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Rat supraspinatus muscle atrophy after tendon detachment
ELSEVIER
Journal of Ort hopaedic Research
Journal of Orthopaedic Research 23 (2005) 259-265
www.elsevier.com/locate/orthres
Rat supraspinatus muscle atrophy after tendon detachment Elisabeth R. Barton
Jonathan A. Gimbel ’, Gerald R. Williams ‘, Louis J. Soslowsky
’
Department of Anatomy and Cell Biology, School of Denial Medicine, 441A Levy Building, 240 S. 40th Street, University of Peimsylvania. Philadelphia, PA, 19104, USA McKay Orthopaedic Research Laboratory, School of Medicine, University of Pennsylvania, Philadelphia, PA, USA a
Received 29 April 2004; accepted 9 August 2004
Abstract
Rotator cuff tears are one of the most common tendon disorders found in the healthy population. Tendon tears not only affect the biomechanical properties of the tendon, but can also lead to debilitation of the muscles attached to the damaged tendons. The changes that occur in the muscle after tendon detachment are not well understood. A rat rotator cuff model was utilized to determine the time course of changes that occur in the supraspinatus muscle after tendon detachment. It was hypothesized that the lack of load on the supraspinatus muscle would cause a significant decrease in muscle mass and a conversion of muscle fiber properties toward those of fast fiber types. Tendons were detached at the insertion on the humerus without repair. Muscle mass, morphology and fiber properties were measured at one, two, four, eight, and 16 weeks after detachment. Tendon detachment resulted in a rapid loss of muscle mass, an increase in the proportion of fast muscle fibers, and an increase in the fibrotic content of the muscle bed, concomitant with the appearance of adhesions of the tendon 10 surrounding surfaces. At 16 weeks post-detachment, muscle mass and the fiber properties in the deep muscle layers returned to normal levels. However, the fiber shifts observed in the superficial layers persisted throughout the experiment. These results suggest that load returned to the muscle via adhesions to surrounding surfaces, which may be sufficient to reverse changes in muscle mass. 0 2004 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved. Keywords: Supraspinatusmuscle; Chronic; Atrophy; Tendon: Shoulder
Introduction Rotator cuff tears of the shoulder represent a common and challenging orthopaedic problem [23]. While many of these tears are asymptomatic, detachment of the tendon can not only lead to pain, but can also lead to atrophy of the associated muscle, accumulation of fat in the areas normally occupied by muscle, and a subsequent loss in shoulder stability and function [13,15,16]. The reason for these changes may be a combination of * Corresponding author. Tel.: + I 215 573 0887; fax: + I 215 573 2324. E-mail address: [email protected] (E.R. Barton).
lack of muscle activity due to the pain of movement, and a lack of force transmission from the muscle to the bone and the potential shortened state of the muscle fibers due to tendon detachment. Subacromial decompression can be performed to remove pain and tendon repair can be performed to restore a strong connection between the bone, tendon, and muscle. However, the delay between the time of injury and surgical repair is thought to inhibit the healing process, resulting in weak, fatty muscles even after repair. Therefore, a more complete understanding of the muscle changes that occur with time from injury to repair is needed to improve the surgical outcome of chronic rotator cuff tears [3,13]. Significant progress toward understanding the changes in the rotator cuff muscle following tendon
0736-0266/$ - see front matter 0 2004 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.orthres.2004.08.018
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injury has been made utilizing a variety of approaches such as non-invasive CT and MRI measurements in patients [11,30]. In addition to these approaches, several animal models (e.g., rabbit and sheep) have been developed to study the changes in the muscle following supraspinatus tendon detachment [1,4,10,19]. In the rabbit, muscle atrophy increased and muscle function decreased with time after tendon detachment and fatty infiltration of the muscle occurred as early as 4 weeks after tendon detachment [l]. Similar results were found in more comprehensive studies using the same animal model [%lo]. More recently, the effect of a delayed repair on the fatty infiltration of the muscle after tendon repair has been studied. It was found that the supraspinatus muscle did not recover after tendon