Interdisciplinary stroke research

I) Vascular mechanisms of stroke protection

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 neuro-protection (i.e. better outcome and reduced cerebral infarct size), these data support 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 neuro-protection in acute as well as in chronic 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.

Measurement of absolute CBF with C14-Iodoantipyrine-autoradiography at 28 days after 30min MCAo.

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II) Neurodegeneration and neuroregeneration

The group aims to dissect molecular mechanisms that may underlie neurodegeneration and which may also confer increased sensitivity to cerebral ischemia. Pathogenetic factors currently under investigation include, for example, folate deficiency and resultant hyperhomocysteinemia, impaired actin filament turnover associated with aging, and psychosocial stress as a risk factor for stroke and worsened stroke outcome.

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. We aim 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 toward gliogenesis.

III) Angiogenesis and vasculogenesis

Imaging angiogenesis in glioma of mice
This projects aims to combine molecular imaging of angiogenesis in glioma using combined MR imaging and near-infrared imaging (NIRF) methods. Targets for neoangiogenesis should not be recruited to the normal vasculature. The alternative spliced variant A of the extracellular matrix protein fibronectin has been identified by the group of Dario Neri in Zurich and the antibody scFv fragment specific to EDA (scF8) was isolated from the human antibody library ETH2 (Cancer Res 2007;67(22):10948–57).

We could demonstrate non-invasive imaging of neo-angiogenesis at the border of the tumor at different time points after intravenous application of scF8 EDA-coupled to Alexa 750 near infrared dye in collaboration with AG Wunder (Jan Klohs). Tumor localization was verified by MR imaging using the small animal 7Tesla scanner. This is a prerequisite to evaluate new methods for the evaluation and stratification of and for new therapeutic paradigms. The final goal is to predict whether a therapeutic action will be beneficial depending on the specific molecular imaging results. Therapeutic regimes and the characterization of the scF8 EDA antibody fragment will be performed in mice breeding 126 glioma using intravital microscopy by the neurosurgery department (Group of Peter Vajkoczy, Marcus Czabanka) and will be combined with Kaplan-Meier-curves for survival and innovative new therapeutic regimes.


  • Christoph Harms, Ulrike Harms, Anna Lena Daetwyler
  • Endres
  • Collaboration with AG Wunder: Jan Klohs
  • Collaboration with AG Vajkoczy: Marcus Czabanka

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IV) Neuronal cell cycle and DNA damage and repair

V) Post stroke depression

Post-stroke depression (PSD) is considered as 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 major 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 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 a functional level is not clear.

Our approach, departing 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 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 mesolimbic pathway are determining contributors to the development of post-stroke psychiatric sequelae.

By therapeutic intervention of the secondary damage evolvement, thus also the correlate of neuro-psychiatric disease, such as PSD could be prevented. To advance to this immediate clinical implication, currently pre-clinical findings are translated into clinical studies to assess the role of the observed secondary brain changes in human.
 

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VI) Stress and Behavior

Chronic stress and Stroke outcome and susceptibility
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 has triggered a growing awareness in the science community that a throughout 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 keys to overcome the so-called translational road block 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 behavior tests which are suitable for in-depth behavioral analysis of post stroke disturbances observed in mice models of stroke. 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 behavior, memory and activity tests are successfully used in our research.

VII) Telemedicine

STROKENET- Implementation of telemedical stroke networks in Berlin
Acute Stroke is a major cause of death and long-term disability. For patients with acute stroke, ambulances should head the closest hospital where patients receive fast and appropriate treatment. Time delays and patients in inappropriate hospitals are major concerns in acute stroke management.

StrokeNet provides the implementation of a telemedical stroke network to improve pre- and early intrahospital stroke care through

  • audiovisual connection between ambulance and Stroke Unit for neurological assessment already in specially equipped ambulances, establishment of telemedical networks for close cooperation between stroke centers and
  • local hospitals without pre-existing specialized stroke wards or 24 hour neurological service. Physicians in these hospitals will be able to contact the stroke centers 24 hours per day for telemetric in-patient consultations and direct CT-data transfer.


“Telestroke Ambulances in Acute Stroke Management”

Telestroke ambulances provide a new approach to improve and accelerate acute stroke management using prehospital telemedical examination and early eligibility evaluation for thrombolysis. Currently the “Telestroke Ambulances In Acute Stroke Management – Telestroke AIM” trial assesses the feasibility and reliability of remote stroke diagnosis in ambulance vehicles. Patients with suspected diagnosis of acute stroke within six hours of symptom onset transported with ambulance vehicles from three corresponding paramedic headquarter are included. Stroke scales (NIHSS, mRS), thrombolysis eligibility and basic times (e.g. arrival-at-scene) are captured during telestroke ambulance transportation and at emergency department arrival.

