Distribution Of Apoptotic Cells Research Articles

Apoptosis in the pathogenesis of cholesteatoma in adults

The aim of this study was to determine the distribution and apoptotic capacity in the various layers of cholesteatoma epithelium compared to the normal skin. Cholesteatomas were collected during surgical procedures of the ear. Normal skin specimens taken from the retroauricular area served as controls. Apoptosis was detected by using highly specific antibodies against APO2.7 antigen. The Membstain Apoptosis kit Direct based on in situ labeling of nuclear DNA fragmentation (Tolt-mediated dUTP nick and labeling TUNEL staining) was used. The distribution of apoptotic cells in the layer of the cholesteatoma epithelium and the epidermis was determined. The percentage of apoptotic cells was then counted per 100 cells of cholesteatoma epithelium and the normal epidermis in three different areas of each section and expressed in terms of mean +/- SD. An automatic analyzing system was used for counting. In normal epidermis, the apoptotic cells were observed in the granular layer of the epidermis. The percentage of these cells was 28.5 +/- 8.1%. A significantly greater number of apoptotic cells was observed in the suprabasal layers of the cholesteatoma epithelium (47.39 +/- 6.2%). A statistically significant difference was obtained for APO2.7-positive cells in the cholesteatoma epithelium as compared to the skin (P < 0.05) as determined by Student's t-test and the Mann-Whitney U test. Apoptotic activity is mainly observed in the suprabasal layer of cholesteatoma epithelium. It is not seen in the basal layer. Apoptosis plays an important role in the pathogenesis of cholesteatoma. It seems to be part of the differentiation and accumulation of keratin debris within the middle ear and expansion of cholesteatoma.

Iris movement mediates vascular apoptosis during rat pupillary membrane regression

In the course of mammalian lens development, a transient capillary meshwork known as the pupillary membrane (PM) forms, which is located at the pupil area; the PM nourishes the anterior surface of the lens and then regresses to make the optical path clear. Although the involvement of apoptotic process has been reported in the PM regression, the initiating factor remains unknown. We initially found that regression of the PM coincided with the development of iris motility, and iris movement caused cessation and resumption of blood flow within the PM. Therefore, we investigated whether the development of the iris's ability to constrict and dilate functions as an essential signal that induces apoptosis in the PM. Continuous inhibition of iris movement with mydriatic agents from postnatal day 7 to day 12 suppressed apoptosis of the PM and migration of macrophage toward the PM, and resulted in the persistence of PM in rats. The distribution of apoptotic cells in the regressing PM was diffuse and showed no apparent localization. These results indicated that iris movement induced regression of the PM by changing the blood flow within it. This study suggests the importance of the physiological interactions between tissues-in this case, the iris and the PM-as a signal to advance vascular regression during organ development, and defines a novel function of the iris during ocular development in addition to the well-known function, that is, optimization of light transmission into the eye.

Patterns of cell proliferation and cell death in the developing retina and optic tectum of the brown trout

We have analyzed the patterns of cell proliferation and cell death in the retina and optic tectum of the brown trout ( Salmo trutta fario) throughout embryonic and postembryonic stages. Cell proliferation was detected by immunohistochemistry with an antibody against the proliferating cell nuclear antigen (PCNA), and apoptosis by means of the TUNEL method. Haematoxylin and DAPI staining were also used to demonstrate apoptotic cells. Photoreceptor cell differentiation was assessed by immunohistochemistry with antibodies against opsins. Throughout embryonic development, PCNA-immunoreactive (PCNA-ir) cells become progressively restricted to the peripheral growth zone of the retina, which appears to be the principal source of new retinal cells from late embryos to adults. However, some PCNA-ir cells are observed secondarily in the differentiated retina, first in the inner nuclear layer of 15-mm alevins and later in the outer nuclear layer of 16-mm alevins, after differentiation of the first rods in the central retina, as demonstrated with opsin immunocytochemistry. Our observations also support the view that the PCNA-ir cells observed secondarily in the INL of the central retina of alevins are photoreceptor precursors. The number and distribution of apoptotic cells in the retina and optic tectum of the trout change throughout development, allowing distinction of several waves of apoptosis. Cell death is detected in proliferating areas at early stages, then in postmitotic or differentiating areas, and later concurring temporal and spatially with the establishment of visual circuits, thus indicating a relationship between apoptosis and proliferation, differentiation and synaptogenesis.

Distribution of apoptotic cells and apoptosis-related molecules in the developing murine palatine rugae.

