Histological changes in connective tissue of rat tails after bipolar radiofrequency treatment.

Radiofrequency (RF) has been included in the techniques used in aesthetic surgery/medicine. To date, no studies have performed a histological assessment of changes in the tissue after application of bipolar radiofrequency (BRF) with low energy and frequency. The aim of this study was to examine changes that are produced in connective tissue, principally in the fibroblasts, following BRF treatment. Four groups of rats received a different number of RF sessions (1, 2, 3 and 5). The following parameters were determined: the number of fibroblasts/unit area (FA), the proliferation index (PI), the Heat shock Protein 47 index (HSPI) and the percentage of connective tissue (PC). For statistical analysis, two subgroups (A and B) were made for the variables FA, PI and PC, and another two subgroups (C and D) for the variable HSPI. Significant differences for FA, PI and PC were observed between subgroups A and B, FA and PI having higher values in A, while PC had higher values in B. The HSPI in subgroup C showed significantly higher values than in D. Low energy and frequency BRF led to an increase in the number, proliferation and biosynthetic activity of fibroblasts. The resulting stress suffered by fibroblasts as a result of heat may be associated with the phenomenon of hormesis.

Proliferation and apoptosis in aged and photoregressed mammalian seminiferous epithelium, with particular attention to rodents and humans.

Imbalances in the proliferation and apoptosis processes are involved in numerous epithelial alterations. In the seminiferous epithelium, normal spermatogenesis is regulated by spermatogonia proliferation and germ cell apoptosis, and both processes are involved in diverse pathological alterations of the seminiferous epithelium. Other physiological phenomena including aging and short photoperiod, in which apoptosis and proliferation seem to play important roles, cause testicular changes. Aging is accompanied by diminished proliferation and increased apoptosis, the latter occurring in specific states of the seminiferous cycle and considered the cause of epithelium involution. However, there is no clear evidence concerning whether proliferation decreases in the spermatogonia themselves or is due to an alteration in the cell microenvironment that surrounds them. As regards the factors that regulate the process, the data are scant, but it is considered that the diminution of c-kit expression in the spermatagonia, together with the diminution in antiapoptotic factors (Bcl-x(L))) of the intrinsic molecular pathway of apoptosis play a part in epithelial regression. A short photoperiod, especially in rodents, produces a gradual involution of the seminiferous epithelium, which is related with increased apoptosis during the regression phase and a diminution of apoptosis during recrudescence. Proliferative activity varies, especially during the total regression phase, when it usually increases in the undifferentiated spermatogonia. In other species showing seasonal reproduction, however, decreased proliferation is considered the main factor in the regression of the seminiferous epithelium. Little is known about how both phenomena are regulated, although data in rodents suggest that both the intrinsic and extrinsic pathways of apoptosis contribute to the increase in this process. In conclusion, regression of the seminiferous epithelium in physiological situations, as in many pathological situations, is a result of alterations in equilibrium between the proliferation and apoptosis of germinal cell types. However, both physiological phenomena showed important differences as regard proliferation/apoptosis and their regulation pathways, probably as a result of their irreversible or reversible character.

Involution of seminiferous tubules in aged hamsters: an ultrastructural, immunohistochemical and quantitative morphological study.

In this study, we examined the age-related changes on morphometric parameters and ultrastructure of seminiferous tubules, and on the expression of extracellular matrix proteins in lamina propria of Syrian hamsters. A significant decrease in the percentage of normal tubules and an increase in the percentage of hypospermatogenic and arrested maturation tubules was observed with aging. Aged animals showed a decrease in tubular diameter, tubular lumen, seminiferous epithelium volume and total tubular volume. However, the total length of seminiferous tubules was significantly increased with aging. The most important ultrastructural changes with aging were the thickening of the lamina propria, the presence of diverse abnormalities in the spermiogenesis process, degeneration of germ cells, and vacuolization and flattening of Sertoli cells showing abundant lipofucsin droplets and residual bodies. Laminin immunoreactivity was found along the lamina propria of seminiferous tubules both in young and aged animals. Fibronectin immunoreactivity was found along the lamina propria and blood vessels. Both laminin and fibronectin total volume of immunostaining per testis was increased in aged hamsters. In conclusion, the age-related changes in seminiferous tubules of hamster include: a decrease in tubular width and an increase in tubular length; widening of the lamina propria caused by a more extensive connective matrix between the peritubular cells and the basal membrane; and a strong disarrangement of the seminiferous epithelium, including germ cell degeneration and important alterations in both spermiogenesis and Sertoli cell structure.

Effect of ageing on the proliferation and apoptosis of testicular germ cells in the Syrian hamster Mesocricetus auratus.

