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Medicine & Science in Sports & Exercise:
doi: 10.1249/MSS.0b013e31823dbff3
Basic Sciences

Postcontusion Polyphenol Treatment Alters Inflammation and Muscle Regeneration

KRUGER, MARIA J.; SMITH, CARINE

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Author Information

Department of Physiological Sciences, Stellenbosch University, Stellenbosch, SOUTH AFRICA

Address for correspondence: Carine Smith, Ph.D., Department of Physiological Sciences, Stellenbosch University, Private Bag X1, Matieland, 7602, South Africa; E-mail: csmith@sun.ac.za.

Submitted for publication June 2011.

Accepted for publication October 2011.

Supplemental digital contents are available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Web site (www.acsm-msse.org).

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Abstract

Purpose: Given the major role that oxidants play in cellular damage, and the recent focus on antioxidants as treatment for muscle injuries, the aim of this study was to examine the effect of short-term postinjury grape seed–derived polyphenol supplementation on muscle inflammation and repair processes after contusion injury.

Methods: Experimental injury of the right gastrocnemius muscle was achieved by drop-mass method (200 g from a height of 50 cm), after which rats were gavaged with either 0.9% saline (placebo—PLA) or 20 mg·kg−1·d−1 of proanthocyanidolic oligomer (PCO) from 2 h after contusion injury, for up to 14 d after injury. Blood samples and injured muscle were collected at 4 h and at days 1, 3, 5, 7, and 14 after injury.

Results: Compared to an uninjured control group, PCO supplementation resulted in an earlier peak in number of activated satellite cells in contusion-injured muscle tissue (4 h for PCO vs day 3 for PLA, n = 4 per time point per group) and fetal myosin heavy chain expression (day 5 for PCO, P < 0.01 with no change in PLA, n = 3 per time point per group), indicative of quicker muscle regeneration. PCO supplementation limited neutrophil infiltration and facilitated earlier macrophage infiltration into the injured area (n = 4 per group). PCO also resulted in an earlier return toward control levels of muscle proinflammatory cytokines on day 3 (P < 0.01 for interleukin 6 and P < 0 05 for tumor necrosis factor α, both n = 3 per group).

Conclusions: Data show that short-term postinjury PCO supplementation was able to quicken muscle regeneration by facilitating earlier recruitment of activated satellite cells and to modulate the immune system in favor of an anti-inflammatory status.

The cellular and molecular mechanisms of normal, unassisted muscle regeneration after injury have been described extensively in the literature, specifically in models of exercise or training intervention-related injury such as after stretch and/or eccentric exercise. The process of soft-tissue wound healing is complex and progresses through the three distinct, yet overlapping phases of destruction, repair, and remodeling, which can take between 14 and 21 d to run to completion (20). Considering that injury results not only in primary damage to muscle cells but also in capillary rupturing and infiltrative bleeding, inflammation, oxidative stress, and fibrosis (24), several targets need to be addressed for optimal healing. The most common clinically prescribed therapy for injuries remains anti-inflammatory drugs (1), both steroidal (19) and nonsteroidal (38). However, most often, this therapy results in delayed or incomplete recovery, which may be attributed to incorrect dosage or timing of administration of anti-inflammatory drugs (prolonged use and therefore prolonged inhibition of inflammation may largely inhibit positive events associated with inflammation which is required for recovery), or insufficient rest, resulting in increased risk of repeat injury and extensive tissue scarring (20).

Other treatment modalities that are used in the treatment of injured skeletal muscle include rest, ice, compression, and elevation (26), therapeutic ultrasound (39), stem cell therapy (14), and hyperbaric oxygen therapy (15). Currently, all treatments are directed to the restoration of full skeletal muscle function, augmenting normal repair and regeneration processes, and limiting inflammation and muscle fibrosis (17), so as to allow for quicker muscle regeneration. Despite numerous efforts with experimental and clinical studies, the scientific proof of effectiveness of these treatment regimens on muscle recovery has been somewhat disappointing and limited (3), mainly because of the different injury models used to test the different treatment options. The type of injury model and the extent of damage will determine to a great extent the choice of both treatment and duration of treatment. We believe that a model of muscle injury that occurs in the absence of confounding factors associated with exercise and that delivers an injury standardized in size and severity may be a less complex model to use for studies wanting to elucidate the time course of inflammation and muscle regeneration, as well as the optimal time frame for interventions aimed at reducing the negative effects associated with inflammation. One such model that we have recently identified is contusion injury (35), which is a simulation of (usually) accidental, blunt force trauma that frequently occurs not only in especially contact sports but also in situations not associated with exercise.