Distribution of apoptotic cells and expression of the apoptosis-related factors p53, bcl-2 and bad during morphogenesis of the murine palatine rugae (PR) were examined histochemically using the terminal deoxynucleotidyl transferase-mediated UTP nick end-labeling (TUNEL) technique and specific antibodies against apoptosis and cell cycle-related molecules. Formation of the PR rudiment was controlled by cell proliferation and apoptosis in the palatal epithelium. TUNEL-positive cells were detected only at the epithelial placode area at 12.5-13.5 days post coitus (dpc), but only a few cells were positive at the protruding PR area at 14.5-16.5 dpc. Bcl-2 protein was expressed mainly in the areas outside of those containing TUNEL-positive cells at 15.5 -6.5 dpc. P53 protein was not detected throughout gestation. Bad was detected in the epithelial layer at 13.5 and 15.5 dpc and overlapping the apoptotic area at 13.5-15.5 dpc. Apoptosis of palatal epithelial cells might therefore involve spatiotemporally regulated expression of bad during murine PR development.

Proliferation, cell death and reelin expression during development of the retina in a teleost, the common trout (Salmo trutta fario)

We have analysed the spatiotemporal pattern of cell proliferation and apoptosis during the development of the retina in a teleost. Distribution of proliferating cells in different developmental stages (embryos, alevins, and juveniles) was analysed with an antibody against proliferating cell nuclear antigen (PCNA). After establishment of the retinal lamination, PCNA-immunoreactive (PCNA-ir) cells were frequently observed in all cell layers, with a high majority of proliferating cells located at the marginal (growing) retina. Clusters of PCNA-ir cells were characteristic of the inner nuclear layer (INL). The distribution of apoptotic cells was studied by the TUNEL method, which has revealed significant cell death before completion of the retinal lamination. We have also used an antibody against Reelin (RELN) as a marker for retinal cells that secrete this glycoprotein to the extracellular space. The pattern of RELN expression, as well as that of proliferating and dying cells was very similar to that described in tetrapods, suggesting that this protein regulates the cell migration and synaptogenesis in the different developmental stages of the retina.

Inhibition of cell proliferation in the embryonic myocardium by A1 adenosine receptor activation.

A1 adenosine receptors (A1ARs) are expressed in the embryonic heart and influence cardiac function at early developmental stages. To test if A1ARs also affect cardiac structural development, we examined the effects of A1AR activation on heart formation. When pregnant mice were treated with the A1AR agonist, N(6)-cyclopentyladenosine (CPA), fetuses showed marked reduction of ventricular size and manifested features of heart failure. Suggesting that these findings represent direct effects on embryos, CPA treatment of cultured whole murine embryos resulted in cardiac hypoplasia. When rates of cell division and cell death in the heart were examined, we found decreases in cardiac cell proliferation following A1AR activation. In contrast, no changes in numbers or distribution of apoptotic cells were seen. Collectively, these data show that A1AR activation inhibits cardiac cell proliferation and can lead to cardiac hypoplasia. These data identify adenosine as a potential regulator of cardiac cell division during early development.

Distribution of apoptotic cells and expression of apoptosis-related proteins along the intrahepatic biliary tree in normal and non-biliary diseased liver.

The cell kinetics and homeostasis of biliary epithelial cells may be maintained differently along the biliary tree. In this study, the role of apoptosis in the maintenance and homeostasis of the intrahepatic biliary tree was evaluated. By counting apoptotic biliary cells and by immunostaining apoptosis-related proteins in normal liver, fatty liver, and those with acute viral hepatitis, chronic viral hepatitis, and hepatitis virus-related cirrhosis, it was found that the larger the intrahepatic bile ducts became, the more biliary epithelial cells underwent apoptosis. bcl-2, an inhibitor of apoptosis, was diffusely expressed in the interlobular bile ducts, but rarely detectable in the large and septal bile ducts. bcl-XL and mcl-1, inhibitors of apoptosis, and bax, a promoter of apoptosis, were diffusely expressed along the intrahepatic biliary tree. CD95, a direct inducer of apoptosis, was present in the large and septal bile ducts, but rarely in the interlobular bile ducts. The ratio of bax to bcl-2, as well as the expression of CD95 which differed at the interlobular versus large and septal bile ducts, may be responsible for the unique distribution of apoptotic biliary cells and involved in the homeostasis of the intrahepatic biliary tree.