The cellular mechanisms implicated in the atrophy of seminiferous epithelium in ageing are currently under debate, although recent reports suggest that apoptosis may be the primary mechanism implicated in aged germ cell loss. Other investigators have suggested that changes in spermatogonial proliferation are also involved. In the present work, the changes in proliferation and apoptosis in the seminiferous epithelium of aged (24 months) Syrian hamsters were examined in concert and compared with those in young (6 months) animals. Proliferation of germ cells was studied by bromodeoxyuridine labelling and apoptosis was assessed by transmission electron microscopy and in situ TUNEL labelling. Aged animals showed a significant decrease in the numbers of total and proliferating spermatogonia plus preleptotene spermatocytes per unit volume and per testis and in the proliferative index (24.8 +/- 1.6%) compared with young animals (30.8 +/- 1.2%) (P < 0.05). The number of apoptotic spermatogonia plus spermatocytes per unit volume and the apoptotic index were significantly higher in aged animals (1.51 +/- 0.23% v. 0.77 +/- 0.04%; P < 0.05). Apoptosis was confirmed by morphological characteristics: condensation of the chromatin and nuclear fragmentation. In aged hamsters, tubular degeneration could be classified into several categories, showing an increase of apoptotic cells in tubular cross-sections characterized by maturation arrest in comparison with all other types. Spermatogonial proliferation was also diminished as seen in tubular cross-sections showing hypospermatogenesis, sloughing off of germ cells and maturation arrest. The results obtained in the present study suggest that the decrease in the proliferation of spermatogonia and the increase in apoptosis constitute two consecutive mechanisms correlated with the ageing of the seminiferous epithelium.

Transforming growth factor beta1 mediates hypopigmentation of B16 mouse melanoma cells by inhibition of melanin formation and melanosome maturation.

Transforming growth factor-beta1 (TGFbeta1) downregulates tyrosinase in B16 melanoma cells by decreasing gene expression and the intracellular half-life of the enzyme, but does not block tyrosinase stimulation by alpha-melanocyte stimulating hormone (alphaMSH). In the presence of both agents, the enzymatic activity is intermediate between the one of cells treated with either agent alone. Here we show that TGFbeta1 equally inhibits the melanogenic activities of melan-a melanocytes and B16 melanoma cells, thus validating the B16 model. In both cell types, TGFbeta1 (10(-10) M, 48 h) inhibited to comparable levels tyrosine hydroxylation and melanin formation from L-tyrosine. Thus, the inhibitory effect is exerted mainly at the rate limiting step of the pathway. By means of quantitative image analysis techniques, we also studied the effects of TGFbeta1 and alphaMSH on melanosome number, volume density and maturation degree. alphaMSH (10(-7) M, 48 h) increased 7-fold melanosome volume density, whereas TGFbeta1 by itself had no significant effect. However, melanosomal volume density was intermediate in cells treated with both agents, as compared to control or alphaMSH-treated cells. Moreover, TGFbeta1 blocked the alphaMSH-elicited increase in the number of melanosomes. Control and alphaMSH-treated melanocytes contained more stage I+II premelanosomes and stage IV, fully melanized organelles than partially melanized stage III melanosomes. TGFbeta1 increased the percentage of stage III melanosomes. This trend was even more marked in cells treated with alphaMSH and TGFbeta1. The accumulation of incompletely melanized melanosomes is consistent with the inhibition of melanin formation activity by TGFbeta1 and with its hypopigmenting effect.

Melanization stimulating factors in the integument of the Mugil cephalus and Dicertranchus labrax.

The pigment pattern expression resides in the chromatoblasts of the embryonic skin. The differentiation of these chromatoblasts is influenced by specific local factors such a melanization inhibiting factor (MIF) and a melanization-stimulating factor (MSF). We reveal the presence of these factors by means of a series of experiments on the skin of the marine species of fish Dicertranchus labrax and Mugil cephalus, each with different pigment pattern, the former having a light skin and the latter a darker one. Media conditioned by exposure to dorsal and/or ventral skin, stimulates the melanization of Xenopus laevis neural crest cells throughout a 3 day assay period. Similarly conditioned culture media tested on B16-F10 murine malignant melanocytes, revealed a considerable influence in enzymatic activities: dopachrome tautomerase (DCT), tyrosine hydroxylase and dopa oxidase. The use of media in a dose response basis suggests that the conditioned media may contain both melanophore stimulating and inhibiting factors. The results obtained may actually reflect the resultant activity of the two factors present.

Ultrastructural and biochemical analysis of epidermal xanthophores and dermal chromatophores of the teleost Sparus aurata.

