Techniques for Visualizing Cell Death in Experimental Cerebral Stroke
At a glance
The neuropathology of stroke is brain cell death, and its degree is one of the main predictors of stroke outcomes and survival. Adjusting for stroke-induced loss of neuronal cells is the ultimate purpose of new stroke drugs, like thrombolysis or neuroprotection. The more we learn about stroke as a disease, the more common it is to see that many different mechanisms can lead to cell damage and death. To diagnose stroke, to grasp how brain tissue can be lost, and to test whether therapies work in the mouse model or in human patients, we have to watch cerebral cell death with unprecedented sensitivity.
A number of techniques are available to identify the various pathological features of cell death in stroke. Specific visualization of cell death using these techniques is of great importance in basic research, and there is great interest in applying them to humans.
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Microscopes for Visualizing Cell Death in Experimental Cerebral Stroke
Light Microscopy and Macroscopy
Light microscopy is used to visualize cell death after ischemic stroke, allowing for the analysis of infarction areas and changes at the cellular level. Differentiating between apoptotic and necrotic cell death is often challenging based solely on morphology, particularly in the debate over delayed neuronal death in hippocampal neurons. Detecting early ischemic changes reliably is another significant challenge. Various staining methods, including routine histology, silver staining, and fluorescence markers, are available, each with its own advantages and disadvantages.
| Staining Methods | Advantages | Disadvantages |
|---|---|---|
Routine histological stains
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Can be quickly and easily performed on tissue sections | The pathology observed can only be reliably interpreted under their specific standardized experimental conditions. |
Silver stains
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Require cryostat material. |
Fluorescence-based techniques
|
|
One basic prerequisite is perfused tissue, otherwise erythrocytes will also be highlighted and interfere with signals from degenerating cells. |
Fig. 1. Light microscopy of cell death in ischemic stroke. (Zille, et al., 2012)
Electron Microscopy
Electron microscopy allows researchers to see ultrastructural details of ischemic brain tissue. Only electron microscopy can clearly distinguish between necrosis and apoptosis, and several morphological features have been accepted as reliable criteria.
However, it cannot be used for high-throughput screening, because the magnifications obtained do not provide an overview, but only a detailed observation of the cells.
Fig. 2. Electron microscopy of cell death in ischemic stroke. (Zille, et al., 2012)
Assays and Immunohistochemical Markers of Cell Death
Standard light and fluorescence microscopy remain the primary methods for evaluating and quantifying damaged or dead cells in experimental stroke. A growing array of biological markers can be utilized to detect molecules at the subcellular level. The following are the most common assays and immunohistochemical markers used to assess cellular damage or death in tissue sections following focal cerebral ischemia.
- Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end labeling (TUNEL): TUNEL is a standard of measure for focal cerebral ischemia and is often the only tool used to detect apoptosis after stroke.
- Markers of mitochondrial damage: Bioluminescence on the luciferin–luciferase reaction can measure Adenosine-5′-triphosphate (ATP). Another hallmark of destroyed mitochondria is a large release of reactive oxygen species. There are many reactive oxygen species sensitive dyes that are visible in microscopy, spectroscopy, and flow cytometry.
- Caspases: Caspase 3 has been described to have a crucial role in focal cerebral ischemia and can be detected in the ischemic tissue using immunoblotting and immunohistochemistry with antibodies against procaspase 3 and the activated form of caspase 3.
- Calpains and cathepsins: They can be detected using immunoblotting and immunohistochemistry with antibodies or by fluorometric assays.
- Phosphatidylserine exposure: Annexin A5 has been suggested to be a marker of cell death by detection of extracellular phosphatidylserine.
- Vital dyes: Loss of cell membrane integrity is assessed by staining cells with compromised cell membrane integrity. Commonly used dyes include PI, nuclear yellow, ethidium homodimer-1, and TOTO-3.
- Poly(ADP-ribose) polymerase (PARP)-1: In focal cerebral ischemia, it has been shown that damage in the DNA that is caused by reactive oxygen species hyperactivates PARP-1.
Fig. 3. Assays and immunohistochemical markers of cell death in ischemic stroke. (Zille, et al., 2012)
Noninvasive Imaging Techniques for Visualizing Cell Death in Experimental Cerebral Stroke
| Noninvasive Imaging Techniques | Description | Advantages |
|---|---|---|
| Magnetic Resonance Imaging (MRI) |
|
Both imaging techniques are strongly correlated with similar measures from tissue sections and infarct volume. |
| Nuclear and Noninvasive Fluorescence Imaging | To detect signals in PET or SPECT imaging, a radiopharmaceutical is injected, while a fluorochrome is used for near-infrared fluorescence imaging (NFI). After injection, a scanner determines the distribution of these compounds. | The strongest advantages of these techniques when compared with MRI are that they can be orders of magnitude more sensitive, and molecules and/or proteins specifically associated with cell death can be labeled with radioactive or fluorescent compounds enabling a direct visualization of cell death after stroke. |
- Zille, M., et al. (2012). Visualizing cell death in experimental focal cerebral ischemia: promises, problems, and perspectives. Journal of Cerebral Blood Flow & Metabolism, 32(2), 213-231.
