Ischemia Research

The focus of this unit is the investigation and development of novel therapeutics and diagnostics for ischemic stroke and myocardial infarction. Preclinical evaluations occur stepwise from cell culture to animal models. The implementation of strict quality standards increases the predictive value of preclinical research and may therefore improve the translation of promising therapies to the clinic.


An adapted and sensitive anesthetic performance is the basis for demanding examinations. Various techniques for local and general short- and long-term anesthesia are established in this unit. Apart from inhalation anesthesia we also employ perfusion anesthesia (in the MRI device). For this purpose we have standard devices for use in human medicine (Primus and Titus A, Draeger), MRI-compatible inhalation systems (Servo 900D, Siemens) and high-precision perfusion pumps (Perfusor® compact S, Braun Melsungen AG). Moreover, the animals are observed by extensive pre-, intra- and post-operative monitoring.

Flow cytometry

Double staining cytotoxic T cells (CD3 / CD8) in rat blood.
© Fraunhofer IZI

Double staining cytotoxic T cells (CD3 / CD8) in rat blood.

We conduct flow cytometric examinations and characterizations of a wide variety of primary and cultivated cells using equipment by Beckman Coulter (FC500, 7 parameters) and Becton Dickinson (FacsCalibur, 6 parameters). We can fall back on many years of experience in establishing multicolor experiments and the analysis of data thus obtained. Furthermore, using the Fraunhofer IZI's high-speed cell sorter MoFlo II (3 lasers, 9 parameters), we are capable of purifying specific subpopulations from heterogeneous cell mixtures in multicolor approaches with up to 7 different fluorochromes.

Experimental cerebral ischemia

Surgically induced stroke.
© Fraunhofer IZI

Surgically induced stroke.

Suitable disease models are required for the development of novel therapeutic approaches and the investigation of pathophysiological processes. The unit has different models at its disposal for simulating a cerebral ischemia. Depending on the localization of the occlusion site, the permanent or transient interruption of blood supply leads to a focal stroke or to global cerebral ischemia, which is typically observed after cardiac arrest.

Determination of excitotoxicity and viability tests

© Fraunhofer IZI
Murine primary neural cells in culture. Different parameters of the cells are measured in an in vitro ischemia model.
© Fraunhofer IZI

Murine primary neural cells in culture. Different parameters of the cells are measured in an in vitro ischemia model.

The cell-specific effects of the substances employed in the test system are examined by means of photometric viability assays. The measurement parameters of both tests serve as a readout system for the viability status of primary cell cultures.

Production of primary cell transplants

Mononuclear cells from bone marrow biopsies as well as cell populations containing stem cells from subcutaneous fat tissue are obtained for regenerative cell therapy. The primary tissues of fat or bone marrow are dissociated mechanically and / or enzymatically to form single cell suspensions and are subsequently purified using different separation techniques (density gradient centrifugation, magnetic cell selection with the aid of specific cell surface markers (MACS® technology; Miltenyi Biotech).

Histological and immunohistochemical stainings

Immunohistochemical staining of neurons (green) and nuclei (blue).
© Fraunhofer IZI

Immunohistochemical staining of neurons (green) and nuclei (blue).

The unit has many years of experience with frozen and paraffin sections. The entire spectrum of histological and immunohistochemical stainings can be performed on different tissues.

In vitro stroke model

Our model comprises the isolation and cultivation of hippocampal and cortical neurons as well as glial cells from the embryonic brain of mice and rats. Using these in vitro systems the complex processes occurring in the brain during a stroke are represented in a simplified, two-dimensional system in which the neural cultures are subjected to a temporary deprivation of oxygen and glucose. The test system is employed for the systemic and cell-specific analysis of potential neurotoxic, neuroprotective and neuroregenerative substances.

Isolation of viable primary immune cells from nerve tissue

Separation of myelin and membrane fragments using density gradient centrifugation.
© Fraunhofer IZI

Separation of myelin and membrane fragments using density gradient centrifugation.

By means of our self-established tissue lysis method and purification using density gradient centrifugation it is possible to isolate resident and infiltrated immune cells from the brain of the rat animal model for use in immunological characterizations. Thus, immunological processes within the framework of CNS pathologies (stroke, Alzheimer's disease, etc.) can be extensively examined on the single cell level, e.g. using four-color flow cytometry.

