The pathogenesis of vascular changes in erythema nodosum-like lesions of Beh?et's syndrome: an electron microscopic study

The probable sequence of microvascular changes affecting endothelial cells in erythema nodosum-like lesions was examined by electron microscopy in 18 patients with typical Beh?et's syndrome. Dark endothelial cells in the superficial and deep dermis exhibited degeneration with contraction changes. Consequently, degenerated endothelial cells showed nuclear and cytoplasmic condensation, vacuolization in the cytoplasm, and rich ribosomes. Moreover, blood vessels which were enveloped by cuffs of lymphocytes simultaneously exhibited both endothelial cell hypertrophy and endothelial cell necrosis. The implied associations between perivascular lymphocytic cuffs and blood vessels exhibiting endothelial cell alterations indicate that microvascular changes in erythema nodosum-like lesions may be closely associated with the delayed-type hypersensitivity reaction.     

Lipopolysaccharide induces apoptosis in a bovine endothelial cell line via a soluble CD14 dependent pathway

We investigated the mode of bovine endothelial cell death induced by lipopolysaccharide (LPS). Inter-nucleosomal DNA fragmentation was observed indicating apoptotic cell death. Apoptosis was confirmed by examining cell morphology using confocal laser scanning and transmission electron microscopy, which revealed characteristic nuclear condensation in LPS-treated endothelial cells. Apoptosis was blocked by a monoclonal antibody against CD14, suggesting that LPS triggers apoptosis via a soluble CD14 (sCD14) dependent mechanism.     

Mechanism of deletion of endothelial cells during regression of the corpus luteum

Endothelial cells are deleted rapidly, and in large numbers, during cyclical regression of the corpus luteum of the guinea pig. This paper reports a study of the mechanisms, structural and causal, by which this deletion occurs. Corpora lutea from guinea pigs were examined by transmission electron microscopy on day 9 (functional stage) and day 16 (regressing) of the estrous cycle. Corpora lutea were also studied at 1, 3, 12, 24, and 48 hours after administration of a synthetic luteolytic substance (cloprostenol), and after temporary occlusion of the ovarian blood vessels for 15, 30, 60, or 120 minutes. Early signs of endothelial cell degeneration included protrusion of some individual cells into capillary lumina and the formation of adherens junctions across the lumen. Intermediate stages of degeneration included nuclear and cytoplasmic condensation and cellular and nuclear lobation or fragmentation in cells either protruding into, or lying within, the lumen. Terminal changes included loss of plasma membrane integrity and cytoplasmic density, together with disruption of cell organelles. Some degenerate endothelial cells were engulfed by viable endothelial cells. Macrophages were not seen to be involved in removal of dead endothelial cells, and integrity of the walls of capillaries was maintained while individual endothelial cells were deleted. Experimental findings were consistent with the hypothesis that cessation or reduction of flow of blood along capillaries plays an etiologic rule in endothelial cell deletion.     

IGF—IR反义硫代磷酸型寡核苷酸诱导胶质瘤细胞凋亡的病理学研究

目的：探讨IGF-IR基因的反义硫代磷酸型寡核苷酸对胶质瘤细胞的形态学影响。方法：根据IGF-IRcDNA序列设计正义,反义寡核苷酸片段,并对其部分碱基进行硫代磷酸修饰。体外培养的胶质瘤细胞分别经正义寡核苷酸和反义寡核苷酸处理,应用倒置显微镜活细胞观察,HE染色光镜观察,透射电镜及DNA琼脂糖凝胶电泳等方法研究IGF-IR反义硫代磷酸型寡核苷酸诱导胶质瘤细胞凋亡作用。结果：经反义寡核苷酸转染的胶质瘤细胞中,呈现典型的凋亡形态学改变,凋亡细胞最早期表现为细胞体积,容量减少,染色质凝聚,继之染色质集聚于核膜下成7新月状或块状;细胞核内染色质可完全固缩成团呈“黑洞”样或萎缩的核破裂形成一些较小的膜包绕的球体位于胞质内,最后出泡形成凋亡小体,DNA琼脂糖凝胶电泳分析经反义寡核苷酸处理的胶质瘤细胞DNA降解片段,可见有明显的小分子量DNA梯状条带,而野生型和正义寡核苷酸处理的胶质瘤细胞未见DNA梯状条带。结论：IGF-IR所介导失发泌环路IGF-I/IGF-IR。在胶质瘤细胞增殖和维持恶性表型中起重要作用。IGF-IR反义硫代磷酸型寡核苷酸能诱导胶质瘤细胞凋亡。     