The bystander effect in photodynamic inactivation of cells

Treatment of MDCK II cells with the lipophilic photosensitizer tetra(3-hydroxyphenyl)porphyrin and light was found to induce a rapid apoptotic response in a large fraction of the cells. Furthermore, the distribution of apoptotic cells in microcolonies of eight cells was found to be different from the binomial distribution, indicating that the cells are not inactivated independently, but that a bystander effect is involved in cell killing by photodynamic treatment. The observation of a bystander effect disagrees with the common view that cells are inactivated only by direct damage and indicates that communication between cells in a colony plays a role in photosensitized induction of apoptosis. The degree of bystander effect was higher for cells dying by necrosis than for cell dying by apoptosis.

In vivo regulation of cell death by embryonic (pro)insulin and the insulin receptor during early retinal neurogenesis.

Programmed cell death is an established developmental process in the nervous system. Whereas the regulation and the developmental role of neuronal cell death have been widely demonstrated, the relevance of cell death during early neurogenesis, the cells affected and the identity of regulatory local growth factors remain poorly characterized. We have previously described specific in vivo patterns of apoptosis during early retinal neurogenesis, and that exogenous insulin acts as survival factor (Díaz, B., Pimentel, B., De Pablo, F. and de la Rosa, E. J. (1999) Eur. J. Neurosci. 11, 1624-1632). Proinsulin mRNA was found to be expressed broadly in the early embryonic chick retina, and decreased later between days 6 and 8 of embryonic development, when there was increased expression of insulin-like growth factor I mRNA, absent or very scarce at earlier stages. Consequently, we studied whether proinsulin and/or insulin ((pro)insulin) action in prevention of cell death has physiological relevance during early neural development. In ovo treatment at day 2 of embryonic development with specific antibodies against (pro)insulin or the insulin receptor induced apoptosis in the neuroretina. The distribution of apoptotic cells two days after the blockade was similar to naturally occurring cell death, as visualized by TdT-mediated dUTP nick end labeling. The apoptosis induced by the insulin receptor blockade preferentially affected to the Islet1/2 positive cells, that is, the differentiated retinal ganglion cells. In parallel, the insulin survival effect on cultured retinas correlated with the activation of Akt to a greater extent than with the activation of MAP kinase. These results suggest that the physiological cell death occurring in early stages of retinal development is regulated by locally produced (pro)insulin through the activation of the Akt survival pathway.

The distributions of apoptotic cells in the medial preoptic areas of male and female neonatal rats.

The medial preoptic area (MPA) of the hypothalamus of the rat contains two sexually dimorphic nuclei, the periventricular preoptic nucleus (PVpo) and the medial preoptic nucleus (MPN). To examine the relationship between sexual dimorphism and neuronal death, we examined the number of apoptotic cells in the subdivisions of the MPA in neonatal rats of postnatal days 1 (P1), 4 (P4), 7 (P7) and 14 (P14). Apoptotic cells in these areas were classified according to their progression into three stages. P1 and P4 rats contained many apoptotic cells in the subfield along the third ventricle, including the PVpo, and their number was significantly larger in P1 males: in particular, the number of early-stage cells was larger in males than females. The number of apoptotic cells in the MPN was increased in P4 and P7 rats, although no significant sexual differences were seen in the total number or in the number of each progressive stage of apoptotic cells. In P14 rats, very few apoptotic cells were seen in the MPA. Our data revealed that the distribution of apoptotic cells in the MPA of developing rats depends on the sexuality, subdivision of the area and postnatal period.

Apoptosis during development of the human retina: Relationship to foveal development and retinal synaptogenesis

Apoptosis in the ganglion cell (GCL) and inner nuclear (INL) layers of human fetal retinae aged 14–35 weeks of gestation (WG) was investigated in relation to synaptogenesis and foveal depression formation. Terminal transferase dUTP-biotin nick end labeling (TUNEL) was used to identify apoptosis, and synapse development was demonstrated by synaptophysin immunoreactivity (-IR). The distribution of apoptotic cells and synaptophysin-IR was studied as a function of eccentricity. Between 14 and 23–24 WG in the GCL, rates of apoptosis were relatively low in central retina. A shallow fovea was detected at 23–24 WG. In the central GCL, the rate of apoptosis was 0.21% of viable cells compared with a higher incidence of 0.79–1.64% peripherally. Apoptosis in the INL was 2–8 times greater than that in the GCL. At 14–15 WG, peak death occurred at the incipient fovea; however, by 20 WG the distribution was bimodal, with peaks at more eccentric locations on either side of the incipient fovea with increasing age. Approximately 90% of INL apoptotic cells were in the middle and outer regions, suggesting that bipolar cells formed the majority of dying neurons. Synaptophysin-IR was present in cones, bipolar cells, and processes in the inner and outer plexiform layers at the incipient fovea at 14 WG and spread peripherally with increasing age. The peripheral margin of synaptophysin-IR coincided with areas of peak INL apoptosis. This pattern suggests that bipolar cell elimination is associated with the onset of synaptogenesis. Apoptosis in the GCL and INL is not a significant factor in foveal depression morphogenesis. J. Comp. Neurol. 413:198–208, 1999. © 1999 Wiley-Liss, Inc.

