Recent molecular studies have revealed genes which regulate cell death, especially apoptosis, or programmed cell death occurring in organogenesis during fetal life, for the maintenance of cell numbers in the body by balancing cell production with cell death, and in tumor cell turnover. As has been suggested, however, cell death is not a single entity, but is heterogeneous in morphological and metabolic mechanisms, circumstances of initiation and biological significance (WYLLIE, 1981); it is at present difficult to understand the entire cascade of intracellular metabolism concerning apoptosis
The present review focuses on the description of precise morphological features characteristic of apoptosis with those of necrosis, particularly inward and outward blebbings of the cell membrane causing cell shrinkage. Special reference is given to inward blebbing, an event which has been shown in various physiological and pathological apoptotic cells. In this process, the blebs form numerous clear vesicles in the cytoplasm, which are degraded together with parts of the cytoplasm by autophagolysosomes.
Analysis of the processes of the death of cells and the determination of whether it is apoptotic or necrotic in nature is important when considering the fact that the hindrance of apoptosis in embryogenesis may cause congenital disorders, whereas its prevention in pathological diseases may open the way to new therapies.

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For nearly 70 years apoptosis has been known to be a form of cell death distinct from necrosis as well as an important regressive event during the normal development of the nervous system. For example, in the chick, mouse, rat and human approxiamately 50% of postmitotic neurons die naturally during embryonic or fetal development. It is generally accepted that neurons die during this period by apoptosis. After the period of naturally occurring cell death, the surviving neurons may undergo degeneration and death due to injury or disease later either during development or in adulthood. Recently, apoptosis has been suggested to be involved in the abnormal neuronal death that occurs following axonal injury or in neurodegenerative diseases such as amyotrophic lateral sclerosis and Alzheimer's. Although little is known about the etiology of these diseases, progress is steadilly being made toward understanding their underlying mechanisms. For diseases of spinal motoneurons, during the past two years gene mutations have been identified in patients with familial amyotrophic lateral sclerosis or spinal muscular atrophy. Furthermore, a number of in vitro, in vivo, and mutant animal models have been developed in order to study the factors which control motoneuron survival and/or death. Here, we review the morphological differences between necrotic and apoptotic cell death and some of the methods used to differentiate the two pathways. We also discuss motoneuron cell death during development, following injury and in disease, and its prevention by different agents, including neurotrophic factors.

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Epithelial cells of the gut are characterized by rapid, constant cell renewal. The death of epithelial cells at the villas tips occurs so regularly that it must be regarded as a well-controlled cell death, designated as apoptosis. However, only limited information has been available on the mechanism of this phenomenon, including the disposal of the effete cells.
In the small intestine of the guinea pig and monkey, macrophages are densely aggregated at the lamina propria of the villas tips and vigorously engaged in the phagocytosis of effete epithelial cells. Intraepithelial lymphocytes possessing cytoplasmic granules, possibly intense in cytotoxicity, are topographically associated with the dying enterocytes, suggesting lymphocyte-mediated killing. After the engulfing of apoptotic enterocytes by macrophages, a thin apical portion of the enterocytes is left within the epithelium, maintaining the epithelial barrier until it is pinched off by the pushing of surrounding enterocytes.
In the rat and mouse, on the other hand, effete enterocytes are exfoliated as a whole from the villas tips into the gut lumen. Macrophages, also numerous at the villas tips in these species, are less intense in phagocytotic activity. At the shoulder region of the villus, subepithelial macrophages extend thick processes deep into the epithelium; the processes appear to push out some enterocytes with typical apoptotic signs into the gut lumen. Lamina propria macrophages in the rat and mouse do not engulf enterocytes, but are believed to be involved in the induction of their apoptosis. The species difference in the mechanism of the apoptosis of enterocytes provides clues for understanding apoptosis.