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VIII) Clinical trials – the Berlin Cream&Sugar study

The Berlin ‘Cream&Sugar’ Study – Prognostic impact of an oral triglyceride tolerance test in
The exact role of triglyceride levels for the risk of secondary stroke is unknown. We hypothesize that the results of an oral triglyceride tolerance test (oTTT) three to seven days after incident ischemic stroke are associated with the risk of recurrent stroke within 12 months. Primary endpoint of this prospective observational study is recurrent fatal or non-fatal stroke within 12 months. Secondary outcomes are myocardial infarction, coronary revascularization, cardiovascular death (death due to any cardiovascular or cerebrovascular event), TIA within 12 month after the qualifying event.The study will show if the oTTT is a useful tool in the sub-acute setting to predict the risk of recurrent stroke.


IX) MRI Imaging in stroke research

Magnet-Resonance-Imaging is a powerful tool in the field of stroke research.  We have access to a 7 Tesla animal scanner (Bruker Biospin - Pharmascan 70/16, Ettlingen, Germany). Mainly we work with mice in our projects. We use different sequences in order to observe the lesion size and the pathophysiology of the brain tissue. For the purpose of imaging the lesion size we acquire T2-Weigthed-Images as well as Diffusion-Weighted-Images (DWI). Especially in the early stage after stroke the DWI shows much better the lesion. We are able to observe quantitatively the perfusion of the tissue. With different kind of contrast agent we observe the blood brain barrier. We also worked with the new method of micro vessel density imaging.   


X) Ischemic Preconditioning

Clinical neurologists face an enormous discrepancy between incidence of ischemic stroke and its treatment options. Ischemic stroke is a leading cause of death and disability worldwide. The only approved treatment for patients with acute ischemic stroke is thrombolysis with recombinant tissue plasminogen activator (rtPA). However, less than 5% of all stroke patients are treated with rtPA. Therefore, there is a pressing need for development of new treatment strategies for patients with ischemic stroke. One approach is to gain further understanding in the mechanisms of endogenous adaptation of brain to cope with a damaging stimulus. This brain-derived neuroprotection is also known as ‘ischemic preconditioning’ or ‘ischemic tolerance’ and describes a phenomenon in which a sublethal noxious stimulus applied to the brain induces a transient resistance against a subsequent lethal ischemic insult. Ischemic tolerance occurs in two different time windows: Early tolerance, which occurs within minutes to a few hours after preconditioning and late tolerance, which needs a latency of a couple of hours to occur. So far, research on ischemic tolerance in brain has focussed on late tolerance. Much less is known on the mechanisms of early ischemic tolerance. Objective of our research activities in the field of preconditioning is to extend our knowledge in the mechanisms of ischemic tolerance particular in the early time window. Many studies have established that ischemic tolerance is associated with genetic, biochemical and neurophysiological changes in neurons and glia that render the brain more resistant to ischemia. Much less is known on the role of cerebral vasculature, i.e. tolerance-induced improvements in cerebral blood flow (CBF) and CBF regulation. Therefore, focus of our investigations is on the contribution of cerebral vasculature and CBF regulation in ischemic tolerance, in particular the role of nitric oxide (NO) and the NO-synthesizing enzymes in this regulation. NO as a potent vasodilator plays a central role in CBF regulation and it has also been demonstrated to participate in the mechanisms of ischemic tolerance.
For induction of ischemic stroke, we transiently occlude middle cerebral artery (MCA occlusion) in mice for 60 min. Ischemic tolerance is induced (1) by sublethal transient global ischemia using bilateral common carotid artery occlusion (BCCAO) (2) by a low dose (0.5 mg/kg, ip) lipopolysaccaride (LPS) injection and (3) by a short (5min) transient MCA occlusion.
In perspective, better comprehension and exploitation of the mechanisms of early ischemic tolerance might enable us to develop new strategies for the treatment of patients with acute ischemic stroke, in particular in patients at high risk for an ischemic insult.



XI) Inflammation

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.


Interleukin-6 (IL-6) is an essential inflammatory mediator, and significant elevations of serum IL-6 levels have been reported in stroke patients within 24 hours of the ischemic event (Waje-. In the clinical setting, circulating IL-6 levels have been shown to correlate with larger infarct volume, increased stroke severity, and less favorable outcome. Although deleterious effects related to IL-6’s pro-inflammatory and pyrogenic actions have been described following ischemia, there is also evidence for direct neuroprotective and neurotrophic actions.
Whereas acutely, a surge of cytokines may induce oxidative stress and endothelial dysfunction, longer-term beneficial effects of IL-6 after stroke have not been investigated. Therefore, we test the hypothesis that this key pro-inflammatory cytokine promotes recovery after ischemic stroke.


