Clinic-oriented Therapy Assessment

Evaluation of therapeutic drugs in transient stroke model in sheep

MRI image of acute stroke in the sheep; green areas reduced or not perfused at all (A) and depiction of the corresponding cerebral lamella following TTC staining; red areas vital tissue, white areas dead tissue (B)
© Fraunhofer IZI

MRI image of acute stroke in the sheep; green areas reduced or not perfused at all (A) and depiction of the corresponding cerebral lamella following TTC staining; red areas vital tissue, white areas dead tissue (B)

The human brain needs a blood supply of 80ml/100g brain tissue and minute. Massive functional and tissue loss occurs by serious restriction of the blood flow (e.g. an occlusion of a cerebral artery) in a circumscribed brain area: A stroke occurs. Brain areas, which are completely interrupted by blood and with this oxygen supply, die very quickly. The affected brain tissue can be rescued from die-off if the oxygen level in the affected area is increased immediately. Hence, an intermediate and sustained accumulation of oxygen recovery is crucial to the patient’s recovery. The current available treatments such as the reopening of the blood vessel have a very limited time window of 4.5 hours after onset and a lot of contraindications. Therefore, approximately only 30 percent of the stroke patients are treated.

The company Omniox Inc. and the Fraunhofer Institute for Cell Therapy and Immunology are striving to improve this situation with a new therapy concept. This concept is based on a treatment that increases the oxygen level specifically in low- or non-perfused tissue at an early stage thereby extending the time window for reopening the blood vessel and limiting the affected stroke area. On the basis of highly diagnostic models, the safety as well as the efficacy of the described procedure is verified. In doing so, a specialized imaging procedure for a localization-based description of blood supply and diffusion capacity and therefore oxygen supply is performed in cooperation with the Clinic of Nuclear Medicine at the University of Leipzig. On the basis of these investigations, the treatment can be optimized accordingly and the development of a therapeutic agent can be driven forward.

External partners
Department for Nuclear Medicine, University Hospital Leipzig
Omniox Inc.

Reference project: Autologous bone marrow transplantation after stroke in a large animal model

The establishment of a therapy protocol for ischemic stroke on the basis of stem cell-containing bone marrow populations was successful only recently. Following successful preliminary experiments in our rat models, the therapy protocol was translated into a large animal model. 24 hours after induction of an ischemic infarction by permanent occlusion of the central brain artery the therapy was performed in the sheep model by intravenous delivery of autologous bone marrow cells. By means of continuous behavioral-phenotypic and imaging monitoring using state-of-the-art examination routines comprising magnetic resonance and positron emission imaging the success of the therapy (reduction of infarction area and functional failures) was validated and histologically confirmed later on. Imaging was conducted in cooperation with partners at the University of Leipzig.

External partners
Department for Nuclear Medicine, University Hospital Leipzig
Department for Neuroradiology, University Hospital Leipzig
Faculty for Veterinary Medicine, University of Leipzig
Vita 34

This project is co-financed by tax revenues on the basis of the budget approved by members of the Saxon state parliament.

Increased cerebral blood flow in a sheep model of stroke

Coronal (left), sagitale (middle) and transversale (right) picture of a sheep brain with 3D T1 MRI (top), 15O-H2O PET (middel) and overlay (bottom).
© Fraunhofer IZI

Coronal (left), sagitale (middle) and transversale (right) picture of a sheep brain with 3D T1 MRI (top), 15O-H2O PET (middel) and overlay (bottom).

Despite the establishment of specialized care units (stroke units), sufficient treatment of the remaining brain tissue (penumbra) after stroke is limited to only a few hours. Herein, we tested the hypothesis that a selective increase in cerebral blood flow could reduce the cerebral damage. Therefore, an inhaled neuroprotectant was tested under clinical-related conditions in a large animal model of stroke. Stroke was induced in adult sheep by occlusion of the middle cerebral artery. Serial imaging of cerebral blood flow with and without treatment was perform­ed within the next 4.5 hours using highly sophisticated imaging procedures together with our partners (positron emission tomography and magnetic resonance imaging). Inhalative treatment results in a selective increase of cerebral blood flow within the penumbra while the proportion of potentially salvageable brain tissue may be increased.