repair [19] and interestingly, fatty infiltration increased following repair. This suggests that although tendons are intact and load is restored, complete muscle repair is not established. While all models have specific advantages and disadvantages, the anatomy and function of the rat rotator cuff most closely resembles that of the human cuff (other than certain non-human primates). Specifically, the rat has an enclosed arch created by the acromion, coracoid, and clavicle, through which the supraspinatus tendon passes repetitively during shoulder motion [25]. Since the role of the acromion and the enclosed arch is believed to be critical and is at a minimum controversial in the pathogenesis of rotator cuff injuries, an animal model that includes this feature has certain advantages for rotator cuff injury and repair studies. This model has been used previously to address a variety of rotator cuff tendon injury and repair problems [26,28]. The objective of this study was to determine the time course of changes to the supraspinatus muscle in an established rat model of rotator cuff tendon detachment injury [25] to understand the nature of muscle atrophy following tendon detachment and to determine if the potential changes in rat muscle recapitulate those found in muscles from human patients. We hypothesize that tendon detachment would lead to a significant decrease in supraspinatus muscle mass, conversions in fiber properties toward those of fast fiber types, and an increase in the proportion of connective tissue within the muscle bed. Materials and methods Surgical injury model
A total of 24 Sprague-Dawley rats were used in this study. Twenty rats were operated upon bilaterally under general anesthesia to fully detach the supraspinatus tendon at its bony insertion site utilizing a previously described procedure [2,12,27]. Briefly, the supraspinatus was exposed and visualized as it passed under the bony arch created by the acromion, coracoid, and clavicle to its insertion on the greater tuberosity of the proximal humerus. The supraspinatus tendon was separated from adjacent tissues and detached sharply at its insertion on the greater tuberosity using a scalpel blade. The attachments of synergist muscles remained intact during the procedure. A 5-0 proline
suture with long tails was attached to the tendon stump to enable identification of the tendon end at sacrifice. The musculotendinous unit was allowed to freely retract without attempt at repair creating a gap approximately 4mm from its insertion. The overlying deltoid muscle and skin was then closed and the rats were allowed unrestricted cage activity. Rats were sacrificed at one, two, four, eight, and 16 weeks post-detachment (n = 4 each timepoint). The remaining four rats did not undergo tendon detachment, and served as uninjured controls for this study. All animal experiments were approved by the University of Pennsylvania’s Institutional Animal Care and Use Committee. Histological assessment of collagen and,fat inJiltration
At sacrifice, the supraspinatus tendon and muscle were exposed and removed for histological analysis. The wet weight of each muscle was measured, after which the muscles were surrounded in embedding medium (Tissue-Tek, Torrance, CA) and rapidly frozen in liquid nitrogen cooled isopentane. Muscles were stored at -80°C for subsequent analysis. Frozen cross-sections (10pmthick) were taken from the proximal, mid-belly and distal portions of the supraspinatus muscle. Masson’s trichrome was utilized to assess the presence of collagen infiltration and fibrosis. Oil red 0 staining was utilized to assess the presence of fat infiltration. Microscopy was performed on a Leitz DMR microscope (Leica Microsystems, Bannockburn, IL). Image acquisition and analysis was carried out using a MicroMAX digital camera system (Princeton Instruments, Inc., Trenton, NJ) and imaging software (OpenLab, Improvision, UK). Six high powered fields were analyzed per muscle cross-section to determine the proportion of collagen or fat infiltration in the muscles. For collagen content, high powered fields were acquired away from the central tendon, which could potentially obscure differences. These images were then quantitatively analyzed to determine the area of fibrotic tissue per high powered field. The analysis was performed by applying a consistent threshold to each image and calculating the area of fibrotic tissue using the above imaging software (OpenLab, Improvision, UK). Immunohistochernistry Fiber size analysis. Immunostaining with polyclonal Laminin antibody (Neomarkers, Fremont, CA) was utilized to measure muscle fiber