One of the main exacerbating factors after primary injury to skeletal muscle, and in particular after contusion injury where the vasculature may be disrupted to a greater extent when compared with other injury models, is the generation of reactive oxygen species (ROS) during the early recovery process, which result in increased oxidative stress and potentially subsequent secondary damage. Given the major role that oxidants play in cellular damage, it is not surprising that myocytes and other cells contain several endogenous antioxidant defense mechanisms to prevent oxidative injury. Two major classes of endogenous protective mechanisms work together to reduce the harmful effects of oxidants in the cell: enzymatic defense (e.g., superoxide dismutase, glutathione peroxidase, and catalase) and nonenzymatic antioxidants (e.g., glutathione, ascorbic acid, α-tocopherol, β-carotene, uibiquinol-10, albumin, ceruloplasmin [copper part of it], and ferritin) (2,33). Although mammalian cells seem to have adequate antioxidant reserves to cope with ROS production under normal physiological conditions, these antioxidant reserves may be inadequate to cope when additional ROS are produced (27). Therefore, supplementing the diet with dietary antioxidants or vitamins might prove beneficial to scavenge the extra ROS generated.

Several studies have been conducted on the properties and effectiveness of dietary antioxidants (which include vitamin C [ascorbic acid], vitamin E [tocopherols, tocotrienols], and vitamin A [retinol], or its precursor, β-carotene) to scavenge free radicals and therefore to decrease oxidative stress. Their importance was highlighted in a study by Basu (2), where deficiencies in some of these antioxidants were associated with increased oxidative stress. Similarly, acute dietary antioxidant supplementation is associated with a decrease in lipid peroxidation (5). Various studies have also provided evidence that antioxidant (vitamin) supplementation may have positive effects on at least some aspects of the muscle response to damage. However, these studies, which were exercise related, only investigated proinflammatory markers such as interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) (37), or indirect indicators of damage, such as plasma creatine kinase (CK) (32), rather than specific markers of recovery. Therefore, studies including some specific markers of regeneration (such as satellite cell (SC) response or fetal isoform of myosin heavy chain formation) and/or recovery (e.g., recovery of muscle force production) to confirm potential effects of antioxidants treatment after injury are still lacking.

Plant-derived polyphenols, which include carotenoids, bioflavonoids, and flavonoids, have well-established antioxidant capacity (2,33). To date, very few studies exist as proof that polyphenols may also be beneficial for muscle repair. Positive effects reported include the ability of polyphenols to decrease muscle necrosis and elevate twitch tension in muscular dystrophy using a mdx mouse model (6,9), as well as the suppression of oxidative stress and lowering force deficit to allow for earlier skeletal muscle recovery in rat skeletal muscle after forced treadmill running (22). Among the polyphenols that have attracted research attention in the context of muscle repair recently, certain flavonols have been shown to prevent collagen breakdown by inhibiting collagenase, which is positively associated with muscle healing (34,40). However, other polyphenols, such as quercetin, showed inhibitory effects on inflammation only at limited dosages (31).

In summary, most often antioxidants such as vitamins C and E are taken chronically as a means of boosting the immune system. Similarly, a previous study by our group have shown that grape seed–derived antioxidant-containing proanthocyanidolic oligomers (PCO) have the ability to hasten muscle regeneration when taken as a preventative strategy before injury (29). However, the effect of postinjury PCO supplementation should also be considered.

The current study investigated whether short-term postinjury PCO supplementation is beneficial for skeletal muscle regeneration. We hypothesized that PCO supplementation would have a positive effect by limiting the inflammatory response, while facilitating earlier muscle fiber regeneration.

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METHODS

Experimental Animals

Adult, male Wistar rats weighing approximately 280 g were used in this study. All animals had access to food and tap water ad libitum and were exposed to a 12-h light–dark cycle (lights on at 6:30 a.m.). Ambient temperature was controlled at 21°C, and rooms were ventilated at 10 changes per hour. All experimental protocols, including death by pentobarbitone sodium overdose, were approved by the Animal Ethics Committee of Sub-Committee B of Stellenbosch University (project no. 2006Smith01) and carried out in accordance with the American College of Sports Medicine animal care standards.

