelial function in acute ischemic stroke: circulating endothelial progenitor cells (EPC), endothelial microparticles (EMP), ENDOPAT (RH- PAT ratio) in two regards: 1) time after ischemic events (< 48h, Days 4-5, day 7 or at discharge) 2) etiological stroke subtypes. It is not known whether these parameters are changed after acute cerebral ischemia and could possibly serve as specific target for treatment.

Migraine and Endothelial Dysfunction (NCT01388699)

Our objectives are to determine whether novel endothelial function methods and novel markers of endothelial activation are associated with migraine in female migraineurs with aura. Migraine is an independent risk factor for stroke, especially in young women with aura symptoms. Endothelial dysfunction is a risk factor for cardiovascular diseases. Recent studies suggest that there is a link between migraine with aura and endothelial activation, dysfunction and impaired vascular reactivity. In this case-control study we examine several novel biomarkers of endothelial function such as endothelial progenitor cells and endothelial microparticles as well as novel methods such as reactive hyperemic peripheral arterial tonometry in female migraineurs with aura and age-matched controls.

PROSCIS-B Prospective Study with Incident Stroke Berlin (NCT01363856 )

Primary aim of this study is to develop a prognostic model of long-term vascular risk after stroke. Secondary aims are natural course of post stroke depression and dementia up to 3 years after first-ever stroke. The protocol and study design of PROSCIS-B study has been implemented in the Institute of Stroke and Dementia Research Stroke Study Munich (PROSCIS-M) that will serve as a validation cohort regarding multiple research questions regarding with a focus on vascular risk and functional outcome after stroke. We are currently finalizing the 3 years follow up in approx. 750 patients with stroke.

Ongoing research project are:

• Characteristics of activating endothelial receptor autoantibodies in stroke and impact on functional outcome at 1 year after ischemic stroke
• Association of Lipoprotein A with outcome after stroke
• Prediction of Vascular Risk at 1 year after stroke – development of a prognostic model
• Impact of socioeconomic status on vascular risk after stroke
• Biomarkers of endothelial activation and inflammation (VCAM, hsCRP) and outcome after stroke

ENDOBASE – Endothelial activation, cerebral small vessel disease and cognition in the Berlin Aging study II Endothelial activation and dysfunction may play a major and causal role in the development of small vessel disease. Small vessel disease is associated with cognitive decline and dementia. White matter lesions and cerebral microbleeds are both established markers for small vessel disease.

As a novelty the proposed study investigates the association of novel blood based markers for endothelial activation such as stromal derived factor-1 of endothelial activation and endothelial microparticles in a large cohort of elderly subjects from the Berlin Aging Study II with the following outcomes:

1. Severity of small vessel disease using MRI markers
2. Kognitive impairment
3. Arterial stiffness

It is not known whether these novel markers of endothelial activation are associated with these outcomes and may serve as preclinical surrogate markers of underlying activity of endothelial dysfunction.

The Berlin Aging Study II (BASE-II) has been designed to identify and characterize factors that are associated with "healthy" versus "unhealthy" aging. Base –II is a multi-disciplinary and -institutional study that aims to include 1,822 elderly subjects. Similar to other industrialized countries, Germany is facing an aging population. While some are cognitive and physically healthy, others suffer from multiple age-related disorders and impairments in older age with reduced life expectancy and quality of life. The proposed study aims to investigate in a cross-sectional design the association between endothelial activation and a variety of outcome variables in 750 patients in this cohort with advanced brain imaging.

Team-Contact

Until now, translation of experimental studies into clinical reality has been sorely lacking. So far, most studies have focused on acute stroke outcome and early treatment paradigms affording neuro-protection. It is increasingly recognized that, in addition, it will be necessary to harness the capacity of the brain for neuro-regeneration after brain ischemia. Therefore, understanding the mechanisms of post-stroke angiogenesis and neurogenesis appear as a promising approach to promote regeneration, thereby improving longer-term stroke outcome. During acute stroke, tissue may be salvaged by blood flow supplied from collateral vessels. In contrast, the formation of functionally intact vessels could re-establish cerebral blood flow in the damaged tissue in the chronic phase after stroke, therefore promoting neuronal regeneration according to the “vascular niche” hypothesis where adult neurogenesis occurs in an angiogenic environment. Here, we were able to show, that physical activity also improves long-term stroke outcome via augmentation of neo-vascularization and cerebral blood flow.

Despite the presence of neural stem cells and ongoing neuroneogenesis in some regions of the adult mammalian brain, neurons are not replaced in most brain regions after injury. Therefore, one aim of our work is to develop cell-based therapeutic strategies for post-stroke brain repair. This will require a deepened understanding of the key cellular players that might be recruited for the neuronal fate and the mechanisms that usually instruct them towards gliogenesis. Recently, we explored and modulated the fate determinants Olig2 and Pax6 after brain ischemia in order to redirect.