Scanning and transmission electron microscopic observation of femoral head feeding vessels in stroke-prone spontaneously hypertensive rats

Stroke-prone spontaneously hypertensive rats (SHRSP) are known to show necrosis of the femoral head with a frequency of about 50%. This rat has thus been used as an animal model for necrosis of the femoral head in many studies. In a detailed investigation of feeding vessel disorders that cause femoral head necrosis, we observed changes over time in the feeding vessels using scanning electron microscopy and transmission electron microscopy. In scanning electron microscopy of vascular casts, abnormal findings in feeding vessels of SHRSP with aging from the immature stage included contortion and bending in the lumen with overall narrowing. Under transmission electron microscopy, decreased numbers of smooth muscle cells and increased amounts of collagen fibers were marked, and these changes with hypertrophy of vascular walls might be similar to those of arteriolosclerosis. The structural changes first revealed by transmission electron microscopic observation might cause the friability of the feeding vessels so that contortion and bending occurred, suggesting transient obstruction of blood flow to the femoral head and subsequent induction of femoral head necrosis. These findings should help in understanding the causes of femoral head necrosis in humans, including Perthes’ disease.     

Morphological changes of cerebral arteries in a canine double hemorrhage model

Cerebral vasospasm is a major cause of morbidity and mortality in patients suffering from subarachnoid hemorrhage (SAH). Despite numerous studies, the pathogenesis of this deadly disorder is not clearly understood. Alterations in endothelial cells are a distinct morphological feature of cerebral vasospasm and some recent studies suggest that apoptosis might play a role in the cells' death. The goal of the present study is to examine the time course of apoptosis in endothelial cells of spastic cerebral arteries following experimental subarachnoid hemorrhage. Fifteen dogs were used in the present study. Twelve of them were divided into three groups (four per group) and subjected to a double-hemorrhage method of SAH. Following SAH, groups were sacrificed respectively on days 3, 5, and 7. Three dogs served as controls without blood injection. The basilar arteries were studied with the transmission electron microscopy and with angiography. Angiographic vasospasm began on day 3 and peaked on day 7. In morphologic studies, control dogs did not demonstrate apoptotic-like changes in endothelial cells of the basilar arteries. Beginning with day 3, apoptotic-like changes were noted in endothelial cells and consisted of condensation of peripheral nuclear chromatin, blebbing of the cell membrane, and condensation of the cytoplasm. Such changes progressed with time and were maximally developed by day 7. This is the first study that demonstrates the time course of apoptotic-like changes in the endothelial cells in the vasospastic basilar artery. Apoptosis might play an important role in the pathogenesis of vasospasm.     

人endostatin体外诱导血管内皮细胞凋亡的实验研究

目的 探讨人endostatin抑制血管内皮细胞增殖的作用机制。方法 在含重组人endostatin蛋白和10%小牛血清的DMEM培养基中，培养人脐静脉内皮细胞ECV304。72h后，采用透射电镜，流式细胞术细胞周期分析和细胞核DNA的1.5%琼脂糖凝胶电泳，检测重组人endostatin蛋白作用后，血管内皮细胞的凋亡。结果 透射电镜下可见实验组ECV304细胞核染色质浓缩，边集，核碎裂及胞浆浓缩等，呈典型的凋亡细胞的形态学表现，流式细胞术细胞周期分析显示，在G1期峰前存在1个凋亡峰（20.6%)，1.5%琼脂糖凝胶电泳显示，细胞核DNA呈梯状，对照组ECV304细胞表达正常。结论 重组人endostatin蛋白可诱导血管内皮细胞凋亡，是其抑制血管内皮细胞增殖的原因之一。     

Scanning and transmission electron microscopic studies of microvascular pathology in the osmotically impaired blood-brain barrier