External partners
University Hospital Leipzig, Department for Nuclear Medicine
University Hospital Leipzig, Department for Neuroradiology
University of Munich, Walter-Brendel Center for Experimental Medicine
University of Leipzig, Faculty of Veterinary Medicine

Labeling and relocalization of ovine mesenchymal stem cells using VSOPs (Very Small Superparamagnetic Iron Oxide Particles)

Iron staining using the Berlin blue method (left) shows the incorporation of VSOPs (blue) into ovine MSCs (red, counterstaining with eosin), resulting in a circumscribed signal loss (red circle).
© Fraunhofer IZI

Iron staining using the Berlin blue method (left) shows the incorporation of VSOPs (blue) into ovine MSCs (red, counterstaining with eosin), resulting in a circumscribed signal loss (red circle).

Various preclinical studies have shown the efficacy of a transplantation of mesenchymal stem cells (MSCs) after stroke. Knowledge regarding the mechanism of action of this therapeutic option, however, is as yet limited. In the present study a labeling protocol involving VSOPs was thus developed for MSCs from sheep, aiming at the localization of the autologously transplanted stem cells in the living organism with the aid of magnetic resonance imaging (MRI). After determining the viability and labeling intensity, different quantities of VSOP-labeled stem cells were stereotactically transplanted into the cerebrum of sheep and the detectability limit was determined using 3T MRI (Siemens) and histological methods. In the further course of the experiment, the autologous VSOP-labeled MSCs from bone marrow are employed after stroke in order to investigate their migration behavior under pathological conditions.

External partners
University Hospital Leipzig, Department for Neuroradiology
University of Leipzig, Faculty of Veterinary Medicine
University of Leipzig, Institute for analytic Chemistry
University of Greifswald, Institute for Diagnostic Radiology and Neuroradiology
Technical University Munich, Institute for Neurosciences

Stroke therapy using NSC: Investigations in the large animal model

Image of fibers (red, DTI MRI) of the brain (T1 MRI) after stroke (light areas, DWI MRI) created by fiber tracking in the three-dimensionally reconstructed brain.
© Fraunhofer IZI

Image of fibers (red, DTI MRI) of the brain (T1 MRI) after stroke (light areas, DWI MRI) created by fiber tracking in the three-dimensionally reconstructed brain.

This investigation aims at verifying the efficacy of neuronal stem cells (obtained before the 2007 deadline) after cerebral ischemia in the large animal (sheep) model over time while accounting for relevant mechanisms. Against the background of the intended application in human regenerative medicine, these cells of the human neural cell line, which were produced according to GMP regulations, were characterized in various small animal models – according to the criteria of the Food and Drug Administration (FDA, USA) and under GMP conditions – by the group around the neurosurgeon Prof. Gary Steinberg of Stanford (Palo Alto, CA, USA). The project aims at investigating questions on the therapeutic efficacy of local neural stem cell transplantation in the large animal (sheep) model, testing the safety of this application after local administration in the gyrencephalic brain over long time periods and validating the feasibility of an individualized (i. e. specifically customized for the present infarction pattern) treatment protocol by (stereotactic) administration of the cells.

External partners
Stanford University, Kalifornien, USA
University of Leipzig, Translational Center for Regenerative Medicine (TRM)
University Hospital Leipzig, Department for Nuclear Medicine
University Hospital Leipzig, Department for Neuroradiology
University of Leipzig, Faculty of Veterinary Medicine

Evaluation of ultrasonic system for the early discrimination between cerebral ischemia and hemorrhage in an ovine model system

T2-weighted MR-image of a cerebral hemorrhage in the right hemisphere. Beside the ischemic model this is a reference object for the ultrasound.
© Fraunhofer IZI

T2-weighted MR-image of a cerebral hemorrhage in the right hemisphere. Beside the ischemic model this is a reference object for the ultrasound.

Currently the only approved treatment of acute ischemic stroke is the recanalization of the blocked vessel by using alteplase for thrombolysis. This approach is restricted to a narrow time window of 4.5 hours after the onset. Beside the general diagnosis of stroke a more detailed characterization to differentiate between ischemia and hemorrhage (strict contra-indication) is mandatory. At present a time-consuming imaging procedure is required (e. g. MRI, CT), but is only available in specialized clinics.

A new kind of ultrasound technology is being evaluated in a large animal model to reach an early diagnosis in acute patients for providing a rapid and distinct diagnostic assessment. Beside the evaluation of a safe administration, the effectiveness and sensitivity is determined.

The stroke model in sheep available in our institute serves as a basis for this study. An MRI-based stereotactic procedure was established for implementing a cerebral hemorrhage model. In a pilot study, we demonstrated the feasibility of the chosen models and procedures for a proof of concept.

External partner
SONOVUM AG

This project is co-financed by tax revenues on the basis of the budget approved by members of the Saxon state parliament.