Cryostat Sections Of Skeletal Muscle Research Articles

Skeletal myopathy in mice over-expressing the human myotonic dystrophy protein kinase (DMPK) gene

Myotonic dystrophy (DM) is caused by the expansion of a trinucleotide repeat located in the 3’-untranslated region of a serine-threonine kinase (DMPK), such that repeat size corresponds with severity of disease and age of onset. The mechanism by which this mutation causes DM remains unclear. Recent reports indicate that over-expression of DMPK in murine C2C12 myoblasts inhibits myogenesis, reminiscent of the marked immaturity observed in DM patient muscle. Accordingly, we generated transgenic mice over-expressing the human DMPK gene with expression enhancing matrix attachment region (MAR) sequences. These mice show substantial over-expression of human DMPK transcript and protein in brain, skeletal muscle, tongue, and eye - tissues typically affected in DM. Cryostat sections of skeletal muscle from these transgenic animals revealed diagnostic hallmarks of DM including increased centronucleation, type 1 fiber atrophy and ringed fibers. Additionally, primary myoblasts established from these mice showed reduced fusion potential indicating a delay or defect in myoblast differentiation. These results suggest that over-expression of the human DMPK gene in these mice confers a skeletal muscle pathology similar to that seen in DM patients.

Modified methods for preparation of cryostat sections of skeletal muscle

We developed two modified methods for the preparation of cryostat sections of skeletal muscle: one using solid carbon dioxide and the other using the ultra-low freezer to prepare 2-methylbutane (isopentane) for freezing of skeletal muscle. The results were quite satisfactory, with good preservation of cellular details and histochemical properties. Freezing artifacts were minimal when compared with the standard liquid nitrogen method.

Proteases in normal and diseased human skeletal muscle: a preliminary histochemical survey.

Seven proteases assumed to be aminopeptidases A, B and M, dipeptidyl peptidases II and IV, esteroproteinase and gamma-glutamyltransferase were localized histochemically, using semipermeable membrane simultaneous coupling techniques, in unfixed cryostat sections of skeletal muscle removed from one healthy volunteer, six patients with disuse muscle atrophy, and 15 patients with some form of muscle disease. Normal muscle fibres showed weak reactions for aminopeptidases A and M and for the dipeptidyl peptidases, but no reactivity for gamma-glutamyltransferase or esteroproteinase. No change was detected in diseased muscle fibres except that low gamma-glutamyltransferase and esteroproteinase activities appeared in some cases. The activities of the seven enzymes were stronger in the intermyosial connective tissue than in the muscle fibres, but were also unchanged in disease. The strongest reactions were found in some interstitial cells (mast cells and macrophages) and these were much increased in diseased muscle, particularly for dipeptidyl peptidases II and IV. The findings are interpreted in terms of the release of proteases from such cells and their subsequent involvement in the breakdown of myofibrillar proteins in muscle disease.

Cryostat sections with tissue amastigotes of Trypanosoma cruzi as a substrate for Chagas' disease serology

Sera from 30 chronic chagasic patients together with 52 control samples (34 with other pathological conditions and 18 from normal individuals) were titrated by the indirect immunofluorescent technique (IFA) on Trypanosoma cruzi amastigotes. Acetone-fixed cryostat sections of skeletal muscle of Rockland mice 10 days post-infection with the RA isolate of T. cruzi were used as substrate. Results were compared with titres obtained by conventional IFA on epimastigotes. All 52 control sera had amastigote litres ⩽2 double dilutions (dd) as compared with epimastigote values. Out of the 30 chagasic samples, differences were ⩾4 dd (⩾1 log) for 22, 3 dd for 5 and ⩽2 dd for the remaining 3, when comparing amastigote and epimastigote titres. These results show that the use of amastigotes in cryostat sections of infected tissue for performing Chagas' serology is a simple, adequate and sensitive method.

A method for myoglobin in cryostat sections of muscle by precipitation with sulfosalicylic acid.

Transverse cryostat sections of skeletal muscle were fixed in a solution containing 1.5% glutaraldehyde and 1.5% sulfosalicylic acid and stained in a solution containing equal volumes of 3% hydrogen peroxide and 50% ethanol saturated with o-tolidine. Myoglobin in the sarcoplasm of muscle fibers was precipitated and stained blue. Applicability of this method to cryostat sections, without glutaraldehyde fixations prior to freezing, allowed the myoglobin content of individual muscle fibers to be correlated with other histochemical characteristics of the same fibers seen in serial sections. In the dark red bovine sternomandibularis muscle, fibers with weak adenosine triphosphatase (ATPase) and strong succinate dehydrogenase (SDH) activity always exhibited strong myoglobin staining. An equal degree of staining was found in many fibers with strong ATPase and intermediate to strong SDH activity. Fibers with strong ATPase and weak SDH activity were less strongly stained than the preceding types.

A method for obtaining longitudinal cryostat sections of living muscle without contraction artifacts.

Longitudinal cryostat sections of skeletal muscle picked up with glass slides onto which a film of ethylenediaminetetracetic acid (EDTA) has been dried after dipping in a 3% EDTA solution do not show the severe retraction artifacts obtained when plain glass slides are used. Histochemical techniques work well on the longitudinal sections after the EDTA has been rinsed away and remaining traces saturated with calcium, Morphologic preservation is good. Regularly distributed histochemical reaction products are found within any one fiber.