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.

Enhanced ERbeta immunoexpression and apoptosis in the germ cells of cimetidine-treated rats

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

Apoptosis in the early developing periodontium of rat molars

Development of the periodontium involves a series of complex steps that result in the formation of root dentine, cementum, bone and fibres of the ligament. These precisely controlled and timed events require the participation of the enamel organ derived epithelial cells of Hertwig's (HRS) and ectomesenchymal cells of the dental follicle. These events involve rapid turnover of the tissues and cells, including disappearance of epithelial cells of HRS. Thus, it seemed likely to us that programmed cell death (apoptosis) may play a role in the development of the periodontium. Fragments of first molars, obtained from 14- and 29-day-old rats, were fixed in glutaraldehyde-formaldehyde and processed for light and electron microscopy. For the TUNEL method for detection of apoptosis, specimens were fixed in 4% formaldehyde and embedded in paraffin. Results confirmed that epithelial cells of HRS maintain a close relationship with the forming dentine root, and that they may become trapped in the dentino-cemental junction. Some of the epithelial cells exhibited ultrastructural features which are consistent with the interpretation that they were undergoing programmed cell death, i.e. apoptosis. Periodontal fibroblast-like cells showed typical images of apoptosis and engulfed apoptotic bodies. TUNEL positive structures were present in all corresponding regions. It seems therefore that apoptosis of epithelial cells of HRS and fibroblast-like cells of the periodontal ligament constitutes an integral part of the developmental process of the tissues of the periodontium. (C) 2000 Wiley-Liss, Inc.

Cimetidine-induced vascular cell apoptosis impairs testicular microvasculature in adult rats

Cimetidine, an H2 receptor antagonist used for treatment of gastric ulcers, exerts antiandrogenic and antiangiogenic effects. In the testes cimetidine impairs spermatogenesis, Sertoli cells and peritubular tissue, inducing apoptosis in the myoid cells. Regarding the importance of histamine and androgens for vascular maintenance, the effect of cimetidine on the structural integrity of the testicular vasculature was evaluated. Adult male rats received cimetidine (CMTG) and saline (CG) for 50 days. The testes were fixed in buffered 4% formaldehyde and embedded in historesin and paraffin. In the PAS-stained sections, the microvascular density (MVD) and the vascular luminal area (VLA) were obtained. TUNEL method was performed for detection of cell death. Testicular fragments embedded in Araldite were analyzed under transmission electron microscopy. A significant decrease in the MVD and VLA and a high number of collapsed blood vessel profiles were observed in CMTG. Endothelial cells and vascular muscle cells were TUNEL-positive and showed ultrastructural features of apoptosis. These results indicate that cimetidine induces apoptosis in vascular cells, leading to testicular vascular atrophy. A possible antagonist effect of cimetidine on the H2 receptors and/or androgen receptors in the vascular cells may be responsible for the impairment of the testicular microvasculature.

Modulação da proliferação, apoptose e autofagia de linfocitos T por RAGE

Pós-graduação em Odontologia - FOAR

Apoptosis and reduced microvascular density of the lamina propria during tooth eruption in rats

During tooth eruption, structural and functional changes must occur in the lamina propria to establish the eruptive pathway. In this study, we evaluate the structural changes that occur during lamina propria degradation and focus these efforts on apoptosis and microvascular density. Fragments of maxilla containing the first molars from 9-, 11-, 13- and 16-day-old rats were fixed, decalcified and embedded in paraffin. The immunohistochemical detection of vascular endothelial growth factor (VEGF), caspase-3 and MAC387 (macrophage marker), and the TUNEL method were applied to the histological molar sections. The numerical density of TUNEL-positive cells and VEGF-positive blood vessel profiles were also obtained. Data were statistically evaluated using a one-way anova with the post-hoc Kruskal-Wallis or Tukey test and a significance level of P ≤ 0.05. Fragments of maxilla were embedded in Araldite for analysis under transmission electron microscopy (TEM). TUNEL-positive structures, fibroblasts with strongly basophilic nuclei and macrophages were observed in the lamina propria at all ages. Using TEM, we identified processes of fibroblasts or macrophages surrounding partially apoptotic cells. We found a high number of apoptotic cells in 11-, 13- and 16-day-old rats. We observed VEGF-positive blood vessel profiles at all ages, but a significant decrease in the numerical density was found in 13- and 16-day-old rats compared with 9-day-old rats. Therefore, the establishment of the eruptive pathway during the mucosal penetration stage depends on cell death by apoptosis, the phagocytic activity of fibroblasts and macrophages, and a decrease in the microvasculature due to vascular cell death. These data point to the importance of vascular rearrangement and vascular neoformation during tooth eruption and the development of oral mucosa.

