Abstract

After 3 wk of immobilization, the effects of free cage activity and low- and high-intensity treadmill running (8 wk) on the morphology and histochemistry of the soleus and gastrocnemius muscles in male Sprague-Dawley rats were investigated. In both muscles, immobilization produced a significant (P < 0.001) increase in the mean percent area of intramuscular connective tissue (soleus: 18.9% in immobilized left hindlimb vs. 3.6% in nonimmobilized right hindlimb) and in the relative number of muscle fibers with pathological alterations (soleus: 66% in immobilized hindlimb vs. 6% in control), with a simultaneous significant (P < 0.001) decrease in the intramuscular capillary density (soleus: mean capillary density in the immobilized hindlimb only 63% of that in the nonimmobilized hindlimb) and muscle fiber size (soleus type I fibers: mean fiber size in the immobilized hindlimb only 69% of that in the nonimmobilized hindlimb). Many of these changes could not be corrected by free remobilization, whereas low- and high-intensity treadmill running clearly restored the changes toward control levels, the effect being most complete in the high-intensity running group. Collectively, these findings indicate that immobilization-induced pathological structural and histochemical alterations in rat calf muscles are, to a great extent, reversible phenomena if remobilization is intensified by physical training. In this respect, high-intensity exercise seems more beneficial than low-intensity exercise.

  • capillary number
  • fiber changes
  • intramuscular connective tissue
  • rats
  • remobilization
  • soleus and gastrocnemius muscles

immobilization is a frequently used treatment for musculoskeletal injuries despite well-documented resulting muscle cell atrophy, intramuscular fibrosis, and loss of muscle extensibility, strength, and endurance (2, 12, 14, 15, 19). The immobilization-induced increase in the intramuscular connective tissue occurs both endomysially and perimysially, separating the individual muscle fibers from each other (12). Simultaneously, the fiber cross-sectional area and the capillary density of the muscle decrease (1, 12, 17, 18, 21). The quality and quantity of the immobilization-induced pathological histological and histochemical alterations within the muscle fibers have not been properly described, to our knowledge.

Compared with the knowledge of immobilization, the effects of various forms of remobilization on the immobilized muscle are less well known, although the question is of utmost importance in exercise physiology and sports medicine (2, 15). The key issue is whether the immobilization-induced degenerative changes are temporary and reparable, or permanent. If the changes are not permanent, we should know whether complete muscle tissue recovery is possible and the best methods for optimal recovery.

The purpose of this investigation was therefore to examine with a randomized, controlled study design the effects of immobilization and three different types of remobilization (free cage activity and low- and high-intensity treadmill running) on intramuscular connective tissue in the soleus and gastrocnemius muscles in rats. Attention was also paid to the changes in the intramuscular capillary density, muscle fiber cross-sectional area, and number of abnormal fibers (fibers with one or more pathological alterations). We tested the hypothesis that daily running would increase the rate of recovery after hindlimb immobilization and that high-intensity treadmill running would be more beneficial than low-intensity running.

MATERIALS AND METHODS

Immobilization and remobilization.

Forty-two male rats of the Sprague-Dawley strain were used in the study. At the beginning, the rats were 9–11 wk old, and their body weight ranged from 320 to 360 g. Four to five animals were housed per cage (18 × 35 × 55 cm), and the animals received laboratory chow and water ad libitum. The experimental animals were kept in the facilities of the Central Animal Laboratory of the University of Tampere (Finland). At all times, the rats were treated in a manner consistent with the “Guiding Principles For Research Involving Laboratory Animals and Human Beings” as approved by the American Physiological Society. In addition, the research scheme with the description of the procedures for immobilization, remobilization, anesthesia, and euthanasia of the animals was accepted by the Ethical Committee for Animal Experiments of the University of Tampere.

With the use of carbon dioxide inhalation, 5 of the 10 control rats were euthanized after 3 wk and the other 5 rats at the end of the experiment (11 wk). Between these two time points, the evaluated histological parameters in the control rats did not show significant group differences, and therefore the data from the control rats were pooled and analyzed together.

The remaining 32 animals were anesthetized with intraperitoneal pentobarbital (30 mg/kg), and the left hindlimb was immobilized in a padded tape from toes to ∼1 cm above the knee. The knee was fixed in 100° flexion and the ankle in 60° plantar flexion so that the calf muscles were relaxed (12). The fixation was checked daily. The right hindlimb was kept free. The immobilization method has been described in detail elsewhere (12).