Experimental animals were randomly divided into three groups, a control placebo, a postinjury placebo-treated, and a postinjury PCO-treated group. All rats were accustomed to gavage before the intervention protocol. Rats in the control placebo group received 0.9% saline for 2 wk, without injury. Rats in the postinjury groups started treatment 2 h after injury and continued for up to 14 d after injury (PI-PLA and PI-PCO, respectively; n = 96, i.e., eight rats per time point per group, sacrifice/collection time points were at 4 h and days 1, 3, 5, 7, and 14 after injury).

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Interventions
PCO treatment

Rats were administered a daily dose of 20 mg·kg−1·d−1 (derived from the recommended daily dose for humans of 140 mg and corrected to allow for the higher metabolic rate of rats) grape seed–derived PCO (kindly provided by Brenn-O-Kem, Wolseley, South Africa), with the first dose administered 2 h after muscle injury by oral gavage. The same procedure was followed for PLA rats, but rats were gavaged with 0.9% saline instead. Oxiprovin, the grape seed–derived PCO used, contains 45% proanthocyanidins and less than 5% monomers; the remainder is made up of long-chain sugars and oligomer-attached glycosides.

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Muscle injury intervention

Before injury, rats were anesthetized with 75 mg·kg−1 ketamine and 0.5 mg·kg−1 medetomidine in 0.9% saline, administered intraperitoneally. Hind limb contusion was achieved using a noninvasive drop-mass injury jig, which facilitated standardized dropping of a weight of 200 g from a height of 50 cm, onto the medial surface of the right gastrocnemius muscle.

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Analyses

After euthanasia by pentobarbitone overdose, blood was collected into a vacutainer tube containing heparin (BD Vacutainer Systems, Plymouth, UK) before the injury (day 0), at 4 h, and on days 1, 3, 5, 7, and 14 after the contusion injury. Plasma was separated by centrifugation at 3000g for 10 min at 4°C (PK121R; ACL International SRL, Milan, Italy), aliquoted into 1.5-mL reaction vials and stored at −80°C until subsequent analysis.

Total plasma CK (n = 8 per time point per group) was determined enzymatically by PathCare pathology laboratory using standard laboratory procedures and automated analyzers (Vergelegen Medi Clinic, Somerset West, South Africa).

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Oxygen radical absorbance capacity assay

The oxygen radical absorbance capacity (ORAC) assay was performed on plasma and muscle tissue homogenate supernatants (n = 8 per time point per group) using the method of Huang et al. (16) no later than 10 d after sample collection. Results for plasma ORAC were expressed as micromole Trolox equivalents per liter, whereas muscle ORAC were corrected for protein content using the Bradford assay (4) and expressed as micromoles of Trolox equivalents per microgram of protein.

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Muscle histology and immunohistochemistry

Gastrocnemius muscles were fixed in 10% formal saline, processed, and embedded in paraffin wax. Five-micrometer cross sections were prepared (CM1850; Leica Microsystems, Nussloch, Germany) and stained with hematoxylin and eosin for qualitative analysis of muscle recovery.

Sections for immunohistochemistry were adhered to poly-L-lysine (P8920; Sigma Aldrich, St. Louis, MO)–coated slides, deparaffinized, rehydrated, and fixed in 0.1% trypsin (Highveld) at 37°C for 30 min, after which it was blocked with 5% donkey serum for 30 min. The following primary antibodies were used: mouse antihuman Pax-7 (1:100; DSHB) to identify SC, rabbit antihuman laminin (1:200, Z 0097; Dako Diagnostic) to identify the basal lamina, mouse antirat His48 (1:200, 554905; BD Biosciences) to identify neutrophils, and goat antimouse F4/80 (1:200, sc-26642; Santa Cruz Biotechnology, Santa Cruz, CA) to identify macrophages. The first primary antibody was left to incubate for 4 h at room temperature, after which sections were revealed with a donkey antigoat Texas Red-conjugated (1:250; Santa Cruz), donkey antimouse fluorescein isothiocyanate–conjugated (1:250; Santa Cruz), goat antimouse fluorescein isothiocyanate–conjugated (1:250; Invitrogen), or goat antirabbit Texas Red–conjugated (1:250; Invitrogen) secondary antibody for 40 min at room temperature. Sections were costained with Pax-7 and laminin. bisBenzimide H 33342 trihydrochloride (Hoechst, 1:200; Sigma Aldrich, B2261) was added to all sections to visualize nuclei.

