RESUMO
The present study investigated the spermatological characteristics of raw semen of Lebranche mullet (Mugil liza), namely pH, and sperm density, and motility; and subsequently evaluated the effects of different times of exposure to cryoprotectants, and the application of an ultra-rapid freezing protocol, on sperm motility and plasma membrane integrity. Semen samples were analyzed undiluted (control) and diluted 1:50 v/v in CF-HBSS + 10% Dimethyl sulfoxide + 30% Ethylene glycol + 94.58 gL-1 Trehalose dehydrate (n = 15). Two treatments - diluted semen samples in cryoprotective medium without ultra-rapid freezing (T1), and diluted semen in cryoprotective medium with ultra-rapid freezing (T2) - were evaluated at 0, 2, 4, 6 and 8 min. The frozen samples were thawed at 37ºC for 60 s. The spermatological characteristics recorded for the semen were: pH: 7.57 ± 0.21; sperm density: 30.4 ± 2.9 × 109 sperm mL-1; motility: 82 ± 4.9%. Sperm motility presented differences after 2 min exposure to cryoprotectants (70.0 ± 2.7%) and ultra-rapid freezing (66.5 ± 5.8%) compared to the control group (98.5 ± 1.9% and 98.5 ± 2.1%, respectively; p < 0.05). On the other hand, the plasma membrane integrity of the spermatozoa after 2 min exposure to cryoprotectants (64.0 ± 8.6%) and ultra-rapid freezing (62.5 ± 5.2%) presented no differences compared to the control group (69.5 ± 3.9% and 70.0 ± 3.5%, respectively p > 0.05); however, differences were observed in the parameters evaluated after longer exposure and cryopreservation times. This is the first report evaluating the effects of different times of exposure to cryoprotectants and direct ultra-rapid freezing in liquid nitrogen on Mugil liza sperm. Our results demonstrated the protocol of sperm ultra-freezing is safe within a time´s window of 2 min of exposure to cryoprotectants, after which a toxicity effect on sperm can be observed.
Assuntos
Preservação do Sêmen , Smegmamorpha , Animais , Criopreservação/métodos , Criopreservação/veterinária , Crioprotetores/toxicidade , Congelamento , Masculino , Preservação do Sêmen/métodos , Preservação do Sêmen/veterinária , Motilidade dos Espermatozoides , EspermatozoidesRESUMO
A dysfunction in the mitochondrial-lysosomal axis of cellular homeostasis is proposed to cause cells to age quicker and to accumulate lipofuscin. Typical protocols to mediate lipofuscinogenesis are based on the induction of the senescent phenotype either by allowing many consecutive cycles of cell division or by treating cells with physical/chemical agents such as ultraviolet (UV) light or hydrogen peroxide. Due to a direct connection with the physiopathology of age-related macular degeneration, lipofuscin that accumulates in retinal pigment epithelium (RPE) cells have been extensively studied, and the photochemical properties of RPE lipofuscin are considered as standard for this pigment. Yet, many other tissues such as the brain and the skin may prompt lipofuscinogenesis, and the properties of lipofuscin granules accumulated in these tissues are not necessarily the same as those of RPE lipofuscin. Here, we present a light-induced protocol that accelerates cell aging as judged by the maximization of lipofuscinogenesis. Photosensitization of cells previously incubated with nanomolar concentrations of 1,9-dimethyl methylene blue (DMMB), severely and specifically damages mitochondria and lysosomes, leading to a lipofuscin-related senescent phenotype. By applying this protocol in human immortalized non-malignant keratinocytes (HaCaT) cells, we observed a 2.5-fold higher level of lipofuscin accumulation compared to the level of lipofuscin accumulation in cells treated with a typical UV protocol. Lipofuscin accumulated in keratinocytes exhibited the typical red light emission, with excitation maximum in the blue wavelength region (~450 nm). Fluorescence lifetime image microscopy data showed that the keratinocyte lipofuscin has an emission lifetime of ~1.7 ns. Lipofuscin-loaded cells (but not control cells) generated a substantial amount of singlet oxygen (1O2) when irradiated with blue light (420 nm), but there was no 1O2 generation when excitation was performed with a green light (532 nm). These characteristics were compared with those of RPE cells, considering that keratinocyte lipofuscin lacks the bisretinoids derivatives present in RPE lipofuscin. Additionally, we showed that lipofuscin-loaded keratinocytes irradiated with visible light presented critical DNA damages, such as double-strand breaks and Fpg-sensitive sites. We propose that the DMMB protocol is an efficient way to disturb the mitochondrial-lysosomal axis of cellular homeostasis, and consequently, it can be used to accelerate aging and to induce lipofuscinogenesis. We also discuss the consequences of the lipofuscin-induced genotoxicity of visible light in keratinocytes.
