Combined TUNEL and TRAP methods suggest that apoptotic bone cells are inside vacuoles of alveolar bone osteoclasts in young rats

Although it is generally accepted that osteoclasts breakdown and resorb bone matrix, the possibility that they may also be able to engulf apoptotic osteoblasts/ lining cells and/or osteocytes remains controversial. Apoptosis of osteoblasts/ lining cells and/or osteocytes and interactions between these cells and osteoclasts are extremely rapid events that are difficult to observe in viva. A suitable in viva model for studying these events is the alveolar bone of young rats because it is continuously. Thus, sections of aldehyde fixed alveolar undergoing intense resorption/remodeling bone of young rats were stained by the combined terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method and the tartrate-resistant acid phosphatase (TRAP) method for the simultaneous visualization of apoptotic cells and osteoclasts in the same section. The combined TUNEL and TRAP reactions, in the same section, greatly facilitated visualization of relationship between osteoclasts and apoptotic bone cells during alveolar bone remodeling. Our results showed that several TRAP-positive osteoclasts exhibited large vacuoles containing TUNEL positive apoptotic structures, probably derived from osteoblasts/lining cells and/or osteocytes. These results support the idea that alveolar bone osteoclasts are able to internalize dying apoptotic bone cells.

Long-term high-fat diet-induced obesity decreases the cardiac leptin receptor without apparent lipotoxicity

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Evaluation of Cell Proliferation and Apoptosis in Placentas of Rats with Severe Diabetes

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Immunosuppressant Prograf (R) (Tacrolimus) Induces Histopathological Disorders in the Peritubular Tissue of Rat Testes

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Structural alterations in the seminiferous tubules of rats treated with immunosuppressor tacrolimus

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Apoptosis during the seasonal spermatogenic cycle of Rana catesbeiana

In the bullfrog Rana catesbeiana, testicular weight is constant throughout the year, but the volume densities of germinative and interstitial compartments undergo inverse changes from winter (non-breeding) to summer (breeding). The occurrence of apoptosis in the seminiferous lobules of bullfrogs was investigated in these two periods using sections stained with haematoxylin and eosin (H&E), the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling) method and transmission electron microscopy. TUNEL-positive cells were observed in the seminiferous lobules, and ultrastructural morphological details confirmed the occurrence of cell death by apoptosis. In summer, the occurrence of several spermatogenic processes (in addition to spermiogenesis and spermiation), and then the overconsumption of Sertoli cell-derived pro-survival factors, could be responsible for the increased density of apoptotic cells. Alternatively, the low apoptotic frequency in winter could be related to the constant homeostasis in the germinative compartment given that most lobules are filled with primary spermatocytes. As volume densities of interstitial and germinative compartments undergo inverse seasonal variations through the year, the incidence of apoptosis (in summer) could play a part in controlling the spermatogenic process, maintaining the lobular size when interstitial tissue is maximally developed. In winter, the low apoptotic cell density leads to spermatogenic recrudescence and, thereby, the production of an adequate quantity of spermatozoa for the next breeding period. Thus, apoptosis may participate not only in the maintenance of spermatogenic homeostasis, but also in the cyclical control of the different spermatogenic processes according to seasonal changes of the testicular compartments as a whole.

Decrease in the number and apoptosis of alveolar bone osteoclasts in estrogen-treated rats

Bone is a mineralized tissue that is under the influence of several systemic, local and environmental factors. Among systemic factors, estrogen is a hormone well known for its inhibitory function on bone resorption. As alveolar bone of young rats undergoes continuous and intense remodeling to accommodate the growing and erupting tooth, it is a suitable in vivo model for using to study the possible action of estrogen on bone. Thus, in an attempt to investigate the possibility that estrogen may induce the death of osteoclasts, we examined the alveolar bone of estrogen-treated rats.Fifteen, 22-d-old female rats were divided into estrogen, sham and control groups. The estrogen group received estrogen and the sham group received corn oil used as the dilution vehicle. After 8 d, fragments containing alveolar bone were removed and processed for light microscopy and transmission electron microscopy. Sections were stained with hematoxylin and eosin and tartrate-resistant acid phosphatase (TRAP)-an osteoclast marker. Quantitative analysis of the number of TRAP-positive osteoclasts per mm of bone surface was carried out. For detecting apoptosis, sections were analyzed by the Terminal deoxynucleotidyl transferase-mediated dUTP Nick-End Labeling (TUNEL) method; TUNEL/TRAP combined methods were also used.The number of TRAP-positive osteoclasts per mm of bone surface was significantly reduced in the estrogen group compared with the sham and control groups. TRAP-positive osteoclasts exhibiting TUNEL-positive nuclei were observed only in the estrogen group. In addition, in the estrogen group the ultrastructural images revealed shrunken osteoclasts exhibiting nuclei with conspicuous and tortuous masses of condensed chromatin, typical of apoptosis.Our results reinforce the idea that estrogen inhibits bone resorption by promoting a reduction in the number of osteoclasts, thus indicating that this reduction may be, at least in part, a consequence of osteoclast apoptosis.