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Image analysis

All imaging data were obtained by analyzing two serial sections from each muscle sample, at each time point for each antibody. Six fields of view per section were imaged using a microscope (Nikon ECLIPSE E400; 400× objective used), equipped with a color digital camera (Nikon DXM1200). All images were overlaid using a computer software program (Simple PCI, version 4.0; Compix Inc., Imaging Systems, Cranberry Township, PA). Where necessary, photos were enlarged, and the color of the stain was enhanced after importation into Simple PCI to assist with identification. The images presented here are only partial images of those taken at 400×. Photos were used to count positively labeled SC per muscle fiber or field of view (350 μm2) in the border zone. In order for a cell to be classified a true SC, it had to comply to a few criteria: (a) Pax-7+ cells had to be in contact with the sarcolemma, (b) the SC had to form an indentation into the muscle fiber, and (c) an elongated nucleus had to be present. If one of these criteria was not present, the cell could not be classified a true SC. Immune cell infiltration were assessed in sections including border zones (areas right next to the severely injured areas, where muscle fibers are intact) and injured areas. Immune cell data were counted manually and expressed as the number of positively labeled immune cells per field of view (350 μm2) in both the injured and the border zone areas. For this analysis, a subgroup of n = 4 per time point per group was randomly selected—these subgroups did not differ from the total groups for any parameter assessed in total groups (n = 8).

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Western blotting

Proteins were extracted from tissue samples and labeled by incubation with primary antibodies for TNF-α (1:1000, sc-1351; Santa Cruz), IL-1β (1:1000, sc-7884; Santa Cruz), and IL-6 (1:1000, sc-1265; Santa Cruz) and fetal myosin heavy chain (MHCf, 1:1000, F1.652; DSHB). β-Actin was used as a loading control (1:1000, sc-81178; Santa Cruz). For detection, horseradish peroxidase secondary antibody (1:4000; Santa Cruz) was used. Antigen–antibody complexes were visualized using ECL detection reagents according to the manufacturer’s instructions (Amersham Life Science, Inc., Arlington Heights, IL) and exposed to an autoradiography film (Hyperfilm ECL, RPN 2103) to detect light emission through a nonradioactive method. Films were densitometrically analyzed (UN-SCAN-IT; Silkscience), and protein values were corrected for minor differences in protein loading (using β-actin), as required. Western blotting experiments were run in triplicate. As for immunohistochemistry and image analysis techniques, a subgroup was also selected for Western blotting analysis. The choice of n = 3 per subgroup per time point was a logistic one, but once again, the subgroups did not differ from the total group (n = 8) for any measure assessed in the total group. (All individuals represented in the subgroup for Western blotting were also represented in the subgroup used for immunohistochemistry and image analysis.)

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Statistical analysis

Values are presented as means ± SD, unless otherwise specified. Factorial ANOVA and Fisher post hoc tests were performed to assess main effects of treatment, time, and treatment–time interaction. All statistical analyses were done using the computer software Statistica version 8 (StatSoft Software, Tulsa, OK). The accepted level of significance was P < 0.05.

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RESULTS

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No difference detected in plasma CK

Main effects ANOVA indicated a trend toward a time effect (P = 0.068), with only a small variable increase in plasma CK in both the PLA and the PCO groups 4 h after injury (548 ± 470 and 732 ± 835 UI·L−1, respectively, vs 213 ± 27 UI·L−1 at baseline). No significant differences were evident between treatment groups over time for CK.

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Acute PCO accelerated muscle ultrastructural recovery

Qualitative histological assessment of muscle damage indicated that, irrespective of treatment, muscle fibers were significantly disrupted and the vasculature damaged, resulting in influx of red blood cells into the interstitial space 4 h after injury (Figs. 1B, H). Edema was evident in both groups as demonstrated by a widening of the interstitial space between fibers. A significant influx of inflammatory cells on day 1 after injury occurred in PLA (Fig. 1B) compared with a more limited, but earlier response in PCO (e.g., Fig. 1H). Immune cells remained visible in the injured area up to day 7 in PLA (Fig. 1F) but disappeared after day 5 in PCO (Fig. 1K). At 7 d after injury, the inflammatory cells had progressed to more peripheral portions of the damaged muscle, and their numbers were reduced in PLA (Fig. 1F) but totally absent at this time point in PCO (Fig. 1L). The appearance of regenerating muscle fibers as identified by centronucleation was only evident on day 14 in PLA (Fig. 1G) but already at day 7 in PCO (Fig. 1L). Two weeks after injury (Figs. 1G, M), muscle in PCO clearly had recovered to a greater extent than that in PLA, although in neither regeneration was complete.