After 3 wk, eight rats were killed by using the above-mentioned technique (the 3-wk immobilization group; IM3). In the remaining 24 animals, the tape was removed and the animals were allowed to remobilize the left hindlimb for 8 wk by using three different exercise protocols. The first eight rats moved freely in the cage, and no additional physical training was used (the free remobilization group; FR11).

The remaining two groups of rats (with 8 animals in each group) were allowed to move freely in their cage for 1 wk, after which they ran on a treadmill twice a day, 5 days/wk, for 7 wk. In the group with a low-intensity running program (LIR11), the speed of the treadmill was 20 cm/s, with an inclination of 10°. During the first week of the program, there was only one 20-min session per day, after which there were two sessions per day (the morning and afternoon sessions at least 5 h apart) for 6 wk. The program was progressive so that the running time increased from 20 min/session in the first 2 wk to 45 min/session in week 7.

In the group with a high-intensity running program (HIR11), the speed of the treadmill was 30 cm/s, with an uphill inclination of 30°. This was achieved by a gradual increase in speed and inclination during the first week of running. As in the LIR11 group, there was only one daily session during the first week, and two thereafter. The running time was similarly progressive, from 20 min/session in the first 2 wk to 45 min/session in week 7.

After the remobilization period of 8 wk, the remobilized animals in the FR11, LIR11, and HIR11 groups were also euthanized with carbon dioxide.

Sample preparation, histochemistry, and histology.

In each animal, the hindlimbs were freed from the overlying skin and disarticulated at the hip. The soleus and the medial gastrocnemius muscles were dissected out, cleared of fat and connective tissue, and transversely divided into two equal-size halves. The proximal half of the muscle was frozen in liquid nitrogen and stored at −35°C until processing and analysis, whereas the distal half was fixed in neutral buffered 6% Formalin (pH 7.4) and embedded in paraffin.

The soleus and gastrocnemius muscles were used because they have different fiber type distribution (2, 4, 12), and both muscles are rather sensitive to immobilization atrophy and known to respond well to physical training (12, 14, 15).

The unfixed serial cryostat cross sections (6 μm in thickness) were obtained from the frozen muscles and stained for myofibrillar ATPase activity, after preincubation at pH 4.2, 4.6, and 10.2 (9). This staining procedure allowed the identification of muscle fibers as type I, type IIA (fast-twitch oxidative glycolytic), or type IIB (fast-twitch glycolytic), measurement of fiber cross-sectional area, and identification of the intramuscular capillaries (13), although it did not allow reliable identification of the type IIX fibers (10). The oxidative enzyme activity of the fibers was demonstrated by the NADH reductase reaction (22). The remaining cryostat sections were stained with periodic acid-Schiff (PAS), with and without diastase pretreatment, for demonstration of the glycogen content of the muscle fibers.

From the paraffin blocks, 5-μm-thick serial sections were cut and stained with hematoxylin-eosin, picrosirius, and phosphotungstic acid-hematoxylin for evaluation of the intramuscular connective tissue and pathological fiber alterations.

Visualization and histometric quantitation.

To minimize any bias on the part of the observer during the analyses described below, all data collection was performed in blind fashion with respect to treatment group assignment. Also, all examinations were performed on a blind basis so that, at the time of the examination, the examining pathologist (L. Jozsa) did not know which group of specimens he was studying.

Percent area of connective tissue.

Picrosirius stained the connective tissue (endo-, peri-, and epimysium) dark red, contrasting well with the pale yellow muscle fibers (23). From each muscle, two to three picrosirius-stained cross sections were examined under a Zeiss microscope and were analyzed with use of a system consisting of a video camera, automatic image analyzer, and image software (Muscle Image Analysis System, IBM-KFKI, Budapest, Hungary). The system was housed in an IBM 486-AT microcomputer. In each section, the connective tissue and muscle fiber areas were recorded by measuring the optical density of 442,400 points in a microscopic field, ∼0.86 mm2 in ×160 magnification. The percent area of connective tissue or the connective tissue-to-muscle fiber ratio was then expressed as the percent ratio of total connective tissue area to muscle fiber area. In calculation of the mean connective tissue area for each muscle, 10–30 images/muscle were analyzed (2–3 sections/muscle including 2–10 fields/section). Fields containing blood vessels other than capillaries were avoided.