We have studied the pigmentary system of the teleost Sparus aurata skin by electron microscopy and chromatographic analysis. Under electron microscopy, we found the dermis to contain the three major types of recognized chromatophores: melanophores, xanthophores and iridophores. Melanophores were more abundant in the dorsal region, whereas the iridophores were more abundant in the ventral region. The most important discovery was that of epidermal xanthophores. Epidermal xanthophores were the only chromatophores in the epidermis, something only found in S aurata and in a teleost species living in the Antartic sea. In contrast, the biochemical analysis did not establish any special characteristics: we found pteridine and flavin pigments located mostly in the pigmented dorsal region. Riboflavin and pterin were two of the most abundant coloured pigment types, but other colourless pigments such as xanthopterin and isoxanthopterin were also detected.

Early hematopoiesis and developing lymphoid organs in the zebrafish.

In zebrafish, the transparent and rapidly developing embryo and the potential for genetic screening offer a unique opportunity to investigate the early development of the vertebrate immune system. Here we describe the initial appearance of various blood lineages and the nature of accumulating hematopoietic tissue in the thymus and kidney, the main lymphoid organs of adult teleosts. The ultrastructure of the first site of hematopoiesis, the intermediate cell mass (ICM), is described from the 5-somite stage, about 11.5 hours post-fertilization (hpf) until 24 hpf. The ICM gives rise to the primitive erythroid lineage, which accounts for all circulating erythrocytes for the first 4 days pf. From 24 to 72 hpf, a few developing granulocytes are seen close to the yolk sac walls and in the caudal axial vein. The heart, previously proposed as an early blood-forming organ in zebrafish, did not contain hematopoietic cells. The thymic primordium, consisting of two layers of epithelial cells, appears at 60 hpf. By 65 hpf, it is colonized by immature lymphoblasts. The thymus gradually accumulates lymphocytes, and the lymphocytes and epithelial cells progressively differentiate for 3 weeks pf. At 96 hr, the pronephros contains hematopoietic cells, mainly developing erythrocytes and granulocytes. The amount of renal hematopoietic tissue increases rapidly; however, lymphocytes were not detected until 3 weeks pf.

The melanogenic system of Xenopus laevis.

Melanin pigments in lower vertebrates are often found in locations other than the skin, thus forming an extracutaneous pigmentary system of unknown function. The cellular and biochemical structure of this system is still poorly characterized. This paper deals with the ultrastructural and biochemical features of the melanogenic system of Xenopus laevis. Melanin containing cells were identified in the dorsal and ventral skin, and in the lung, spleen, liver and connective tissue surrounding blood vessels. The pigment cells in the skin and the lungs appeared to be typical melanocytes. The spleen contained isolated melanocyte-like cells, but most of the pigment cells present in this organ were associated with melanomacrophage centers. Conversely, the liver appeared devoid of melanocytes and only displayed melanomacrophage centers. Tyrosinase activity was found in all pigment-containing organs except the liver. All organs containing tyrosinase activity also displayed melanin formation potential from L-tyrosine. Therefore, tyrosine hydroxylase and melanin formation activities could be detected only in those organs containing typical melanocytes but not in locations such as the liver, where only melanomacrophages centers were found.

[Anatomic features of the middle ear in the gerbil. Scanning electronic microscopic study]. / Aspectos anatómicos del oído medio del jerbo. Estudio con microscopia electrónica de barrido.

The surgical anatomy of the middle ear was studied in 20 adult gerbils (Meriones unguiculatus) by microdissection and scanning electron microscopy. The most significant finding was a well pneumatized tympanic bulla, which facilitated the identification of useful anatomical landmarks for middle and inner ear dissection. The typical features of middle ear structures in the gerbil are described.

Melanocytes in the stria vascularis and vestibular labyrinth of the Mongolian gerbil (Meriones unguiculatus).

Inner ear melanocytes are mainly present in the cochlea, vestibular organ, and endolymphatic sac, but their exact biological function has not been determined. In this investigation, we study the pigment cells in the membranous labyrinth of the gerbil. The inner ear melanocytes of M. unguiculatus show an irregular dendritic shape with cytoplasmic processes. These cells are disposed following the distribution of strial marginal and vestibular dark cells that have an important metabolic activity. Gerbil inner ear melanocytes are characterized by the presence of melanosomes, which are homogeneously dense organelles, of variable size and shape, that are surrounded by a membrane. In these cells, the Golgi apparatus plays a important role in melanin synthesis. When melanocytes were incubated in L-DOPA solution, the vesicles and cisterns of the Golgi apparatus exhibited a positive tyrosinase reaction. An interesting observation is the relation between melanocytes and inner ear capillaries. Sometimes, near to sensory vestibular areas, the melanocytes were in contact with Schwann cells and with myelinated fibres of vestibular nerve. The ultrastructural findings of this investigation are consistent with the hypothesis that melanocytes may have functional significance in the inner ear.

