Automated Methods for the Analysis of Skeletal Muscle Fiber Size and Metabolic Type - International Review of Cell and Molecular Biology - page 283

Of the metabolic enzymes present within skeletal muscle, COX is likely
the most widely studied. Interestingly, the combination of muscle staining
for COX activity and visualization of capillaries with alkaline phosphatase
was used by Romanul (1964) to show that number of capillaries surrounding
each fiber is proportional to the oxidative capacity of the fiber. Millis et al.
(1985) used staining for COX activity to monitor the effect of decreased
oxygen on muscle fibers during ischemic muscle injury. Muller-Hocker
(1990) evaluated age-related changes in the accumulation of COX-deficient
muscle fibers in human limb and diaphragm muscles. Age-related dimin-
ished COX activity, which manifests as an increase in the percentage of
fibers that are COX deficient, was also reported for human extraocular mus-
cles ( Muller-Hocker et al., 1992 ). COX activity was used by Schubert et al.
(2007) to evaluate skeletal muscles of caveolin null mice. COX activity in
muscle biopsies of patients with myasthenia gravis were evaluated by
Martignago et al. (2009) .
Many studies have utilized staining of serial sections from skeletal muscle
with different histochemical enzymes; this is a form of multiplexing as the
same fibers can often be identified in the neighboring serial sections. Most
commonly, serial sections from the same tissue have been assayed for mAT-
Pase and SDH staining ( Blanco et al., 1995; Bredman et al., 1990; Lind and
Kernell, 1991; Nicol and Bruce, 1981; Sieck et al., 1995; Soukup, 1976;
Ustunel and Demir, 1997 ), or for SDH and COX ( Aspnes et al., 1997;
Ferraris et al., 2008; Guida and Zorzetto, 2000; Miro et al., 1998; Rifai
et al., 1995 ). Indeed, some groups have also assayed (in addition to SDH
and COX) neighboring sections by such methods as amylase-PAS method
to quantify capillaries ( Andersen and Henriksson, 1977 ), Gomori trichrome
staining to visualize ragged red fibers ( Reichmann et al., 1996 ), and Sudan
Black to evaluate lipids ( Pachter and Colbjornsen, 1983 ).
4.2. Skeletal muscle lipid content
Triglyceride-containing lipid droplets are a common feature of skeletal mus-
cle ( Schrauwen-Hinderling et al., 2006 ), as fatty acids, derived by lipolysis
from the lipid droplets, are an important fuel source for ATP production via
oxidative phosphorylation. Consistent with their metabolic phenotype,
Type I and IIA fibers express more lipid droplets (termed “intramyocellular
lipids”) than Type IIB or Type IIX fibers. Intramyocellular lipid droplets are
often very closely juxtaposed to mitochondria ( Tarnopolsky et al., 2007 ),
suggesting an intimate interaction between the organelles for metabolic