Apoptosis during development of the human retina: Relationship to foveal development and retinal synaptogenesis

Apoptosis in the ganglion cell (GCL) and inner nuclear (INL) layers of human fetal retinae aged 14-35 weeks of gestation (WG) was investigated in relation to synaptogenesis and foveal depression formation. Terminal transferase dUTP-biotin nick end labeling (TUNEL) was used to identify apoptosis, and synapse development was demonstrated by synaptophysin immunoreactivity (-IR). The distribution of apoptotic cells and synaptophysin-IR was studied as a function of eccentricity. Between 14 and 23-24 WG in the GCL, rates of apoptosis were relatively low in central retina. A shallow fovea was detected at 23-24 WG. In the central GCL, the rate of apoptosis was 0.21% of viable cells compared with a higher incidence of 0.79-1.64% peripherally. Apoptosis in the INL was 2-8 times greater than that in the GCL. At 14-15 WG, peak death occurred at the incipient fovea; however, by 20 WG the distribution was bimodal, with peaks at more eccentric locations on either side of the incipient fovea with increasing age. Approximately 90% of INL apoptotic cells were in the middle and outer regions, suggesting that bipolar cells formed the majority of dying neurons. Synaptophysin-IR was present in cones, bipolar cells, and processes in the inner and outer plexiform layers at the incipient fovea at 14 WG and spread peripherally with increasing age. The peripheral margin of synaptophysin-IR coincided with areas of peak INL apoptosis. This pattern suggests that bipolar cell elimination is associated with the onset of synaptogenesis. Apoptosis in the GCL and INL is not a significant factor in foveal depression morphogenesis.

Ontogeny of the thymus in a teleost fish, Cyprinus carpio L.: developing thymocytes in the epithelial microenvironment

A monoclonal antibody, WCL9, specific for membrane molecules of a thymocyte subpopulation was used to detect these cells in situ during the ontogeny of thymus. Cryo-sections revealed WCL9 + cells in the rudiment of the thymus (day 4 post fertilization); thereafter, the positive cells were observed exclusively in the cortex from the first appearance of thymic regionalization (week 4 post fertilization) until adult age. Whole-mount immunostaining of the thymus with WCL9 revealed the three-dimensional structure of the cortex by specific staining. The presence and distribution of apoptotic cells during thymus development was studied by in situ end-labelling of fragmented DNA. From week 4 post fertilization onwards, apoptotic cells were more frequently detected in the cortex than medulla, suggesting a continuous selection of thymocytes in the cortex. Ultrastructural studies confirmed the presence of numerous cortical apoptotic cells inside macrophages. Electron microscopy provided evidence for the existence of epithelial heterogeneity in the thymus. During the ontogeny, the differentiation of epithelial cells was followed from the first weeks until the juvenile age. Cell types were classified on the basis of their localization and cytological characteristics as: i) limiting epithelial cells located in subcapsular, perivascular and peritrabecular zones; ii) reticular epithelial cells situated in medullary and cortical zones; iii) nurse-like cells at the border between the cortex and medulla, iiii) Hassalls body-like structures localized in the medulla. This study could suggest the occurrence of a wide range of lympho-epithelial interactions throughout thymocytes differentiation.

Temporal and spatial profile of apoptotic cell death in transient intracerebral mass lesion of the rat.