size distribution. Muscle cryosections (10pm thick) were incubated in 5%) bovine serum albumin (BSA) in PBS for 20min. and then incubated in the same solution plus anti-laminin (1:200) overnight at 4°C. After several washes in PBS, the sections were incubated in Rhodarnine-conjugated anti-rabbit (1:200) (Jackson lmmunoresearch Laboratories, Inc., West Grove, PA) in 5%BSA for 1 h a t room temperature. After several more washes in PBS, sections were mounted in aqueous mounting medium containing DAPI (Vector Laboratories, Burlingame, CA) to counterstain the nuclei. Stained sections were visualized on an epifluorescent microscope, as described above. For each muscle cross-section, six high power fields (HPF) were acquired for analysis. The number of fibers per HPF was counted to determine if there is a significant change in fiber size. For example, if the fibers decrease in size, then the number per HPF will increase. Fiber t.vpe composition. Immunohistochemistry was also used to determine myosin heavy chain composition [24]. Primary antibody dilutions were as follows: Type I myosin (BA-FS), 150; Type IIa myosin (SC-71), l:lO; Type IIb myosin (BF-F3), 1:3; Embryonic myosin (BF45). 150.FITC-conjugated, goat anti-mouse IgM antibodies and donkey anti-mouse IgG (H + L) (Jackson lmmunoresearch Laboratories, Inc., West Grove, PA) were used as secondary antibodies. Microscopy was performed on an epifluorescent microscope, as described above. Preliminary experiments revealed an asymmetric fiber type distribution across the supraspinatus muscle. Therefore, the fiber type compositions in the superficial and deep layers were analyzed independently, with images of five high powered fields acquired for each region. Statistical analysis
Between group differences were compared using an ANOVA followed by Fisher’s post-hoc test. Statistical significance was set at p < 0.05.
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Results
m,
All of the animals tolerated the surgery well. The animals initially favored their arms and shoulders for the first week after surgery, but appeared normal after that time. Gross inspection of the specimens at the time of dissection revealed that scar was present throughout the rotator cuff region. The musculotendinous units were retracted to the glenoid (approximately 4 mm) and scar filled the space between the bony insertion site and the tendon stump, Scar tissue adhesions to the acromion, infraspinatus, and subscapularis were also apparent. Less scarring was observed with time but the scar remained throughout all time points studied. Despite the scar tissue in all injured specimens, the end of the tendon stump could easily be identified by the marking suture attached during detachment surgery. Muscles exhibited a statistically significant loss in mass initially following tenotomy. By one week after tendon detachment the mass was 13% below that of uninjured controls (Fig. 1). Muscle mass remained decreased by this amount until four weeks. Consistent with the loss in muscle mass, fiber size was also significantly reduced at these time points, resulting in an increase in the number of fibers per HPF (Fig. 2). However, by eight weeks post-detachment, muscle mass returned to near-control levels, and this trend continued until 16 weeks after tendon detachment. In fact, by 16 weeks, muscle mass and fiber size were not significantly different from muscles isolated from age- and weight-matched control animals. There was no evidence of muscle repair as indicated by the lack of central nuclei in the fibers (data not shown). Therefore, rapid but transient muscle atrophy was observed in association with surgical detachment of the supraspinatus tendon.
800,
*
T
1
*
*
11 2
1
4
1-L16
Tim post teootomy (weeks)
Fig. 1. Mass of supraspinatus muscle after tendon detachment. Significant loss of muscle mass was observed by one week after tendon detachment. Mass remained at this level until four weeks after detachment, after which muscle mass returned to control values. Data (mean f SD) represents n = 4 muscles for each condition.
*
26 1
*
150.
100
-
50-
0, 0
I
2
4
8
16
Time post teootomy (weeks)
Fig. 2. Muscle fiber size analysis after tendon detachment. The number of fibers per high powered field (HPF) was utilized as an index of fiber size. A decrease in fiber size results in an increase in the number of fibers per HPF. Fiber size remained depressed until four weeks after tendon detachment. By sixteen weeks, there was no significant difference in the fiber size compared to images from untreated control muscles. Data (mean f SD) represents n = 4 muscles for each condition.