XII) Clinical Research Group KFO213 (Harms/ Endres)

Clinical Research Group KFO213, TP5, DFG/HA5471-1 (Harms, Endres)undefined

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Multimodal imaging of inflammation and angiogenesis in experimental glioma and stroke using monomeric ultra-small iron oxide particles (USPIOs)

The goal of this project is to visualize inflammation and angiogenesis in stroke and experimental glioma. The Clinical Research Group KFO213 has developed innovative citrate coated ultrasmall iron oxide particles (USPIOs) which are coupled to a near-infrared fluorescent dye (Cy5.5.), too. The basic hypothesis is that it is possible to detect cellular migration of inflammatory cells and endogenous neuronal stem cells towards the lesion (experimental glioma or focal ischemia) and that distinct populations of cells can be tracked depending on the mode of application, i.e. intracerebroventricular or intravenous administration. Both, spatiotemporal resolution using repeated MRI scans and non-invasive and invasive near-infrared fluorescence imaging (NIRF) will be used to verify the signal within inflammatory cells. These qualitative data will be further validated by a new susceptrometric approach for specific detection of nanoparticles in collaboration with the Physikalisch- Technische Bundesanstalt (PTB),. This will allow to differentiate the USPIO signal from iron “contamination” (eg from blood in haemorrhagic transformations) and to absolutely quantify tissue particle concentration.

In addition, target-specific USPIOs coupled to antibodies or antibody fragments against inflammatory (CD40) or neo-angiogenic epitopes will be generated and characterized using multimodal imaging approaches. Our vision is to pave the way for a clinical use of these compounds for applications in man.

  • Collaboration with the AG Wunder/Dirnagl: Jan Klohs
  • Collaboration with the Physikalisch-Technische Bundesanstalt: AG Lutz Trahms, Frank Wiekhorst
  • Collaboration within the Clinical Research Group: AG Schellenberger

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Funding

  • Deutsche Forschungsgemeinschaft
  • Volkswagen-Stiftung
  • Bundesministerium für Bildung und Forschung
  • ARISE
  • EU-Stroke
  • Industry

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Selected references

Kronenberg G, Harms C, Sobol RW, Cardozo-Pelaez F, Linhart H, Winter B, Balkaya M, Gertz K, Gay SB, Cox D, Eckart S, Ahmadi M, Juckel G, Kempermann G, Hellweg R, Sohr R, Hörtnagl H, Wilson SH, Jaenisch R, Endres M.
Folate deficiency induces neurodegeneration and brain dysfunction in mice lacking uracil DNA glycosylase, J Neurosci. 2008 Jul 9;28(28):7219-30

Hauck L, Harms C, An J, Rohne J, Gertz K, Dietz R, Endres M, von Harsdorf R.
Protein kinase CK2 links extracellular growth factor signaling with the control of p27(Kip1) stability in the heart. Nat Med. 2008 Mar;14(3):315-24. Epub 2008 Mar 2. Erratum in: Nat Med. 2008 May;14(5):585. An, Junfeng

Ji S, Kronenberg G, Balkaya M, Färber K, Gertz K, Kettenmann H, Endres M. Acute neuroprotection by pioglitazone after mild brain ischemia without effect on long-term outcome. Exp Neurol. 2009 Apr;216(2):321-8.

Endres M, Engelhardt B, Koistinaho J, Lindvall O, Meairs S, Mohr JP, Planas A, Rothwell N, Schwaninger M, Schwab ME, Vivien D, Wieloch T, Dirnagl U.
Improving outcome after stroke: overcoming the translational roadblock. Cerebrovasc Dis. 2008;25(3):268-78. Review.

Endres M, Laufs U, The medical case for the development of an intravenous statin formulation--beyond ischemic stroke. Cerebrovasc Dis. 2008;25(6):593-4.

Prinz V, Laufs U, Gertz K, Kronenberg G, Balkaya M, Leithner C, Lindauer U, Endres M. Intravenous rosuvastatin for acute stroke treatment: an animal study, Stroke. 2008 Feb;39(2):433-8

Leithner C, Gertz K, Schröck H, Priller J, Prass K, Steinbrink J, Villringer A, Endres M, Lindauer U, Dirnagl U, Royl G. A flow sensitive alternating inversion recovery (FAIR)-MRI protocol to measure hemispheric cerebral blood flow in a mouse stroke model. Exp Neurol. 2008 Mar;210(1):118-27.