Melanization stimulating activity in the skin of the gilthead porgy, Sparus auratus.

The presence of a melanization-stimulating factor (MSF) was discovered in dorsal and/or ventral skin of Sparus auratus. Skin from this marine species was used to condition Steinberg's balanced salt solution (BSS), which was subsequently tested with the neural tube assay. BBS conditioned by dorsal and/or ventral skin of S. auratus at 25% and 50% concentrations had a profound stimulatory effect on the percentage of melanization of neural crest cells throughout the 3-day assay period. In some cases 90% melanization occurred within the first 24 hr. Such stimulated cells showed a doubling of the number of dendrites per cell. To assess the effects of MSF on other indices of melanization, dorsal and/or ventral skin was used to condition MEM used in the culture of B16-F10 murine melanoma cells. During the first 24 hr, B16-F10 murine melanoma cells responded to conditioned media by demonstrating a considerable increase in activities of tyrosine hydroxylase, dopa oxidase, and dopachrome tautomerase, but no effect was observed on melanin content. In contrast, melanin content increased after 48 hr of incubation, whereas the enzymatic activities were inhibited during this period. It seems that MSF activity, expressed in several ways, may be present generally among marine species.

Intrinsic pigment-cell stimulating activity in the catfish integument.

In keeping with the concept that local factors in the vertebrate integument affect the expression of pigment cells, the present study was directed toward demonstrating the existence of such factors in the skin of the channel catfish, Ictalurus punctatus. This species has a dark dorsal surface in marked contrast to an almost white midventral surface. Pieces of skin from these two surfaces were used to condition culture media, which were in turn bioassayed using the Xenopus neural tube explant system (Fukuzawa and Ide, 1988, Dev. Biol. 129:25). A certain number of neural crest cells grow out from the explant, and many of these are melanized in a culture medium of Steinberg's basic salt solution (BSS). When the BSS was conditioned with either dorsal or ventral skin, a profound increase in both the number of crest cells emigrated from the neural tubes and the percentage of melanized cells was observed. The effects of dorsal skin were stronger than those of ventral skin and were evident on a dose/response basis. Initial fractionation of conditioned BSS with DEAE ion exchange chromatography produced fractions of particular potency in the stimulation of melanogenesis. A similarly conditioned medium based upon Leibovitz's L-15 was used in the primary culture of mature chromatophores, namely, melanophores, iridophores, and xanthophores from tadpoles of Rana pipiens. Both dorsal and ventral conditioned media stimulated iridophores and xanthophores, but seemed to have little or no effect on tadpole melanophores. A melanization inhibiting factor (MIF) from the ventral surface of adult frogs has been suggested as the basis for the light colored ventrum of amphibians, and although the present experiments were not designed to study catfish MIF, the possible existence of such a factor in this species was supported by the results. The total results of this investigation are discussed in the light of the possible presence of a melanization inhibiting factor (MIF) of greater prevalence in the ventrum and a melanization stimulatory factor (MSF) of greater prevalence in the dorsal integument. It is suggested that the light-colored ventral surface of the catfish and other poikilotherms may result from the presence of higher levels of MIF than MSF. Thus, the expression of melanophores is inhibited while that of iridophores is enhanced. In contrast, higher levels of MSF over MIF in the dark dorsal surface would result in melanophore stimulation and inhibition of iridophore expression.

Preliminary biological characterization of a melanization stimulating factor (MSF) from the dorsal skin of the channel catfish, Ictalurus punctatus.

A two step fractionation of conditioned media made from the darkly pigmented dorsal skin of the channel catfish, Ictalurus punctatus, has produced fractions that contain a melanization stimulating factor (MSF). Isolated neural tubes of Xenopus laevis embryos exposed to conditioned media and to specific fractions exhibit greater melanization (increased numbers of melanized cells and elevated percentages of melanized cells), a greater number of dendrites per melanized cell, and a greater number of emigrated neural crest cells than control neural tubes. The presence of MSF activity in the darkly pigmented dorsal integument suggests a role for a molecule or molecules in the development and maintenance of the dorsal/ventral pigment pattern of this piscine species and possibly of other vertebrates.

Distribution of extracutaneous melanin pigment in Sparus auratus, Mugil cephalus, and Dicertranchus labrax (Pisces, Teleostei).