Molecular biological analysis of mechanisms of action

Curve of amplification qRT-PCR.
© Fraunhofer IZI

Curve of amplification qRT-PCR.

Potential mechanisms of action of a cell-based therapy are analyzed by establishing profiles of transcription (qRT-PCR) and protein expression (SDS PAGE / Western Blot).

Perioperative physiological monitoring in small animals

Original registration perioperative monitoring.
© Fraunhofer IZI

Original registration perioperative monitoring.

As required, a continuous registration of ECG, oxygen saturation and arterial blood pressure (invasive or non-invasive) as well as a selective control of the arterial blood gases is conducted in addition to monitoring and regulation of body temperature and, optionally, pressure- or volume-controlled ventilation.

Preclinical study design

The implementation of quality criteria is of crucial importance for the predictive value of preclinical studies. The members of this unit thus set a high value on recommendations of the relevant expert groups. These include, among others, the a priori calculation of group sizes, the randomized assignment to experimental groups, the selection of independent and suitable end points, the blinded analysis of end points and the cooperation with biomathematicians for study planning and evaluation.

Protein biochemical detection methods

Expression of the astrocyte marker GFAP rises up to 72h after oxigen glucose deprivation. C (Control), OGD (Oxygen-Glucose Deprivation).
© Fraunhofer IZI

Expression of the astrocyte marker GFAP rises up to 72h after oxigen glucose deprivation. C (Control), OGD (Oxygen-Glucose Deprivation).

Further examination of post-ischemic regulatory mechanisms is implemented using immunofluorescent stainings and FACS analyses. Moreover, protein characterizations by means of immunoprecipitation and Western blot are employed. These methods facilitate quantitative and qualitative analyses of cells and proteins with the aid of specific antibodies.

Quantification of neurological deficits

The assessment of therapeutic success in experimental animal models of neurological diseases is dependent on the quantification of functional deficits. The unit has various neurological test systems at its disposal by means of which it is possible to measure and compare sensomotoric and cognitive abilities.


Stereological counts are the gold standard for error-free quantitative analyses in tissues. By using different algorithms it is possible to quantify object frequencies, surfaces and volumes as well as lengths and branching degrees of structures.

Stereotactic interventions in small rodents

After systemic delivery, certain substances are incapable of passing the blood-brain barrier and therefore have to be delivered directly into the brain tissue. Stereotaxy allows the targeted application of substances or cells to selected areas of the brain.

Behavioral / physiological examinations

The clinically neurological evaluation of stroke in the large animal model is based on established neurological tests. The alterations are video-documented and evaluated in a blinded manner.

Isolation and characterization of cells from lymphatic organs and blood

Pathological alterations of the central nervous system (stroke, Alzheimer's disease, MS) are in most cases associated with significant systemic effects. Above all, interactions with the peripheral immune system play a significant role in this context. For the examination of immunological processes, e.g. after stroke, we employ tissue lysis and / or density gradient centrifugation for the isolation of primary immune cells from various tissues (spleen, lymph nodes, bone marrow, blood) for flow cytometric analyses and cell assays, respectively. Moreover, we are capable of purifying specific subpopulations by means of different methods (including methods based on magnetic beads, FACS).

