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Correspondence: School of Anatomy and Human Biology, The University of Western Australia, 35 Stirling Hwy.,
Crawley, Western Australia, Australia 6009. E-mail:
mgrounds@anhb.uwa.edu
doi: 10.1096/fj.03-1024com
0892-6638/04/0018-0676 FASEB
Histological assay
Inflammatory cell infiltration, muscle necrosis, and myotube
formation were analyzed using H&E stained 5 m frozen
and/or paraffin sections using light microscopy. TA muscle
for each parameter was examined in transverse section using
the following scale: 1 indicating 15% of total tissue area
affected, 2 representing 6 15%, 3 for 16 30%, 4 for 31 60%,
and 5 for 61100%. Inflammatory cell infiltration was identified by cells with basophilic nuclear staining and little cytoplasm located within the endomysium and/or necrotic tissue.
Muscle necrosis was identified by breakdown of sarcolemma
and fragmented sarcoplasm, and myotubes (indicating regeneration) were identified as plump cells with central nuclei. In
older (12 wk) mice, myofibers with persisting central nuclei
are a cumulative index of previous cycles of necrosis/regeneration (39).
RESULTS
Overall, there was no significant difference in weight
gain between Remicade injected and control MSA
injected mdx mice during the study period. The mean
gram body weight (and standard error) for the Remicade and control mdx mice, respectively, was 7.25
(/0.07) and 8.04 (/1.83) on day 7, 8.25
(/0.22) and 9.26 (/2.27) on day 14, 10.27
(/0.30) and 6.62 (/1.79) on day 21, 14.44
(/0.83) and 10.9 (/2.05) on day 28, and 32.03
(/0.41) and 30.83 (/0.6) at 12 wk. Only day 21
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TNF(/) mice, where the overall extent and pattern of damage resembled the control mdx mice (although the peak of inflammation and necrosis was
delayed by several days) (Fig. 2, compare panels b, c,
and a). The striking delay in onset of muscle breakdown in the Remicade-treated mdx mice was further
supported by analysis of EBD staining in TA muscles
from 22-day-old mice, where many EBD-positive myofibers (indicating membrane damage) were conspicuous
in control mdx mice; such myofibers but were rare in
the Remicade-treated mice (Fig. 4). Statistical analysis
of the quantitative data related to the area occupied by
necrotic tissue (Fig. 3) shows highly significant
(P0.05) reduced necrosis in Remicade-treated dystrophic muscles at 21 days compared with control
(MSA) mdx muscles.
The onset of necrosis was followed 3 days later by
the appearance of myotubes, as expected, since myotubes are not formed until 23 days after regeneration
starts (42). In control mdx mice (Fig. 2a), myotubes
were dramatically increased on day 24, 3 days after the
peak of necrosis (at 21 days). In Remicade-treated
mdx mice, myotube numbers increased gradually after
day 24 and the rise in numbers seen at 27 days with a
further increase at 28 days is 3 4 days after (the much
smaller) peaks of necrosis evident at 23 and 25 days.
The timing of myotube formation (with respect to
myofiber necrosis) appears normal and there is no hint
of impaired myogenesis in the Remicade-treated mdx
mice. This is further endorsed by the many regenerated
dystrophic myofibers (identified by central myonuclei)
in 12-wk-old Remicade-treated mdx TA muscle
In 12-wk-old mice, the Remicade-treated muscles
generally had features similar to control mdx muscles
with many central nuclei and small areas of necrosis
sometimes associated with small myotubes. The extent
of necrosis was low and similar in muscles of both
groups, being only 0.185% (/0.19) and 0.245%
(/0.294), respectively, for control mdx and Remicade-treated mice. There was no difference in EBD
staining between control and Remicade-treated mdx
mice with only a few individual or small groups of
EBD-positive myofibers in TA muscle sections; these
Figure 3. Quantitative analysis of the extent of necrosis and of myotube formation in TA muscles (n4 mice) from
control and Remicade-treated mice are
shown for samples at 21 and 28 days.
These data strongly endorse the semiquantitative data in Fig. 2. *The lack of
necrosis at 21 days in Remicade-treated
compared with control mdx mice is
highly significant (P0.05).
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