Assuntos
Lipofuscina , Degeneração Macular , Humanos , Queratinócitos , Lisossomos , Raios UltravioletaRESUMO
Previous studies have shown that the renin-angiotensin system (RAS) is affected by adverse maternal nutrition during pregnancy. The aim of this study was to investigate the effects of a maternal low-protein diet on proinflammatory cytokines, reactive oxygen species and RAS components in kidney samples isolated from adult male offspring. We hypothesized that post-weaning losartan treatment would have beneficial effects on RAS activity and inflammatory and oxidative stress markers in these animals. Pregnant Sprague-Dawley rats were fed with a control (20% casein) or low-protein diet (LP) (6% casein) throughout gestation. After weaning, the LP pups were randomly assigned to LP and LP-losartan groups (AT1 receptor blockade: 10 mg/kg/day until 20 weeks of age). At 20 weeks of age, blood pressure levels were higher and renal RAS was activated in the LP group. We also observed several adverse effects in the kidneys of the LP group, including a higher number of CD3, CD68 and proliferating cell nuclear antigen-positive cells and higher levels of collagen and reactive oxygen species in the kidney. Further, our results revealed that post-weaning losartan treatment completely abolished immune cell infiltration and intrarenal RAS activation in the kidneys of LP rats. The prevention of augmentation of angiotensin (Ang II) concentration abolished inflammatory and fibrotic events, indicating that Ang II via the AT1 receptor is essential for pathological initiation. Our results suggest that the prenatal programming of hypertension is dependent on the up-regulation of local RAS and presence of immune cells in the kidney.
Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Dieta com Restrição de Proteínas , Rim/fisiologia , Losartan/farmacologia , Fenômenos Fisiológicos da Nutrição Materna , Sistema Renina-Angiotensina , Animais , Citocinas/metabolismo , Feminino , Rim/imunologia , Rim/metabolismo , Masculino , Distribuição Aleatória , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Regulação para CimaRESUMO
The oral denticles of some elasmobranchs are found on the surface of the oral cavity and are homologous to those on the body surface, being well developed, independent and non-growing, with varying morphology and distribution depending on the species. The structural and three-dimensional characteristics of oral denticles from the rostro-ventral surface of the sharpnose shark Rhizoprionodon lalandii were described following imaging by both light and scanning electron microscopy. The light microscopy results showed that the triangular shape of the denticles consisted of a base and an apex. Picrosirius staining showed the arrangement of collagen fibres and oral denticles, and a predominance of type-I collagen was found in both structures under polarized light. There was a broad homogeneous distribution of denticles on the ventral surface, forming a leaf-like shape with the cusp facing the caudal region. Interlocking, hexagonal, geometric structures on its rostral side and ridges on the rostral side of the oral denticles were observed under increased magnification. We concluded that the denticle morphology found in R. lalandii differ of others analysed species, and the descriptions of these structures therefore provide important information for the classification of the species. In this species, the main functions can be assigned to help reduce hydrodynamic drag, particularly by this being a species that uses ram ventilation, and to protect the epithelium of the oropharynx of abrasion and parasites.
Assuntos
Calcificações da Polpa Dentária/veterinária , Mucosa Bucal/anatomia & histologia , Tubarões/anatomia & histologia , Animais , Calcificação Fisiológica , Colágeno Tipo I/análise , Colágeno Tipo I/ultraestrutura , Calcificações da Polpa Dentária/patologia , Dentina/química , Masculino , Microscopia Eletrônica de Varredura/veterinária , Mucosa Bucal/química , Mucosa Bucal/ultraestruturaRESUMO
In most anatomical studies developed with mammals, the tongue is described as highly differentiated among different species. However, studies on the tongue of aquatic mammals are still limited as compared to those on terrestrial mammals. The aim of this study was to describe the tongue morphology of the Franciscana dolphin (Pontoporia blainvillei) using macroscopic observations, light, and scanning electron microscopy. Microscopically, the dorsal surface was covered by a keratinized stratified epithelium. Salivary gland acini were found on the middle and caudal third of the tongue. The dorsal surface was totally covered by filiform papillae with a connective tissue core and a connective tissue structure round in shape in the middle and caudal regions.