  • Balseanu AT, Buga AM, Catalin B, Wagner DC, Boltze J, Zagrean AM, Reymann K, Schaebitz W, Popa-Wagner A. Multimodal approaches for regenerative stroke therapies: combination of granulocyte colony-stimulating factor with bone marrow mesenchymal stem cells is not superior to G-CSF alone. Frontiers in Aging Neuroscience. 2014 Jun 23;6:130.DOI
  • Boltze J, Ayata C, Wagner DC, Plesnila N. Preclinical phase III trials in translational stroke research: call for collective design of framework and guidelines. Stroke. 2014 Feb;45(2):357. DOI
  • Boltze J, Lukomska B, Jolkkonen J; MEMS–IRBI consortium. Mesenchymal stromal cells in stroke: improvement of motor recovery or functional compensation? Journal of Cerebral Blood Flow and Metabolism. 2014 Aug;34(8):1420-1. DOI
  • Diederich K, Schmidt A, Beuker C, Strecker JK, Wagner DC, Boltze J, Schäbitz WR, Minnerup J. Granulocyte colony-stimulating factor (G-CSF) treatment in combination with transplantation of bone marrow cells is not superior to G-CSF treatment alone after cortical stroke in spontaneously hypertensive rats. Frontiers in Cellular Neuroscience. 2014 Dec 4;8:411. DOI
  • Fronz U, Deten A, Baumann F, Kranz A, Weidlich S, Härtig W, Nieber K, Boltze J, Wagner DC. Continuous adenosine A2A receptor antagonism after focal cerebral ischemia in spontaneously hypertensive rats. Naunyn Schmiedeberg's Archives of Pharmacoloy. 2014 Feb;387(2):165-73. DOI
  • Kaiser D, Weise G, Möller K, Scheibe J, Pösel C, Baasch S, Gawlitza M, Lobsien D, Diederich K, Minnerup J, Kranz A, Boltze J, Wagner DC. Spontaneous white matter damage, cognitive decline and neuroinflammation in middle-aged hypertensive rats: an animal model of early-stage cerebral small vessel disease. Acta Neuropatholica Communications. 2014 Dec 18;2(1):169. DOI
  • Minnerup J, Wagner DC, Strecker JK, Pösel C, Sevimli-Abdis S, Schmidt A, Schilling M, Boltze J, Diederich K, Schäbitz WR. Bone marrow-derived mononuclear cells do not exert acute neuroprotection after stroke in spontaneously hypertensive rats. Frontiers in Cellular Neuroscience. 2014 Jan 8;7:288. DOI
  • Möller K, Boltze J, Pösel C, Seeger J, Stahl T, Wagner DC. Sterile inflammation after permanent distal MCA occlusion in hypertensive rats. Journal of Cerebral Blood Flow and Metabolism. 2014 Feb;34(2):307-15. DOI
  • Pieroh P, Koch M, Wagner DC, Boltze J, Ehrlich A, Ghadban C, Hobusch C, Birkenmeier G, Dehghani F. Temporal dynamics of glyoxalase 1 in secondary neuronal injury. PLoS One. 2014 Feb 3;9(2):e87364. DOI
  • Pösel C, Scheibe J, Kranz A, Bothe V, Quente E, Fröhlich W, Lange F, Schäbitz WR, Minnerup J, Boltze J, Wagner DC. Bone marrow cell transplantation time-dependently abolishes efficacy of granulocyte colony-stimulating factor after stroke in hypertensive rats. Stroke. 2014 Aug;45(8):2431-7.DOI
  • Pösel C, Uri A, Schulz I, Boltze J, Weise G, Wagner DC. Flow cytometric characterization of brain dendritic cell subsets after murine stroke. Experimental and Translational Stroke Medicine. 2014 Nov 4;6(1):11. DOI
  • Wagner DC, Pösel C, Schulz I, Schicht G, Boltze J, Lange F, Scheibe J, Möller K, Weise G. Allometric dose retranslation unveiled substantial immunological side effects of granulocyte colony-stimulating factor after stroke. Stroke. 2014 Feb;45(2):623-6. DOI
  • Weise G, Lorenz M, Pösel C, Maria Riegelsberger U, Störbeck V, Kamprad M, Kranz A, Wagner DC, Boltze J. Transplantation of cryopreserved human umbilical cord blood mononuclear cells does not induce sustained recovery after experimental stroke in spontaneously hypertensive rats. Journal of Cerebral Blood Flow and Metabolism. 2014 Jan;34(1):e1-9. DOI