Capillary density.

In each muscle, 300–500 consecutive neighboring capillaries and the number of simultaneously occurring muscle fibers were calculated from the above-described ATPase-stained sections, with the sections preincubated at pH 4.2 (1, 13). In the description of the capillary density of the muscle, the number of capillaries per 1,000 muscle fibers was reported.

Fiber cross-sectional area.

The mean cross-sectional area (μm2) was calculated for each muscle and each fiber type, using in the analysis the above-described automatic image-analysis system and 2,000–2,500 fibers of an entire soleus preparate and 29,000–30,000 similar fibers of an entire gastrocnemius preparate. The analysis was made from the ATPase-stained sections (pH 4.2 and 4.6), allowing the differentiation between type I and type II fibers. In the soleus muscle, the cross-sectional area was determined for type I fibers, and, in the gastrocnemius muscle, it was determined for type I and type II fibers.

Pathological fiber alterations.

The number (%) of fibers with a pathological morphological and histochemical alteration was determined by analyzing 500 consecutive neighboring fibers from each control and experimental muscle, type I fibers from the soleus muscle and type II fibers from the gastrocnemius muscle. The above-described NADH reductase, PAS, ATPase, and phosphotungstic acid-hematoxylin preparates were used for these analyses.

According to their characteristic histological and histochemical features, the alterations were classified as follows: a moth-eaten fiber (referring to spiral-type deformation and destruction of the myofibrillar network of the fiber, the term being derived from the microscopic moth-eaten appearance of the fiber); central core formation within the fiber (referring to abnormally increased oxidative enzyme activity and abnormal aggregation of the myofibrils in the central area of the fiber); loss of oxidative enzyme activity in the central part of the fiber (referring to reduced number of mitochondria and thus reduced aerobic energy production in that area of the fiber); increased oxidative enzyme activity in the peripheral areas of the fiber (referring to increased number of mitochondria and thus increased aerobic energy production in that area of the fiber); a shell-like fiber (referring to shell-like degradation and degeneration of the myofibrillar network of the fiber, the term being derived from the microscopic shell-like appearance of the fiber); fiber splitting; any other (undetermined) alteration; and multiple alterations. The total percentage of fibers with a pathological alteration was also calculated for each control and experimental muscle.

Statistical analysis.

In the continuous outcome variables, the statistical comparisons were first done by using a two-way ANOVA, the rat group and hindlimb side being the grouping variables. When the two-way ANOVA indicated significant (P < 0.05) group and side differences and significant (P< 0.05) group × side interactions, Tukey’s post hoc analyses were used for pairwise comparisons. In the frequency outcome variable (percentage of pathological fiber alterations), the groups were compared with the χ2 test. The sample size (8 rats/group with both of the hindlimbs analyzed) required to detect a 10% difference in muscle morphology between the experimental and control groups was based on a power analysis by using alpha = 0.05 and beta = 0.20 (power 0.80) (20).

An alpha level of <5% (P < 0.05) was considered significant. The given significance levels refer to two-tailed tests.

RESULTS

Percent area of connective tissue.

The effects of immobilization, free remobilization, and low- and high-intensity treadmill running on the percent area of intramuscular connective tissue are presented in Fig. 1. In this parameter, the two-way ANOVA indicated significant group and side differences in both the soleus muscle (P < 0.001 for both differences) and gastrocnemius muscle (P < 0.001 for both differences), as well as a significant group × side interaction (P < 0.001 for both muscles), and therefore, Tukey’s post hoc analyses were also performed (see below).

Fig. 1.

Percent area of intramuscular connective tissue in rat soleus (A) and gastrocnemius muscles (B) after immobilization and remobilization. Values are means ± SD. C, control; IM3, 3-wk immobilization; FR11, 3-wk immobilization plus 8-wk free remobilization (free cage activity); LIR11, low-intensity remobilization (3-wk immobilization plus 1-wk free remobilization and 7-wk low-intensity treadmill running); HIR11, high-intensity remobilization (3-wk immobilization plus 1-wk free remobilization and 7-wk high-intensity treadmill running). Significant immobilized-to-nonimmobilized hindlimb differences, ** P < 0.01 (Tukey’s post hoc analysis of two-way ANOVA). For other comparisons, see text.