Summary The present investigation focused on the structural events occurring in endothelial cells lining the lumina of brain microvessels in rats subjected to a single intracarotid injection of hypertonic 1.8m l (+) arabinose solution with or without intravenous injection of horseradish peroxidase. Blood vessels from cerebral cortex and thalamus were evaluated by transmission and scanning electron microscopy. After short-term exposure (10–12 min) there was widespread flooding of peroxidase into the brain neuropil of the ipsilateral hemisphere. Peroxidase tracer was frequently observed within vesiculo-tubular profiles, and occasionally within widened interendothelial junctional clefts. Partially fragmented, necrotic endothelial cells appeared to be in the process of desquamation. Individual endothelial cells appeared to be shrunken with widened interendothelial spaces. Some healthy endothelial cells appeared to be involved in repair processes, manifested by the extension of thin cellular processes towards the area of vessel injury. Other pathological alterations included a conspicuous increase in the number of endothelial cell microvilli, large crater-like invaginations of the endothelial plasma membranes and muscular blood vessels in the process of spasm. We also observed a platelet reaction with or without endothelial cell necrosis and attached microthrombi in some arterial segments.     

Endothelial changes of hypertensive rat mesenteric arteries

The mesenteric arteries of hypertensive rats with bilaterally constricted renal arteries were observed by light, scanning electron and transmission electron microscopy. In the early stage of hypertensive arterial lesions, many leukocytes adhered to the endothelial surface, and large or small and polygonal endothelial cells were irregularly arranged. Sporadically brightened and atrophied endothelial cells were distributed on the injured surface. These atrophied cells were transmission electron microscopically confirmed to have undergone coagulation necrosis. In the surrounding of the atrophied cells were observed opened endothelial cell junctions and enlarged intercellular spaces to which many leukocytes and platelets adhered. Because of loosening of connection with neighboring cells, atrophied endothelial cells became solitary or being denuded. Opened endothelial cell junctions were also observed between the uninjured endothelial cells as well as in areas penetrated by leukocytic pseudopods. Where light microscopy disclosed the deposition of a large amount of fibrinoid substance in the intima and media, scanning electron microscopy showed opened endothelial cell junctions and denudation of endothelial cells. From the results of the present experimental study, it was suggested that in the genesis of the hypertensive rat arterial lesions, the degeneration, necrosis and denudation of endothelial cells, opened endothelial cell junctions between these cells, and insudation of blood plasma through the opened junctions or denuded areas might play important roles.     

Stromal cell organisation in the mouse lymph node. A light and electron microscopic investigation using the zinc iodide-osmium technique

The organisation of the stromal cell compartment in the mouse lymph node was studied by light and electron microscopy after tissue impregnation by the zinc iodide-osmium (ZIO) method. Fibroblastic reticular cells (FRCs) represented the main stromal cell population. These cells were located both in the cortical region and in the medulla and exhibited various configurations. In the cortex, FRCs were fusiform in shape and came into close proximity with the floor of the subcapsular sinus. In the medulla, the FRCs were shaped like irregular dendritic cells which formed a complex 3-dimensional network. The FRCs surrounded vascular structures such as capillaries and/or high endothelial venules; in these instances they were organised in a discontinuous sheath-like fashion around the vessel wall. By light and electron microscopy, FRCs have been observed to come in close spatial relationship with a number of cells in the lymph node, including sinus endothelial cells, the endothelium of high endothelial venules and capillaries, various types of lymphocytes, follicular dendritic cells and interdigitating cells. These microanatomical features are consistent with the proposal that FRCs may be involved in the communicative networks between the different lymph node compartments. In particular, the FRCs may be involved in the transport of molecules from the sinus compartment to the high endothelial venules or to the distinct cell populations in the lymphoid parenchyma.     

ENDOTHELIAL CHANGES OF HYPERTENSIVE RAT MESENTERIC ARTERIES

The mesenteric arteries of hypertensive rats with bilaterally constricted renal arteries were observed by light, scanning electron and transmission electron microscopy. In the early stage of hypertensive arterial lesions, many leukocytes adhered to the endothelial surface, and large or small and polygonal endothelial cells were irregularly arranged. Sporadically brightened and atrophied endothelial cells were distributed on the injured surface. These atrophied cells were transmission electron microscopically confirmed to have undergone coagulation necrosis. In the surrounding of the atrophied cells were observed opened endothelial cell junctions and enlarged intercellular spaces to which many leukocytes and platelets adhered. Because of loosening of connection with neighboring cells, atrophied endothelial cells became solitary or being denuded. Opened endothelial cell junctions were also observed between the uninjured endothelial cells as well as in areas penetrated by leukocytic pseudopods. Where light microscopy disclosed the deposition of a large amount of fibrinoid substance in the intima and media, scanning electron microscopy showed opened endothelial cell junctions and denudation of endothelial cells. From the results of the present experimental study, it was suggested that in the genesis of the hypertensive rat arterial lesions, the degeneration, necrosis and denudation of endothelial cells, opened endothelial cell junctions between these cells, and insudation of blood plasma through the opened junctions or denuded areas might play important roles.     