Harms C, Blei K, Harms U, Seidel K, Hauck L, An J, Muselmann C, Ruscher K, Meisel A, Kronenberg G, Dirnagl U, von Harsdorf R, Endres M*, Hörtnagl H* (2007)
PI3-Akt-kinase-dependent phosphorylation of p21 as a novel mechanism of neuroprotection by glucocorticoids. J Neurosci, 27:4562-4571

Gertz K, Priller J, Kronenberg G, Winter B, Schröck H, Ji S, Milosevic M, Harms C, Böhm M, Dirnagl U, Laufs U, Endres M (2006), Physical activity improves long-term stroke outcome via eNOS-dependent augmentation of neo-vascularization and cerebral blood flow. Circulation Res 99: 1132-1140

Endres M, Laufs U, Liao JK, Moskowitz MA (2004), Targeting eNOS for stroke protection. Trends Neurosci 27:283-289

Endres M, Laufs U. Effects of statins on endothelium and signaling mechanisms. Stroke. 2004 Nov;35(11 Suppl 1):2708-11.

Endres M, Biniskiewicz D, Sobol RW, Harms C, Ahmadi M, Lipski A, Katchanov J, Mergenthaler P, Dirnagl U, Wilson SH, Meisel A, Jaenisch R (2004)
Increased postischemic brain injury in mice deficient in uracil DNA glycosylase. J. Clin. Invest. 113:1711-1721

Winter B, Bert B, Fink H, Dirnagl U, Endres M (2004)
Dysexecutive syndrome after mild cerebral ischemia? Mice learn normally but have deficits in strategy-switching. Stroke 35:191-195

Endres M, Gertz K, Lindauer U, Katchanov J, Schultze J, Schröck H, Kuschinsky W, Dirnagl U, Laufs U (2003)
Physical activity protects from stroke in mice via eNOS-dependent mechanisms. Ann Neurol, 54:582-590

Gertz K, Laufs U, Nickenig G, Böhm M, Dirnagl U, Endres M (2003)
Withdrawal of statin treatment abrogates stroke protection in mice. Stroke, 34:551-557

Katchanov J, Harms, C, Gertz K, Priller J, Waeber C, Hirt L, Brück W, Moskowitz MA, Bhide P, Dirnagl U, Endres M (2001)
Mild cerebral ischemia induces loss of cyclin dependent kinase inhibitors and activation of cell cycle machinery prior to delayed neuronal cell death. J Neurosci, 21:5045-5053

Endres M, Meisel A, Namura S, Prass K, Ruscher K, Lipski A, Jaenisch R, Moskowitz MA, Dirnagl U (2000)
DNA methyltransferase contributes to delayed ischemic brain injury. J Neurosci, 20:3175-3181

Endres M, Fink K, Zhu J, Stagliano NE, Bondada V, Geddes JW, Azuma T, Mattson MP, Kwiatkowski DJ, Moskowitz MA (1999)
Neuroprotective effects of gelsolin and analogues during murine stroke. J Clin Invest 103:347-354

Endres M, Laufs U, Huang Z, Nakamura T, Huang PL, Liao JK, Moskowitz MA (1998)
Stroke protection by 3-hydroxy-3 methylglutaryl (HMG)-CoA reductase inhibitors mediated by endothelial nitric oxide synthase. Proc Natl Acad Sci USA 95:8880-8885

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Team Endres

Project Leader
Endres, Matthias, MD

Project Team
Baldinger, Tina, Biologist
Böhmerle, Wolfgang, MD
Ebinger, Martin, MD
Gertz, Karen, MD
Jekili, Jan
Harms, Christoph, MD (group leader)
Harms, Ulrike, MD
Kronenberg, Golo, MD
Kunz, Alexander, MD
Liman, Thomas, MD
Prinz, Vincent, MD
Winter, Benjamin, MD

Students:
Balkaya, Mustafa
Dätwyler, Anna-Lena
Hetzer, Anna-Maria
Hübner, Denise
Lee, Sabrina

Technicians:
Herrmann, Bettina
Strahl, Nadja

COOPERATIONS

International:
M.A. Moskowitz, Boston, USA
R. Jaenisch, Camebridge, USA
R. Sobol, Pittsburgh, USA
C. Waeber, Boston, USA
R. von Harsdorf, Toronto, Canada
E.  Dejana, Milan, Italy
I. Torres-Aleman, Madrid, Spain
P. Bhide, Boston, USA

National:
U. Laufs, Homburg Saar
K. Fink, Bonn
M. Götz, Neuherberg/München
W. Huttner, Dresden
G. Kempermann, Dresden
W. Kuschinsky, Heidelberg
J. Schulz, Aachen
R. Stumm, Magdeburg

Berlin Area:
D. Schmitz, Charité
H. Kettenmann, Charité
I. Buschmann, Charité
J. Priller, Charité
P. Vaykoczy, Charité
H. Hörtnagl, Charité
AL. Pina, Charit
P.U. Heuschmann
M. Taupitz, Charité
R. Veh, Charité
J. Schwab, Charité
H. Audebert, Charité
I. Häuser, Charité