Morphological assessment of apoptosis

Abstract

Apoptosis is implicated in biological processes ranging from embryogenesis to ageing, from normal tissue homoeostasis to many human diseases. Apoptotic cells share a number of common features such as cell shrinkage, membrane blebbing, chromatin cleavage, nuclear condensation and formation of pyknotic bodies of condensed chromatin. In the final stages of apoptosis these pyknotic or apoptotic bodies are rapidly engulfed by neighbouring cells. Necrotic cells on the other hand exhibit loss of membrane integrity, cellular and nuclear swelling and an associated inflammatory response. Such characteristics demonstrate that apoptosis is an orderly genetic programme, which could potentially be manipulated or controlled at various points, while necrosis is a form of cell death that lacks these control points. These distinctive morphological differences form the basis of some of the most widely used techniques for the identification and quantification of apoptosis and thus morphologic description using light or electron microscopy remains one of the best ways to define apoptosis and contrast it with necrosis. However, the field of apoptosis or cell death research is advancing rapidly and it is becoming increasingly evident that apoptosis and necrosis represent two extremes of cell death and that many variations now exist. There is often a continuum of apoptosis and necrosis in response to high and low doses of the same stimulus and features of both apoptosis and necrosis may coexist in the same cell. Therefore, it is clear that an increasing amount of care must be taken when assigning the label ‘apoptosis’ to a dying cell on the basis of morphology.

Introduction

Programmed cell death or apoptosis has, since its first description in 1972, become an important area of research due to the fact that it plays a pivotal role in embryonic development and in pathological processes. Apoptotic cells were initially distinguished from healthy cells and necrotic cells by certain morphological features and these characteristics remain an important tool in apoptosis research. These features include cell rounding and shrinkage, membrane blebbing, chromatin condensation and nuclear fragmentation [1]. Indeed, Kerr et al., coined the term apoptosis to describe a particular morphological aspect of cell death and it remains a morphological description. Furthermore, guidelines laid down by the Nomenclature Committee on Cell Death advise against using biochemical analyses such as DNA ladders to define apoptosis because the degree to which DNA becomes fragmented, is noticeably different depending on the cell type, potentially resulting in false negatives [2]. Therefore, methods which evaluate morphological characteristics truly determine the presence of apoptosis and are widely used in the study of tissue development and a variety of diseases.

Cell shrinkage is one of the most ubiquitous characteristics of cell death, occurring in almost all incidences of apoptosis independent of the stimulus [3]. The primary determinant of cell volume is water and this in turn is controlled by alterations in osmotically active particles such as K⁺, Na⁺, and Cl⁻ ions. Under physiological conditions ion gradients are maintained by a diverse range of ion channels and pumps. Early increases in intracellular Na⁺ have been observed, which are transient and appear to control early signalling events in the initiation of apoptosis and in downstream cell shrinkage [3]. As cell shrinkage proceeds there is a loss of both Na⁺ and K⁺ ions and inhibition of the Na⁺/K⁺-ATPase and Ca²⁺-dependent potassium channels reduce this shrinkage event [4]. Importantly, movement of these ions appears to play a role in regulating the progression of the apoptotic process not just in the shrinkage event itself.

As cells shrink they lose contact with and detach from neighbouring cells or the extracellular matrix (ECM). Once released from ECM attachments, focal adhesions are re-organized and the cell adopts a more rounded morphology. This is followed by formation of non-retracting blebs at the cell surface due to separation of the plasma membrane from the cytoskeleton. This was originally thought to result from cleavage of cell–cell contact factors by proteases such as caspases. However, evidence now suggests that membrane blebbing requires activation of myosin light-chains by phosphorylation [5] and rearrangement of the actin cytoskeleton [6]. Inhibition of ROCK-I, a Rho effector protein involved in phosphorylation of myosin light-chains, prevents bleb formation. However, apoptotic cells without membrane blebs readily undergo phagocytosis by macrophages, indicating that blebs, unlike other cell surface changes do not contribute to recognition and engulfment [7]. One interesting consequence of bleb repression is that fragmented DNA no longer redistributes from the nuclear region into blebs and apoptotic bodies, suggesting that rearrangement of the actin cytoskeleton necessary for bleb formation may also be required to break down the nuclear envelope and laminar matrix [8].

Blebbing continues until finally cells enter the condensation stage, characterized by formation of apoptotic bodies. It may be that eventually excessive invagination causes a portion of the plasma membrane to pinch off forming these sealed membrane vesicles termed apoptotic bodies which are impermeable to vital dyes. These events correlate with dissolution of polymerised actin and microtubule disassembly/degradation [9]. Rapid removal of these apoptotic bodies by macrophages or by non-professional phagocytes in vivo prevents the late apoptotic event of cell lysis we see in many culture systems. Thus, apoptosis is described as a ‘clean’ process, since leakage of potentially toxic or immunogenic cellular contents which could lead to inflammation is prevented.