Assuntos
Golfinhos/anatomia & histologia , Língua/anatomia & histologia , Língua/citologia , Animais , Histocitoquímica , Microscopia , Língua/ultraestruturaRESUMO
The presence of lingual papillae and the nerve endings in the middle region of the tongue mucosa of collared peccary (Tayassu tajacu) were studied using scanning electron microscopy and light microscopy, based upon the silver impregnation method. The middle region of tongue mucosa revealed numerous filiform and fungiform papillae. The thick epithelial layer showed epithelial cells and a dense connective tissue layer containing nerve fibre bundles and capillaries. The sensory nerve endings, intensely stained by silver impregnation, were usually non-encapsulated and extended into the connective tissue of the filiform and fungiform papillae very close to the epithelial cells. In some regions, the sensory nerves fibres formed a dense and complex network of fine fibrils. The presence of these nerve fibrils may characterize the mechanisms of transmission of sensitive impulses to the tongue mucosa.
Assuntos
Gengiva/ultraestrutura , Mamíferos/anatomia & histologia , Terminações Nervosas/ultraestrutura , Língua/ultraestrutura , Animais , Tecido Conjuntivo/irrigação sanguínea , Tecido Conjuntivo/inervação , Células Epiteliais , Feminino , Gengiva/inervação , Masculino , Microscopia Eletrônica de Varredura , Coloração pela Prata/métodos , Língua/inervaçãoRESUMO
This study verified the effect of unilateral teeth extraction on the periodontal ligament in gerbils (Meriones unguiculatus). Ten adult male gerbils weighing about 50 g had induced occlusal alterations by upper left molar extractions while the other ten animals, only submitted to surgical stress, were considered as controls. The periodontal ligament was characterized by qualitative and quantitative analysis, histological description and histomorphometric quantification. Significant alterations were observed on the left side of the experimental group (P < 0.05), the hypofunctional region, when it was compared with the contralateral side and the corresponding region of the control group. Two months after occlusal alterations induced by unilateral teeth extraction, atrophic histological alterations and a decrease in the periodontal space on the ipsilateral side characterized the periodontal ligament. In this study it was possible to conclude that the gerbil can be used in experimental models attempting to correlate the periodontium's biological response to various mechanical stresses, as the periodontal ligament was shown to be highly sensitive to occlusal alterations.
Assuntos
Análise do Estresse Dentário/veterinária , Gerbillinae , Ligamento Periodontal/patologia , Extração Dentária/veterinária , Animais , Modelos Animais de Doenças , Masculino , Distribuição Aleatória , Extração Dentária/efeitos adversosRESUMO
El objetivo de esta investigación fue estudiar las fibras sensitivas mielinizadas y amielínicas localizadas en la lámina propia subepitelial de la mucosa lingual de ratas. Se usó el método de impregnación argéntica, microscopía MET y mediciones morfométricas. Los resultados revelaron que los fascículos de fibras subepiteliales de las regiones anterior, media y posterior de la lengua provenían de su capa muscular profunda. Los grupos de fibras fueron localizados dentro del tejido conectivo de la lámina propia. Estas fibras nerviosas se ramificaron varias veces y en el tejido conectivo de las papilas formaron terminaciones simples o ramificados. Las papilas fungiformes y valadas contenían numerosas terminaciones nerviosas. La ultraestructura demostró en el axoplasma la presencia de neurofilamentos, mitocondrias y microtúbulos; aunque los registros morfométricos de las fibras mielinizadas mostraron que alrededor del 44 por ciento tenían un diámetro entre 3 y 4 µm, el valor promedio fue de 4.5 µm. El diámetro mayor fue de 12 µm y el menor de 1.4 um. Los rangos de menores diámetros fueron de 1 a 3 µm, siendo el promedio de 2.33 µm. En relación al espesor de las vainas de mielina, los valores obtenidos fueron de 0.2 a 0.8 um siendo el valor promedio de alrededor de 0,5 µm, en el 90 por ciento de ellas. En las fibras amielínicas los mayores diámetros (62 por ciento) variaron entre 0.25 y 0.75 µm. El valor promedio fue de 0.6 µm, siendo el valor máximo 3.17 µm y el mínimo 0.2 µm. El menor diámetro fue obtenido en 44 por ciento de las fibras amielínicas y los rangos variaron entre 0.2 y 0.4 µm. El valor máximo obtenido fue de 1 µm y el mínimo 0.12 µm.