Ventral ectodermal ridge and ventral ectodermal groove: two distinct morphological features in the developing rat embryo tail

The ventral ectodermal ridge (VER) is a thickening of the surface ectoderm on the ventral side of the embryonic tail which resembles the apical ectodermal ridge of the limb bud. The morphological characteristics of the ventral part of the embryo tail were investigated in 10.5- to 14-day rat embryos by light microscopy of serial semithin sections and by scanning and transmission electron microscopy. In 10.5- to 11.5-day embryos the thickening of the ventral surface ectoderm includes the complete ventral midline of the tail and can be divided into two parts. The posterior part is elevated and represents the ventral ectodermal ridge. The anterior part is, in contrast to the ridge, concave, and we have termed it the ventral ectodermal groove (VEG). The cloacal membrane is located at its anterior end. Contacts between the VER and the mesenchymal cells are visible until an intact basal lamina is formed at 11.5 days. Similarly, the VEG is connected by elongated cell processes with the ventral part of the tail gut. Gap junctions are present between the apical parts of ridge and groove cells. The VEG flattens and disappears in 12-day embryos. At this stage the ridge is at its maximum height, simultaneously undergoing extensive cell death. The VER is no longer visible in 14-day rat embryos.     

Observation on the basal lamina of duodenal mesothelial cells during metamorphosis of Xenopus laevis

Morphologic changes in the basal lamina of duodenal mesothelial cells during metamorphosis of Xenopus laevis were observed. In the prometamorphosis stage (Stage 56-59), the basal lamina was almost completely flat; the lamina densa of the basal lamina was a 50 nm layer of high electron density. In the early stages of metamorphic climax (Stage 60-62), the basal lamina showed occasional slight folding (stage 60), with the lapse of time, the folding became continuous and deeper. The development of an additional thin basal lamina was observed in areas where the folded basal lamina was separated from mesothelial cells, viz. on the side adjacent to the mesothelium. The lamina densa in this stage was approximately twice the thickness of the prometamorphosis stage and exhibited high electron density. In the later stages of metamorphic climax (stage 63-66), the basal lamina just under mesothelium became more apparent. The folded basal lamina shifted from the mesothelium into the subserosa and gradually disappeared, and the basal lamina became a single layer. The thickness of the lamina densa was almost the same as in the prometamorphosis stage. Since the timing of the folding of the basal lamina coincides with the shortening of the digestive tract and the marked narrowing of the lumen, we suggest that physical changes in the digestive tract during metamorphosis may play an important role in these morphologic changes of the basal lamina.     

Innervation of blood vessels in the rat incisor pulp: A scanning electron microscopic and immunoelectron microscopic study

Although two types of nerve endings have been proposed to innervate blood vessels in the dental pulp, the precise innervation pattern is not well understood. This is mainly due to the lack of information regarding the positional relationships of nerve fibers with blood vessels at the electron microscopic level. The rat incisor pulp was investigated by scanning electron microscopy (SEM) after connective tissue digestion and by transmission electron microscopy after immunohistochemical localization with polyclonal anti-PGP 9.5 antibody. SEM specimens revealed that unmyelinated nerve fibers passed through the tunica adventitia of the blood vessel in the center of the pulp and that these fibers then entered the tunica media of the smaller arterioles. The nerve fibers divided into many collaterals, and these terminated on the surface of smooth muscle cells and endothelial cells. The fibers extended toward smaller vessels. The terminal fibers then reached the subodontoblastic capillary plexus and terminated on pericytes, while the odontoblastic capillary plexus had no direct innervation. In the pulpal venules, nerve fibers were located adjacent to the pericytes and endothelial cells, and they extended toward postcapillary venules. The same results were confirmed by immunoelectron microscopy. The present study demonstrated that in the rat incisor pulp the microvasculature as well as larger vessels were directly innervated by free nerve endings, suggesting that the local regulation of blood flow could take place not only at larger vessels but also at the level of the microvasculature in this tissue. Anat. Rec. 251:384–391, 1998. © 1998 Wiley-Liss, Inc.     

