High-intensity eccentric muscle contractions constitute a large component of dynamic sports activities (14). The risk of eccentric contraction-induced injury is increased when dynamic sports activities are introduced for the first time or reintroduced after an off-season or a prolonged injury-related break (14). In many cases, eccentric contraction-induced injury has pathological characteristics similar to a myopathic condition, including fiber degeneration and inflammation.
Intramuscular leukocyte infiltration is a common inflammatory process that occurs in response to various eccentric contraction protocols involving humans (6,12,15) or rodents (18). Significant leukocyte infiltration was evident as early as 45–120 min after eccentric contraction-induced injury in human quadriceps muscles (3,12) or as late as 9–14 d in human biceps brachii and gastrocnemius muscles (6,15). In contrast, leukocyte infiltration in murine extensor digitorum longus (EDL) and soleus muscles was evident at 24–48 h after eccentric contractions (11,16). At these time points, peak fiber infiltration by F4/80-, CD11b-, and/or acid phosphatase-leukocytes occurred in the EDL or soleus muscles (11,16). These temporal differences between humans and rodents may be related to the nature of the eccentric contraction protocols, and are not necessarily related to species variability.
Injuring eccentric contractions also induce alterations in fiber cytoskeletal proteins, such as desmin. Desmin is the predominant intermediate filament of mature skeletal muscle and is important for muscle structure and function (reviewed in Capetanaki and Milner (2)). Several studies have shown that desmin loss, as evidenced by decreased or lack of antidesmin staining, occurs in fibers of eccentric contraction-injured rabbit EDL and TA muscles (5,9,10). Desmin loss in rabbit EDL muscle was evident as early as 5 min (10), peaked at 3 d (9), and declined between 7 and 28 d after eccentric contractions (9). At 3 d, more than 30% of the fibers in rabbit EDL muscle exhibited desmin loss, whereas less than 1% of fibers exhibited desmin loss at 28 d (9). These findings suggest that desmin loss may be a significant cytoskeletal alteration associated with eccentric contraction-induced injury (8).
As part of a larger study, Fridén and Lieber (5) examined the timing of both desmin loss and inflammatory cell infiltration in eccentric contraction-injured rabbit EDL. Desmin loss occurred in large fibers at 1, 3, 7, and 28 d. Inflammatory or mononuclear cellular infiltration was observed in large fibers at 1, 3, and 7 d but not at 28 d. Peak intramyofiber mononuclear cellular infiltration occurred at 3 d. Although the mononuclear cells were not specifically identified in this study, the morphology and timing of infiltration suggests that these cells were leukocytes.
Taken together, the rabbit EDL desmin loss and leukocyte infiltration data (5,9,10) indicate a sequence of events regarding the timing of desmin loss and leukocyte infiltration: 1) eccentric contractions result in leukocyte infiltration and intramyofiber leukocyte infiltration occurs within 24 h after the start of desmin loss; 2) desmin loss and intramyofiber leukocyte infiltration peak at similar time points (3 d); and 3) intramyofiber leukocyte infiltration decreases as desmin loss declines. This sequence of events suggests that the time courses of intramyofiber leukocyte infiltration and desmin loss are closely linked. Such coordination between leukocytes and desmin changes might be beneficial for promoting recovery after eccentric contraction-induced injury by ensuring that leukocytes promptly attend to and remove damaged cellular material. The purpose of this study was to determine the desmin characteristics of fibers infiltrated by CD11b-leukocytes in fast- and slow-twitch murine muscles after eccentric contractions.
Animals and eccentric contraction-induced injury protocol.
Ten male and 11 female C57BL/6 mice (4–6 wk old) were purchased from Harlan Laboratories (Indianapolis, IN) and allowed to acclimate in the animal housing area for 1–3 wk in a 12-h light/12-h dark cycle. Animals received water and food ad libitum.
