Experimental Imaging


Modern imaging procedures for diagnostics and preclinical research

Visualization of a stroke in a 3D model of a rat’s brain
© Fraunhofer IZI
Visualization of a stroke in a 3D model of a rat’s brain.
3D model of astrocytes based on immunohistochemical staining
© Fraunhofer IZI
3D model of astrocytes based on immunohistochemical staining.
Evidence of calcifications in the aorta of a rat with chronic kidney disease
© Fraunhofer IZI
Evidence of calcifications in the aorta of a rat with chronic kidney disease.

The field of life sciences presents us with a number of imaging methods. Procedures applied in this field draw on a broad span of the electromagnetic spectrum, ranging from short-wave roentgen radiation (computer tomography) to light that is visible to humans (microscopy), right over to the radio frequency range (magnetic resonance imaging). Each one of these procedures is able to pinpoint structures or biological processes in the living organism with great precision. Data can be collected here to create a virtual 3D rendering of the investigated structures.

Based on this image, pathological processes such as those which emerge in the case of a stroke can be precisely quantified. With the aid of magnetic resonance imaging (MRI), different contrast methods and special segmentation algorithms, the damaged area can be depicted in vivo (image 1). Far-reaching rebuilding processes take place in the affected areas of the brain once brain tissue is damaged, for instance due to hypoxia during a stroke. To be able to depict regeneration on a microscopic level, the respective region is marked immunohistochemically and scanned using a confocal laser scanning microscope (LSM). This makes it possible to specifically describe the number and morphology of cells, their interaction with other cells, and their changes over the course of time (image 2). The processes facilitate the quantification of pathological changes following brain damage and therefore lend themselves well to verifying the efficacy of new therapeutic procedures.

Beyond stroke research, other diseases and their progression can also be monitored through experimental imaging procedures. For instance, in the case of chronic kidney disease, both an increase in kidney volume and also calcifications in the aorta emerge as time goes by. In a study, both these disease markers were able to be diagnosed (image 3) with the aid of computed tomography (CT). The next step here is to conduct investigations to identify a more effective treatment for chronic kidney disease.

In future, the competencies of the Experimental Imaging Unit are to be pooled further with those of the Image Analysis of Cell Function Unit and merged into the central imaging and image evaluation facility.

Magnetic resonance imaging description of infarct growth following a stroke in the rat

Various surrogate parameters are required to scientifically describe an effective stroke treatment. One of the quantification procedures frequently used in the literature defines the change in the spread of the stroke over time in the living animal. In addition, magnetic resonance imaging T2-weighted sequences are primarily surveyed and a volumetric evaluation of the affected brain area then carried out. Until now, these analyses were carried out using a 1.5T MR scanner. However, only a limited resolution could be achieved in examinations on small animals. Using the high-field MRI with up to a 7 Tesla field strength can significantly improve this resolution restriction. Besides examinations into the development of the stroke, further surrogate parameters can, of course, also be determined on the living animal.

Stereological evaluation of diaschisis following stroke in the rat

Following a stroke, damage occurs not only in the primary infarct area, but also in more distant regions of the brain – an occurrence referred to as diaschisis. This is how, for example, following ischemic damage in the primary sensorimotor cortex through axonal fibre connectivity, a selective die-off of cells takes place in the thalamus' more distant, ventral, posterior nucleus. The number of secondarily damaged neurons in this core region was determined as part of the project. Precise quantification took place via a stereological analysis using the program Stereo Investigator from MicroBrightField. This program allowed statements to be made on the total number of mortified neurons in the defined region. A stereological analysis is being demanded from an increasing number of scientific publishers as the gold standard for quantifications.

Investigations into distribution kinetics following cell transplantation in the rat

One of the main issues following the application of cells relates to the fact that these cells remain in the recipient organism. In order to shed light on this issue there are currently a range of procedures available, the majority of which involve the examined cells being marked. For this project, tumor cells were marked using a luciferase vector. After administering luciferin, a photochemical reaction occurs in the affected cells, which can be illustrated in the bioluminescence imager. This showed that the affected cell population builds up in the lung following intravenous administration and later likely continues to be redistributed.

Quantification of glial cells following brain tissue damage

Brain tissue damage caused by trauma or hypoxia results in far-reaching changes in the affected areas of the brain. The rebuilding processes do not only affect the vulnerable nerve cells, but also the brain‘s connective and supporting tissue. These cells, referred to by Rudolf Virchow as glia (Greek for ”glue”) have extremely varied tasks to fulfill. They surround the nerve cells and provide them with nutrients, thus con­tribute to the forwarding of information and maintenance of homeostasis in the brain.

Following brain damage, some glial cells experience an en­largement of cells (hypertrophy) and an increase in the number of cells (hyperplasia). This can go so far that it becomes impossible to differentiate between certain glial cells (such as so-called astrocytes) on histological stainings, as they form a tight network of cell bodies and overlapping processes. In spite if this, in order to be able to describe the cells, processes are applied at the Fraunhofer IZI which transform them into definable three-dimensional objects. This makes it possible to quantitatively describe the number of cells, their morphology, interaction with other cells, and their changes over the course of time. For this purpose, the affected tissue is immunohistochemically stained and scanned using confocal laser scanning microscopy. The resulting dataset is processed and rendered into a 3D structure. Overlaps (colocalization) of selectively stained cells can then be projected on top of each other, allowing individual cells and cellular components to be segmented. This allows the subsequent count to determine exactly which segments should be recorded and which should be excluded.

This process enables a precise quantification of pathological changes following brain damage and is, for that reason, suitable for verifying the efficacy of new therapeuticprocedures. Additionally, not only the above-mentioned astrocytes, but also any desired cell in any desired histological section can be analyzed. The procedure is currently being adjusted to be able to describe microglial cells and nerve cell inter­actions in more detail.

Immunohistochemical staining of astrocytes
© Fraunhofer IZI
Immunohistochemical staining of astrocytes.
Rendered 3D models of immunohistochemical stained astrocytes
© Fraunhofer IZI
Rendered 3D models of immunohistochemical stained astrocytes.
Determination of colocalization
© Fraunhofer IZI
Determination of colocalization.