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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online August 7, 2002 as doi:10.1096/fj.02-0187fje. |
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in traumatic muscle injury 1
Department of Physical Therapy, Georgia State University, Atlanta, Georgia, USA; and
* Toxicology and Molecular Biology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, West Virginia, USA
2Correspondence: NIOSH 1095 Willowdale Road, Morgantown WV 26505, USA. E-mail: phs9{at}cdc.gov
SPECIFIC AIMS
We hypothesized that tumor necrosis factor 
(TNF-) contributes to the degenerative and regenerative processes after traumatic muscle injury. Transgenic mice deficient in TNF receptors I and II and wild-type mice administered neutralizing antibodies to TNF- were used to modulate TNF- response. At specified times after freeze injury of the tibialis anterior (TA) muscle, muscle strength was measured and compared to local expression of TNF- and myogenic factors including MyoD, myogenin, and Myf6. IL-6 was evaluated as a mediator of TNF-associated responses in injured muscle.
PRINCIPAL FINDINGS
1. Expression of TNF- and interleukin 6 (IL-6) in injured skeletal muscle
RNA isolated from control or injured TA muscles was examined for TNF- and IL-6 mRNA transcripts by TaqMan real-time PCR after muscle freeze injury. Uninjured muscle expressed low constitutive levels of TNF- and IL-6 mRNA. An increase in TNF- expression occurred within 5 h after injury and was observed to be maximally increased at 24 h after injury. The rapid increase of mRNA TNF- transcript levels was followed by a gradual reduction and a return to control levels by day 13. The time course of IL-6 expression in TA postinjury was similar to that for TNF- expression.
Immunohistochemical analysis detected TNF- in the damaged muscle region adjacent to the deep uninjured region. TNF- was localized primarily within infiltrating inflammatory cells as granular cytoplasmic staining on days 2 and 3. On days 5 and 7, inflammatory cell staining was decreased whereas slight cytoplasmic staining appeared in the regenerating myofibers. There was no detectable TNF- staining in the TA muscles on day 13 postinjury.
2. Histopathology and immunohistochemistry in wild-type and TNF-deficient mice are similar
H&E staining indicated that by day 2 postinjury, considerable infiltration of inflammatory cells was apparent in the injured muscles. The inflammatory cells demonstrated immunostaining for Mac-1 (or Cd11b/CD18), a member of the ß2-integrin family of receptors involved in leukocyte recruitment and Mac-3, a marker of activated monocytes/macrophages. By day 3 postinjury, the number of infiltrating inflammatory cells in the damaged muscle reached near-peak levels as did the number of myoblast nuclei in the infiltrated region that stained for MyoD and myogenin. By days 5–7 postinjury, the inflammatory cells had progressed to more peripheral portions of the damaged muscle whereas the presumptive myoblast activation paralleled the peripheral migration. By day 13 after injury, the injured muscle showed minimal signs of inflammation or degenerating fibers. There were no remarkable differences between wild-type and TNFR-DKO mice in routine histopathology at any time examined after muscle injury. Similarly, there were no apparent differences between the two groups in the expression of the myogenic regulatory factors, MyoD, or myogenin based on immunohistology.
3. MyoD expression is reduced in TNF--deficient but not IL-6-deficient mice
Although not detectable immunohistologically when examined by conventional RT-PCR and semiquantitative real-time PCR SYBR green, expression of MyoD was moderately but consistently reduced in TNFR-DKO compared to wild-type mice. Figure 1
A, B shows MyoD expression at day 3 postinjury, the day of peak response, using PCR and SYBR green methods, respectively. To confirm that the effect is associated with TNF- and not with differences in the genetic background of the knockout mice, neutralizing antibodies to TNF- were administered to wild-type mice before freeze injury. Similar to TNFR-DKO, MyoD expression was reduced by neutralizing antibodies to TNF- (Fig. 1B
). In contrast to the TNF response-deficient mice, MyoD expression in injured TA muscles of IL-6 KO mice was similar to that of the wild-type mice (Fig. 1A
). These results demonstrate that the TNF- deficiency leads to moderate but consistent depression of MyoD expression. Myogenin and MRF-4 expression measured by SYBR green was increased in TA muscles at 3 and 5 days postinjury, but to a similar degree in the wild-type and TNF-deficient mice.