  • Boltze J, Kleinschnitz C, Reymann KG, Reiser G, Wagner DC, Kranz A, Michalski D. Neurovascular pathophysiology in cerebral ischemia, dementia and the ageing brain - current trends in basic, translational and clinical research. Exp Transl Stroke Med. 2012 Aug 10;4(1):14.
  • Boltze J, Reich DM, Hau S, Reymann KG, Strassburger M, Lobsien D, Wagner DC, Kamprad M, Stahl T. Assessment of neuroprotective effects of human umbilical cord blood mononuclear cell subpopulations in vitro and in vivo. Cell Transplant. 21 (2012), 4, S. 723-737. DOI
  • Boltze J, Schmidt UR, Reich DM, Kranz A, Reymann KG, Strassburger M, Lobsien D, Wagner DC, Förschler A, Schäbitz WR. Determination of the therapeutic time window for human umbilical cord blood mononuclear cell transplantation following experimental stroke in rats. Cell Transplant. 21 (2012), 6, S. 1199-211. DOI
  • Michalski D, Heindl M, Kacza J, Laignel F, Küppers-Tiedt L, Schneider D, Grosche J, Boltze J, Löhr M, Hobohm C, Härtig W. Spatio-temporal course of macrophage-like cell accumulation after experimental embolic stroke depending on treatment with tissue plasminogen activator and its combination with hyperbaric oxygenation. Eur J Histochem. 2012, 56(2):e14. DOI
  • Möller K, Stahl T, Boltze J, Wagner DC. Isolation of inflammatory cells from rat brain tissue after stroke. Exp Transl Stroke Med. 4 (2012), 1:20. DOI
  • Pösel C, Möller K, Fröhlich W, Schulz I, Boltze J, Wagner DC. Density gradient centrifugation compromises bone marrow mononuclear cell yield. PLoS One. 7 (2012), 12:e50293. DOI
  • Taubert, J. Therapeutischer Einfluss der Transplantation syngener regenerativer Zellen aus dem Fettgewebe nach experimentellem ischämischem Schlaganfall in Spontan Hypertensiven Ratten. 2012, Universität Leipzig, Dissertation
  • Terpolilli NA, Kim SW, Thal SC, Kataoka H, Zeisig V, Nitzsche B, Klaesner B, Zhu C, Schwarzmaier S, Meissner L, Mamrak U, Engel DC, Drzezga A, Patel RP, Blomgren K, Barthel H, Boltze J, Kuebler WM, Plesnila N. Inhalation of nitric oxide prevents ischemic brain damage in experimental stroke by selective dilatation of collateral arterioles. Circ Res. 110 (2012), 5, S. 727-38. DOI
  • Wagner DC, Bojko M, Peters M, Lorenz M, Voigt C, Kaminski A, Hasenclever D, Scholz M, Kranz A, Weise G, Boltze J. Impact of age on the efficacy of bone marrow mononuclear cell transplantation in experimental stroke. Exp Transl Stroke Med. 2012 Aug 24;4(1):17.
  • Wagner DC, Deten A, Härtig W, Boltze J, Kranz A. Changes in T2 relaxation time after stroke reflect clearing processes. Neuroimage.61 (2012), 4, S. 780-5. DOI
  • Weise G, Stoll G. Magnetic resonance imaging of blood brain/nerve barrier dysfunction and leukocyte infiltration: closely related or discordant? Front Neurol. 2012; 3:178. DOI
  • Boltze, J. Neurobiological and clinical aspects of cell therapies for ischemic stroke – an interdisciplinary and translational study. 2011, Universität Leipzig, Dissertation
  • Boltze J, Kranz A, Wagner DC, Reymann K, Reiser G, Hess DC. Recent advances in basic and translational stroke research. Expert Rev Neurother. 2011 Feb;11(2):199-202.
  • Boltze J, Reich DM, Hau S, Reymann KG, Strassburger M, Lobsien D, Wagner DC, Kamprad M, Stahl T. Assessment of neuroprotective effects of human umbilical cord blood mononuclear cell subpopulations in vitro and in vivo. Cell Transplant. 2011 Sep 16. DOI [Epub ahead of print]