Eccentric contractions were induced in the mice using a procedure similar to one previously developed (1) and described (16). The mice were anesthetized with inhalation methoxyflurane throughout the procedure. The left hindlimb was shaved and cleaned with Betadine scrub (Purdue Frederick, Norwalk, CT), sterile water, Betadine solution (Purdue Frederick), and 70% isopropyl alcohol (Purdue Frederick). Then, the foot was strapped into a shoe attached to a servomotor (Model 305B, Aurora Scientific, Aurora, Ontario, Canada). The knee was immobilized with a C-shaped bar attached to a platform without compressing the joint. Two needle electrodes were inserted percutaneously near the tibial nerve. The tibial nerve was activated for 400 ms by a 150-Hz, 4-V electrical stimulus (Harvard Dual Impedance Electrical Stimulator, Harvard Apparatus, Holliston, MA). During the final 200 ms of tibial nerve activation, the servomotor rotated the ankle 17° in opposition to the plantarflexor muscles. This ankle rotation induced eccentric contractions in the plantarflexor muscles. Then, the ankle was passively returned to its original position. This protocol was repeated 450 times, with 5-min rest periods after the 150th and 300th eccentric contractions. Servomotor control and data acquisition were accomplished with a Macintosh G3 computer (Apple Computer, Cupertino, CA), GW Instruments PCI card (Somerville, MA), Superscope II software (GW Instruments), and instruNet network device (GW Instruments).
After completion of eccentric contractions, mice were returned to their cages. No postprocedure analgesia was necessary. Twenty-four hours later, the mice were anesthetized with inhalation methoxyflurane, and the eccentric contraction-injured soleus and medial gastrocnemius muscles were surgically isolated. This sampling time point was selected because our previous study showed that the number of CD11b-positive fibers peaked in soleus muscle at 24 h after eccentric contractions (16). Both the soleus and medial gastrocnemius muscles were selected for study for two reasons: 1) we have observed CD11b-positive fibers in both muscles (16; unpublished observations); and 2) these muscles are examples of slow- and fast-twitch muscles, respectively. After muscle dissection, the anesthetized mice were euthanized by cervical dislocation. This study protocol was approved by the University of Wisconsin–Madison Institutional Animal Care and Use Committee and follows animal care standards of the American College of Sports Medicine.
Immediately after dissection, the eccentrically contracted muscles were frozen in melting isopentane cooled by liquid nitrogen and then were stored at −70°C. Ten-micron-thick muscle cross-sections were cut on a Leica (Deerfield, IL) cryostat at −20°C and applied to poly-l-lysine–coated glass slides (Sigma Chemical Co., St. Louis, MO). Slides were stored at −70°C.
Frozen medial gastrocnemius and soleus cross-sections were air dried for 10–15 min and then fixed in acetone at room temperature for 10 min. Sections were dried again at room temperature for 15 min, and then washed in phosphate-buffered saline (PBS), pH 7.6 for 8 min before 2% bovine serum albumin (Vector Laboratories, Burlingame, CA) in PBS was applied for 20 min. After an 8-min PBS rinse, one section was stained with the mouse anti-human desmin primary antibody (Clone DE-R-11; 1:10 and 1:20 for medial gastrocnemius and soleus muscles, respectively; Vector Laboratories) for 2 h and a serial section was stained with the primary antibody, biotinylated rat anti-mouse CD11b (Clone M1/70; 1:5; Caltag Laboratories, South San Francisco, CA) for 2.5 h. (For one soleus muscle, the CD11b-stained section was two sections away from the desmin-stained section.) The desmin-stained slides were then washed in PBS and incubated with biotinylated anti-mouse IgG Fab1 2 (1:200; Caltag Laboratories) for 30 min. Then, all slides were washed in PBS. Sections were quenched with 0.3% hydrogen peroxide in methanol for 20 min followed by a PBS rinse. The Vectastain ABC peroxidase reagent (Vector Laboratories) was applied to the sections for 30 min. After rinsing the slides in PBS, the sections were incubated with a diaminobenzidine substrate/nickel chloride solution (Vectastain DAB substrate kit, Vector Laboratories) for 3 min for the desmin-stained slides and 7 min for the CD11b-stained slides. The reaction was stopped by rinsing the slides in distilled water. Sections were dehydrated in 95% and 100% ethanol and cleared with Hemo De (Fisher Scientific, Chicago, IL). Glass coverslips were applied using the nonaqueous medium Cytoseal (VWR Scientific, Bridgeport, NJ). Nonspecific binding of the antidesmin and anti-CD11b antibodies was evaluated by omitting these antibodies on some sections during the immunohistochemistry procedure. After staining was completed, each slide was numerically coded so that the mouse’s identity of each section would not be known during analysis.