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4. In contrast to IL-6, TNF deficiency is associated with reduced muscle strength recovery
Before injury induction, anterior crural muscle strength as assessed by the maximal isometric tetanic torque measurements was 16–19% lower in TNFR-DKO mice compared to C57BL/6 wild-type mice and independent of administration of TNF- neutralizing antibodies (i.e., 2.12±0.07 N mm for the TNFR-DKO mice vs. 2.53±0.08 and 2.63±0.11 N. mm for the C57/BL and C57/BL+TNF antibody-treated mice, respectively). These differences occurred even though body weights were similar in all three groups (P=0.67). Two minutes after injury induction, muscle strength was reduced by 68% (Fig. 2
). There were no significant differences between the three groups in this early strength loss (P=0.83). By day 5 postinjury, strength had begun to recover to a similar degree among the three groups (P=0.38). By 13 days postinjury muscle, strength in the C57BL/6 mice had recovered substantially and was only 13% lower than before injury. On the other hand, strength in the TNFR-DKO and TNF- antibody-treated mice had not recovered to the same degree (P=0.006) and was still 27–31% below pre-injury levels (Fig. 2A
). In experiments conducted on IL-6 KO mice, muscle strength recovered from injury at a rate identical to that of the wild-type mice (P=0.88) (Fig. 2B
).
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CONCLUSIONS
In the current studies we demonstrate, using real-time PCR, that traumatic skeletal muscle injury induced by freezing is accompanied by an early increase in TNF- and IL-6 expression. TNF- is a major regulator of inflammatory responses and influences the inflammatory cell influx in part by activating chemokines and adhesion molecules. In the present study, the influx of inflammatory cells in TA freeze injury model was not significantly affected by inhibiting TNF-’s action, suggesting a minor role at best in regulating monocyte/macrophage accumulation in muscle damage or that compensatory mediators are present. We originally hypothesized that, similar to liver injury, TNF- would exacerbate the early degenerative process and enhance the subsequent regenerative process. For example, the hepatotoxins amantadine and actinomycin D, which function as transcriptional inhibitors, mediate liver toxicity directly through excess TNF- activity whereas TNF- stimulates hepatocyte proliferation and liver repair after partial hepatectomy or CCl4-induced hepatotoxicity. However, we observed that only the repair process was affected by TNF- after muscle injury. TNF- provides growth modulatory and differentiation activities for many cell types. Repair processes associated with TNF- normally occur indirectly through the ability of TNF- to stimulate growth promoting factors. In this respect, IL-6, which is induced by TNF-, is mitogenic for both hepatocytes and keratinocytes, being required for normal liver regeneration and wound healing, respectively. Based on the observation of a normal strength recovery in the IL-6 KO mice, IL-6 does not appear to play a significant role in the repair process, although the cytokine is expressed at high levels after injury in wild-type mice; its role in muscle injury/repair remains unknown.
Increasing evidence from in vitro studies suggests a role for TNF- in modulating myogenesis. TNF- has been shown to enhance proliferation of muscle precursor cells and is required during the early period of myoblast differentiation. Consistent with these in vitro data, the present studies demonstrate that TNF- plays a role in the recovery of muscle function although it is not evident by histological evaluation. Previous studies using TNF-/- transgenic mice also failed to identify histological differences in muscle regeneration when compared to wild-type mice, and it is possible that moderate dysregulation in myogenic transcription factor expression or muscle metabolism might account for contractile dysfunction without significant differences in muscle histopathology. Nonetheless, the current studies indicate that TNF- is involved in the recovery of muscle function after traumatic muscle injury and this effect might be associated with modulation of muscle regulatory genes, including MyoD (Fig. 3
). However, the absence of TNF- only moderately affects the muscle injury processes, which would suggest that redundant mechanisms and/or factors may exist. An important question that remains to be answered is the role that inflammatory processes and mediators play in the muscle regeneration and functional recovery.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0187fje; to cite this article, use FASEB J. (August 7, 2002) 10.1096/fj.02-0187fje 
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), and anti-TNF-
) mice after injury. Each value represents mean ± SE of 6–8 mice per group. *Significantly different from injured/wild-type mice at P < 0.05. B) Strength of the left anterior crural muscles in C57BL/6 wild-type (•) (n=12) and IL-6 KO (
) (n=10) mice after injury.