Cell surface and nuclear changes during TNF-α-induced apoptosis in WEHI 164 murine fibrosarcoma cells

 Tumour necrosis factor (TNF)-α-induced apoptosis is associated with several nuclear and cell surface alterations, in particular with the condensation of chromatin and the fragmentation of the cell nucleus, formation of blebs on the cell surface and breakdown of the plasma membrane. However, there is little information about the relationship between the cell surface alterations and the nuclear changes during apoptosis. To study this, cultured WEHI cells were exposed to TNF-α over different time periods. The cytological changes were studied using a correlative approach, which allowed observation of the same cell consecutively under light, scanning and transmission electron microscopy. The earliest sign of cell alteration was a reduction of the number of microvilli after 15 min of TNF-α exposure. This reaction was reversible (reappearance of microvilli) and took place during the first hour, in which neither nuclear alterations nor plasma membrane breakdown were observed. The changes in the nucleus began with condensation of chromatin after approximately 1 h of TNF-α-exposure. After 4–5 h the microvilli disappeared again, particularly in areas where the formation of blebs (blebbing) was observed. Strikingly, cell surface alterations (bleb formation) were detected only in those cells that presented with condensed chromatin, and not in cells with a normal chromatin pattern, proving at least a close correlation between nuclear and cell surface changes during the process of apoptosis. Received: 7 October 1997 / Accepted: 23 March 1998     

鸡胚脾脏凋亡细胞核及其环孔板的超微结构

目的：着重观察鸡胚脾脏凋亡细胞核及其环孔板的形态学变化。方法,用放线菌酮处理第１５天胚龄鸡胚,建立鸡胚脾脏细胞凋亡模型,用透射电镜进行观察。结果：凋亡细胞为各类幼稚血细胞,以幼稚淋巴细胞为主,巨噬细胞未见凋亡。凋亡细胞核在裂解分离为核碎块的过程中出现环孔板,凋亡细胞核完全裂解分离后,环孔板消失。结果：环孔板参与凋亡细胞核的分割以及核碎块的包囊,提示凋亡细胞核裂解成为核碎块形凋记录上体的过程是一个主     

Development of the connective tissue in the digestive tract of the larval and metamorphosing Xenopus laevis

Developmental changes, until the completion of metamorphosis, of the connective tissue of the digestive tract in the South African clawed frog Xenopus laevis were examined by light and scanning electron microscopy. During the pre- and prometamorphosis (stages 50 through 59) the layer of connective tissue was prominent in the typhlosole, while that in the rest of the intestine and stomach remained very thin. Most of the connective tissue cells were fibroblasts and were concentrated mainly in the typhlosole, the boundaries of which were distinct. In the anterior part of the typhlosole just behind the stomach, the layers of connective tissue and muscle were the thickest and the boundary between these 2 layers was obscure. Although the mitotic cells of the connective tissue were rare, if any, and no migrating cells were observed on the surface of the connective tissue, the connective tissue of the intestine gradually increased in cell number during the pre- and prometamorphosis. In the stomach, on the other hand, there was no such increase. At the beginning of the metamorphic climax (stages 60 and/or 61), the layer of connective tissue suddenly became thick in both the stomach and the intestine. The boundaries of the typhlosole became less distinct. Mitotic index of the connective tissue became high, and its cells dramatically increased in number and contained many undifferentiated mesenchyme-like cells. In the stomach, these connective tissue cells invaded the glands of the larval epithelium into just below the surface epithelium and mitotic index of the entire stomach epithelium increased. In the intestine, in contrast, mitotic epithelial cells were localized in the islets which invaginated into the connective tissue and later formed the adult epithelium. With the growth of islets, the intestinal folds were formed as straight rows and, after stage 63, were modified into a zigzag pattern and had mitotic cells localized in the troughs of folds. The present results indicate that dramatic changes of the connective tissue of both the stomach and the intestine occur at the beginning of the metamorphic climax, concomitantly with those of the epithelium. Moreover, the results also show that, before the climax, even when there were no indications of metamorphic changes in the epithelium, cells of the connective tissue gradually increased in number in the intestine (but not in the stomach). It is also suggested that the muscle cells of the typhlosole just behind the stomach may participate in this gradual increase of connective tissue cells.     

