Histochemical Evaluation of Lung Co...

Histochemical Evaluation of Lung Collagen contentment in Acute and Chronic Interstitial Diseases (*1)

Interstitial lung diseases show a group of pulmonary disorders of multiple causation that may cause marked alterations of extracellular matrix of pulmonary parenchyma. In the past ten years, great attention has been focused onward the study of the biology of collagen in these disorders in order to understand the natural story of ILD and to establish prognostic and therapeutic practices in more rational bases. [1-8]

In the ready investigation, we performed a practical and inexpensive histochemical evaluation of the collagen contented and its state of aggregation using slides stained with Sirius r F3BA. This color has been shown to stain collagen specifically and is suitable and permits quantitative analysis of this protein. [9-14] This cogitation was carried out in interpret chest lung biopsies in three collections of interstitial lung diseases that near distinctive morphologic and clinical pictures: a rapidly fibrosing ILD exhibited by ARDS in its proliferative stage, a granulomatous ILD as the sarcoidosis, and pulmonary fibrosis associated to CTD and IPF.

MATERIAL AND METHODS

Patient Characteristics

In this work were studied make open chest lung biopsies of patients divided in three clusters as follow: (a) ten patients with ARDS; (b) six patients with pulmonary sarcoidosis: and (c) seven patients with pulmonary fibrosis (three with IPF and four with CTD) The diagnosis above was based in succession clinical, laboratory, and histologic findings. For controlling intentions the results were compared with those obtained in eight samples of histologically normal lung parenchyma obtained from necropsy (five samples) or from areas of normal parenchyma during lung cancer resection.

Collagen Measurements

Technical Considerations: The analyses were performed in formalin-fixed, paraffin-embedded tissue sections stained with Sirius r F3BA. This stain has been used since 1964 for staining collagen in histologic specimens and was demonstrated to enable quantitative analysis of collagen separated through sodium dodecyl sulfate (SDS)-acrylamide gel electrophoresis and also in paraffin sections. Sirius r F3BA was shown to bind selectively to collagen stamps I, II and III, giving colored outcomes in which the bound shade is proportional to the amount of collagen near [9-14] Quantitative studies using Sirius r F3BA may be performed below two circumstances as follow: the measurement of its color intensity, which is proportional to the collagen concentration; and next to the first measurement of the emitted light during observation using polarized light. These sum of two units procedures reflect different analytical approaches. The color intensity measurement thinks the amount of collagen, independent of aggregation; upon the other hand, the birefringence observ when the slides were viewed using polarized light contemplates not only the collagen make easy but also the degree of parallel orientation and state of aggregation of the collagenous erections Collagen synthesis involves several complexus steps of molecular aggregation, involving one as well as the other intracellular and extracellular phases. All collagens have the public feature of being composed by the agency of a triple helix of alpha polypetide chains. This indivisible particle is known as procollagen and is the monomer which will constitute the collagen fibrils in consequence of extracellular aggregation. Collagen fibrils (which are identifiable by dint of electron microscopy) aggregate in order to form collagen fibers, which are visible in routine histologic preparations. The collagen representations differ in their alpha-chain amino acid arrangement [15] and these differences influence the aggregation of the ultimate particle s of collagen to form fibrils. [16] Ultrastructural observations forward the connective tissue of several organs in representative species of the main vertebrate classes disclose brace distinct populations of collagen fibrils, which could be recognized forward the basis of their diameter, and either unfasten or compact arrangement. [17] Collagen archetype III fibrils present a relatively small and uniform diameter when compared to collagen sign I. [17] In addition, scanning electron microscopy of the in vitro reconstitution of collagen polymer demonstrated the formation of thick packets of pure collagen type I and thin isolated fibers of sign III, showing that the chemical constitution of collagen determines their physical aggregation. [15] Considering these facts, increased collagen refringence may think changes in type I/type III collagen ratio and/or higher measure of collagen cross-linkings as a come of natural aging of cicatricial proces The enhancement of collagen birefringence promot at the Sirius red polarization mode is specific for collagen, disclosing its distinct patterns of physical aggregation. [17] In other words, measurements of collagen birefringence in slides stained with Sirius r F3BA throw backs not only the amount, nevertheless also the macromolecular collagen arrangement in the extracellular matrix.