With prolonged periods of disuse, muscle can exhibit an increase in fibrotic tissue [5,18]. Cryosections were stained with Masson's trichrome to assess whether this occurred after tendon detachment. Intermuscular collagen could be observed in muscle sections throughout the period of tendon detachment (Fig. 3A). The proportion of fibrotic tissue in the muscles was significantly higher than controls at one, two, and four weeks post-detachment (Fig. 3B). At eight and 16 weeks post-detachment, there was no significant quantifiable difference in collagen content between control and detached muscles, even though fibrosis could be observed in these samples. In human rotator cuff injuries, there is a significant increase in fatty infiltration of the muscles [5]. Therefore, proportion of fat in the rat supraspinatus muscle was assessed after tendon detachment. Oil red 0 staining revealed a slight increase in the appearance of fat in the muscles from the eight and 16 week post-detachment group (Fig. 4), although the presence was not significantly different than control muscles. Lastly, muscle fiber type has been shown to shift toward faster fiber types when there is a lack of muscle activity [17,20,22]. For the control specimens, the deep layers contained a mixture of slow (MHC I) and fast oxidative (MHC IIalx) and fast glycolytic (MHC IIb) fibers, whereas the superficial layer was predominantly fast glycolytic fibers, and there were no slow fibers (Fig. 5, Table 1). In the superficial layer, there was a significant shift toward the fastest fiber type by two weeks after tendon detachment. Specifically, there was an increase in MHC IIb fibers, a comparable loss in the IIa/x population, and no appearance of MHC I fibers. The shift in fiber type was maintained for the entire length of the study. In the deep layers, there was also
E. R. Barton et al. I Journal of Orthopaedic Reseurch 23 (2005) 259-265
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I
1
Fig. 4. Assessment of fat infiltration after tendon detachment by Oil Red 0 staining. Intermuscular fat was detected in cross-sections of supraspinatus muscles after eight weeks detachment (panel B, black arrow) compared to control muscles (panel A), which had no apparent intermuscular fat. Scale bar, 100pm.
T
0
16
2
Time post tenotomy (weeks) Fig. 3. Assessment of collagen content after tendon detachment. (A) Representative images of muscles stained with Masson’s trichrome from control (left panel) and eight weeks (right panel) after tendon detachment showed increases in fibrosis (white arrows) between the muscle fibers. Scale bar, 100pm. (B) Image analysis of collagen content. The region of positive collagen staining in a high powered field (HPF) was utilized to determine collagen content. At one, two and four weeks after tendon detachment, there was a significant increase in collagen content compared to untreated control muscles. Data (mean f SD) represents n = 4 muscles for each condition.
a shift toward fast fiber types, which was detectable at two weeks after tenotomy. Specifically, a loss in MHC I fibers was reflected in an increase in the MHC IIa/x population. By 4 weeks, there was also an increase in the proportion of MHC IIb fibers and a further decrease in the MHC I fibers. However, the shift reversed between four and eight weeks after tenotomy, and by 16
Superfkial Layer MHC IIb
MHC I
weeks, the fiber type distribution in the deep layer was not significantly different than that found in control muscles. Therefore, tendon detachment resulted in a persistent shift toward fast fiber types in the superficial regions of the supraspinatus muscle, and a transient shift in the fiber type distribution in the deep layers of the detached muscle.
Discussion This study determined the histological modifications to the rat supraspinatus muscle that occur with time following tendon detachment. We hypothesized that tendon detachment would result in a decrease in muscle mass. Our results support this hypothesis in that the supraspinatus muscle underwent a rapid and statistically significant decrease in muscle mass and fiber size. However, the loss of muscle mass was not persistent, and by 16 weeks after detachment, muscle mass and fiber size returned to control levels. This transient loss in muscle mass suggests that there was a return of load to the muscle. Even though the muscle was in a significantly shortened state due to the separation of the tendon from its original insertion site, scar tissue adhesions to the
Deep Layer MHC IIb
MHC I
Fig. 5. Immunohistochemistry of myosin heavy chain (MHC) isoforms in the superficial (left panels) and deep (right panels) layers of the rat supraspinatus muscle. The distribution of MHC isoforms differed in the regions within the muscle, such that MHC 1 fibers were found only in the deep layers of the supraspinatus muscle. An antibody directed at laminin was used to outline the muscle fibers (in red). and antibodies which recognized specific MHC isoforms identified muscle fibers which were MHC IIA, Ilalx, and I (in green).