Quantification of the intramyofiber CD11b-leukocyte infiltration.
Because the amount of infiltration by CD11b-leukocytes varies among fibers at a single time point, the percentage of intramyofiber CD11b-leukocyte infiltration was quantified. The CD11b-stained cross-sections initially were viewed using a light microscope. CD11b-positive fibers were easily visualized because CD11b-negative fibers are absent of the purplish substrate reaction product. Computer-generated images were made of these CD11b-positive fibers. The images were generated using a 20× objective, a Photometrics monochrome CCD videocamera (Tucson, AZ), a Scion capture board (Frederick, MD), Macintosh G3 computer (Apple Computer), and the National Institutes of Health Image program (Bethesda, MD). All images were of a similar light intensity. Most images were made so that only one to three CD11b-positive fibers were present in a single image. The CD11b-positive fibers that were analyzed met the following criteria: complete identifiable borders, nonoverlapping with surrounding fibers, and present in a desmin-stained serial section. CD11b-positive fibers were excluded if they were located on the section periphery.
With each CD11b-positive fiber, the entire cross- sectional area was selected and saved as a separate image file from the original image. Separate images were created so that each fiber could be quantified individually. The percentage of intramyofiber CD11b-leukocyte infiltration was calculated using the following formula: (the number of pixels added at a threshold of 95 ÷ the number of pixels added at a threshold of 1) × 100. The threshold value of 95 was chosen because only the most intense anti-CD11b staining was detected at this value.
Identification of the desmin characteristics of CD11b-positive fibers.
After identifying the CD11b-positive fiber in the serial desmin-stained section, an image of the desmin-stained fiber was made. The image was generated using the same method for producing the CD11b-stained images. However, because desmin is found in uninjured fibers and the intensity of desmin staining can vary across muscles, the light intensity setting could not be standardized across all images. Instead, the light intensity was set for each image so that all images contained high contrast. By setting the light intensity with high contrast, areas devoid of staining or interstitial spaces appeared white-like. Whenever possible, images were made so that they corresponded to the CD11b-stained images. In addition, each desmin-stained image was made so that a nearby CD11b-negative fiber that did not show any granularity or other abnormal morphological features was included. Only those desmin-stained fibers that had complete identifiable borders, were nonoverlapping with surrounding fibers, and were not located on the section periphery were analyzed.
Before identifying the desmin characteristics of each CD11b-positive fiber, the normal desmin characteristic of a nearby CD11b-negative fiber was determined. The sarcolemma was excluded from the analysis because of intense desmin staining. The sarcoplasmic area that was selected was referred to as a fiber. Next, the selection was saved into a separate image file from the original image so that each fiber could be analyzed individually. The threshold was set at 1. By setting the threshold at 1, the minimum and maximum threshold values for the CD11b-negative fiber image were given. The minimum threshold represents the value at which the greatest number of black pixels would be added to the entire selected CD11b-negative fiber image, whereas the maximum threshold represents the value at which the least number of pixels would be added to the image. The normal desmin characteristic was represented by these values and the values between them. The possible range of threshold values was 1 to 255.