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Various lines of evidence indicate the involvement of DNA strand breaks (DSB) in the regulation of physiological states of cells, especially in cell death. Currently, cell death is divided into two categories, apoptosis and necrosis. As lysosomal integrity is maintained in apoptosis, while disrupted in necrosis, it is possible to assume that necrotic chromatin is exposed to digestion by various lysosomal enzymes. We have therefore investigated whether apoptotic DSB and necrotic DSB can be discriminated by in situ nick translation (INT) under various conditions of protease pretreatment. Used models of apoptosis and necrosis were the rat thymus with an intraperitoneal injection of hydrocortisone (10mg/100g body weight (b. w.)) and rat liver with an intraperitoneal injection of CCl₄ (100μl/100g b. w.), respectively. As results, we found that necrotic DSB was readily detected by INT without protein digestion, whereas apoptotic ones were not. These results indicate that the environment around DSB and/or the nature of DSB in apoptosis differs from that of necrosis, and that INT is a convenient molecular histochemical tool to discriminate both types of cell death in frozen sections.

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Apoptosis was originally defined as shrinkage necrosis, as a distinct mode of cellular death from coagulative necrosis in the rat liver lobes after portal branch ligation. To reveal the functional role of the apoptosis of hepatocytes in the volume reduction and recovery of normal hepatic architecture in portal-deprived liver lobes, we ligated the portal vein branch supplying the left and median liver lobes in Wistar rats. The liver lobes were perfusion-fixed with glutaraldehyde solution via the proximal site of the ligated portion of the portal vein on Days 1, 2, 4, 7 and 14 after operation and examined light and electron microscopically. On Day 2 after ligation, massive necrosis of hepatocytes occurred in the central to intermediate zones of the liver lobule, and apoptotic hepatocytes were observed in the boundary region between necrotic and normal areas. Such necrotic area-associated apoptosis of hepatocytes was most frequent on Day 2, declining thereafter. The sequential changes of the cell organelles in apoptotic hepatocytes were distinct from those in necrotic hepatocytes. On Day 7, when necrotic areas had almost disappeared the apoptosis of hepatocytes occurred mostly between intact hepatocytes in the “combined hepatic cell cords” which included no obvious sinusoidal lumen between hepatic cell cords. Such necrotic area-non-associated apoptosis began to increase in frequency on Day 4, reached a peak on Day 7 and was gone by Day 14, when normal hepatic architecture was recovered. The present study suggests that both necrotic area-associated and non-associated apoptosis of hepatocytes may be induced by mild ischemia and contribute in part to the volume reduction of ligated liver lobes. It further reveals that the necrotic area-non-associated apoptosis of hepatocytes plays a role in reconstructing the architecture of hepatic cell cords after necrotic hepatocytes have undergone dissolution in the liver lobule.

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Apoptotic cells were histochemically demonstrated by the TdT-mediated biotinylated dUTP nick end-labeling (TUNEL) method in formalin-fixed and paraffin-embedded sections of the human endometrium and placental villi. In 53 endometrial biopsy specimens, labeled nuclei were identified in 16 samples showing a desquamating change, associated with menstruation, functional bleeding or adenocarcinoma. Cells in the normal proliferative and secretory phases were unlabeled. The labeled nuclei in the gland and stroma corresponded well to the so-called apoptotic bodies. Placental tissues at various stages of gestation were obtained by spontaneous abortion, intrauterine fetal death or normal delivery. Syncytiotrophoblastic cells in an early gestational stage (7-12 weeks) and in the term placenta were focally labeled, and the labeled cells possessed pyknotic nuclei and densely eosinophilic cytoplasm. In the early gestational chorionic villi with marked hydropic degeneration or in hydatidiform mole, the stromal cells were frequently labeled. Villous cells in coagulation necrosis (infarction) also revealed strong signals. The apoptotic bodies were not recognizable histologically in these labeled villi. The placenta at the 20th to 33rd week of gestation lacked labeling. From a technical point of view, it should be noted that cells in the foci showing ischemia or coagulation necrosis were labeled positively.