Figure 1
Figure 1
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Plasma and muscle ORAC was increased after PCO supplementation

ANOVA indicated a main effect of time (P < 0.001) and a treatment–time interaction effect (P < 0.05) for plasma ORAC. A significant but transient decrease is evident in plasma ORAC on day 3 in PLA, with recovery by day 5, and a second decrease on day 14 (Fig. 2A). No significant change over time was observed in PCO. Plasma ORAC in PCO was significantly higher when compared with PLA on day 14, mainly as a result of the depressed ORAC seen in PLA only at this time point. In the gastrocnemius muscle, there was a main effect of both time and a treatment–time interaction effect (P < 0.001 for both). Muscle ORAC in PCO increased significantly earlier (day 3) when compared with PLA (day 7; Fig. 2B). After a transiently decreased ORAC on day 5 in both groups, PLA ORAC was significantly higher than PCO on day 7, but the opposite was seen on day 14, when PCO ORAC was again significantly greater than that of PLA.

Figure 2
Figure 2
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PCO supplementation accelerated the SC response

ANOVA indicated a significant effect of time, as well as a treatment–time interaction (P < 0.001 for both). Three days after injury, a significant increase in the number of SC expressing Pax-7 was evident in PLA. In contrast, Pax-7+ SC numbers were already significantly elevated (when compared with both control and PLA) 4 h after injury and had already returned to values seen in the control group by day 3 (Fig. 3A). Representative immunofluorescence images of these data are also available as Supplemental material 1, http://links.lww.com/MSS/A147.

Figure 3
Figure 3
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As a consequence of the low number of intact myofibers resulting from excessive fiber swelling, in the injured area of muscle in the PLA, the number of Pax-7+ SC per area (field of view = 350 μm2) was also considered (Fig. 3B). An ANOVA main effect of treatment, time, and a treatment–time interaction was evident (all P < 0.05). The number of Pax-7+ SC per muscle area was unchanged over time in PLA, whereas Pax-7 expression in PCO was similar to that seen when Pax-7 were expressed per myofiber.

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Fetal myosin heavy chain expression is accelerated in response to PCO

PCO supplementation resulted in a significant increase in MHCf on day 5 after injury, which was not evident in the PLA group (Fig. 4).

Figure 4
Figure 4
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Acute PCO treatment blunted the neutrophil response, whereas macrophages infiltrated the injured area earlier

Repeated-measures ANOVA indicated a similar and significant effect of treatment (P < 0.01), time, and treatment–time interaction (both P < 0.001) for neutrophils in both the border zone and the injured area. No neutrophils were evident in the control groups, whereas injury resulted in a significant (>20-fold) elevation in neutrophils on day 1 after injury in PLA, which normalized on day 3 (Fig. 5). In contrast, PCO supplementation resulted in a significantly blunted neutrophil response. Representative immunofluorescence images of neutrophil infiltration in muscle from both placebo and supplemented groups are available as Supplemental material 2, http://links.lww.com/MSS/A148.

Figure 5
Figure 5
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Main effects of time and treatment–time interaction (both P < 0.001) were evident for macrophages in the injured area, and in the border zone, a main effect of treatment (P < 0.001), time, and treatment–time interaction (both P < 0.01) was evident. No macrophages were present in the gastrocnemius muscle of uninjured rats (Fig. 5). Similar gradual elevations in macrophage infiltration occurred in PLA, but somewhat earlier in the border zone than in the injured area. PCO supplementation resulted in relatively earlier invasion of macrophages in the injured area (4 h after injury) when compared with PLA. This initial peak was followed by a similar gradual increase up to day 5, at which time PCO had a significantly higher degree of macrophage infiltration than PLA. In contrast to PLA, PCO rats exhibited a largely suppressed macrophage response in the border zone. As for neutrophil infiltration, macrophage infiltration is illustrated by representative immunofluorescence images provided as Supplemental material 3, http://links.lww.com/MSS/A149.

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Cytokine responses were shorter with PCO supplementation

No significant changes were observed for IL-1β, IL-6, or TNF-α in PLA over time. In contrast, PCO displayed a significant early peak for IL-1β, with levels significantly higher than PLA on day 1 and a subsequent return to control levels by day 3 (Fig. 6B). IL-6 levels in PCO were significantly reduced on days 3 and 5, when compared with both PLA and earlier time points (Fig. 6D). Similar results were observed for TNF-α, with PCO exhibiting significantly lower levels than PLA (Fig. 6F).