To identify and quantify the desmin characteristics of a CD11b-positive fiber, the sarcoplasm of the CD11b-positive fiber in the desmin-stained section was selected. The sarcolemma was excluded in the selection. Next, the selection was saved into a separate image file from the original image so that each CD11b-positive fiber could be analyzed individually. The percentage of increased desmin was calculated using the following formula: (the total number of pixels added to the CD11b-positive fiber at the maximum threshold + 1 value of the CD11b-negative fiber image ÷ the total number of pixels added to the CD11b-positive fiber at a threshold value of 1) × 100. The percentage represents the portion of the CD11b-positive fiber that is determined to have increased desmin staining only.
The percentage of decreased desmin was calculated using the following formula: [(the total number of pixels added to the CD11b-positive fiber image at a threshold value of 1 − the total number of pixels added to the CD11b-positive fiber image at a threshold value equal to the minimum threshold value of the CD11b-negative fiber) ÷ the number of pixels added to the CD11b-positive fiber at a threshold value of 1] × 100. The percentage represents the portion of the fiber that is determined to have decreased desmin staining only. The decreased desmin formula was set up this way because the Image program only counts pixels at and above a certain threshold value. In other words, the program does not count simultaneously the number of pixels at a predetermined low- and high-threshold value range.
Mean and standard error were determined. The difference in the percentage of intramyofiber CD11b-leukocyte infiltration among infiltration patterns was tested using the ANOVA procedure. Pearson’s correlation and Fisher’s exact test were also used to determine the association between 1) intramyofiber CD11b-leukocyte infiltration and the increased desmin characteristic and 2) intramyofiber CD11b-leukocyte infiltration and decreased desmin. The ANOVA procedure also was used to compare the percentages of increased desmin and decreased desmin among intramyofiber CD11b-leukocyte infiltration patterns. The influence of sex and muscle type on the percentages of intramyofiber CD11b-leukocyte infiltration, increased desmin, and decreased desmin was tested using the two-sample t-test. For all statistical procedures, α was set at 0.05.
Mice and eccentric contraction-injured muscles.
Adequate cross-sections suitable for analysis were obtained from 20 injured medial gastrocnemius muscles (10 males and 10 females) and 17 injured soleus muscles (eight males and nine females). All cross-sections stained with the anti-CD11b antibody were examined for CD11b-positive fibers with complete identifiable borders. When such a CD11b-positive fiber was identified, the presence of the same fiber was assessed in a serial cross-section stained with the antidesmin antibody. Corresponding fibers in the desmin-stained cross-sections had to meet the same criteria as those in the CD11b-stained cross-sections to be included in the analysis. As a result, we identified 40 and 26 CD11b-positive and desmin-stained fibers in medial gastrocnemius and soleus muscles, respectively, of males and females combined.
Intramyofiber CD11b-leukocyte infiltration.
After identifying 66 CD11b-positive fibers, the percentage of CD11b-leukocyte infiltration was determined for each fiber. There was only one fiber that was identified as positive via microscopic inspection that was determined as having no CD11b-leukocyte infiltration by the computer image analysis procedure. The overall mean percentage of intramyofiber CD11b-leukocyte infiltration was 34 ± 3%. The percentage of intramyofiber CD11b-leukocyte infiltration was 50% or greater for almost one third of the CD11b-positive fibers.
Three distinct infiltration patterns were observed (Fig. 1). There was an infiltration pattern in which distinct CD11b-leukocytes localized at or near the sarcolemma only (Fig. 1A). Fibers with this infiltration pattern were designated as EDGE fibers (N = 10). A second infiltration pattern was the localization of distinct CD11b-leukocytes at or near the sarcolemma and deep inside the sarcoplasm (Fig. 1B). Fibers with this second infiltration pattern were designated as EDGE/INSIDE fibers (N = 27). The third infiltration pattern consisted of distinct CD11b-leukocytes localized inside the sarcoplasm, or indistinct CD11b-leukocytes localized near the sarcolemma and deep inside the sarcoplasm (Fig. 1C). Fibers with the third infiltration pattern were designated as INSIDE fibers (N = 29).
