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Review
. 2012 Feb;32(2):213-31.
doi: 10.1038/jcbfm.2011.150. Epub 2011 Nov 16.

Visualizing cell death in experimental focal cerebral ischemia: promises, problems, and perspectives

Marietta Zille  1 , Tracy D Farr, Ingo Przesdzing, Jochen Müller, Clemens Sommer, Ulrich Dirnagl, Andreas Wunder
Affiliations
Review

Visualizing cell death in experimental focal cerebral ischemia: promises, problems, and perspectives

Marietta Zille et al. J Cereb Blood Flow Metab. 2012 Feb.

Abstract

One of the hallmarks of stroke pathophysiology is the widespread death of many different types of brain cells. As our understanding of the complex disease that is stroke has grown, it is now generally accepted that various different mechanisms can result in cell damage and eventual death. A plethora of techniques is available to identify various pathological features of cell death in stroke; each has its own drawbacks and pitfalls, and most are unable to distinguish between different types of cell death, which partially explains the widespread misuse of many terms. The purpose of this review is to summarize the standard histopathological and immunohistochemical techniques used to identify various pathological features of stroke. We then discuss how these methods should be properly interpreted on the basis of what they are showing, as well as advantages and disadvantages that require consideration. As there is much interest in the visualization of stroke using noninvasive imaging strategies, we also specifically discuss how these techniques can be interpreted within the context of cell death.