Effects of environmental complexity on neurogenesis in the hippocampal dentate gyrus

Introduction. Postnatal neurogenesis in the hippocampal dentate gyrus, can be modulated by numerous determinants, such as hormones, transmitters and stress. Among the factors positively interfering with neurogenesis, the complexity of the environment appears to play a particularly striking role. Adult mice reared in an enriched environment produce more neurons and exhibit better performance in hippocampus-specific learning tasks. While the effects of complex environments on hippocampal neurogenesis are well documented, there is a lack of information on the effects of living under socio-sensory deprivation conditions. Due to the immaturity of rats and mice at birth, studies dealing with the effects of environmental enrichment on hippocampal neurogenesis were carried out in adult animals, i.e. during a period of relatively low rate of neurogenesis. The impact of environment is likely to be more dramatic during the first postnatal weeks, because at this time granule cell production is remarkably higher than at later phases of development. The aim of the present research was to clarify whether and to what extent isolated or enriched rearing conditions affect hippocampal neurogenesis during the early postnatal period, a time window characterized by a high rate of precursor proliferation and to elucidate the mechanisms underlying these effects. The experimental model chosen for this research was the guinea pig, a precocious rodent, which, at 4-5 days of age can be independent from maternal care. Experimental design. Animals were assigned to a standard (control), an isolated, or an enriched environment a few days after birth (P5-P6). On P14-P17 animals received one daily bromodeoxyuridine (BrdU) injection, to label dividing cells, and were sacrificed either on P18, to evaluate cell proliferation or on P45, to evaluate cell survival and differentiation. Methods. Brain sections were processed for BrdU immunhistochemistry, to quantify the new born and surviving cells. The phenotype of the surviving cells was examined by means of confocal microscopy and immunofluorescent double-labeling for BrdU and either a marker of neurons (NeuN) or a marker of astrocytes (GFAP). Apoptotic cell death was examined with the TUNEL method. Serial sections were processed for immunohistochemistry for i) vimentin, a marker of radial glial cells, ii) BDNF (brain-derived neurotrofic factor), a neurotrophin involved in neuron proliferation/survival, iii) PSA-NCAM (the polysialylated form of the neural cell adhesion molecule), a molecule associated with neuronal migration. Total granule cell number in the dentate gyrus was evaluated by stereological methods, in Nissl-stained sections. Results. Effects of isolation. In P18 isolated animals we found a reduced cell proliferation (-35%) compared to controls and a lower expression of BDNF. Though in absolute terms P45 isolated animals had less surviving cells than controls, they showed no differences in survival rate and phenotype percent distribution compared to controls. Evaluation of the absolute number of surviving cells of each phenotype showed that isolated animals had a reduced number of cells with neuronal phenotype than controls. Looking at the location of the new neurons, we found that while in control animals 76% of them had migrated to the granule cell layer, in isolated animals only 55% of the new neurons had reached this layer. Examination of radial glia cells of P18 and P45 animals by vimentin immunohistochemistry showed that in isolated animals radial glia cells were reduced in density and had less and shorter processes. Granule cell count revealed that isolated animals had less granule cells than controls (-32% at P18 and -42% at P45). Effects of enrichment. In P18 enriched animals there was an increase in cell proliferation (+26%) compared to controls and a higher expression of BDNF. Though in both groups there was a decline in the number of BrdU-positive cells by P45, enriched animals had more surviving cells (+63) and a higher survival rate than controls. No differences were found between control and enriched animals in phenotype percent distribution. Evaluation of the absolute number of cells of each phenotype showed that enriched animals had a larger number of cells of each phenotype than controls. Looking at the location of cells of each phenotype we found that enriched animals had more new neurons in the granule cell layer and more astrocytes and cells with undetermined phenotype in the hilus. Enriched animals had a higher expression of PSA-NCAM in the granule cell layer and hilus Vimentin immunohistochemistry showed that in enriched animals radial glia cells were more numerous and had more processes.. Granule cell count revealed that enriched animals had more granule cells than controls (+37% at P18 and +31% at P45). Discussion. Results show that isolation rearing reduces hippocampal cell proliferation but does not affect cell survival, while enriched rearing increases both cell proliferation and cell survival. Changes in the expression of BDNF are likely to contribute to he effects of environment on precursor cell proliferation. The reduction and increase in final number of granule neurons in isolated and enriched animals, respectively, are attributable to the effects of environment on cell proliferation and survival and not to changes in the differentiation program. As radial glia cells play a pivotal role in neuron guidance to the granule cell layer, the reduced number of radial glia cells in isolated animals and the increased number in enriched animals suggests that the size of radial glia population may change dynamically, in order to match changes in neuron production. The high PSA-NCAM expression in enriched animals may concur to favor the survival of the new neurons by facilitating their migration to the granule cell layer. Conclusions. By using a precocious rodent we could demonstrate that isolated/enriched rearing conditions, at a time window during which intense granule cell proliferation takes place, lead to a notable decrease/increase of total granule cell number. The time-course and magnitude of postnatal granule cell production in guinea pigs are more similar to the human and non-human primate condition than in rats and mice. Translation of current data to humans would imply that exposure of children to environments poor/rich of stimuli may have a notably large impact on dentate neurogenesis and, very likely, on hippocampus dependent memory functions.

