Elastic system of the rat ventral prostate and its modifications following orchiectomy.

BACKGROUND: The extracellular matrix (ECM) has important roles in prostatic development, and marked stromal changes take place in the rat ventral prostate (VP) after androgen deprivation. However, little knowledge exists about individual ECM components. METHODS: The distribution of elastic fibers (EF) and elastic-related fibers (ERF) in the VP of castrated and control rats was investigated, using histochemistry and transmission electron microscopy (TEM). RESULTS: EF are barely detected in the prostatic stroma, but ERF are relatively abundant. Castration results in a relative increase in the number and thickness of ERF. TEM showed an open network of ECM microfibrils throughout, the stroma and thin and short EF, which increase in number and thickness after orchiectomy. CONCLUSIONS: The presence of elastic system components in the rat VP warrants the deformability required for the secretion exclusion under the action of smooth muscle cells, and the castration-induced modification may be related to the contraction of the tissue and maintenance of peculiar arrangements of other ECM components.

Collagen type VI is a component of the extracellular matrix microfibril network of the prostatic stroma.

Stroma-epithelium relationships are of great relevance in prostatic morphogenesis and physiology. However, little knowledge exists about either stromal cells or extracellular matrix composition and arrangement in this system. Ultrastructural analysis revealed the existence of a microfibrillar system which occupies large areas of the rat prostatic stroma. In this work, we have applied immunocytochemistry and an ATP treatment for the ultrastructural identification of collagen type VI microfibrils, aiming at examining its participation in the prostatic microfibrillar network. Immunocytochemistry was also extended to a human case of prostatic nodular hyperplasia. Both methods succeeded in identifying collagen type VI in the rat ventral prostate. Collagen type VI is evenly distributed throughout the stroma but mainly associated with the basal lamina, collagen fibrils, and around the stromal cells. The use of ATP treatment allowed for the discrimination between collagen type VI and elastin-associated microfibrils, and demonstrated that these two classes of microfibrils establish an extended, mixed, and open network. The same aspects of association with the basal lamina, with stromal cells (particularly with smooth muscle cells), and with fibrillar components of the stroma were observed in the human tissue. We suggest that the collagen type VI and elastin-associated microfibril system may be involved in the control of some aspects of cellular behavior and may also play a structural role, maintaining the organ integrity after the deformations occurring under smooth muscle contraction.

Fluorescence and confocal laser scanning microscopy imaging of elastic fibers in hematoxylin-eosin stained sections.

We have studied the possibility of associating fluorescence microscopy and hematoxylin-eosin staining for the identification of elastic fibers in elastin-rich tissues. Elastic fibers and elastic laminae were consistently identified by the proposed procedure, which revealed itself to be easy and useful for the determination of such structures and their distribution. The fluorescence properties of stained elastic fibers are due to eosin staining as revealed by fluorescence analysis of the dye in solution, with no or only minor contribution by the elastin auto-fluorescence. The main advantage of this technique resides in the possibility of studying the distribution of elastic fibers in file material without further sectioning and staining. The use of the confocal laser scanning microscope greatly improved the resolution and selectivity of imaging elastic fibers in different tissues. The determination of the three-dimensional distribution and structure of elastic fiber and laminae using the confocal laser scanning microscope was evaluated and also produced excellent results. used to discriminate calcified bone and elastic fibers using the fluorescence microscope (Bradbeer et al. 1994). The selective fluorescence exhibited by these two main tissue components is based on the very faint eosin staining, while intensely eosinophilic substrates and those stained with the highly absorbing basic dyes show no fluorescence or just a very faint signal. Considering the possibility of using eosin fluorescence for the selective identification of some tissue components, we have extended this approach to the study of elastic fibers in hematoxylin-eosin (H&E) preparations. This paper then describes the results of observations with the fluorescence microscope of some H&E stained sections and evaluates the use of the confocal laser scanning microscope to image elastic fibers in the same preparations. Furthermore, we have also studied absorption and fluorescence spectra of eosin and phloxine, a closely related dye, in solution, to correlate them with the fluorescence detected in tissue sections.

Basement membrane associated changes in the rat ventral prostate following castration.