Figure 6
Figure 6
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DISCUSSION

Data presented demonstrate that acute PCO supplementation was able to result in quicker muscle fiber regeneration when compared with PLA. After contusion-related muscle fiber disruption and vascular rupture, PCO facilitated earlier Pax-7 expression on SC and greater MHCf expression on muscle fibers, both suggesting quicker skeletal muscle regeneration. Other important findings with acute PCO supplementation were 1) altered ORAC status, 2) a blunted proinflammatory response which included a greatly suppressed neutrophil response, and 3) an earlier macrophage response in the injured area, which was associated with a blunted macrophage response in the border zone.

CK, which is still used as gold standard indicator of muscle damage (30), was not affected by either PLA or PCO treatment, except for a similar tendency for increased CK 4 h after injury in both groups. Because the responses in the two groups were similar in both the degree of intragroup variability and the time course, one can assume that the experimental contusion intervention indeed produced a repeatable injury across groups, so that major differences between groups as reported above may indeed be ascribed to the PCO treatment. In further support, albeit on a qualitative level, the histology sections at 4 h after injury revealed a similar ultrastructural disruption.

Turning attention to recovery from this damage, the process of SC activation in muscle regeneration has been described in detail in many recent studies, as basic assessment for the initiation of muscle regeneration (12,18). However, many different markers are used to indicate SC activation, the time course of which differs depending on the type and the severity of injury or disease. In a benchmark study by Kuang et al. (25), it was established that SC expression of the activation marker, Pax-7, is very important in the muscle recovery process, and its absence was associated with severely inhibited muscle regeneration.

In the current study, acute PCO supplementation resulted in a very early increase in Pax-7+ SC numbers per myofiber in the injured muscle, suggesting not only early activation of SC (11) but also significant recruitment of resident SC into the injured area. A study by Dumont et al. (10) on hindlimb unloading/reloading showed that depletion of neutrophils might spare the muscle from excessive membrane damage. It is therefore possible that because PCO had its effect through limiting neutrophil infiltration, it reduced sarcolemmal damage, thus allowing more SC to migrate to the injured area. This is unlikely in our model at the 4-h postinjury time point, given the fact that no neutrophils were evident in the injured area at this point in either group. This explanation could however hold true for the increase in SC on day 1 in the PCO group. These latter early increases did not correspond to any changes in macrophage infiltration or cytokine levels assessed, although IL-6 has been linked to SC activation (28). The effect of PCO on the contribution of other potential role players in SC activation, such as neutrophil or macrophage-derived NO and growth factors (released by injured fibers and macrophages) including hepatocyte growth factor, fibroblast growth factor, and insulin-like growth factor, remains to be investigated. Nevertheless, on day 3, Pax-7+ SC numbers in the PCO group only were similar to those of the uninjured muscle, which suggests that terminal differentiation to form new muscle fibers or fusion with injured fibers had occurred. The fact that MHCf was significantly higher on day 5 in the PCO group compared with the PLA group further supports this interpretation, as does the fact that IL-6 levels in PCO, but not PLA, decreased from day 3.

Because muscle fibers in the PLA group were relatively more swollen, resulting in few intact muscle fibers per field of view, the possibility exists that SC numbers may be falsely elevated when expressed per myofiber. When Pax-7+ SC numbers were expressed per area, the peak on day 3 for PLA indeed disappears. However, the finding of earlier peak in PCO is still valid. This “discrepancy” in interpretation remains an interesting technical point, which should receive more attention by researchers covering a variety of injury models.

SC are not the only cellular role players in muscle regeneration. The inflammatory immune cells with their related, but different time courses, greatly affect the speed of recovery. Acute PCO administration was responsible for blunting the neutrophil response 1 day after injury, potentially as a result of reduced adhesion molecule expression—a different grape seed–derived PCO product, activin, was reported to decrease circulating soluble adhesion molecule and selectin expression in plasma of patients with systemic sclerosis (21). An associated result was that of a relatively low number of macrophages in PCO in the border zone, which may suggest that there was less neutrophil-associated secondary damage when compared with PLA. In contrast, in PCO, relatively more macrophages were present in the injured area at the 4 h and day 5 time points. Because both M1 and M2 macrophages are important role players in inflammation, our data may suggest that both M1 and M2 macrophage subpopulations may be present in our sample, i.e., that there may have been a shift to the left for time course, with anti-inflammatory macrophage subtypes present in the tissue much earlier. However, a more detailed analysis of macrophage subpopulation distribution is required to confirm this.