Cell death and quantitative reduction of rests of Malassez according to age

Background and Objective: Rests of Malassez are clusters of epithelial cells that remain in the periodontal ligament throughout life. However, it has been reported that the number of these structures decreases with age, and some epithelial cells undergo apoptosis in rests of Malassez of young and adult rats. Therefore, the purpose of the present study was to investigate the incidence of epithelial cell death and the quantitative changes in the rests of Malassez in rat molars of different ages.Material and Methods: Fragments containing the upper molars of rats aged 29, 45 and 120 d were fixed, decalcified and embedded for analysis by light microscopy. In the sections stained by hematoxylin and eosin, the number of rests of Malassez and the number of nuclei of these epithelial structures were obtained. Moreover, the nuclei exhibiting typical features of cell death were also counted in each rest of Malassez. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method for detection of cell death was also carried out.Results: In all groups examined, some rests of Malassez exhibited epithelial cell nuclei with typical features of apoptosis and some of them were also TUNEL positive. From 29 to 120 d of age in rats, the quantitative analysis showed a significant decrease in the total number of rests of Malassez in the cervical, middle and furcation regions of the periodontal ligament. Moreover, a significant decrease of epithelial cell nuclei was concomitant to an increase in the frequency of cell death in the oldest rats.Conclusion: These results suggest that epithelial cell death by apoptosis may be, at least in part, responsible for the reduction in the number of rests of Malassez according to age.

Vitamin B-12 Supplement Exerts a Beneficial Effect on the Seminiferous Epithelium of Cimetidine-Treated Rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Morphological evidences indicate that the interference of cimetidine on the peritubular components is responsible for detachment and apoptosis of Sertoli cells

Cimetidine, referred as antiandrogenic agent, has caused alterations in the seminiferous tubules, including alterations in the peritubular tissue and death of myoid cells by apoptosis. Regarding the structural and functional importance of the peritubular tissue for the maintenance of Sertoli cells (SC), we purpose to investigate the SC-basement membrane interface, focusing the morphological features of SC and their interaction with the basement membrane in the affected tubules by cimetidine. Ten animals were distributed into two groups, control (CG) and cimetidine (CmG) which received saline solution and 50 mg of cimetidine per kg of body weight, respectively, for 52 days. The testes were fixed, dehydrated and embedded for analyses under light and transmission electron microscopy. Paraffin sections were submitted to the TUNEL method; sections of testes embedded in glycol methacrylate were submitted to PAS method and stained by H&E for morphological and quantitative analyses of Sertoli Cells. In the CmG, the SC nuclei were positive to the TUNEL method and showed typical morphological alterations of cell death by apoptosis (from early to advanced stages). A significant reduction in the number of Sertoli Cells was probably due to death of these cells by apoptosis. A close relationship between SC nuclear alterations (including a high frequency of dislocated nuclei from the basal portion) and damage in the peritubular tissue was observed. The ultrastructural analysis showed a parallelism between the gradual advancement of apoptotic process in SC and detachment of the anchoring sites (hemidesmosomes) of SC plasma membrane from the lamina densa. The presence of portions of lamina densa underlying the detached hemidesmosomes indicates a continuous deposition of lamina densa, resulting in the thickening of the basal lamina. The results indicate a possible disarrangement of the SC cytoskeleton, including the focal adhesion structure. These alterations are related to SC apoptosis and probably result from disturbs induced by cimetidine on the peritubular tissue.

Neonatal Gonocyte Differentiation in Mongolian Gerbil Meriones unguiculatus Involves Asynchronous Maturation of Seminiferous Cords and Rapid Formation of Transitional Cell Stage

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Apoptotic bone cells may be engulfed by osteoclasts during alveolar bone resorption in young rats

The alveolar bone is a suitable in vivo physiological model for the study of apoptosis and interactions of bone cells because it undergoes continuous, rapid and intense resorption/remodelling, during a long period of time, to accommodate the growing tooth germs. The intensity of alveolar bone resorption greatly enhances the chances of observing images of the extremely rapid events of apoptosis of bone cells and also of images of interactions between osteoclasts and osteocytes/osteoblasts/bone lining cells. To find such images, we have therefore examined the alveolar bone of young rats using light microscopy, the TUNEL method for apoptosis, and electron microscopy. Fragments of alveolar bone from young rats were fixed in Bouin and formaldehyde for morphology and for the TUNEL method. Glutaraldehyde-formaldehyde fixed specimens were processed for transmission electron microscopy. Results showed TUNEL positive round/ovoid structures on the bone surface and inside osteocytic lacunae. These structures - also stained by hematoxylin - were therefore interpreted, respectively, as osteoblasts/lining cells and osteocytes undergoing apoptosis. Osteoclasts also exhibited TUNEL positive apoptotic bodies inside large vacuoles; the nuclei of osteoclasts, however, were always TUNEL negative. Ultrathin sections revealed typical apoptotic images - round/ovoid bodies with dense crescent-like chromatin - on the bone surface, corresponding therefore to apoptotic osteoblasts/lining cells. Osteocytes also showed images compatible with apoptosis. Large osteoclast vacuoles often contained fragmented cellular material. Our results provide further support for the idea that osteoclasts internalize dying bone cells; we were however, unable to find images of osteoclasts in apoptosis. (C) 2001 Harcourt Publishers Ltd.