Team-Contact

Post-stroke depression (PSD) is considered the most frequent and important neuropsychiatric consequence of stroke, since approximately one-third of stroke survivors experience major depression. Moreover, this condition can have an adverse effect on cognitive function, functional recovery and survival. While an integrated bio-psycho-social model including both biological and psychosocial aspects of post-stroke depression seems warranted, a number of studies clearly suggest that biological mechanisms play a crucial role in the development of post-stroke depression.

• Stroke patients show a higher rate of depression compared to orthopedic patients with disabilities of comparable severity.
• Several studies proposed an association with specific lesions (left anterior and basal ganglia lesions and lesions close to the frontal pole) and occurrence of post-stroke depression.
• Some studies reported an association between post-stroke mania and right orbital frontal, basotemporal, basal ganglia lesions.
• It has been shown that patients with anosognosia who are unaware of their disability still develop post-stroke depression.

Despite this evidence, the association of post-stroke depression to specific brain lesions is still vague and needs replication from various independent groups. Furthermore the cause of post-stroke depression at the functional level is not clear. Our approach, starting from observations in our animal model of stroke (mice show “depressive-like“behavior after stroke which can be reversed by antidepressant treatment) is to study delayed functional, biochemical and structural changes, due to a specific ischemic lesion and to replicate these findings in a group of selected stroke patients.

It might well be that these secondary changes, such as exofocal delayed degeneration of monaminergic nuclei and consecutive changes in monoamine levels of the neuronal circuits of the basal ganglia and the mesolimbic pathway are determining contributors to the development of post-stroke psychiatric sequelae.

Team-Contact

Chronic stress and Stroke outcome and susceptibility

The bodily stress response is crucial in managing allostatic mechanisms, however, chronic activation of this system creates “allostatic load” which may in turn lead to disease. The link between cardiovascular disease and stress is well established. Epidemiologic studies indicate that classical vascular risk factors are exacerbated by stress. However the exact mechanisms by which stress may increase stroke incidence is far from being understood. By using a 28 day chronic stress paradigm, we examine whether chronic stress induces endothelial dysfunction and increases stroke susceptibility. Our findings so far indicate a deleterious effect of chronic stress on the endothelium in association/relation with hypertenstion, which may increase stroke susceptibility.

Post Stroke behavioral testing

For decades, histological assessments were used as the only end point in stroke research, typically using lesion size determination to evaluate the potential therapeutic effectiveness of an agent in in-vivo stroke models. Failures in clinical trials have triggered a growing awareness in the scientific community that a thorough re-examination of the models and techniques used in stroke research is not only necessary but inevitable. According to this new approach, one of the key strategies to overcome the “translational road block” in stroke research is to use behavior testing to assess functional outcome as a relevant end point. In line with this concept we have established a wide range of behavioral tests which are suitable for in-depth behavioral analysis of post stroke disturbances observed in mouse models. Seven different sensory-motor tests ranging from Pole test to Corner test, anxiety tests such as plus maze and light dark box, tests for depression-like behaviors, memory and activity tests are successfully used in our research.

Team-Contact

Ischemic tolerance describes a phenomenon in which a sublethal noxious stimulus applied to the brain induces a transient resistance against a subsequent lethal ischemic insult. Focus of this research project is to gain further understanding in the mechanisms of ischemic tolerance in the early time window. Early ischemic tolerance occurs within minutes to a few hours after the preconditioning stimulus. Experiments are being performed using widely established murine in vivo-models of ischemic tolerance and transient focal cerebral ischemia. Particular interest in this field is to further elucidate the contribution of the potent vasodilator nitric oxide (NO) as well as its isoenzymes and signaling pathways involved in acutely preconditioned brains.

Team-Contact

Peroxisome proliferator-activated receptors (PPARs) have emerged as important regulators of metabolic and inflammatory signaling. PPARs belong to nuclear receptors, a family of ligand-activated transcription factors, and consist of at least three subtypes, i.e. PPARα, PPARδ and PPARγ. The ability of these receptors to link metabolism to inflammatory signaling makes them potentially attractive targets for the treatment of human diseases such as type 2 diabetes and atherosclerosis. Furthermore, animal studies on ischemic stroke have recently discovered that activation of PPARs suppresses post-ischemic inflammation in the brain and thereby reduces ischemic brain injury. Most studies on the role of PPARs in ischemic brain injury have focused on PPARα and PPARγ, much less is known on the role of PPARδ and its activation by synthetic ligands in experimental ischemic stroke. Objective of this research project is to characterize a highly potent and selective PPARδ agonist, as a potential neuroprotective agent in a murine ischemic stroke model. Furthermore, it is the aim to elucidate mechanisms how PPARδ and its activation by synthetic ligands acts neuroprotective in ischemic stroke.

