Structural organisation of connective tissue

The mammalian skeletal muscle cell is very well investigated and the spatial arrangement of the molecules organizing it into sarcomeres, myofilaments, myofibril and myofibres can be considered as completely understood. It is also known that the structural integrity of muscle fibres is maintained by three layers of intramuscular connective tissue: the endomysium that surrounds the individual muscle fibre, the perimysium that bundles a group of muscle fibres and the epimysium that enfolds the whole muscle. On the other hand, much less is known about the hierarchical organisation of the structural components within these intercellular tissues. To make matters even more complicated, in these intercellular spaces, muscle tissue is also mixed with vascular and nervous tissues. Using chemical methods, the molecular components of these tissues are not distinguishable from the muscle tissue. Scanning electron microscopy (SEM) can be used to get an overall view of the localisation and distribution of the vascular and nervous tissue. The further use of confocal laser scanning microscopy (CLSM) in combination with immuno histochemistry allows for identification and localisation studies of molecular components. Introduction If one considers the contractile machinery of muscle in the context of interrelated factors, then suddenly one is faced with a very complex area of study. At the one level it is essential that one can combine contractile units in such a way as to augment and optimize force production, but at another level one needs also to provide space for an ancillary network to facilitate the supply of nutrients and the removal of waste compounds. It is this interconnection both between the anatomical and physiological parameters that comprise muscle and between the fundamental requirements of contraction versus the continued supply and removal of compounds that makes muscle a fascinating subject to research. The traditional way of looking at connective tissue in skeletal muscle focuses primarily on the structural integrity of muscle, which is thought to be maintained by three layers of intramuscular connective tissue: the endomysium that surrounds the individual muscle fibre, the perimysium that bundles a group of muscle fibres and the epimysium that enfolds the whole muscle. Moreover, since the most plentiful and widespread protein component of the connective tissue is collagen, there has been a great deal of focus on the identification and localization of collagenous structures. However, the connective tissues are also the place where vascular and nervous structures are embedded. Vascular structures in particular, are highly abundant in skeletal muscle and could therefore considerably contribute to the quality of meat and meat products. There is a lack of knowledge concerning these structures, which is grounded in a lack of methodology. However, with the development of confocal laser scanning microscopy it is possible to study the molecular composition and behaviour of such structures. Material and methods Muscle samples from M. biceps femoris were collected at 24 h post-mortem and consequently snap frozen in liquid nitrogen. Antibodies against Desmin DE-R-11, LYVE 1 and Collagen I were purchased at Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, U.S.A.). The antibody against laminin came from the Developmental Studies Hybridoma Bank (University of Iowa, Dept. of Biological Sciences, Iowa City, IA 52242 U.S.A.). The phallotoxin Atto 647N-Phalloidin and fluorescently labelled wheat germ agglutinin (WGA) from Fluka was purchased via Sigma-Aldrich Inc. (St. Louis, MI, U.S.A.). Secondary antibodies conjugated to Alexa 488, 555 and 647 and the lipid stain Bodipy were acquired from Invitrogen, Molecular Probes (Eugene, OR, U.S.A.). Immuno-histochemistry was performed as previously described in Brüggemann and Lawson (2005). A CLSM (SP2, Leica Laser Technik GmbH, Heidelberg, Germany) was used for imaging. In order to avoid false positives by collecting fluorescence emission from adjacent fluorophore channels, a sequential confocal imaging technique was used. The images shown are the result of a projection from the stack of images in the vertical direction using the maximum intensity mode. The