Avaliação da técnica imunohistoquímica como método de deteção da apoptose a nível celular

A apoptose constitui um processo fisiológico de morte celular, caracterizado por alterações morfológicas distintas e mecanismos bioquímicos e moleculares bem definidos. O seu papel de destaque em numerosos eventos biológicos e importantes processos patológicos conduziu a um crescente interesse na investigação dos mecanismos celulares que regulam o processo apoptótico. A aplicação de metodologias capazes de identificar células apoptóticas despoletou um enorme desenvolvimento de técnicas. No entanto, as propriedades demonstradas por estes ensaios nem sempre se aplicam ao estudo de amostras tecidulares, pelo que a escolha dos diferentes métodos deverá ser criteriosamente avaliada, tendo em conta a aplicação pretendida e as alterações morfológicas que se pretendem detetar. Das várias técnicas disponíveis para deteção da apoptose em tecidos, muitos investigadores recomendam o método TUNEL, o qual se baseia na marcação de produtos endonucleossómicos resultantes da fragmentação do DNA. Outros métodos histoquímicos também disponíveis incluem a deteção do citocromo c, libertado da mitocôndria ou a deteção das proteínas pró e anti-apoptóticas, Bax, Bidm e Bcl-2, envolvidas nos mecanismos intrínsecos da apoptose. Mais recentemente, a marcação de produtos específicos resultantes da clivagem de proteínas alvo pelas caspases, tem vindo a ser considerada uma abordagem promissora. Como principal objectivo deste trabalho pretendeu-se avaliar a técnica imunohistoquímica como método de deteção da apoptose a nível celular, em tecidos animais, tendo por base o método TUNEL, o qual permite a deteção de fragmentos de DNA. Os resultados obtidos permitiram concluir que, apesar do método TUNEL possuir as suas limitações ao nível da sensibilidade e especificidade, o mesmo constitui um mecanismo imunohistoquímico útil na deteção de células apoptóticas. Contudo, segundo opinião de vários autores, adverte-se para a necessidade da aplicação de pelo menos dois métodos imunohistoquímicos como forma de validar a ocorrência do processo apoptótico, razão pela qual se optou pela deteção do citocromo c citosólico como método complementar, uma vez que a sua libertação para o espaço citosólico se encontra implicada na ativação da apoptose.

Serum levels of lycopene, beta-carotene, and retinol and their correlation with sperm DNA damage in normospermic and infertile men

Background: Oxidative stress in reproductive system leads to sperm DNA damage and sperm membrane lipid peroxidation and may play an important role in the pathogenesis of male infertility, especially in idiopathic cases. Antioxidants such as carotenoids function against free radical damages. Objective: The aim of this study was to determine the levels of lycopene, beta-carotene and retinol in serum and their relationship with sperm DNA damage and lipid peroxidation in infertile and normospermic males. Materials and Methods: Sixty two infertile men and 71 normospermic men participated in this study. Blood and semen samples were collected from all subjects. Sperm DNA damage was measured using TUNEL method. Carotenoids, retinol, and malonedildehyde in serum were also determined. Results: DNA fragmentation was higher in infertile group comparing to control group. Serum levels of lycopene, beta-carotene and, vitamin A in infertile men were significantly lower than normospermic men (p< 0.001, =0.005, and =0.003 respectively). While serum MDA was not significantly different between two groups, MDA in seminal plasma of infertile men was significantly higher than control group (p< 0.001). Conclusion: We concluded that lycopene, beta-carotene, and retinol can reduce sperm DNA fragmentation and lipid peroxidation through their antioxidant effect. Therefore the DNA fragmentation assay and determination of antioxidants factors such as lycopene, beta-carotene and retinol, along with sperm analysis can be useful in diagnosis and treatment of men with idiopathic infertility.