Because proinflammatory cytokines are released as a result of injury (7), it is important to note specific changes over time. In a previous study by our group, it was evident that chronic PCO supplementation was able to blunt the proinflammatory response not only in circulation but also at the muscle level (29). In this study, short-term postinjury PCO supplementation resulted in a shift to the left for the time course of the proinflammatory response in the muscle, indicating that PCO is beneficial both as remedial and as preventative therapy.

Proinflammatory cytokines, neutrophils, and M1 macrophages are all involved in the generation of free radicals and subsequent contribution to secondary damage. After injury, a large amount of reactive species are formed as a result of neutrophil or macrophage respiratory burst. SC, upon activation, are also responsible for the formation of reactive species. PCO administration in the current study prevented significant changes in plasma ORAC over time, and on day 14, a significantly higher ORAC compared with PLA group was observed. This suggests that the PCO group had a better ability to respond to free radical production, possibly as a result of an increased antioxidant enzyme activity (13). This result corresponds to a study in humans, which reported improved ORAC after acute postexercise supplementation with vitamin C (12.5 mg·kg−1 body weight) and N-acetyl-cysteine (10 mg·kg−1 body weight) (8).

Most studies on the effect of antioxidants investigate ORAC in plasma only, without any indication of what occurs at muscle level. The current study aimed to bridge this gap by also considering changes in ORAC in the skeletal muscle compartment. Muscle ORAC can be expected to differ from plasma ORAC because a host of different cells, including injured muscle fibers, contribute to antioxidant (and thus ORAC) status. In muscle, PCO supplementation resulted in a significant increase in ORAC on day 3 (an earlier response was probably prevented by the fact that the first PCO dosage was administered only 2 h after injury). A study by Kayali et al. (23) demonstrated that β-glucan, an antioxidant, worked as a scavenger and had suppressive effects on lipid peroxidation after spinal cord contusion injury. Furthermore, a review by Teixeira (36) concluded that PCO has the ability to limit oxidative stress via several mechanisms, including decreased tissue degradation by inhibition of proteolytic enzymes, limiting the production of oxygen free radicals and limiting hypoxia by improving local tissue circulation. In the current study, a low number of macrophages and neutrophils were present on day 3 in both the injured area and the border zone in PCO, supporting the idea of a limited free radical production by these relatively fewer cells. On day 5, ORAC in the PCO group was decreased again compared with day 3, possibly as a result of SC fusing (supported by MHCf and Pax-7 data).

In conclusion, it may be of interest to further put our results in the context of a previous work using the same supplement. As mentioned earlier, results reported here are in accordance with results reported after chronic supplementation with PCO (29). However, these results were more pronounced when using a chronic protocol. In the current study, PCO supplementation (which was administered by oral gavage) could only be started 2 h after injury when rats were conscious again. Although this may roughly correspond to the time that could pass after sports injury before supplementation is administered, especially if ultrasound or other diagnostics are performed first, this may have resulted in a delay in the bioavailability of the PCO, so that very early responses occurring immediately after injury may not have been influenced optimally. This stresses the importance of optimizing the timing of remedial treatment to maximize positive outcomes.

Nevertheless, PCO has shown positive effects when administered after injury by blunting neutrophil infiltration while allowing for earlier macrophage infiltration at the tissue level. It is therefore important to further consider and research possible mechanisms of action whereby PCO might exert these effects because it will not only improve our understanding of muscle recovery but also provide a potential natural alternative to anti-inflammatory treatment. Perhaps a limitation of the current study is the absence of functional assessments—inclusion of assessment of muscle functional recovery in future studies will make results more widely applicable and also more specifically include recovery from exercise and training intervention–induced damage.

The authors thank Geraldine Pretorius for technical assistance.

This work was financially supported by the South African National Research Foundation.

No author personally benefited financially as a result of this. All authors declare no conflict of interest.

The results of the present study do not constitute endorsement by the American College of Sports Medicine.

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Keywords:

CONTUSION; INFLAMMATION; MACROPHAGE; NEUTROPHIL; PROANTHOCYANIDIN; REGENERATION

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