This study focuses on the basement membrane associated modifications that take place after androgen blockade, by studying some of its main components, through histochemical, immuno-histochemical and Western blotting tests, and its ultrastructural aspects. It was demonstrated that laminin and collagen type IV remain associated with a thickened basement membrane and that there is an apparent increase in heparan sulfate content 21 days after castration. Ultrastructurally, basal lamina appeared extensively folded and pleated. It was also observed that detachment of epithelial cells is not dependent of basal lamina degradation and that the free basal lamina surfaces are folded by the action of adjacent cells. We have also observed some aspects of smooth muscle cell degeneration and death, that lead to modifications of the associated basal lamina. In this case, residual basal lamina also shows extensive folding. The results suggested that degradation of excess basement membrane does not occur or is a very slow process within the period examined, and that basement membrane is left re-organized but ultrastructurally and compositionally unaffected.

The applicability of hematoxylin-eosin staining plus fluorescence or confocal laser scanning microscopy to the study of elastic fibers in cartilages.

This study focuses on the use of hematoxylin-eosin staining plus fluorescence microscopy for the investigation of elastic fibers in some elastic cartilages. We have observed that elastic fibers are consistently imaged by the proposed procedure and the resolution attained is similar to that obtained with the classical Weigert's fuchsin-resorcin. The results also demonstrate that elastin autofluorescence gives little or no contribution to the final fluorescence and that the use of the confocal laser scanning microscope adds to the resolution, permits the use of thicker sections and reveals of minute structural at features. We conclude that this is a relevant tool in elastin research.

The elastic system of a pressure-bearing tendon of the bullfrog Rana catesbeiana.

Elastic system components have been described in the pressure-bearing tendon of the bullfrog, Rana catesbeiana, as a result of histochemical tests and transmission and scanning electron microscopy. The tension region was shown to possess microfibril bundles, some of which exhibited central deposits of amorphous material. The use of ANS-butanol plus fluorescence microscopy enormously facilitated the identification of elastic system components in both tension and compression regions of the frog tendon. The compression region exhibited pre-elastic and mature elastic fibers, which were shown to be associated with the surface of the convoluted collagen bundles. Thin fibrils were observed in the compression region after ANS treatment. The visceral paratenon had an increased number of elastic fibers located between the collagen bundles and close to the cells. Congo red plus polarization microscopy failed to impart birefringence to the elastic fibers, but they could be identified by their intense staining and isotropic appearance against the bright background of birefringent collagen fibers. SEM demonstrated the three-dimensional aspects of the elastic fibers. They are composed of fibrils of a sinuous nature. The use of ruthenium red in the fixative allowed for the observation of an intimate association of proteoglycan granules with the microfibril bundles. The elastic components identified in the pressure-bearing tendon are assumed to be important for the tissue supramolecular organization, especially in the maintenance of the convoluted state of the collagen fibers in the compression region and their crimp morphology in the tension region. The elastic system must also play an important role in the restoration of the resting shape of the tendon after the deformation achieved during mechanical stimulation.

Understanding the biomechanics of tendon fibrocartilages.

A direct analogy with the function of large proteoglycans in articular cartilage has led to the assumption that the presence of such components in tendon fibrocartilages is a major factor enabling these structures to resist pressure. However, the association of collagen fibrils in bundles and their tensional state as verified through the existence of crimp under polarized light in tendon fibrocartilage raises doubts as to whether the large proteoglycans are kept under osmotic pressure, which is an essential condition for their physiological role in articular cartilage. This suggests that tendon fibrocartilage has distinct mechanisms to resist pressure. In tendon fibrocartilages, the compressive forces must be transferred to the inextensible collagen fibers which lie in many directions in different planes, before reaching the parallel fibers of the tension region. In this sense, the large proteoglycans are likely to have the function of providing a viscous and cohesive environment in which collagen fibers could get stretched to reinforce the tissue.

Microfibrils: neglected components of pressure-bearing tendons.

Some tendons wrap around joints and receive compressive forces besides transferring the tension forces from muscle to bone. These tendons develop a fibrocartilaginous structure which enables them to withstand pressure. This article describes the existence and distribution of microfibrils (or preelastic fibers) in the pressure-bearing tendons of rabbits and dogs by the application of histochemical assays and transmission electron microscopy. Rabbit and dog tendons possess no mature elastic fibers. The rabbit tendon exhibits some response to Weigert's method prior to oxidation which indicates the existence of the so-called elaunin fibers, especially in the pressure zone. Oxidation with peracetic acid or oxone discloses intricate aspects of the oxytalan fiber distribution in both tension and pressure zones of the dog and rabbit tendons. Bundles of 12 nm microfibrils were demonstrated in the rabbit tendon by electron microscopy after fixation in the presence of tannic acid. The existence of preelastic fibers in the pressure-bearing tendons has been neglected and they are assumed to have importance in the microarchitecture of the tissue and in the ability of the tendon to support tension and compression forces.