  • Boltze J, Schmidt UR, Reich DM, Kranz A, Reymann KG, Strassburger M, Lobsien D, Wagner DC, Förschler A, Schäbitz WR. Determination of the therapeutic time window for human umbilical cord blood mononuclear cell transplantation following experimental stroke in rats. Cell Transplant. 2011 Dec 13. DOI [Epub ahead of print]
  • Glocke, I. Immunhistochemische Differenzierung der astroglialen Reaktion nach experimentellem Schlaganfall in der Ratte. 2011, Universität Leipzig, Bachelorarbeit
  • Jaklin, M. Der Einfluss von Astrozyten auf die Neuroprotektion nach Glucose-Sauerstoff-Entzug. 2011, Universität Leipzig, Bachelorarbeit
  • Michalk, S. Stereologische Quantifizierung der sekundären neuronalen Degeneration im Thalamus nach experimentellem Schlaganfall. 2011, Universität Leipzig, Bachelorarbeit
  • Wagner, DC. Einfluss des Transplantationsweges auf die Effektivität von Zelltherapien nach fokaler zerebraler Ischämie. 2011, Universität Leipzig, Dissertation
  • Wagner DC, Riegelsberger UM, Michalk S, Härtig W, Kranz A, Boltze J. Cleaved caspase-3 expression after experimental stroke exhibits different phenotypes and is predominantly non-apoptotic. Brain Res. 2011 Mar 24;1381:237-42.
  • Michalski D, Pelz J, Weise C, Kacza J, Boltze J, Grosche J, Kamprad M, Schneider D, Hobohm C, Härtig W. Early outcome and blood-brain barrier integrity after co-administered thrombolysis and hyperbaric oxygenation in experimental stroke. Exp Transl Stroke Med. 3 (2011), 5, 9 S. DOI
  • Riegelsberger UM, Deten A, Pösel C, Zille M, Kranz A, Boltze J, Wagner DC. Intravenous human umbilical cord blood transplantation for stroke: impact on infarct volume and caspase-3-dependent cell death in spontaneously hypertensive rats. Exp Neurol. 2011 Jan;227(1):218-23. Epub 2010 Nov 16.
  • Skardelly M, Gaber K, Burdack S, Scheidt F, Hilbig H, Boltze J, Förschler A, Schwarz S, Schwarz J, Meixensberger J, Schuhmann MU. Long-term benefit of human fetal neuronal progenitor cell transplantation in a clinically adapted model after traumatic brain injury. J Neurotrauma. 28 (2011), 3, S. 401-14. DOI
  • Fricke S, Fricke C, Oelkrug C, Hilger N, Schönfelder U, Kamprad M, Lehmann J, Boltze J, Emmrich F, Sack U. Characterization of murine non-adherent bone marrow cells leading to recovery of endogenous hematopoiesis. Cell Mol Life Sci. 67 (2010), 23, S. 4095-4106.
  • Kranz A, Wagner DC, Kamprad M, Scholz M, Schmidt UR, Nitzsche F, Aberman Z, Emmrich F, Riegelsberger UM, Boltze J. Transplantation of placenta-derived mesenchymal stromal cells upon experimental stroke in rats. Brain Res, 1315 (2010), S. 128-36.
  • Nitzsche, F. Analyse einer murinen Astrozytenkultur vor und nach Oxygen-Glucose-Deprivation. 2010, Universität Leipzig, Bachelorarbeit
  • Fisher M, Feuerstein G, Howells DW, Hurn PD, Kent TA, Savitz SI, Lo EH; STAIR Group. Update of the stroke therapy academic industry roundtable preclinical recommendations. Stroke, 40 (2009), 6, S. 2244-2250.
  • Fricke S, Ackermann M, Stolzing A, Schimmelpfennig C, Hilger N, Jahns J, Hildebrandt G, Emmrich F, Ruschpler P, Pösel C, Kamprad M, Sack U. Allogeneic non-adherent bone marrow cells facilitate hematopoietic recovery but do not lead to allogeneic engraftment. PLoS One. 2009 Jul 7;4(7):e6157.
  • Fronz, U. Multimodal evaluation of the neuroprotective potential of CSC in a rodent model of focal cerebral ischemia. 2009, Universität Leipzig, Diplomarbeit
  • Härtig W, Reichenbach A, Voigt C, Boltze J, Bulavina L, Schuhmann MU, Seeger J, Schusser GF, Freytag C, Grosche J. Triple fluorescence labelling of neuronal, glial and vascular markers revealing pathological alterations in various animal models. J Chem Neuroanat, 37 (2009), 2, S. 128-138.