Apoptosis is involved in the pathogenesis of cerebral ischemia. Previous studies have confirmed that the brain surrounding an intracerebral hematoma develops ischemia. We investigated the number and distribution of cells exhibiting DNA fragmentation with apoptotic morphology in the transient intracerebral mass lesion to determine whether apoptosis contributed to the lesion progress after intracerebral hemorrhage (ICH). Transient intracerebral mass was created by inflation of a microballoon for 10 min (group A) or 2 h (group B) in the caudoputamen in rats, and brains were examined 1, 3, 6, 24, and 48 h after microballoon deflation. The lesion volume was calculated using parallel coronal sections with cresyl violet staining. Terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine (dUTP)-biotin nick end labeling (TUNEL) was used to detect cells undergoing DNA fragmentation. Immunohistochemistry for Fas antigen was also done to ascertain molecular mechanisms of apoptosis. Histological examination of hematoxylin and eosin-stained sections showed the typical appearance of neuronal necrosis in the caudoputaminal lesion. Lesion volume in the caudoputamen gradually increased as time advanced from 1 to 48 h. Cells stained heavily by TUNEL with apoptotic morphology were detected in the lesion, but not in the inner boundary zone of the lesion. The number of these cells significantly increased from 6 to 24 h in each experimental group (p < 0.05). The cells with positive immunoreactivity for Fas antigen was prominently observed in the lesion at 6 h. The distribution of apoptotic cells and the rapid increase in the number of apoptotic cells after 24 h propose that apoptotic cell death may contribute to lesion core formation but not to gradual development of the lesion.

Apoptosis in metastatic carcinoma of the central nervous system

High apoptotic indices have been described in pseud‐opalisading areas of glioblastoma multiforme (astrocytoma grade 4). In order to explore further the role of apoptosis in regulating the proliferation in central nervous system (CNS) tumors, we used TdT‐mediated dUTP‐nick endlabeling (TUNEL) methods to compare the frequency and distribution of apoptotic cells in 32 cases of metastatic carcinoma and 54 cases of primary diffuse astrocytomas. Frequencies of apoptotic cells and Bcl‐2 positive cells in metastatic carcinoma were low compared with those observed in high‐grade diffuse astrocytomas and did not correlate with the expression of the p53 protein. These findings suggest that apoptosis does not have a major role in regulating proliferation in metastatic carcinoma of the CNS.

Fas ligand, tumor necrosis factor-alpha expression, and apoptosis during allograft rejection and tolerance.

Cytotoxic T cells can induce target cell lysis and apoptosis by different pathways. The interactions of CD95 antigen (Fas) with its ligand (CD95L) and of tumor necrosis factor (TNF)-alpha with its receptor (TNF-R1) lead to apoptotic cell death. Recently, conflicting studies have been published concerning the expression and the role of CD95L in allograft rejection and tolerance. In this study, the intragraft expression of CD95/CD95L and TNF-alpha and the frequency and distribution of apoptotic cells were compared in a model of heterotopic cardiac allograft in the rat in which recipients were either not treated (acute rejection) or pretreated with donor-specific blood transfusion (tolerant). In the acutely rejected allografts, a peak in the expression of CD95L and TNF-alpha and in the number of apoptotic cells was observed during the first week after transplantation; apoptotic cells were confined to graft-infiltrating cells. In the tolerated allografts, however, levels of graft-infiltrating cell apoptosis and CD95L and TNF-alpha expression during the same period of time were dramatically lower. The expression of Fas was constitutive and was not modulated during acute rejection or tolerance. This down-regulation of CD95L and TNF-alpha in allografts rendered tolerant by donor-specific transfusion suggests a role for apoptosis-inducing pathways in acute allograft rejection.

Insulin-like growth factor binding protein 5 is associated with involution of the ventral prostate in castrated and finasteride-treated rats.

Insulin-like growth factor binding protein (IGFBP)-5 has been proposed as a signal for apoptosis in the ovary. To determine the relationship between IGFBP-5 and apoptosis during regression of the androgen-deprived prostate, rats were castrated or treated with the 5alpha-reductase inhibitor finasteride for 4, 9, 14, 21, and 28 days. Ventral prostate tissue was immunostained for IGFBP-5, and apoptotic cells were identified by in situ end-labeling of fragmented DNA (TUNEL). To compare the distribution of IGFBP-5 with the distribution of apoptotic cells, mirror-image serial sections of prostate tissues from normal and day 4 finasteride-treated rats were examined. In normal rats, 4+/-1% of prostate epithelial cells stained positively for IGFBP-5, and 0.1+/-0.03% demonstrated DNA fragmentation. IGFBP-5 staining peaked at day 9 with 93 +/-2% and 64+/-13% of epithelial cells staining positively in castrated and finasteride-treated rats, respectively. In contrast, DNA fragmentation peaked at day 4 in tissues from both castrated and finasteride-treated rats with 7+/-1% and 0.7+/-0.3% of epithelial cells, respectively, staining. In the serial sections, TUNEL and IGFBP-5 staining were not usually expressed in the same cells. Prostatic involution involves both programmed cell death and inhibition of cell growth. Because of the distribution of staining and the delayed expression of IGFBP-5 relative to initiation of apoptosis, we postulate that IGFBP-5 functions as an inhibitor of cell proliferation rather than as a signal for apoptosis.

