Clinic-oriented Therapy Assessment

© Photo Clinic and Polyclinic for Nuclear Medicine & Department of Neuroradiology, University of Leipzig

The Clinic-oriented Therapy Assessment Unit tests and develops innovative diagnosis and therapy procedures for ischemic stroke. As the possibility of being able to transfer findings from current laboratory rodent models to human patients is sometimes only very limited, a globally unique large-animal model was established for the translational approach. Using this model means that a therapeutic or diagnostic principle can be tested under conditions which come close to patient treatment in a clinical setting. Both the gyrencephalic brain structure and the size of the brain much more closely resemble the human situation in the sheep model as opposed to in the small animal.

Both active ingredients and cell-therapeutic approaches can be developed and evaluated using this model. Functional characterization occurs through a sensory-motor behaviour test developed especially for this purpose. Imaging in a clinic-oriented environment (CT, MRI, PET in cooperation with the University of Leipzig) permits structural and morphological analyses which can be supplemented by comprehensive histological examinations.

In addition, an MRI-based stereotactic system for intracranial applications is available, which is used for therapeutic purposes (e.g. intracranial stem cell administration) or for inducing pathological conditions (e.g. brain haemorrhage model).

Anesthesia

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 our unit. Apart from inhalation anesthesia we also employ perfusion anesthesia (at MRI). 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.

Imaging examination using clinical scanners and its evaluation

3D-reconstruction of skin and bone from head of a sheep (CT data set).
© Photo Fraunhofer IZI

3D-reconstruction of skin and bone from head of a sheep (CT data set).

In cooperation with partners of the University of Leipzig and the Experimental Imaging Unit, state-of-the-art imaging techniques are established for the large animal (sheep) model. Alterations occurring after stroke can be specifically detected by means of anatomical (computed tomographic imaging (CT)) and anatomical-functional (magnetic resonance imaging (MRI)) examinations at the Clinic for Radiology, Department of Neuroradiology (Prof. K.-T. Hoffmann). In addition, metabolic processes can be visualized by means of positron emission tomography (PET) at the Clinic for Nuclear Medicine (Prof. H. Barthel). A wide range of quantifying evaluation routines allows for an exact evaluation of alterations.

DNA RNA isolation

RNA and DNA can be isolated and processed from nucleated cells. Therefore different isolation methods can be used and adapted to diverse questions. Thereafter the prepared nucleic acids can be utilized directly for gene expression analysis.

Experimental cerebral ischemia

Surgical induced stroke in sheep.
© Photo Fraunhofer IZI

Surgical induced stroke in sheep.

Suitable disease models are required for developing novel therapeutic approaches and investiging pathophysiological processes. The unit has a permanent, transcranial model in sheep by the occlusion of cerebral middle artery (MCAO) for simulating a cerebral ischemia. Depending on the localization of the occlusion (one artery branch, two artery branches or total MCAO), the interruption of blood supply leads to a low-grade or moderate focal stroke.

Determination of excitotoxicity and viability tests

The effects of chemicals or other substances to cellular test systems are examined by vitality and toxicity test (LDH and MTT-Assays). The measurement parameters of both photometric tests serve as a readout system for the viability status of cell cultures.

Histological and immunohistochemical stainings

Immunohistochemical staining of astrocytes (green) and nuclei (blue) in the brain of sheep.
© Photo Fraunhofer IZI

Immunohistochemical staining of astrocytes (green) and nuclei (blue) in the brain of sheep.

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.

Confocal laser scanning microscopy

In this unit, the qualitative and quantitative analysis of fluorescence-dyed samples is conducted using a confocal microscope (Zeiss LSM710) and is provided in collaboration with the Experimental Imaging Unit. This microscopic technology allows non-overlapping recordings of specific signals in tissues or cell cultures. For further processing and 3D image analysis, the acquisition of images is complemented by the complex software package Imaris.

Cultivation, differentiation and characterization of pluripotent cell populations for cell therapeutic applications

With respect to cell therapeutic applications, a cultivation of multipotent cells and progenitors is conducted and established in present protocols for the proliferation and differentiation which can be transferred to other cell systems. Diverse proliferation assays are processed as well as differentiation of neural progenitors to neurons and glial cells.

Molecular biological analysis

By using polymerase chain reaction (PCR) the subsets of DNA can be amplified and analyzed. Genetic or disease-related alterations in different tissues or cellular test systems can be detected. Furthermore responses of cell cultures to other factors can be decoded and described.

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).

Neurosurgical operations and monitoring

Surgical interventions take place under sterile conditions.
© Photo Fraunhofer IZI

Surgical interventions take place under sterile conditions.

For examining therapeutic effects in large animals we established a transcranial MCAO model in sheep. The sensitive operation techniques facilitate long-term examinations over several weeks. Moreover, our range of capabilities comprises various neurosurgical operation techniques. The unit has a wide range of equipment  at its disposal for neurosurgery and soft tissue surgery, including a surgical drill system (microspeed® uni, Aesculap) and an electrosurgical system (KLS Martin). Furthermore a continuous registration of ECG, oxygen saturation, expiratory CO2, body temperature, arterial blood pressure (invasive or non-invasive) as well as punctual control of the arterial blood gases is conducted.

Protein biochemical detection methods

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.

Investigation of neurological deficits

The clinical neurological evaluation of stroke in the large animal model is based on the quantification of functional deficits. Therefore the group has established a sensomotoric, neurological test based on the neurological analysis of dogs. The alterations are video-documented and evaluated in a blinded manner.

Stereotactic neuronavigation in large animals

Pre-operative planning of the stereotactic approach on the computer.
© Photo Fraunhofer IZI

Pre-operative planning of the stereotactic approach on the computer.

Local minimal invasive interventions with the lowest possible interference with the brain are based on state-of-the-art imaging techniques using computed tomographic or magnetic resonance imaging. A specific stereotactic system (Brainsight®) for large animals facilitates accurate and safe neuronavigation, close to the human situation.

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

Logo EFRE

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

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).
© Photo 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).
© Photo 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.
© Photo 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.
© Photo 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.

Logo EFRE

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

  • Cleveland Clinic, Department of Neurological Surgery
  • Lake Bioscience
  • Sonovum AG, Leipzig, Germany
  • Stanford University, Department of Neurosurgery
  • University Medical Center of the Johannes Gutenberg University Mainz, Institute for Microscopic Anatomy and Neurobiology, Research Group Molecular Imaging and Optogenetics,
  • University of Greifswald, Clinic for Neurology
  • University of Leipzig, Clinic for Neurology
  • University of Leipzig, Clinic for Nuclear Medicine
  • University of Leipzig, Clinic for Radiology
  • University of Leipzig, Institute for Medical Informatics, Statistics and Epidemiology
  • University of Leipzig, Institute for Veterinary Pathology
  • University of Leipzig, Institute of Veterinary Anatomy, Histology and Embryology
  • University of Leipzig, Large Animal Clinic for Surgery
  • University of Leipzig, Large Animal Clinic for Theriogenologie and Ambulatory Services
  • University of Leipzig, Paul-Flechsig-Institute for Brain Research
  • University of Leipzig, Translational Centre for Regenerative Medicine (TRM) Leipzig
  • University Hospital Carl-Gustav-Carus Dresden, Department for Neuropathology
  • VITA 34 International AG

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