In the soleus muscle of the nonimmobilized hindlimbs, the group differences in the amount of connective tissue were small and nonsignificant, the percent area averaging 3.5% in the control group, 3.6% in the IM3 group, 3.9% in the FR11 group, 4.4% in the LIR11 group, and 4.5% in the HIR11 group, respectively (Fig.1 A). In contrast, immobilization of the left hindlimb for 3 wk created a large and significant (P < 0.01) side-to-side difference in the mean percent area of the connective tissue, the immobilized left hindlimb percentage being 18.9%. Free remobilization and especially low- and high-intensity treadmill running for 8 wk significantly (P < 0.01 for all groups) restored this value toward control levels (14.8, 7.7, and 7.4%, respectively), the restorative effect being significantly better in the running groups (P < 0.01 for both groups) than in the FR11 group. Despite this apparent benefit of running, the side-to-side difference was still significant in all three remobilization groups (P < 0.01 for each group) (Fig.1 A).

In gastrocnemius muscle, the connective tissue results were in line with those in the soleus muscle (Fig.1 B), except that free remobilization could not reduce the amount of intramuscular connective tissue from the level during immobilization [15.3% in IM3 vs. 14.4% in FR11; not significant (NS)] and high-intensity running (HIR11group) produced significantly better results than low-intensity running (LIR11 group) (9.7 vs. 13.1%,P < 0.01) (Fig.1 B). Still, as was the case in the soleus muscle, the side-to-side difference was significant in all remobilization groups (P < 0.01 for each group) (Fig. 1 B).

Capillary density.

The changes in capillary density of the soleus and gastrocnemius muscles are reported in Fig. 2. As above, this parameter also showed significant group and side differences in both soleus and gastrocnemius muscles (P < 0.001 for all differences) and significant group × side interaction (P < 0.001 for both muscles), and, therefore, Tukey’s post hoc analyses were also performed (see below).

Fig. 2.

Capillary density of rat soleus (A) and gastrocnemius muscles (B) after immobilization and remobilization. Values are means ± SD. Group names are as defined in Fig. 1. Significant immobilized-to-nonimmobilized hindlimb differences, ** P < 0.01 (Tukey’s post hoc analysis of two-way ANOVA). For other comparisons, see text.

In the soleus muscle of the nonimmobilized hindlimbs, the capillary densities were significantly different among the groups as expected so that the LIR11 and HIR11 groups had higher mean capillary density than did the control group, the IM3 group, and the FR11 group (P < 0.01 for both running groups) (Fig. 2 A). Immobilization for 3 wk created a significant (P < 0.01) side-to-side difference in the soleus muscle capillary density, the density of the immobilized left hindlimb being only 63% of that of the nonimmobilized right hindlimb (and 67% of the control). Free remobilization for 8 wk did not significantly improve the situation (left hindlimb density was still only 70% of that of the right hindlimb, P < 0.01, and 74% of the control), but after low- and high-intensity treadmill running the capillary density of the once-immobilized left hindlimbs reached the control level (LIR11 group: left hindlimb capillary density 96% of that in the control, NS) or even exceeded it (HIR11 group: the left hindlimb capillary density 103% of that in the control, NS) (Fig.2 A). In the LIR11 and HIR11 groups, however, the side-to-side differences were significant (P < 0.01 andP < 0.01) because of the above-noted positive effect of running on the capillary density of the nonimmobilized right hindlimbs (Fig.2 A).

In the gastrocnemius muscle, the capillary density results were very similar to those in the soleus muscle (Fig.2 B).

Fiber cross-sectional area.

The changes in the fiber cross-sectional area are reported in Fig.3. As previously, this parameter also showed significant group and side differences in both soleus and gastrocnemius muscles (P < 0.001 for all differences) and significant group × side interaction (P < 0.001 for both muscles, exceptP < 0.01 for gastrocnemius type I fibers), and, therefore, Tukey’s post hoc analyses were also performed (see below).

Fig. 3.