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Recent in vivo and in vitro studies have suggested that medial edge epithelial (MEE) cells covering the lateral palatine shelves do not undergo cell death, but migrate into the oral and nasal epithelium or transform into mesenchymal cells. We, therefore, reexamined the fate of MEE cells during palatal fusion in rat embryos by in situ 3′ nick end labeling of dUTP (TUNEL), electron microscopy, and immunohisto/cytochemistry. TUNEL staining revealed positive nuclei in the medial edge epithelium immediately prior to contact, in epithelial triangles formed between the epithelial seam and nasal or oral epithelium, in epithelial pearls, and in mesenchymal tissue near the epithelium. However, these TUNEL-positive cells were rarely present in the epithelial seam. Electron microscopy revealed MEE cells showing nuclear chromatin condensation and cell shrinkage, and apoptotic bodies in the fusing epithelium; these often contained apoptotic body-like structures as heterophagosomes. By double staining using a laser scanning microscope, TUNEL-positive nuclei were co-localized with lysosomal cysteine proteinases, cathepsin B or L in MEE and mesenchymal cells adjacent to the epithelium. These results suggest that MEE cells undergo apoptosis during the palatal formation, even though they migrate into epithelial triangles or transform into mesenchymal cells. Moreover, apoptotic bodies and cellular debris were phagocytosed by adjacent MEE cells or mesenchymal cells and digested by lysosomal enzymes.

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Until recently, little has been known about the morphological features of dying enterocytes at the villus tips of the human small intestine. The present study aimed to show the exfoliating processes of effete enterocytes at the villus tips. Cellular elements of the duodenal lumen and jejunal tissue in humans were fixed and processed for DNA nick end labeling (TUNEL), and transmission and scanning electron microscopy (TEM and SEM). Most cellular elements in the duodenal lumen were enterocytes having TUNEL-positive nuclei. By SEM, protruding enterocytes were discerned at the villus tips. Using the SEM samples embedded in epoxy resin, protruding enterocytes were observed at the villus tips by TEM; they were shrunk by forming numerous clear and autophagic vacuoles, took dome-like profiles, and possessed nuclei with chromatin condensation. The intercellular spaces beneath these protruding or effete enterocytes were often occupied by large lymphocytes. By TUNEL reaction, positive stainings appeared in the epithelium not only at the tip of the villi but also around the site. The results suggest that effete enterocytes at the villus tips of human small intertine are first shrunk by forming clear and autophagic vacuoles, and showed that their nuclei exhibit chromatin condensation immediately before being exfoliated into the lumen.

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It has been established that pancreatic exocrine acinar cells disappear after pancreatic duct obstruction. This study aimed to examine the relationship between the disappearance of the acinar cells and apoptosis after pancreatic duct ligation of the splenic lobe in dd-mice, six weeks of age. In some mice, the ligature was removed after two or three days. In addition to general light and electron microscopic examinations on the pancreatic tissues, paraffin sections stained with the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method were observed to detect nuclear DNA fragmentation.
Pancreatic acinar cells underwent apoptosis initiated with nuclear DNA damages three days after duct ligation and were completely deleted by seven days. Due to the elevation of the intraluminal pressure, the acinar cells showed interrupted secretion of their zymogen granules and disorganization of their rough endoplasmic reticulum, causing the cessation of granule formation before apoptosis started. These cytoplasmic changes prior to apoptosis are reversible, as observed after removal of the ligation. Apoptosis of the acinar cells was indentified by TUNEL-labeling of the nuclei, the condensation and margination of nuclear chromatin, and round fragmentation of cell bodies, all irreversible changes. Apoptosis of acinar cells seemed to stimulate the proliferation of duct cells, which comprised the main cell components in the exocrine pancreas after the disappearance of acinar cells.