Phosphorylation of Tau, Aβ-Formation, and Apoptosis After In Vivo Inhibition of PP-1 and PP-2A

Chronic inhibition of protein phosphatases 1 and 2A in vivo was induced by infusion of okadaic acid into lateral ventricles of rat brain for up to 4 months. Cytoskeletal pathology, alterations of the amyloid precursor protein, and apoptotic cell death induced by this treatment followed a certain sequence and spatial distribution. Changes in the expression, phosphorylation, and subcellular distribution of neurofilament proteins and tau, as well as first signs of apoptotic cell death, occurred already after about 2 weeks. The distribution of apoptotic cells, however, was different from those revealing a high accumulation of hyperphosphorylated tau, indicating that those cytoskeletal pathology had no obvious sequelae for the viability of these neurones. A continuation of treatment for longer than 2 weeks induced diffuse deposits of both hyperphosphorylated tau and Aβ-amyloid-immunoreactive material in white matter areas that increased in size and number over time. Because τ-phosphorylation is a regulator of the dynamic stability of microtubules, the pathology observed in the present experimental paradigm in the white matter might be viewed as an indication of a disturbed axonal transport. It is hypothesized that perturbations of the axonal transport might also be critically involved in the formation of paired helical filaments and amyloid deposits in Alzheimer’s disease.

Mouse molar morphogenesis revisited by three-dimensional reconstruction. II. Spatial distribution of mitoses and apoptosis in cap to bell staged first and second upper molar teeth.

Tooth morphogenesis is a complex multifactorial process in which differential mitotic activities and cell death play important roles. Upper first (m1) and second (m2) molars from mouse embryos were investigated from early cap to bell stage. m2 differed from m1 by delayed origin of the enamel grooves delimiting the protrusion of the cap bottom towards the dental papilla, and retardation of the enamel knot formation. The width of the m2 enamel organ was conspicuously smaller during cap formation and length remained smaller throughout the period of observation. Formation of the cap depression was comparable in m1 and m2, however margins delimiting the enamel organ cavity arose in m1 and m2 as mirror images. Attempts were made to correlate changes in the distribution of apoptotic cells and bodies and/or mitoses with morphogenesis. These cellular activities were recorded from histological sections and represented in space using computer-assisted three-dimensional reconstructions. Mitoses in the epithelial compartment were associated with the development of the cervical loop. In the mesenchyme of m1 at early bell stage, a postero-anterior increasing gradient of mitoses was observed which might be correlated with the anterior growth of the molar. Cells in the enamel knot demonstrated a high level of apoptosis, retarded in m2, but absolutely no division. Apoptotic processes were also involved in the anterior delimitation of the m1 epithelium. Apoptosis might correspond to the programmed destruction of cells whose function had to be suppressed or whose potential activity had to be avoided.

A transient phase of cell death in the developing medial forebrain of the perinatal ferret.

A transient bilateral population of cells immediately rostral to the ferret corpus callosum was examined at weekly intervals between embryonic day 28 and postnatal day 7. This region is tentatively identified as the medial forebrain apoptosis zone (MFAZ) because of the specific nature of cell death, and the limited area or zone in which it was observed. No other region within the brain or retina exhibited a similar pattern or amount of cell death. Only scattered apoptotic cells were found throughout the remainder of the brain-including the cerebral cortical plate, subplate, and white matter-with the exception of the ventricular zone of the lateral ventricles which contained a significant population of apoptotic cells. This study addressed three questions about apoptosis in the MFAZ: (1) does apoptotic cell death in this region signal the appearance of phagocytic macrophages, (2) does cell degeneration and phagocytosis in this region lead to the formation of an extracellular space analogous to the cavum septi pellucidi of rodents, and (3) what is the duration of degeneration, or clearance rate, of cell death in this defined region. The MFAZ was first found to contain apoptotic nuclei and macrophages late in gestation, at E34. Numbers of apoptotic nuclei and macrophages peaked one week later at birth in this area, but disappeared early in postnatal life. During this period, formation of a space rostral to the corpus callosum due to the removal of apoptotic cells was not observed. Finally, presence of apoptotic cells in the MFAZ over a period of > or = 9 days suggests a clearance rate of many hours. The close spatiotemporal correlation of the distribution of apoptotic cells and their removal by macrophages suggests that these are interrelated, and perhaps interdependent events.