There was a statistically significant difference in the percentage of intramyofiber CD11b-leukocyte infiltration among these three infiltration patterns: EDGE fiber group mean was 14 ± 5%; EDGE/INSIDE fiber group mean was 27 ± 4%; and INSIDE fiber group mean was 47 ± 5% (P < 0.001). This difference in percentage confirmed that the three infiltration patterns are distinct from one another and that the percentage is proportional to the infiltration patterns.
Desmin characteristics of CD11b-positive fibers.
Three desmin characteristics of CD11b-positive fibers were identified using a computer image analysis procedure: normal, increased, and decreased (Fig. 2). Table 1 shows the number of CD11b-positive fibers foreach desmin characteristic. Less than one tenth of the fibers were determined to have one desmin characteristic. This desmin characteristic was normal. Almost three fourths of the fibers were determined to have the increased desmin characteristic in combination with the normal and/or decreased desmin characteristic.
The mean percentage of increased desmin was 34 ± 4%. However, one third of all the CD11b-positive fibers had a percentage of increased desmin less than 1%. About another one third of CD11b-positive fibers had a percentage of increased desmin 50% or greater.
The mean percentage of decreased desmin was 5 ± 2%. Eighty-six percent of the CD11b-positive fibers had a percentage of decreased desmin less than 10%. Only 1 of 66 fibers was determined to have a percentage of decreased desmin greater than 50%.
Intramyofiber CD11b-leukocyte infiltration and increased desmin.
The association between the percentage of intramyofiber CD11b-leukocyte infiltration and increased desmin was determined using Pearson’s correlation. There was no significant correlation between CD11b-leukocyte infiltration and increased desmin (R2 = 0.03, P = 0.18).
However, the percentage of increased desmin was skewed to the left because one third of the fibers had a percentage of increased desmin less than 1%. To address this issue of a skewed distribution, the values of the percentage of intramyofiber CD11b-leukocyte infiltration and increased desmin were dichotomized into high and low groups using a 50% cutoff. Forty-six and 43 values were less than the cutoff for the percentage of intramyofiber CD11b-leukocyte infiltration and increased desmin, respectively. When the high and low groups were compared using Fisher’s exact test, a significant association between the percentage of intramyofiber CD11b-leukocyte infiltration and increased desmin was found (P = 0.005).
To explain further the association between the percentage of intramyofiber CD11b-leukocyte infiltration and increased desmin, the percentage of increased desmin was compared among the intramyofiber CD11b-leukocyte infiltration patterns (Table 2). A significant difference was found among the infiltration patterns. Therefore, the percentage of increased desmin declines as infiltration progresses. Figures 1 and 3 show a set of fibers with increasing leukocyte infiltration and declining amounts of desmin, respectively.
Intramyofiber CD11b-leukocyte infiltration and decreased desmin.
The association between the percentage of intramyofiber CD11b-leukocyte infiltration and decreased desmin was determined using Pearson’s correlation. There was no significant correlation between decreased desmin and CD11b-leukocyte infiltration (R2 = 0.03, P = 0.16).
However, the percentage of decreased desmin was skewed to the left because 65% (43 of 66) of the fibers had a percentage of decreased desmin equal to 0, and 86% (57 of 66) had a decreased desmin percentage less than 10%. To address this issue of a skewed distribution, the values of the percentage of intramyofiber CD11b-leukocyte infiltration and decreased desmin were dichotomized into high and low groups. For the CD11b percentages, the cutoff was 50%, and thus 46 values were less than the cutoff. For the decreased desmin percentages, the cutoff was 0, and thus only 23 values were greater than 0. When the high and low groups were compared using Fisher’s exact test, no association was found between the percentage of intramyofiber CD11b-leukocyte infiltration and decreased desmin (P = 0.399).
