These are the fine delicate fibers that are found connected to the coarser and stronger collagenous fibers (Type I fibers). They provide the bulk of the supporting framework of the more cellular organs (e.g. spleen, liver, lymph nodes, etc.), where they are arranged in a three-dimensional network to provide a system of individual cell support (Fig. 11.2). On light microscopic examination, reticular fibers are weakly birefringent, the weak reaction being attributed to their lack of physical size and the masking effect of the interfibrillar substance. They are seen to branch frequently and appear indistinct in H&E-stained preparations. The characteristics of reticulin fibers in human kidney cortex have been studied using immunohistochemical means. Antibodies directed against Type I and Type III collagens, their corresponding amino peptides and decorin (PG-II) revealed that in this organ the reticulin fibrils consist of hybrids of Type I and Type III collagens. Double immuno-electron microscopy shows that 20–25 nm fibrils consist mainly of Type I collagen, whereas the larger fibrils, 30–35 nm, label simultaneously for Type I and Type III collagens. Most fibrils larger than 40 nm in diameter label for Type III collagen. Reticular fibers may be demonstrated distinctly, in paraffin sections, using one of the many argyrophil-type silver impregnation techniques available or, in frozen section, by the periodic acid-Schiff technique. Both methods of demonstration are dependent upon the reactive groups present in the carbohydrate matrix, and not upon the fibrillar elements of the fiber.

Figure 11.2 Reticular fiber pattern in normal liver by the Gordon and Sweets (1936) silver impregnation method.

Elastic fibers

The elastic system fibers (i.e. oxytalan, elaunin, and elastic fibers) have, respectively, a fibrillar, amorphous, or mixed structure. The elastic fibers may be found throughout the body but are especially associated with the respiratory, circulatory, and integumentary systems. Their appearances under the light microscope may vary considerably according to location, from fine, single fibers, as in the upper dermis, to membrane-like structures (‘internal and external elastic laminae’) in the large arteries. In the latter situation, the elastic membranes are interrupted by minute holes called fenestrae (Latin ‘fenestra’ – window) which permit diffusion of materials through the otherwise impermeable membrane. Recent high-resolution electron microscopic examination has demonstrated that elastic fibers consist of two quite distinct components. There is an amorphous substance which, biochemically, is consistent with the protein elastin, and also a second component, which shows a periodicity of 4–13 nm, that is microfibrillar in nature, and has been termed elastic fiber microfibrillar protein (EFMP). These micro-fibrils, sometimes also called elastin-associated microfibrils (EAMF), are ubiquitous connective tissue structures that are believed to provide tensile strength and flexibility to numerous tissues. They may also act as a scaffold for elastin deposition.

When viewed in transverse section, the central core of the elastic fiber is seen to be composed of the amorphous protein elastin, surrounded by a ring or band of EFMP. The proportions of the two components seem to alter with the age of the fiber (and probably also with the age of the subject). In young fibers, the dominant fraction is the microfibrillar protein. In older fibers, the amorphous protein accounts for over 90% of the fiber content. The basic molecular unit of elastin is a linear polypeptide with a molecular weight of approximately 72 kilodaltons (kDa). This subunit has been referred to as ‘soluble elastin’ or ‘tropoelastin’. One of the characteristic features of elastic fibers is the presence of cross-linking which binds the polypeptide chains into a fiber network. Desmosine and its isomer, isodesmosine, are the cross-linking compounds involved. The polypeptides are transported out of the fibroblasts or smooth muscle cells and the cross-linking occurs in the extracellular spaces.

Elastic fiber microfibrillar protein has an amino acid content that is quite distinct, biochemically, from that of elastin protein. It is particularly rich in amino acids, which are lacking or present in only small quantities in elastin. The content of cysteine in EFMP is high, reflecting the presence of numerous disulfide linkages that will be of significance when the staining properties of elastic fibers are considered later. Associated with EFMP are a number of carbohydrate complexes, termed ‘structural glycoproteins’ (Cleary & Gibson 1983); the significance of these in the staining of elastic fibers will also be considered later. For a more detailed account of elastic fiber composition and biochemistry, reference should be made to the work of Cleary and Gibson (1983), Uitto (1979), or Bailey (1978). Elastic fibers are acidophilic, congophilic, and refractile. Following oxidation, they are quite strongly basophilic due to the formation of sulfonic acid groups from the disulfide linkages of the EFMP. Young fibers with a high content of EFMP show a positive periodic acid-Schiff reaction. They may be seen in routine H&E- stained sections, but, for exacting studies, numerous more selective techniques are available. These may be relatively simple, e.g. the Taenzer-Unna orcein method, or more lengthy and complex, e.g. Weigert resorcin-fuchsin methods. With increasing age of the elastic fibers, physical and biochemical changes are seen to occur. These may include splitting and fragmentation, alteration of the ratio of EFMP to elastin, and increases in the levels of glutamic and aspartic acids and calcium. These changes are readily visible in the skin of the subject, which becomes wrinkled and ‘loose-fitting’. A more serious problem occurs with the loss of elasticity of the elastic arteries.

Oxytalan fibers

Oxytalan fibers were first described by Fullmer and Lillie (1958) in periodontal membranes. More recently they have been demonstrated in a wide variety of tissues, both normal and abnormal (Alexander & Garner 1977; Cleary & Gibson 1983; Goldfischer et al. 1983). On light microscopic examination, oxytalan fibers may be distinguished from mature elastic fibers by their failure to stain with aldehyde fuchsin solutions, unless they have been previously oxidized by potassium permanganate, performic acid, or peracetic acid. They have also been reported to remain unstained following Verhöeff’s hematoxylin, with or without prior oxidation. Following electron microscopic examination by a number of workers, it has been suggested that oxytalan fibers are similar to, if not identical to, EFMP fibers. They appear to be composed of microfibrillar units, 7–20 nm in diameter, with a periodicity of 12–17 nm. Their periodicity is made more conspicuous by pretreatment with ruthenium red. From their morphology, localization, and staining properties, it seems possible that oxytalan fibers may represent an immature form of elastic tissue. It has also been suggested by Goldfischer et al. (1983) that microfibrils and oxytalan fibers may have a role beyond that of elastogenesis and may involve ‘anchoring’ mechanisms between collagen fibers, stromal cells, lymphatic capillary walls, mature elastic fibers, muscle cells, etc.

Elaunin fibers

Gawlik (1965) first described elaunin fibers; the term ‘elaunin’ is derived from the Greek ‘I stretch’. Unlike oxytalan fibers, elaunin fibers stain with orcein, aldehyde fuchsin, and resorcin–fuchsin without prior oxidation, but do not stain with Verhöeff’s hematoxylin.