For this project, the murine in vivo-model of focal cerebral ischemia as well as established in vitro-techniques of ischemia, for example oxygen glucose deprivation (OGD) in cell culture, are being used.

Team-Contact

Both salutary and detrimental effects of neuroinflammation in response to ischemic injury have been described. The 'Janus face' of inflammation after brain ischemia is best exemplified by the fact that the same factors may contribute toward tissue damage or protection depending on the temporo-spatial context of their expression.

To understand the complex responses of particularly resident immune cells to brain ischemia, we are following different research strategies in vitro and in vivo.

• Studying the molecular pathways influencing microglia activation.
• Interfering with microglia activation and describing the resulting phenotypes of activated microglia.
• Describing diversity after stroke by dissecting the responses of resident microglia and invading blood borne monocytes to brain ischemia.

In a recent project, we investigated the complex role of the pro-inflammatory cytokine interleukin-6 (IL-6) for post-stroke regeneration. We found that interleukin-6 promotes post-stroke angiogenesis and thereby affords long-term histological and functional protection. Our data show that a single molecule can exert differential effects on stroke outcome and underscore the need for complex endpoint analyses after stroke. As an interesting phenomena, we found that invading mononuclear cells of the phagocyte system are not able to contribute to IL-6 expression in the brain (Gertz et al., 2012).

Based on our findings, we currently investigate the diverse properties of resident microglia and invading blood borne monocytes after brain ischemia.

By using genetic and pharmacological manipulations, we studied the effects of alterations in actin dynamics on microglia effector functions. We found, that the dynamic state of the actin cytoskeleton profoundly and distinctly affects microglia behaviors. Disruption of actin dynamics attenuates M2 polarization by inhibiting transcription of alternative activation genes. In classical activation, the role of actin remodeling is complex, does not relate to gene transcription, and shows a major divergence between cytokines following conventional and unconventional secretion (Uhlemann et al., 2015).

Team-Contact

Neurotoxic phenomena are amongst the most common side effects of many antineoplastic agents and frequently necessitate alterations or termination of treatment, preventing optimal medical care. Furthermore they significantly increase the burden of disease for cancer patients by severely affecting quality of life.

Neurotoxicity in the peripheral nervous system: chemotherapy induced peripheral neuropathy (CIPN)

Many chemotherapeutic substances induce a predominantly sensory axonal peripheral neuropathy often presenting with sensory deficits and pain. Using dorsal root ganglia and Schwann cell cultures we characterize pathomechanisms of neurotoxicity and identify molecular targets for preventive strategies. Increasing evidence suggests that an altered intracellular calcium (Ca2+) homeostasis plays an important role in the pathogenesis of CIPN. For example, the cytostatic drug Paclitaxel modulates Ca2+ release from the endoplasmatic reticulum whereas salinomycin triggers Ca2+ influx across the plasma membrane and Ca2+ release from mitochondria. Animals treated with these agents develop distinct behavior, electrophysiological and morphological changes that can be prevented by targeted pharmacological interventions.

In addition to preclinical research, CIPN is studied in patients to identify risk factors for CIPN development and to develop tools for clinical management.

Neurotoxicity in the central nervous system: post-chemotherapy cognitive impairment (PCCI)

In recent years, changes of cognitive function in patients treated with chemotherapy have gained increased clinical and scientific focus. Patients often describe a reduction of visual-spatial and verbal memory as well as executive functions. The pathophysiology of those alterations, in non-scientific literature termed “chemobrain”, is completely unknown. Among many proposed pathomechanisms we focus on alterations of neurogenesis using neural stem cell (NSC), microglia and neuronal cultures. Many antineoplastic substances are highly toxic to NSC triggering caspase-dependent cell death. Animals treated with chemotherapy show impaired visual-spatial memory and reduced hippocampal neurogenesis which could be prevented by pharmacological treatments.

Clinical studies of PCCI include:

• CICARO (Chemotherapy induced cognitive alterations in recruits with ovarian cancer)
• TAVAB (Treatment of antibody related autoimmune diseases with bortezomib)

For more information on CIPN and PCCI please also visit the webside research consortium neurotoxicology.

Inflammation in the peripheral nervous System

Acute and chronic immune mediated inflammatory disorders of the peripheral nervous system lead to high morbidity and significantly impair patient’s quality of life. In contrast to autoimmune inflammatory disorders of the central nervous system, much less scientific effort is allocated to these diseases. Our group is interested in the underlying disease mechanisms which are studied in animal models of acute and chronic autoimmune mediated polyneuropathies. In a second step these models are used to devise and test novel treatments for these disorders.

Team-Contact