The percentage of decreased desmin also was compared among the intramyofiber CD11b-leukocyte infiltration patterns (Table 2). There was no significant difference among the infiltration patterns. The difficulty in relating these two variables is that the decreased desmin percentages are extremely small in the majority of CD11b-positive fibers.
Influence of sex and muscle type.
The influence of sex on the percentages of intramyofiber CD11b-leukocyte infiltration, increased desmin, and decreased desmin was analyzed. This analysis was performed by comparing the mean percentage of intramyofiber CD11b-leukocyte infiltration obtained from male mice with that of female mice and by comparing the mean percentages of increased desmin and decreased desmin from male mice with that of female mice. There were no significant differences in these mean values between male and female mice (Table 3).
The influence of muscle type on the percentages of intramyofiber CD11b-leukocyte infiltration, increased desmin, and decreased desmin was analyzed by comparing the mean percentage of intramyofiber CD11b-leukocyte infiltration obtained from soleus fibers with that obtained from the medial gastrocnemius fibers and by comparing the mean percentages of increased desmin and decreased desmin from the soleus fibers with that of the medial gastrocnemius fibers. There were no significant differences in these mean values between the soleus and medial gastrocnemius fibers (Table 4).
By developing a computer image analysis procedure to quantify desmin characteristics of CD11b-positive fibers, we found that the majority of CD11b-positive fibers in slow- and fast-twitch muscles 24 h after eccentric contractions exhibited increased desmin. Fibers that showed the greatest percentage of increased desmin had a CD11b-leukocyte infiltration pattern in which distinct CD11b-leukocytes were located only at or near the sarcolemma and not deep inside the sarcoplasm (designated as EDGE fibers). Additionally, by using our procedure, we noted that these EDGE fibers had the least amount of CD11b-leukocyte infiltration.
Previous investigations of leukocyte infiltration in human and rodent slow- and fast-twitch muscles after eccentric contractions have observed different leukocyte types (e.g., neutrophils, macrophages, lymphocytes), but have not quantified the percentage of intramyofiber leukocyte infiltration (3,11,13,15,16,18). Using our image analysis procedure, we were able to determine the intramyofiber percentage of CD11b-leukocytes in the medial gastrocnemius and soleus muscles. This procedure provided an objective and quantitative approach by which we might establish the stage of intramyofiber leukocyte infiltration in these muscles. With a mean intramyofiber CD11b-leukocyte infiltration percentage of about one third, it is likely that leukocyte infiltration was in an early stage at 24 h after eccentric contractions.
We observed three intramyofiber leukocyte infiltration patterns: EDGE, EDGE/INSIDE, and INSIDE. Although we used a subjective approach to distinguish infiltration patterns, this classification scheme was confirmed quantitatively. The mean percentage of CD11b-leukocyte infiltration was significantly different among these patterns. However, the development of more objective approaches for distinguishing infiltration patterns is a topic for future research.
This investigation is the first study to report the desmin characteristics of CD11b-positive fibers in slow- and fast-twitch muscles 24 h after eccentric contractions using a computer image analysis procedure. Fridén and Lieber (5) and Lieber et al. (9) have reported a significant presence of desmin-negative fibers in rabbit EDL and tibialis anterior muscles after eccentric contractions. In contrast, we found only approximately one tenth of the CD11b-positive fibers to have 10% or more of decreased desmin in both the soleus and medial gastrocnemius muscles. The discrepancy may be attributable to methodological differences. For instance, we might have excluded many fibers having decreased desmin because we included only fibers with completely identifiable borders in our analysis. This inclusion criterion may be a limitation of our study, but does provide a reproducible technique for the quantification of fiber morphology.