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Figures

Figure 1
Figure 1
Rough guidelines for the appropriate application of the most common techniques to visualize cell death in the infarct or peri-infarct area in moderate-to-severe MCAO. H&E, hematoxylin and eosin; MCAO, middle cerebral artery occlusion; MR, magnetic resonance; PS, phosphatidylserine; TTC, 2,3,5-triphenyltetrazolium hydrochloride; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end labeling.
Figure 2
Figure 2
Light microscopy of cell death in ischemic stroke. (A) Reliable demarcation of the infarct can be seen routinely in H&E-stained cryosections (triangles), as shown 4 hours after focal ischemia (permanent middle cerebral artery occlusion (MCAO) in rat). (B) Typical pyknotic neurons (triangle in top panel) compared with morphologically intact neurons (triangle bottom panel) can be seen in H&E-stained paraffin-embedded sections 4 hours after permanent rat MCAO. (C) Similar morphological alterations can be observed in humans; it must be noted that the spectrum ranges from eosinophilic neurons (triangle) to completely shrunken ones with darkly stained pyknotic nuclei (arrow), which complicates exact determination of the postischemic time point solely based on morphology. (D, E) A frequently occurring artifact is the so-called ‘dark neuron' (panel D, encircled; panel E, arrows). It must be noted that in contrast to true eosinophilic degeneration, neurons are homogeneously stained; furthermore, the neurophil looks normal when compared with panels B (top panel) and C. (F) Single scattered degenerating neurons, e.g., in remote areas, are easily detectable by Fluoro-Jade B staining (arrow) (transient global ischemia, rat). (G) Silver stains are ideal for infarct volumetry because of their high contrast (triangles) (mouse, 24 hours after transient MCAO). (H) Similarly, TTC staining is a well-established method for detection of the infarcted area, although distinction of white matter in the infarct is problematic (arrow) (mouse, 24 hours after transient MCAO). H&E, hematoxylin and eosin; TTC, 2,3,5-triphenyltetrazolium hydrochloride.
Figure 3
Figure 3
Electron microscopy of cell death in ischemic stroke. Ultrastructural changes in damaged neurons in the ischemic cortex of rats. EM showed cellular and subcellular alterations of damaged neurons in the ischemic region. (A) Normal cortical neuron. (B) Severely shrunken neurons 8 hours after ischemia. Dark nuclei and condensed and fragmented chromatin (arrow) are evident in many cells. Meanwhile, some membrane and cytosolic disruption were also obvious in these cells. (C, D) Ultrastructural changes 16 hours after ischemia. Damaged cells show smaller cell bodies, apoptotic nuclear and chromatin condensation, and fragmentation (arrow). Formation of apoptotic bodies (arrowhead) was seen in some cells such as the example in panel D. However, the necrotic alterations took place in the cell cytoplasm, indicated by the presence of large vacuoles (*), disappearance of cell organelles, and collapsed membranes. (E, F) Neuronal damage 24 hours after ischemia. Apoptotic nuclear changes (arrow) and formations of apoptotic bodies (arrowhead) continued and the classic apoptotic ‘half-moon' nuclear morphology (unfilled arrow) was seen in some cells. Meanwhile, even more advanced necrotic deterioration developed in the cytoplasm and the cell membrane. Figure with permission from Wei et al, 2006. EM, electron microscopy.
Figure 4
Figure 4
Assays and immunohistochemical markers of cell death in ischemic stroke. (A) Microscopic images of brain tissue taken from a MCAO mouse 4 hours after intravenous injection of annexin A5 and PI. After fluorescence inspection, the section was stained with TUNEL and H&E, and again inspected with fluorescence and light microscopy. The majority of cells in the ischemic area were positive for annexin A5, PI, and TUNEL (white circle). (B) Caspase 3, TUNEL, and Hoechst staining can also be performed on the same tissue section. The cell in the white circle is positive for all three stains, the cell in the blue circle is only positive for Hoechst, the cell in the red circle is only positive for TUNEL and Hoechst, and the cell in the green circle is only positive for caspase 3 and Hoechst. (C) Ex vivo fluorescence image of a mouse brain after intravenous injection of TOTO-3. The strong uptake of TOTO-3 in the ischemic area (arrow) must be noted. Excitation and emission wavelengths of the respective dyes are indicated in brackets. Images in panel A are taken with permission from Bahmani et al, 2011. H&E, hematoxylin and eosin; MCAO, middle cerebral artery occlusion; PI, propidium iodide; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end labeling.
Figure 5
Figure 5
Magnetic resonance imaging of damage in ischemic stroke. MR images indicate temporal differences in estimations of lesion volume in mice with MCAO. T2-weighted images (10 milliseconds) and corresponding T2 maps of a mouse (A) before and at (B) 14 and (C) 21 days after 30 minutes of MCAO. T2 values (milliseconds) are indicated in the color scale bar below the images. The reduction in overall size of the hyperintense T2 area between 14 and 21 days, as well as the appearance of small regions with apparently normal T2 values must be noted. The images are courtesy of JA Adamczak (MPI Cologne). MCAO, middle cerebral artery occlusion; MR, magnetic resonance.
Figure 6
Figure 6
Noninvasive fluorescence imaging of cell death in ischemic stroke. Noninvasive fluorescence images of the heads of ischemic mice, corresponding ex vivo images of the brains, and of brain sections at 48 hours after MCAO (left hemisphere) and 4 hours after injection of either active (binds to PS) or inactive (no affinity for PS) annexin A5 labeled with the near-infrared fluorochrome Cy5.5. The noninvasive images of the heads show that after injection of both compounds, strong fluorescence from vessels, especially from the sinus confluens (indicated by the asterisk), were detected. Strong fluorescence signals were only seen over the stroke area of mice injected with active annexin A5 (see dotted line, arrow). Ex vivo images of the brains and the brain slices show that after injection of inactive annexin A5, only slightly higher fluorescence intensities were detected over the ischemic hemisphere compared with the contralateral side, which is attributed to the disruption of the blood–brain barrier (BBB). After injection of active annexin A5, considerably higher fluorescence intensities were observed over the ischemic hemisphere than over the contralateral side. Areas of higher fluorescence intensities corresponded closely with areas of the infarction appearing as pale in TTC-stained slices. Images adapted with permission from Bahmani et al, 2011. MCAO, middle cerebral artery occlusion; PS, phosphatidylserine; TTC, 2,3,5-triphenyltetrazolium hydrochloride.
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