Apoptosis in the epithelial cells of the rests of Malassez of the periodontium of rat molars

Background and Objectives: Epithelial rests of Malassez are clusters of cells derived from Hertwig's root sheath that remain in the periodontal ligament throughout life. Although it is known that the cells of Malassez proliferate, there are no studies showing that they undergo programmed cell death, i.e. apoptosis. In most tissues, proliferation is balanced by apoptosis. Thus we examined regions of the periodontium of young and adult rat molars in the hope of detecting apoptosis.Methods: Wistar rats aged 29, 45 and 120 days were killed with chloral hydrate (600 mg/kg). Fragments containing maxillary molars were removed and fixed in formaldehyde, decalcified, and embedded in paraffin and glycol methacrylate. Sections were stained with hematoxylin/eosin and the Terminal deoxynucleotidyl transferase-mediated dUTP Nick End Labeling (TUNEL) method for detection of apoptosis. Specimens were also fixed in glutaraldehyde-formaldehyde, decalcified and processed for transmission electron microscopy.Results: Epithelial rests of Malassez containing round/ovoid basophilic dense bodies and TUNEL-positive structures were found in all specimens examined. Ultrastructural examination revealed that some cells of Malassez contained masses of condensed peripheral chromatin and a shrunken cytoplasm exhibiting intact organelles - images typical of apoptosis. Moreover, round/ovoid electron-opaque structures appeared to be in the process of being engulfed by neighboring epithelial cells of Malassez.Conclusions: Our results demonstrate that epithelial cells of Malassez's rests undergo apoptosis in the developing and adult periodontium. Apoptosis may, together with proliferation, be part of the mechanism of turnover/remodelling of the cells of Malassez.

Osteoblasts engulf apoptotic bodies during alveolar bone formation in the rat maxilla

During bone formation, as in other tissues and organs, intense cellular proliferation and differentiation are usually observed. It has been described that programmed cell death, i.e., apoptosis, takes place in the control of the cellular population by removing of the excessive and damaged cells. Although it is generally accepted that apoptotic bodies are engulfed by professional phagocytes, the neighboring cells can also take part in the removal of apoptotic bodies. In the present study, regions of initial alveolar bone formation of rat molars were examined with the aim to verify whether osteoblasts are capable of engulfing apoptotic bodies, such as professional phagocytes. Rats aged 11-19 days were sacrificed and the maxillary fragments containing the first molar were removed and immersed in the fixative solution. The specimens fixed in glutaraldehyde-formaldehyde were processed for light microscopy and transmission electron microscopy. For the detection of apoptosis, the specimens were fixed in formaldehyde, embedded in paraffin, and submitted to the TUNEL method. The results revealed round/ovoid structures containing dense bodies on the bone surface in close contact to osteoblasts and in conspicuous osteoblast vacuoles. These round/ovoid structures showed also positivity to the TUNEL method, indicating that bone cells on the bone surface are undergoing apoptosis. Ultrathin sections showed images of apoptotic bodies being engulfed by osteoblasts. Occasionally, the osteoblasts exhibited large vacuoles containing blocks of condensed chromatin and remnants of organelles. Thus, these images suggest that osteoblasts are able to engulf and degrade apoptotic bodies. (c) 2005 Wiley-Liss, Inc.

Amifostine protective effect on cisplatin-treated rat testis

Cisplatin is a potent drug used in clinical oncology but causes spermatogenesis damage. Amifostine is a drug used against toxicity caused by ionizing irradiation and chemotherapeutic drugs. Since cisplatin provokes fertility and induces germ cell apoptosis and necrosis, we proposed to evaluate the amifostine cytoprotective action on testes of cisplatin-treated rats. Thirty-day-old prepubertal Wistar rats received a single cisplatin dose of 5 mg/kg and were killed after 3, 6, and 12 hr. The hematoxylin-eosin stained testicular sections were submitted to histological, morphometric, and stereological analysis. The terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling (TUNEL) method was used to label apoptotic cells. TUNEL-positive and TUNEL-negative germ cells with abnormal nuclear morphology (ANM) were scored. Significant alterations of greater part of the parameters occurred in the cisplatin-treated group (CE) compared to the group that received amifostine before the cisplatin-treatment (ACE); however, testicular weight and volume did not vary between these groups. Tubular diameter was reduced in CE in comparison to ACE rats, while interstitial tissue and lymphatic space volume and volume density were significantly higher in CE rats; interstitial testicular edema probably occurred in cisplatin-treated rats. CE rats showed important histological alterations, which were more accentuated than in ACE rats. The numerical densities of apoptotic germ cells and TUNEL-negative cells with ANM were lower in ACE than in CE rats. In conclusion, the amifostine previously administered to prepubertal rats reduced the testicular damage caused by cisplatin. We conclude that amifostine partially protected the rat seminiferous epithelium against cisplatin toxicity.