Cytoplasmic shrinkage is usually accompanied by nuclear events such as nuclear condensation/pyknosis and DNA fragmentation. During chromatin condensation the nuclear envelope remains morphologically intact but components of the nuclear matrix and lamina are degraded, causing a loss of structural integrity which allows the chromatin to aggregate. Once nuclear chromatin has collapsed, it adopts a striking morphology that of a crescent or ‘half-moon’ shape, against the nuclear membrane. Activation of Acinus, a factor which is localized to the nucleus can also induce condensation of chromatin [10]. As chromatin condensation progresses, the entire nucleus shrinks and fragments into one or more or dense granular spheres surrounded by nuclear envelope [11]. This fragmentation of DNA usually involves nuclear import of effector proteins called endonucleases, such as caspase-activated DNase (CAD) and apoptosis-inducing factor (AIF). CAD, uses its DNase activity to cleave chromatin at boundaries between nucleosomes generating stretches of DNA about 200 bp long which are indicative of apoptosis [12].

Here, we describe techniques which allow morphological examination of cells undergoing apoptosis. As a preliminary estimation of cell death it can be useful to examine plasma membrane integrity. Viable cells exclude certain dyes such as trypan blue, erythrosin, naphthalene black, propidium iodide and ethidium bromide, whereas non-viable cells stain due to a breakdown in their cell membranes. This is a rapid but rather crude estimation of dead versus live cells. Microscopy is a much more valuable and accurate tool for the assessment of apoptosis permitting identification of the classical hallmarks of apoptosis such as cell shrinkage, membrane blebbing and formation of apoptotic bodies. Examination of stained cells by light microscopy is probably the most widely used technique with which to assess apoptosis by morphology (Fig. 1). There are many commercially available stains for example; the Romanowski group of stains and hematoxylin–eosin are general purpose stains routinely used for the examination of cells or tissue sections. The Romanowski stains are defined based on the formation of a blue/black precipitate as a result of combining aqueous solutions of methylene blue and eosin, dissolved in methanol. This staining technique was designed to incorporate cytoplasmic (pink) staining with nuclear (blue) staining and fixation as a single step. Hematoxylin and eosin (H&E), commonly used to stain tissue sections, are both salts that dissociate in water. The negative ion of eosin interacts with positively charged regions of cytoplasmic proteins resulting in red/pink stain while the positive ion of hematoxylin combines with negatively charged regions of the cell, in particular the phosphate groups of nucleic acids staining them blue.

Fluorescence microscopy is another technique used extensively in apoptosis research. Dapi (4,6-diamidino-2-phenylindole-dihydrochloride) and the Hoechst stains 33258 and 33342 are fluorescent dyes that stain the nuclei of cells. Each stain is equally useful but Hoechst 33342 has slightly higher membrane permeability than Hoechst 33258. These dyes are excited by UV light at around 350 nm, emitting blue fluorescence light at 461 nm. Preferential binding to AT regions makes them highly selective for DNA and they are often used to distinguish the compact chromatin of apoptotic nuclei from that of normal cells [13]. Apoptotic nuclei may appear slightly smaller than normal nuclei [14] and condensed, aggregated chromatin will be visualized as bright fluorescence at the nuclear membrane due to the highly concentrated nature of the DNA. It is also possible to visualize nuclear fragmentation in this way (Fig. 2).

In general both light and fluorescence microscopy can provide both qualitative and quantitative data. However, scanning electron microscopy (SEM) provides detailed information about the cell surface in particular the membrane, enabling visualization of membrane blebs [15]. Transmission electron microscopy (TEM), on the other hand, allows the analysis of sectioned specimens providing internal images of the cell [16]. Where detection of morphological features of apoptosis by regular microscopy is difficult such as in whole tissue sections, TEM can facilitate. In addition, the shape adopted by the condensed chromatin can be visualized by TEM providing information about the biochemical nature of the pathway. Leist and Jaattela describe the different morphology adopted by chromatin depending on the type of death programme initiated [17]. Caspase-dependent apoptosis mostly induces strong chromatin compaction in crescent shaped masses at the nuclear periphery, while caspase-independent apoptosis (sometimes called programmed necrosis) often results in lumpy, incomplete chromatin condensation (Fig. 3, reproduced with permission from [18] © (2001), The John Hopkins University Press). However, TEM has disadvantages one of which is that only a small area of tissue can be analysed at once, making the process of counting apoptotic cells rather tedious. Thus, an initial screen under a light microscope can be useful to identify potentially interesting areas and the tissue then trimmed for more detailed observations.

It is probably wise to identify more than one morphological characteristic in order to confirm apoptotic cell death, since there are certain areas of overlap between the death programmes of apoptosis, autophagy and necrosis. Cells that die by necrosis often exhibit changes in nuclear morphology but not the organized chromatin condensation and formation of 200 bp DNA fragments that is characteristic of apoptosis, therefore, careful examination should easily distinguish the type of death in this case [19]. However, other studies have shown that characteristics of more than one death pathway can be displayed at one time. Cell shrinkage, a morphological feature of classical cell death was observed in neuronal cells treated with tryptamines, alongside features which are characteristic of autophagy, such as large intracellular granules and vacuoles and swollen mitochondria [20]. MCF-7 cells treated with a chemotherapeutic vitamin D analogue exhibit chromatin condensation and DNA fragmentation, in addition to characteristics of autophagy [21]. This illustrates that the long-standing view of two distinct death programmes; apoptosis and necrosis is a simplification of a much more complex process and this should be considered when performing morphological examination of cell death.