  • Kränkel, A. Modulation der Wachstumsfaktorexpression durch Zelltransplantation nach experimentellem Schlaganfall in der Ratte. 2009, Hochschule Zittau / Görlitz, Bachelorarbeit
  • Meier H, Bullinger J, Marx G, Deten A, Horn LC, Rassler B, Zimmer HG, Briest W. Crucial role of interleukin-6 in the development of norepinephrine-induced left ventricular remodeling in mice. Cell Physiol Biochem, 23 (2009), 3-4, S. 327-334.
  • Saver JL, Albers GW, Dunn B, Johnston KC, Fisher M, STAIR VI Consortium. Stroke Therapy Academic Industry Roundtable (STAIR) recommendations for extended window acute stroke therapy trials. Stroke, 40 (2009), 7, S. 2594-2600.
  • Stroh A, Boltze J, Sieland K, Hild K, Gutzeit C, Jung T, Kressel J, Hau S, Reich D, Grune T, Zimmer C. Impact of magnetic labeling on human and mouse stem cells and their long-term magnetic resonance tracking in a rat model of Parkinson disease. Mol Imaging, 8 (2009), 3, S. 166-78.
  • Wechsler L, Steindler D, Borlongan C, Chopp M, Savitz S, Deans R, Caplan L, Hess D, Mays RW, Boltze J, Boncoraglio G, Borlongan CV, Caplan LR, Carmichael ST, Chopp M, Davidoff AW, Deans RJ, Fisher M, Hess DC, Kondziolka D, Mays RW, Norrving B, Parati E, Parent J, Reynolds BA, Gonzalez-Rothi LJ, Savitz S, Sanberg P, Schneider D, Sinden JD, Snyder E, Steinberg GK, Steindler D, Wechsler L, Weiss MD, Weiss S, Victor S, Zheng T. Stem Cell Therapies as an Emerging Paradigm in Stroke Participants. Stem Cell Therapies as an Emerging Paradigm in Stroke (STEPS): bridging basic and clinical science for cellular and neurogenic factor therapy in treating stroke. Stroke, 40 (2009), 2, S. 510-5.
  • Boltze, J. Experimentelle Zelltherapie des ischämischen Schlaganfalls unter Nutzung stammzellhaltiger Populationen des humanen Nabelschnurbluts in der Ratte. 2008, Universität Leipzig, Dissertation
  • Focke A, Schwarz S, Foerschler A, Scheibe J, Milosevic J, Zimmer C, Schwarz J. Labeling of human neural precursor cells using ferromagnetic nanoparticles. Magn Reson Med. 2008 Dec;60(6):1321-8.
  • Hau S, Reich DM, Scholz M, Naumann W, Emmrich F, Kamprad M, Boltze, J. Evidence for neuroprotective properties of human umbilical cord blood cells after neuronal hypoxia in vitro. BMC Neurosci (2008), 9, S. 30.
  • Reich DM, Hau S, Stahl T, Scholz M, Naumann W, Emmrich F, Boltze J, Kamprad M. Neuronal hypoxia in vitro: investigation of therapeutic principles of HUCB-MNC and CD133+ stem cells. BMC Neurosci (2008), 9, S. 91.
  • Förschler A, Boltze J, Waldmin D, Gille U, Zimmer C. MRI of experimental focal cerebral ischemia in sheep. Rofo 179 (2007), 5, S. 516-24.
  • Schmidt A, Haas SJ, Hildebrandt S, Scheibe J, Eckhoff B, Racek T, Kempermann G, Wree A, Pützer BM. Selective targeting of adenoviral vectors to neural precursor cells in the hippocampus of adult mice: new prospects for in situ gene therapy. Stem Cells. 2007 Nov;25(11):2910-8. Epub 2007 Jul 19.
  • Boltze J, Kowalski I, Forschler A, Schmidt U, Wagner D, Lobsien D, Emmrich J, Egger D, Kamprad M, Blunk J, Emmrich F. The stairway: a novel behavioral test detecting sensomotoric stroke deficits in rats. Artif Organs, 30 (2006), 10, S. 756-763.
  • Boltze J, Kowalski I, Geiger K, Reich D, Gunther A, Buhrle C, Egger D, Kamprad M, Emmrich F. Experimental treatment of stroke in spontaneously hypertensive rats by CD34+ and CD34-cord blood cells. Ger Med Sci, 3, Doc09 (2005), 20051110.