The morphological and biochemical characteristics of pigment accumulations found in the kidney, liver, spleen, and mesentery of three different species of teleost fishes have been studied. There are significant differences in number, distribution, and morphology of pigment accumulations in different organs of the three species. Biochemical studies have shown the existence of tyrosinase activity in the mesentery of Mugil cephalus and in the kidney and mesentery of Sparus auratus. No tyrosinase activity was found in any internal organs of Dicertranchus labrax. That activity was assayed using three methods: tyrosine hidroxylation, dopa oxidation, and melanin formation. The morphological and biochemical observations are in agreement. In those organs in which we have demonstrated melanin synthetic activity, the pigment cells are morphologically and like melanophores, while in the organs that show no melanin synthetic activity, the pigment cells resemble macrophages.

Haemopoiesis in the head kidney of Sparus auratus.

The haemopoiesis of Sparus auratus is formed by the following series: erythro-thrombopoietic, granulopoietic, lymphoplasmapoietic and monocytes. All the cells which form these series originate from one cell called the stem-cell, there being a network of reticular cells and melanomacrophage centres amongst these cells. The erythropoietic series comprises erythroblast, proerythrocytes, polychromatocytes, reticulocytes and erythrocytes. The morphological changes which occur during the maturing process are: heterochromatinization of the nuclei, the gradual decrease of organelles in the cytoplasm and the increase of the haemoglobin. We also observed thrombocytes characterized by the presence of numerous vacuoles and abundant glycogen in the cytoplasm. In the lymphoplasmapoietic series are seen lymphoblasts, lymphocytes and plasma cells. The lymphocytes are cells of different sizes with small microvilli in the cell surface and scanty cytoplasm. Outstanding in the plasma cell is the well developed granular endoplasmic reticulum. The monocytes are large cells with an indented nucleus and cytoplasm containing numerous vesicles of different sizes and also a few lysosomes.

Occurrence of melanin granules and melanosynthesis in the kidney of Sparus auratus.

An optical, ultrastructural, and biochemical study of the melanin accumulation nodules found in the kidney of the teleost fish Sparus auratus is presented. These nodules are randomly distributed in the interstitium of the renal tissue. They are formed by large aggregates of cells replete with melanin granules. The melanin granules occur singly or also in aggregates inside the cells. Most of the granules are electron-dense, but sometimes small electron-lucent spaces within them can be seen. Some secondary lysosomes and dendritic processes can also be observed. Biochemical studies have proved for the first time the existence of measurable tyrosinase activity in those nodules. That activity was assayed using three methods: tyrosine hydroxylation, dopa oxidation, and melanin formation. Furthermore, inhibitors of well-characterized plant and animal skin tyrosinases were effective agents for inhibiting those activities in fish kidney preparations. This finding supports the notion of the existence of true tyrosinase in the melanin accumulation nodules of this tissue. Taking into account the results obtained, the origin and functions of the melanin-containing cells found in the teleost fish kidney are discussed.

Granulopoiesis in the head kidney of Sparus auratus.

The head kidney of Sparus auratus (teleost) is comprised of erythropoietic, granulopoietic and lymphopoietic cells. This study describes the ultrastructure of the granulopoietic series of cells. Heterophils, eosinophils and basophils were found at various stages of development. The most numerous cells are the neutrophils, the cells of the basophilic series being very scarce. The most outstanding characteristics of the neutrophil series are the eccentric and slightly segmented nucleus and cytoplasm with numerous, homogeneously dense granules. The eosinophils show a lobed nucleus and two types of granules in the cytoplasm. In the basophils, the cytoplasmic granules are large with fibrillar contents.

Ultrastructure of the tubular nephron of Testudo graeca (Chelonia). A comparison between hibernating and non-hibernating animals.

The tubular nephron of hibernating and non-hibernating specimens of Testudo graeca (Chelonia) was studied by means of conventional light and electron microscopy and histochemistry. The tubular nephron was composed of proximal, intermediate, distal and collecting tubules in both hibernating and non-hibernating animals. The cells of the proximal tubule showed long microvilli, cytoplasmic vacuoles, a developed endoplasmic reticulum and abundant mitochondria. Fat droplets were also observed. The intermediate segment was lined by ciliated and non-ciliated cells. The lining cells of the distal tubule presented few microvilli, abundant dense mitochondria and clear vesicles of mucous appearance in the terminal portion. Collecting ducts are composed of mucous and non-mucous cells. Mucous cells presented strong reaction to the histochemical techniques detecting sialo- and sulpho-mucins. During hibernation, a progressive vacuolar degeneration of the endoplasmic reticulum was observed in all the segments of tubular nephron, which may be caused by a massive intake of extracellular water into the cell.