The majority of the CD11b-positive fibers at 24 h after eccentric contractions were determined to have increased rather than decreased desmin. We ruled out this finding as nonspecific staining of the anti-mouse secondary antibody through our antibody control experiments. In human vastus lateralis muscle, Fridén et al. (4) observed increased desmin staining between Z-lines 3 d after eccentric contractions. On the basis of the timing of their observation, these investigators hypothesized that the increased desmin staining might be important for myofibrillar reorganization associated with repair. However, an alternative explanation is warranted for our finding because we have detected increased desmin staining at a time point associated with fiber damage.
Additional studies need to be conducted to determine the mechanism by which desmin is increased at 24 h after eccentric contractions. Findings from a cardiac myocyte study may suggest one possible mechanism. Simpson et al. (17) reported the accumulation of desmin, myosin heavy chain, and actin proteins in response to 24 h of static stretch. This stretch-induced protein accumulation was associated with the suppression of both myosin heavy chain and actin degradation (17). No association was observed for desmin degradation (17). Nevertheless, a temporary stretch-induced suppression of normal desmin degradation might explain why increased desmin was observed at 24 h after eccentric contractions. If normal desmin degradation were suppressed by eccentric contractions, then this would most likely occur before the onset of desmin degradation by activated calcium-dependent proteases (8).
We found a significant difference in the percentage of increased desmin among the CD11b-leukocyte infiltration patterns of CD11b-positive fibers. A greater portion of a fiber with an EDGE infiltration pattern was determined to have increased desmin compared with fibers with an EDGE/INSIDE or INSIDE infiltration pattern. This finding suggests that desmin is increased before maximal intramyofiber CD11b-leukocyte infiltration. Further research is needed to determine whether the timing of intramyofiber CD11b-leukocyte infiltration is dependent in part on this desmin change.
We determined that sex and muscle type had no influence on the percentage of intramyofiber CD11b-leukocyte infiltration. In an earlier study, we observed no difference in the number of CD11b-positive fibers in eccentric contraction-injured soleus muscles of male and female mice (16). Therefore, not only are a similar number of fibers infiltrated by CD11b-leukocytes in muscles of male and female mice, but the process by which these leukocytes infiltrate fibers also appears to be independent of sex and muscle type.
We also found that sex had no influence on the desmin characteristics of CD11b-positive fibers. This finding contrasts with that of the study by Komulainen et al. (7) in which fewer desmin-negative fibers in the quadriceps femoris muscle of female rats were observed in comparison with that of male rats after downhill running. The discrepancy between the current and previous studies may be because of differences in the eccentric contraction protocols.
Although much is known about the consequences of eccentric contraction-induced injury, less is known about the specific inflammatory mechanisms, such as leukocyte infiltration, that are activated to promote recovery. The findings from the current study expand our understanding in this area by suggesting that intramyofiber leukocyte infiltration may 1) be an incremental process and not an all-or-none phenomenon and 2) occur in response to distinct changes in the cytoskeletal protein, desmin. Therefore, the activation of leukocyte infiltration and other inflammatory processes may be dependent on the timing of specific fiber changes, such as increased and/or decreased desmin, so that damaged cellular material can be removed and recovery proceeds in a timely manner.
This study was supported by funds from the University of Wisconsin–Madison School of Nursing and Graduate School.
Address for correspondence: Barbara St. Pierre Schneider, D.N.Sc., R.N., University of Wisconsin–Madison School of Nursing, 600 Highland Avenue, Room K6/364, Madison, WI; E-mail: firstname.lastname@example.org.
1. Ashton-Miller, J. A., Y. He, V. A. Kadhiresan, D. A. Mccubbrey, and J. A. Faulkner. An apparatus to measure in vivo biomechanical behavior of dorsi- and plantarflexors of mouse ankle. J. Appl. Physiol. 72: 1205–1211, 1992.
2. Capetanaki, Y., and D. J. Milner. Desmin cytoskeleton in muscle integrity and function. Subcell. Biochem. 31: 463–495, 1998.
