Automated Methods for the Analysis of Skeletal Muscle Fiber Size and Metabolic Type - International Review of Cell and Molecular Biology - page 302

7.1. Mitochondrial disease
Mitochondrial disease is the most common neurometabolic disease of chil-
dren and is characterized by impaired energy production resulting from
genetically based oxidative phosphorylation dysfunction. It has a notoriously
variable clinical presentation resulting from over 100 pathogenic point
mutations and 200 deletions, insertions, and rearrangements of the mito-
chondrial genome ( Dimauro and Davidzon, 2005; Greaves et al., 2012;
Haas et al., 2007; Naviaux, 2000 ). Altered assembly of components of the
electron transport chain (ETC), as opposed to mutations to the catalytic sites,
may be responsible for the majority of mitochondrial disorders ( Capaldi
et al., 2004 ). Because of mutations in the mitochondrial-encoded DNA
respiratory chain complexes, there is disrupted electron flow, resulting in
increased generation of reactive oxygen species yielding oxidative damage
( Ishii, 2007; Richter, 1992; Wanagat et al., 2001 ) and apoptosis
( Adhihetty et al., 2005; Aure et al., 2006; Chabi et al., 2008 ). Many of
the clinical symptoms observed in children resemble abnormalities seen dur-
ing aging ( Mita et al., 1989; Newsholme et al., 2012; Payne et al., 2011;
Trifunovic, 2006; Wallace, 2010 ). Another area of concern relevant to
mitochondrial dysfunction in modern society is HIV; anti-HIV treatments
typically inhibit mitochondrial replication ( Koczor and Lewis, 2010 ) and,
therefore, function and treatment schedules must be adjusted, accordingly
( Apostolova et al., 2011 ).
Since defects in ETC enzyme complexes account for the majority of
mitochondrial disorders, most clinical diagnostics for mitochondrial disease
are based on assay of catalytic activity of metabolically relevant enzymes pre-
sent in the mitochondria ( Chen et al., 2011 ). Skeletal muscle biopsies are
routinely collected, as skeletal muscle contains a high content of mitochon-
dria. Electron transport assays (most commonly for COX, SDH, and
NADH dehydrogenase) are performed on frozen tissue sections mounted
on histology slides, using the assays discussed above with regard to metab-
olism. In most cases, researchers, physicians, and pathologists working with
such specimens visually inspect the biopsies and record a qualitative assess-
ment of the enzymatic activity, but there is little standardization among lab-
oratories in sample processing and assay methods ( Chen et al., 2011 ) and
little attempt to derive fiber-by-fiber quantification of the results.
To illustrate how automated analysis of skeletal muscle fibers can be
applied to colorimetric assays of mitochondrial function, an image rep-
resenting the bright-field COX assay, in which increased DAB staining