Structure and histochemistry of a pressure-bearing tendon of the frog.

The plantaris longus tendon of the bullfrog Rana catesbeiana has been structurally and histochemically characterized. The tendon has been shown to wrap around the distal tibio-fibular joint, extending into an aponeurosis. The region under the joint exhibits a large cartilaginous pad. In contrast to the mammalian flexor digitorum profundus tendons, the compression region of the frog tendon is not typically fibrocartilaginous but presents a unique arrangement of convoluted collagen bundles and fibers associated with a large amount of glycosaminoglycans. The sulfated nature of the glycosaminoglycans in the compressed region was determined through enzyme susceptibility as well as through CEC procedures. Cells of the modified zone exhibited morphological variation, from fibroblastic to condrocyte-like. Polarized light analysis revealed an intricate array of collagen bundles which crossed the tendon in different directions and connected the tension and compression regions. The ability of the tendon to resist compressive forces is assumed to be due to the large amount of glycosaminoglycans in the modified region as well as to the complex arrangement of collagen bundles in the tendon as a whole.

The unique fibrillar arrangement of the bullfrog pressure-bearing tendon as an indicative of great functional deformability.

The fiber distribution and ultrastructure in the plantaris longus pressure-bearing tendon of the bullfrog were investigated. The tension region of the tendon showed a predominant parallel distribution of collagen fibers, but three main zones with different crimp parameters were identified with the use of the polarizing microscope. The compression region showed collagen fibers with aspects of disaggregation and were composed of disperse and undulating fibrils. These collagen fibers establish a three-dimensional network but showed a preferential distribution in planes disposed perpendicularly to the tendon's main axis. It is assumed that the convoluted and disaggregated collagen fibers must be distended before exerting any reinforcement on the tissue and that this only occurs after a great deformation of the tendon. Groups of 5-6 fibrils not associated in fibers are also dispersed in the compression region. The tissue is assumed to have a highly viscous fluid nature allowing for the deformation needed for collagen fibrils to reinforce the tendon structure. The convoluted and crimped structure of collagen fibers would be especially useful when the tendon is submitted to the sudden and strong mechanical loading expected to occur during jumping and to provide the tendon with the capacity of great functional deformability necessary for the high amplitude of feet movements attained on jumping and swimming.

Aggregational state and molecular order of tendons as a function of age.

Form or textural birefringence (FB) depends on the geometrical characteristics of rod-like molecules, like collagen. The degree of packing and ordered aggregation are factors that also play important roles in the FB. Since current information reports increases in the diameters of collagen fibrils according to age, a search to determine variations of intrinsic birefringence (IB) and FB of collagen in Achilles and tail tendons of rats as a function of age was carried out. The findings support the conclusion that there are increases on FB and IB as the animals age. These increases are a consequence of higher molecular order and aggregation of collagen in tendons of older animals. Furthermore, an accurate methodology was developed to detect and to control the effect of section thickness variation of the material used in this research.

Hydrophobia, lectin binding, and molecular order in the stratum corneum of adult and neonatal rats and in human breast cells in culture.

A search for differences due to ANS staining (hydrophobia), Con A and PNA binding capacity, and birefringence was carried out on stratified epithelia of rat skin and human breast cells (HBC) in culture. Microfluorimetric measurements confirm that the ANS fluorescence of the stratum corneum from adults is higher than that of newborns. HBC exhibited an unexpected deep ANS-fluorescence. Differences in the binding capacity of the epithelial layers to Con A and PNA were detected with advancing age. Retardation measurements revealed that the form birefringence of the stratum corneum is higher in adult animals specially as revealed by the fact that its form birefringence curve branch from n = 1.414 to n = 1.479 is steeper, i.e. depict higher values. The strong birefringence of the cytoplasmic tonofilaments presented by cultured human breast cells was considered an unexpected finding and attributed to changes that the cells underwent following the in vitro conditions.