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This study used an experimentally induced myocarditis model in rats to demonstrate the formation and fate of multinucleated giant cells which are known to occur in different kinds of inflammatory lesions. Multinucleated giant cells were frequently recognized in the inflammatory foci, being intermingled with numerous ED1-positive inflammatory macrophages rich in phagosomes. The giant cells reacted with the ED1 antibody but not with ED2, and ultrastructurally resembled the inflammatory macrophages. Multinucleated giant cells possessing less than 5 nuclei in an ultrathin section were rich in phagosomes, whereas those with more nuclei contained numerous lipid droplets and only few phagosomes in their cytoplasm.
Light microscopic observation of hematoxylin-eosin stained sections revealed that some multinucleated giant cells displayed variously sized dark bodies which likely corresponded to condensed and fragmented nuclear chromatin. Such multinucleated giant cells were positively stained with the TUNEL method. Under the electron microscope, all nuclei in one multinucleated giant cell showed an eccentric mass of homogeneously condensed chromatin.
These observations suggest that multinucleated giant cells are formed by aggregation and also likely by fusion of inflammatory macrophages; gradually loosing the phagocytotic activity characteristic of the latter cells, they then die by apoptosis.

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Two categories of cell death related to antigens, apogens and engulfens, have been reported to be expressed by apoptotic cells and the cells involved in their engulfment in the immune system, and in mesenchymal tissue in the limb of the chick embryo (ROTTELLO et al., 1994). To determine whether these antigens are also expressed during the process of neuronal death, the distribution of immunoreactivity to both anti-apogen and anti-engulfen antibodies was examined in the spinal cord and the dorsal root ganglia of the chick embryo.
Anti-apogen antibodies labeled a sub-population of the profiles of dying cells in regions where cell death was occurring. The extent of labeling by anti-apogens varied from 3% to 70% of the total number of dying profiles depending on the specific antibody used and the neuronal region examined. Immunoreactive labeling by the anti-engulfen antibodies mainly involved large cells that contained debris of dead cells.
These results indicate that at least some dying neuronal cells express common antigens that are shared by dying mesenchymal cells during programmed cell death, and that phagocytotic cells of the immune system are involved in the engulfment of neuronal cells that have undergone programmed cell death.

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Most thymocytes are known to be depleted from the thymus during T cell development, with the process of thymocyte death considered to be apoptosis. In this study we examined the mechanism of thymocyte death in the thymus of 6-week-old mice by using terminal deoxynucleotidyl transferase to detect DNA fragmentation or double strand breaks (TUNEL method). The TUNEL positive thymocytes were scattered throughout the cortex. Double staining of the section with the TUNEL method and acid phosphatase (ACP) activity showed that all the TUNEL positive cells were phagocytosed by ACP positive macrophages. An ultra-structural study revealed the presence of a substantial number of extremely small, unphagocytosed thymocytes throughout the cortex. These small unphagocytosed thymocytes were apparently dead cells, as based on several morphological features: 1) The majority were much smaller than red blood cells; 2) the nuclei were also considerably small; and 3) the extent of chromatin condensation was enormous. Importantly, these unphagocytosed dead thymocytes were TUNEL negative. These results indicate that: 1) DNA fragmentation, which is detected by the TUNEL method, is not involved in the cell death process of small unphagocytosed dead thymocytes shown in the present study; and that 2) typical apoptosis, which is characterized by DNA fragmentation, is not the dominant type of cell death in the normal murine thymus. Processes of cell death other than typical apoptosis taking place in most thymocytes require further investigation.

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Using rabbit ovaries, this study investigated the fate of follicular epithelial cells undergoing apoptosis in follicle atresia, especially in atretic Graafian follicles. This is the first report to apply the TUNEL method and histochemistry for acid phosphatase in order to demonstrate macrophages immigrating into the follicles to eliminate the apoptotically dead epithelial cells.
At initial stages of atresia, a few epithelial cells solitarily revealed the condensation of nuclear chromatin characteristic of apoptotis, simultaneously reacting positive to the TUNEL method. At advanced stages, chromatin condensation and separation as well as cytoplasmic fragmentation spread to the entire follicular epithelium.
In such an advanced atretic follicle, large round cells appeared in the follicular lumen; these could be identified as macrophages by their morphologies and positive reactions for acid phosphatase. These macrophages were adhering to and internalizing apoptotic epithelial cells and their fragments until the lumen became completely free of dead cells and fragments.

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