Developmental changes in the structure of the rat fetal lung,with special reference to the airway smooth muscle and vasculature

Structural changes in the developing rat lung were studied by a combined use of light microscopy including immunohistochemistry for a-smooth muscle actin (alpha-SMA) and scanning electron microscopy (SEM) using the KOH-collagenase digestion method. In the embryonic stage (E11-E13), the lung bud appeared as an outgrowth from the ventral wall of the foregut which grew caudally into the splanchnic mesoderm to form a pair of bronchial buds at the end. At E13, the airway smooth muscle cells first appeared around the bifurcation of the trachea. These smooth muscle cells were restricted to the dorsal surface of the tracheal epithelium, suggesting a difference in character between the dorsal and ventral sides of the mesenchymal cells in this region. During the pseudoglandular stage (E13-E18.5), the bronchial buds repeatedly gave off branches in the mesenchymal tissue. The smooth muscle cells in the bronchioles were spindle-shaped and arranged completely circularly around the epithelial tube, except that the terminal bud of bronchioles lacked the smooth muscles. The neck of the terminal bud was constantly surrounded by flat and irregularly-shaped immature smooth muscle cells, representing an early event in the smooth muscle cell differentiation from mesenchymal cells. In the canalicular to saccular stages (E18.5 to birth), the terminals of bronchioles became saccular, thus forming prospective alveolar acini. At birth, the alveolar wall became thinner than before birth, and the individual smooth muscle cells in bronchioles were elongated like a tape. As to the blood vessel differentiation, various sized sinusoidal spaces indicating the primitive blood vessels were already present in the mesenchymal tissue at E11.5. The endothelial cells of these sinusoidal spaces were irregularly shaped and sometimes extended their processes into the lumen. The network of tubular vessels appeared from E14.5. These vessels had tapering ends as well as transluminal trabeculae, suggesting that capillary growth proceeds by both the sprouting and partitioning (i.e., intussusception) of vessels in the pseudoglandular stage.     

Altered pericyte-endothelial relations in the rat retina during aging: implications for vessel stability

Mural cells (smooth muscle cells and pericytes) regulate blood flow and contribute to vessel stability. We examined whether mural cell changes accompany age-related alterations in the microvasculature of the central nervous system. The retinas of young adult and aged Wistar rats were subjected to immunohistofluorescence analysis of -smooth muscle actin (SMA), caldesmon, calponin, desmin, and NG2 to identify mural cells. The vasculature was visualized by lectin histochemistry or perfusion of horse-radish peroxidase, and vessel walls were examined by electron microscopy. The early stage of aging was characterized by changes in peripheral retinal capillaries, including vessel broadening, thickening of the basement membrane, an altered length and orientation of desmin filaments in pericytes, a more widespread SMA distribution and changes in a subset of pre-arteriolar sphincters. In the later stages of aging, loss of capillary patency, aneurysms, distorted vessels, and foci of angiogenesis were apparent, especially in the peripheral deep vascular plexus. The capillary changes are consistent with impaired vascular autoregulation and may result in reduced pericyte–endothelial cell contact, destabilizing the capillaries and rendering them susceptible to angiogenic stimuli and endothelial cell loss as well as impairing the exchange of metabolites required for optimal neuronal function. This metabolic uncoupling leads to reactivation of “physiological hypoxia” and angiogenesis in CNS aging.     

Docetaxel-induced apoptosis in the mitotic phase: electron microscopic and cytochemical studies of human leukemia cells

We induced apoptosis in cells of the human leukemia cell line HL-60 using an antitumor agent, docetaxel (Taxotere), and investigated apoptosis in various aspects using in situ end-labeling (ISEL) of DNA, DNA fragmentation assay, flow cytometry, and electron microscopy. Because it inhibits depolymerization of tubulin, docetaxel is thought to arrest the cell cycle at the mitotic stage and to exert an antitumor effect. In this study, accumulation of docetaxel-treated cells at the G2/M phase was detected using flow cytometry. On ISEL of DNA, DNA fragmentation was observed at the mitotic stage. On electron microscopy, the nuclei of apoptotic cells lost their nuclear membranes, as do cells at mitosis, demonstrating that the cells were arrested mainly at the M phase in the cell cycle.