Cross-sectional area of type I fibers in rat soleus muscle (A) and type I (B) and type II (C) fibers in rat gastrocnemius muscle after immobilization and remobilization. Values are means ± SD. Group names are as defined in Fig. 1. Significant immobilized-to-nonimmobilized hindlimb differences, ** P < 0.01 (Tukey’s post hoc analysis of two-way ANOVA). For other comparisons, see text.

In both the soleus (Fig. 3 A) and gastrocnemius (Fig. 3 B) muscles of the nonimmobilized hindlimbs, the mean cross-sectional area of the type I fibers was, as expected, significantly higher in the LIR11 group (P < 0.01) and HIR11 group (P < 0.01) than in the other groups. In soleus, there was also a significant difference between the LIR11 and HIR11 groups, in favor of the latter (P < 0.05). Three weeks of immobilization created a significant side-to-side difference, the mean left hindlimb cross-sectional area being only 69% (soleus) and 70% (gastrocnemius) of that in the right hindlimb (P < 0.01 for both muscles). Free remobilization did not improve the situation (left hindlimb areas were 63 and 69% of that in the right hindlimb,P < 0.01 for both muscles), whereas after low- and high-intensity treadmill running, the left hindlimb cross-sectional area was 92 (LIR11soleus, P < 0.05), 111 (LIR11 gastrocnemius,P < 0.05), and 100 (HIR11 soleus, NS), and 103% (HIR11 gastrocnemius, NS) of that in the control rats (Fig. 3, A andB). In the LIR11 and HIR11 groups, the side-to-side soleus differences were significant (P< 0.01 for both) because of the above-noted positive effect of running on the cross-sectional area of type I fibers in the nonimmobilized right hindlimbs. In gastrocnemius, this was the case for the HIR11 group (P < 0.01).

In the gastrocnemius muscle of the nonimmobilized hindlimbs, the cross-sectional area of type II fibers was, as expected, higher in the LIR11 and HIR11 groups than in the other groups (Fig. 3 C). In both groups, the difference was significant compared with in the IM3 group (LIR11,P < 0.05; HIR11,P < 0.01). Immobilization produced a significant (P < 0.01) side-to-side difference, the left hindlimb cross-sectional area being 63% of that in the right hindlimb. Free remobilization could not improve the situation, the left hindlimb value being 58% of that in the right hindlimb (P < 0.01), whereas after low- and high-intensity running the mean cross-sectional area of the type II fibers was comparable with that in the control rats (92% in the LIR11 group and 99% in the HIR11 group, NS for both groups). In the LIR11 group, the side-to-side differences was still significant (P < 0.01) because of the above-noted positive effect of running on the cross-sectional area of the type II fibers in the nonimmobilized right hindlimbs.

Pathological fiber alterations.

The effects of immobilization, free remobilization, and low- and high-intensity treadmill running on the occurrence of abnormal fibers (fibers with pathological alterations) are presented in Tables1 (soleus) and 2 (gastrocnemius).

View this table:
Table 1.

Percentage of type I muscle fibers with a pathological alteration of all fibers examined in immobilized or immobilized-remobilized soleus muscles

View this table:
Table 2.

Percentage of type II muscle fibers with a pathological alteration of all fibers examined in immobilized or immobilized-remobilized gastrocnemius muscles

In both soleus and gastrocnemius muscles in the control rats, the total number of fibers with a pathological alteration was very low (6 and 4%, respectively) (Tables 1 and 2). Immobilization increased the occurrence of the pathological fibers considerably (P < 0.001) (soleus 66% and gastrocnemius 37%), and the situation was unchanged in the free remobilization group (65 and 36% for soleus and gastrocnemius, respectively). In the LIR11 group, and especially in the HIR11 group, the number of soleus and gastrocnemius fibers with a pathological alteration was clearly lower than that in the IM3 and FR11 groups (33 and 13% in LIR11 group and 17 and 10% in HIR11 group, respectively), although the above-described control level of 6 and 4% was not compeletely reached (Tables 1 and 2).

DISCUSSION

In this randomized, controlled study we tested the hypothesis that daily running would increase the rate of recovery after hindlimb immobilization and that high-intensity treadmill running would be more beneficial than low-intensity running. Our findings indicated that both parts of this hypothesis were correct: greater than normal activity (i.e., greater than free cage activity) was needed to restore the immobilization-induced morphological and histochemical changes in rat soleus and gastrocnemius muscles to normal, and there seemed to be a dose-response relationship so that high-intensity running produced better effects than low-intensity running. These observations have not been made previously, to the best of our knowledge.