3. Fielding, R. A., T. J. Manfredi, W. Ding, M. A. Fiatarone, W. J. Evans, and J. G. Cannon. Acute phase response in exercise: III. Neutrophil and IL-1β accumulation in skeletal muscle. Am. J. Physiol. 265: R166–R172, 1993.
4. Fridén, J., U. Kjörell, and L.-E. Thornell. Delayed muscle soreness and cytoskeletal alterations: an immunocytological study in man. Int. J. Sports Med. 5: 15–18, 1984.
5. Fridén, J., and R. L. Lieber. Segmental muscle fiber lesions after repetitive eccentric contractions. Cell Tissue Res. 293: 165–171, 1998.
6. Jones, D. A., D. J. Newham, J. M. Round, and S. E. Tolfree. Experimental human muscle damage: morphological changes in relation to other indices of damage. J. Physiol. 375: 435–448, 1986.
7. Komulainen, J., S. O. A. Koskinen, R. Kalliokoski, T. E. S. Takala, and V. Vihko. Gender differences in skeletal muscle fibre damage after eccentrically biased downhill running in rats. Acta Physiol. Scand. 165: 57–63, 1999.
8. Lieber, R. L., and J. Fridén. Mechanisms of muscle injury after eccentric contraction. J. Sci. Med. Sport 2: 253–265, 1999.
9. Lieber, R. L., M. C. Schmitz, D. K. Mishra, and J. Fridén. Contractile and cellular remodeling in rabbit skeletal muscle after cyclic eccentric contractions. J. Appl. Physiol. 77: 1926–1934, 1994.
10. Lieber, R. L., L. E. Thornell, and J. Fridén. Muscle cytoskeletal disruption occurs within the first 15 min of cyclic eccentric contraction. J. Appl. Physiol. 80: 278–284, 1996.
11. Lowe, D. A., G. L. Warren, C. P. Ingalls, D. B. Boorstein, and R. B. Armstrong. Muscle function and protein metabolism after initiation of eccentric contraction-induced injury. J. Appl. Physiol. 79: 1260–1270, 1995.
12. MacIntyre, D. L., W. D. Reid, D. M. Lyster, and D. C. McKenzie. Different effects of strenuous eccentric exercise on the accumulation of neutrophils in muscle in women and men. Eur. J. Appl. Physiol. 81: 47–53, 2000.
13. MacIntyre, D. L., W. D. Reid, D. M. Lyster, I. J. Szasz, and D. C. McKenzie. Presence of WBC, decreased strength, and delayed soreness in muscle after eccentric exercise. J. Appl. Physiol. 80: 1006–1013, 1996.
14. Mchugh, M. P. Can exercise-induced muscle damage be avoided? West. J. Med. 172: 265–266, 2000.
15. Round, J. M., D. A. Jones, and G. Cambridge. Cellular infiltrates in human skeletal muscle: exercise induced damage as a model for inflammatory muscle disease? J. Neurol. Sci. 82: 1–11, 1987.
16. Schneider, B. S., L. A. Correia, and J. G. Cannon. Sex differences in leukocyte invasion in injured murine skeletal muscle. Res. Nurs. Health 22: 243–250, 1999.
17. Simpson, D., W. Sharp, T. Borg, R. Price, L. Terracio, and A. Samarel. Mechanical regulation of cardiac myocyte protein turnover and myofibrillar structure. Am. J. Physiol. 270: C1075–C1087, 1996.
18. Stauber, W. T., V. K. Fritz, D. W. Vogelbach, and B. Dahlmann. Characterization of muscles injured by forced lengthening: I. Cellular infiltrates. Med. Sci. Sports Exerc. 20: 345–353, 1988.
Keywords:© 2002 Lippincott Williams & Wilkins, Inc.
SKELETAL MUSCLE INJURY; LEUKOCYTES; QUANTITATIVE IMAGE ANALYSIS