Clinic-oriented Therapy Assessment

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)
© 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
© 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
© 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
© 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
© 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.