Intramuscular connective tissue.

The drastic effects of treadmill running on the percent area of intramuscular connective tissue (Fig. 1) can be partly explained by the fact that the immobilization-induced accumulation of connective tissue is not only absolute but also relative (because of the simultaneous decrease in fiber size). Immobilization produces an increase in the hydroxyproline (an indicator of collagen concentration in the studied tissue) content per muscle volume or mass (14, 16), but this increase is partly relative because the total hydroxyproline content does not change because of immobilization, whereas the total muscle weight and volume and the fiber size clearly decrease as a consequence of rapid net degradation of muscular fibrillar, noncollagenous proteins (8, 14). In intensified remobilization, the fiber size returns to normal (Fig.3), reducing the percent volume of intramuscular connective tissue, respectively.

Capillary density and muscle fibers.

In this study, the intensified remobilization by treadmill running seemed to be especially beneficial in restoring the capillary density of the rat calf muscles so that, in the HIR11 group, the capillary density of the once-immobilized hindlimbs exceeded that in the control rats (Fig. 2). Kvist et al. (18) made a similar observation when studying the capillary density of the rat myotendinous junction and speculated that the running-induced increase in the capillary density occurred because the lumina of the obliterated capillaries opened and new capillaries developed. Also, in injured skeletal muscle myofiber regeneration is dependent on the recovery of the blood supply to the muscle, and the normal architecture and size of injured myofibers are restored more quickly and more completely if active remobilization instead of immobilization is used as the postinjury treatment (11). Thus in our study adequate restitution of blood supply was likely to be a prerequisite for normalization of the muscle fibers, their size and internal structure.

Pathological fiber alterations.

In both the soleus and gastrocnemius muscles, immobilization induced many pathological fiber alterations (Tables 1 and 2). Free remobilization did not change the situation, whereas after low-intensity, and especially high-intensity, treadmill running, the number of these pathological fibers was clearly reduced although still higher than in the control. The good recovery of the fiber population by exercise indicated that most likely many of these immobilization-induced abnormal fiber features were not structurally and functionally detrimental to the muscle itself. It must be kept in mind that we used not only routine histology but also some very specific histochemical techniques to demonstrate and define fiber pathology, and, therefore, in our immobilization group the percentage of fibers showing one or more of the pathological changes was clearly higher than that in the previous hindlimb suspension or immobilization studies in rats (6, 7, 17, 19, 21, 24-26). In other words, our large and sensitive scale of fiber screening resulted in a high number of fibers with some abnormal feature, however, still well maintaining the study design and above-described group comparisons validly and reliably.

Histologically, the above-noted pathological fiber changes bore a resemblance to those seen in muscular dystrophy and neurogenic atrophy or after strenous muscle activity (3, 17), and all of them could be classified as degenerative. It remained, however, unknown whether some forms of these pathological fibers could recover during the period of remobilization, and, if so, to what extent. The alternative option was complete fiber degradation and cell death, followed by de novo synthesis of new fibers by satellite cell activation (5, 15).

In summary, our study gave evidence that immobilization-induced accumulation of intramuscular connective tissue, capillary loss, reduction in fiber size, and accumulation of fibers with pathological morphological and histochemical alterations are, in great part, reversible phenomena, if remobilization is intensified by physical training. Further (longer) remobilization experiments are needed, however, to determine whether full recovery is possible, especially in terms of the functional properties of the once-immobilized muscles. Further studies are also needed to identify the mechanisms behind the activity-induced muscle recovery.

Acknowledgments

We thank Mirja Ikonen and Maria Suba for excellent technical assistance.

Footnotes

  • Address for reprint requests: P. Kannus, The President Urho Kekkonen Institute for Health Promotion Research, Kaupinpuistonkatu 1, FIN-33500 Tampere, Finland (E-mail: klpeka{at}uta.fi).

  • This work was supported by grants from the Research Council for Physical Education and Sports, the Finnish Ministry of Education, the Medical Research Fund of Tampere University Hospital, and the Sigrid Juselius Foundation.

REFERENCES

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