Morphologic criteria and detection of apoptosis

Abstract

Apoptosis is an organized, energy dependent process, which leads to cell death. Its definition is based on distinct morphological features [10] and demonstration of internucleosomal DNA degradation [27], executed by selectively activated DNAses [4, 22]. The morphologic hallmarks of apoptosis include chromatic margination, nuclear condensation and fragmentation, and condensation of the cell with preservation of organelles. The process is followed by fragmentation of the cell into membrane-bound apoptotic bodies, which undergo phagocytosis by nearby cells without associated inflammation [10, 11]. Apoptosis characteristically occurs in isolated single cells. The duration of apoptosis is estimated to be from 12 to 24 hours, but in cell culture visible morphologic changes are accomplished in less than two hours [10, 16]. Non-apoptotic cell death, a prototype of which is cell death due to ischemia (oncosis), is characterized by depletion of intracellular ATP stores, swelling of the cell with disruption of organelles and rupture of the plasma membrane [15]. Groups of necrotic cells and inflammation are found in tissues [10, 15]. The significance of apoptosis has mostly been studied using the TUNEL assay that detects DNA strand breaks in tissue sections and allows quantification of apoptotic cells by light microscopy [6]. Common experience seems to be that the TUNEL assay is prone to false positive or negative findings. This has been explained by the dependence of the staining kinetics on the reagent concentration [17], fixation of the tissue [2] and the extent of proteolysis [17]. Active RNA synthesis [12] and DNA damage in necrotic cells [17, 19] may cause non-specific staining. To obtain reliable and reproducible results, TUNEL assay should be carefully standardized by using tissue sections treated with DNAse (positive control of apoptosis). Quantification of apoptosis should include enough microscopic fields and identification of the cell type undergoing apoptosis. The specificity of the results can be substantiated by combining other methods with TUNEL, such as assessment of the pattern of DNA fragmentation or evaluation of the morphological features. Even though there is high variation in the results obtained in consecutive studies under the same circumstances, increasing evidence shows that TUNEL-positive cardiomyocytes and internucleosomal DNA fragmentation are associated with various cardiac diseases, including acute myocardial infarction and heart failure [reviewed in 5,9]. Some morphological features of apoptosis have been observed in TUNEL-positive cardiomyocytes using light microscopy (Figure 1) or confocal microscopy [20]. Electron microscopic evidence of apoptosis has been found in the degenerating conduction system [7], in experimental heart failure [23], and in human hibernating myocardium [3]. In acutely ischemic myocardium the interpretation of the findings remains controversial, since only non-apoptotic cell morphology has been found in electron microscopy [8, 19]. One explanation might be abortion of the apoptotic program due to the lack of ATP before the morphologic features are fully evident [14]. Another explanation is the possibility that non-apoptotic cell death and apoptosis share common mechanisms in the early phases of the processes [14, 19]. The exact mechanisms of ischemic cell death remain to be clarified and the classification between apoptosis and non-apoptosis cell death to be specified. Recently, caspase activation has emerged as the central molecular event leading to apoptosis, preceding DNA degradation and the development of apoptotic morphology [22, 25]. New methods have been developed to demonstrate caspase activation [1, 13]. Inhibition of caspase may be an efficient way to prevent apoptotic cardiomyocyte death as well as to define and specifically probe the significance of apoptotic cell death in cardiac diseases. Die Apoptose ist ein strukturierter, energieabhängiger Prozeß, der zum Zelluntergang führt. Histomorphologisch ist er klar definiert und zeigt eine internukleosomale DNA-Degradation, die durch selektiv aktivierte DNAsen vollzogen wird. Die morphologischen Charakteristika der Apoptose umfassen die Margination von Chromatin, die Kernkondensation und Fragmentation und die Kondensation der Zellen unter Erhalt der Organellen. Diesem Vorgang folgt eine Fragmentation der Zellen in sogenannte apoptopische Körperchen, die später von benachbarten Zellen, meist von Makrophagen phagozytiert werden, ohne daß eine Entzündungsreaktion nachweisbar wäre. Apoptose findet in der Regel an Einzelzellen statt. Die Dauer eines apoptotischen Vorgangs beträgt etwa 12 bis 24 Stunden, wobei in Zellkultur die morphologischen Veränderungen der Apoptose bereits in weniger als zwei Stunden vorliegen können. Der nicht-apoptotische Zelluntergang läßt sich ganz besonders gut als Folge der Ischämie nachweisen und wird Onkose genannt. Bei ihm werden die intrazellulären ATP-Speicher entleert, die Zellen schwellen an, die Organellen rupturieren ebenso wie die Plasmazellmembran. Gruppen nekrotischer Zellen und eine Entzündungsreaktion finden sich hier als morphologisches Charakteristikum. Bei der lichtmikroskopischen Untersuchung von Geweben wird meist der sogenannte TUNEL-Test angewendel, der DNA-Strangbrüche zeigt und eine Quantifizierung apoptotischer Zellen ermöglicht. Nach Erfahrung verschiedener Zentren ist der TUNEL-Test jedoch nicht selten falsch positiv oder falsch negativ. Dies hängt unter anderem mit methodischen Problemen zusammen, die auf der Färbungskinetik, der Konzentration der Reagenzien, der vorbestehenden Fixation der Gewebe und dem Ausmaß der Proteolyse beruhen. Eine aktive Synthese von RNA und DNA-Schäden in nekrotischen Zellen rufen gleichfalls eine unspezifische TUNEL-Reaktion hervor. Um verläßliche und reproduzierbare Ergebnisse zu...