Decrease of the DNA damage levels after sperm preparation by layering method

The aim of this prospective study was to determine the DNA fragmentation levels before and after sperm preparation by layering method. A total of 78 patients submitted to assisted reproduction technology (ART) for infertility treatment were evaluated. Ejaculated spermatozoa were obtained by masturbation on the day of ART procedure. The evaluation of DNA fragmentation was performed in the fresh semen and after preparation by a layering method, respectively. After washing with PBS, the sperm pellets were smears and then processed for the terminal deoxyribonucleotidyl transferase (TdT)-mediated dUTP nick-end labelling (TUNEL) assay that was performed using a Cell Death Detection Kit with tetramethylrhodamine-labelled dUTP. For quantitative evaluation, 200 spermatozoa in randomly selected areas on microscope slides were evaluated and the percentage of TUNEL positive spermatozoa was determined. If ≥20% of selected sperm were TUNEL positive, the exam was considered abnormal. The mean percentage of DNA sperm fragmentation before sperm preparation was 17±8.3% and after 7.8±6.5% (p<0.0001). The exam was considered normal in 49 patients before preparation and in 73 patients after (p<0.0001). The sperm preparation with a layering method for the ART procedure is effective to select sperm with a significant decrease of the DNA damage.

A Highly Improved Method For Sensitive Determination Of Amitriptyline In Pharmaceutical Formulations Using An Unmodified Carbon Nanotube Electrode In The Presence Of Sulfuric Acid.

The present paper describes a novel, simple and reliable differential pulse voltammetric method for determining amitriptyline (AMT) in pharmaceutical formulations. It has been described for many authors that this antidepressant is electrochemically inactive at carbon electrodes. However, the procedure proposed herein consisted in electrochemically oxidizing AMT at an unmodified carbon nanotube paste electrode in the presence of 0.1 mol L(-1) sulfuric acid used as electrolyte. At such concentration, the acid facilitated the AMT electroxidation through one-electron transfer at 1.33 V vs. Ag/AgCl, as observed by the augmentation of peak current. Concerning optimized conditions (modulation time 5 ms, scan rate 90 mV s(-1), and pulse amplitude 120 mV) a linear calibration curve was constructed in the range of 0.0-30.0 μmol L(-1), with a correlation coefficient of 0.9991 and a limit of detection of 1.61 μmol L(-1). The procedure was successfully validated for intra- and inter-day precision and accuracy. Moreover, its feasibility was assessed through analysis of commercial pharmaceutical formulations and it has been compared to the UV-vis spectrophotometric method used as standard analytical technique recommended by the Brazilian Pharmacopoeia.

Influence Of Delivery Method On Neuroprotection By Bone Marrow Mononuclear Cell Therapy Following Ventral Root Reimplantation With Fibrin Sealant.

The present work compared the local injection of mononuclear cells to the spinal cord lateral funiculus with the alternative approach of local delivery with fibrin sealant after ventral root avulsion (VRA) and reimplantation. For that, female adult Lewis rats were divided into the following groups: avulsion only, reimplantation with fibrin sealant; root repair with fibrin sealant associated with mononuclear cells; and repair with fibrin sealant and injected mononuclear cells. Cell therapy resulted in greater survival of spinal motoneurons up to four weeks post-surgery, especially when mononuclear cells were added to the fibrin glue. Injection of mononuclear cells to the lateral funiculus yield similar results to the reimplantation alone. Additionally, mononuclear cells added to the fibrin glue increased neurotrophic factor gene transcript levels in the spinal cord ventral horn. Regarding the motor recovery, evaluated by the functional peroneal index, as well as the paw print pressure, cell treated rats performed equally well as compared to reimplanted only animals, and significantly better than the avulsion only subjects. The results herein demonstrate that mononuclear cells therapy is neuroprotective by increasing levels of brain derived neurotrophic factor (BDNF) and glial derived neurotrophic factor (GDNF). Moreover, the use of fibrin sealant mononuclear cells delivery approach gave the best and more long lasting results.

Uv-vis Spectra As An Alternative To The Lowry Method For Quantify Hair Damage Induced By Surfactants.

It is well known that long term use of shampoo causes damage to human hair. Although the Lowry method has been widely used to quantify hair damage, it is unsuitable to determine this in the presence of some surfactants and there is no other method proposed in literature. In this work, a different method is used to investigate and compare the hair damage induced by four types of surfactants (including three commercial-grade surfactants) and water. Hair samples were immersed in aqueous solution of surfactants under conditions that resemble a shower (38 °C, constant shaking). These solutions become colored with time of contact with hair and its UV-vis spectra were recorded. For comparison, the amount of extracted proteins from hair by sodium dodecyl sulfate (SDS) and by water were estimated by the Lowry method. Additionally, non-pigmented vs. pigmented hair and also sepia melanin were used to understand the washing solution color and their spectra. The results presented herein show that hair degradation is mostly caused by the extraction of proteins, cuticle fragments and melanin granules from hair fiber. It was found that the intensity of solution color varies with the charge density of the surfactants. Furthermore, the intensity of solution color can be correlated to the amount of proteins quantified by the Lowry method as well as to the degree of hair damage. UV-vis spectrum of hair washing solutions is a simple and straightforward method to quantify and compare hair damages induced by different commercial surfactants.