The purpose of this paper was to study the sensory myelinated and unmyelinated nerve fibers found in the subepithelial lamina propria of tongue mucosa of aging rats. It was used the silver impregnation, transmission electron microscopy methods and morphometric measurements. The results revealed that subepithelial nerve fiber bundles of the anterior, medium and posterior regions of tongue were arise from deep muscular layer of tongue. The nerve fiber bundles were verified inside of the connective tissue of lamina propria. These nerve fibers branched several times and into the connective tissue papillae they form a single or ramified sensory nerve endings. The fungiform and vallate papillae contain numerous nerve terminals. The fine structure demonstrated that in the axoplasm were noted the presence of neurofilaments, mitochondria and microtubules. Although the morphometric data of myelinated fibers showed that about 44% having larger diameter between 3 to 4 µm and the mean value was 4.5 µm. The largest diameter was 12 µm and the smaller was 1.4 um. The smallest diameter ranges from 1 to 3 µm, being that the mean value was 2.33 µm. Concerning to the thickness of myelin sheets were revealed the values ranging from 0.2 to 0.8 µm being that the 90% present the values around 0.5 µm. The unmyelinated fibers showed the largest diameter (62%) varying from 0.25 to 0.75 µm. The mean value was 0.6 µm being the maximum value was 3.17 µm and the minimum was 0.2 µm. The smallest diameter was obtained in 44% of unmyelinated fibers which the diameter ranges from 0.2 to 0.4 µm.Then maximum value obtained was 1 µm and the minimum was 0.12 µm.
Assuntos
Animais , Pré-Escolar , Ratos , Mucosa Bucal/anatomia & histologia , Mucosa Bucal/inervação , Fibras Nervosas Mielinizadas , Fibras Nervosas Amielínicas , Envelhecimento/fisiologia , Ratos/anatomia & histologia , Ratos/fisiologiaRESUMO
BACKGROUND: Data associating palatine mucosa, microvascular architecture, aging and diabetes mellitus are few, so the aim of the present study was to examine these conditions structurally and ultrastructurally. METHODS: We used 18 female rabbits, aged about 2 years at the beginning of the experiment, distributed into 2 groups: aging diabetic and aging animals, prepared by three different methodologies: light microscopy, scanning electron microscopy and transmission electron microscopy. The samples were stained with hematoxylin-eosin for light microscopy, and for scanning electron microscopy, we used Mercox Cl-2B vascular corrosion casts. For transmission electron microscopy, the specimens were fixed and embedded in Epon 812 resin. RESULTS: Under light microscopy, we noted small amounts of underdeveloped connective papillae and significant flattened areas through the epithelium-connective tissue interface in the aging diabetic group. Larger blood vessels were deeply located in the palatine mucosa, branching off in the direction of the epithelium-connective tissue interface. Capillaries followed the interface contour. In both groups, vascular corrosion casts revealed capillaries sprouting out in disorganized rows but parallel between themselves. 'Hair-pin' capillary loops and convoluted capillary loops were noted. CONCLUSION: The aging diabetic group showed rarefying microvasculature areas with complex tortuous capillary loops. Transmission electron microscopy results showed that the aging diabetic group presented small cytoplasmatic projections directed to the vessel lumen and micropinocytic vesicle, i.e. caveolas. Epithelium-connective tissue interface, connective papillae, microangioarchitecture, and information about endothelial cells alterations were observed in the aging diabetic and aging animals.
Assuntos
Envelhecimento , Diabetes Mellitus/patologia , Mucosa Bucal/irrigação sanguínea , Mucosa Bucal/patologia , Palato , Fatores Etários , Animais , Capilares/patologia , Capilares/ultraestrutura , Feminino , Microscopia , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Mucosa Bucal/ultraestrutura , CoelhosRESUMO
In this paper, the distribution of NADH-positive and somatostatin (SOM) immunoreactive neurons in the myenteric plexus of the colon of mice infected with Trypanosoma cruzi was studied. Ten young, male, BALB/c mice were inoculated with the Y strain of T. cruzi, 60 days previously (chronic phase of the infection). Another 10 mice were uninfected controls. Distal and proximal colonic neurons from five chronically infected mice and their controls were stained using the NADH-diaphorase method. Quantitative results showed a significant decrease of 39% in the number of neurons in the proximal colon of infected mice and 58% in the distal colon (p<0.05). SOM was localized in five animals from each group by light microscopy, using an indirect immunofluorescence technique. It was observed that there were far fewer nerve cells and fibres and less intensely stained neuron bodies and varicose SOM-positive nerve fibres in both, control and chronic infected mice. These findings could be related to the disturbances in intestinal motility observed in patients in the chronic phase of Chagas' disease.