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263
Table 1 Myosin heavy chain composition in the supraspinatus muscle Time (weeks) Control 1 2 4 8 16
Deep MHC (YO)
Superficial MHC (Yn) IIb
IIdx
I
IIb
IIalx
68.6 f 0.7 72.5 f 5.2 82.9 ? 5.0' 78.7 f 3.0' 82.7 f 2.1' 86.9 f 1.1'
31.3 f 0.7 27.5 f 5.1 17.1 f 5.0' 21.3 f 3.0' 17.3 f 2.1' 13.1 f 1.1'
0 0 0 0 0 0
22.6 f 3.3 35.1 f 7.2 28.1 f. 3.6 42.5 f 2.7' 36.8 f. 3.4' 21.6 f 3.9
58.6 f 52.2 f 58.9 f 49.0 f 45.5 f 61.9 f
I 4.3 5.4 3.2 3.5' 6.1' 4.7
18.7? 1.0 12.7? 3.2' 13.0 k 2.0' 8.5 ? 1.7' 17.7 f 3.7 20.2 f 3.0
Data represents mean k SD for six high powered fields in each superficial and deep layer from n = 4 muscles per timepoint. p < 0.05 compared to control.
acromion, humerus, and other surrounding structures may have resulted in a return of load to the muscle and may have borne sufficient load to allow the muscle to regain mass. A similar timecourse of histological and functional changes have been observed in the tendon in this detachment model [ 121. While other potential mechanisms exist that could lead to transient effects of tendon detachment to muscle (and to the tendon), the high correlation between muscle and tendon remodeling and the scar formation leads us to conclude that the most likely basis for our observations are due to the return of load to the muscle-tendon complex via scar adhesions. The accumulation of fibrotic tissue and fat in the supraspinatus muscle is a problem that plagues individuals with rotator cuff injuries [6,11,14,23,29]. We hypothesized that there would be an increase in collagen and fat with time post-detachment. Histological evidence of increased collagen content was observed in the supraspinatus muscle by one week post-detachment, and began to decrease by eight weeks after tenotomy (Fig. 3). Although not addressed in this study, an increase in fibrotic tissue in muscle has been shown to be associated with a decrease in muscle function [7,20,21] and is thought to increase the passive tension of the musculotendinous unit after prolonged tendon rupture [15]. An increase in collagen can also have important implications to muscle function which may adversely affect the post-operative healing of the muscle. In other rodent models of muscle atrophy, such as hindlimb unloading, there is little significant collagen accumulation until after load has been returned 10 the muscle or if the muscle is stretched, which suggests that fibrosis can occur when muscles experience acute changes increases in load or length [181. It is also possible that the increase in collagen content at one week post-detachment is triggered by the onset of adhesion formation to adjacent structures, thereby changing load on the muscle. While not examined in this study, it is possible that further increases in collagen might be found after tendons are reattached, and the muscle is subjected to normal length and load. Future studies using this model are needed to determine whether collagen content increases after tendon repair.
In chronic tendon tears in humans, fat accumulation can occur within the muscle and can remain even after repair [11,14]. Despite a small increase in fat, there was not a significant amount of fat accumulation in the rat as in the human, nor was its appearance uniform across samples. No quantification of fat was pursued because changes on this scale are not detectable by such measures. The lack of significant fat accumulation is not surprising based on other unloading studies in the rat, where there was no fat accumulation in rat muscles even after 6 months of unloading [7]. Fat accumulation within muscle appears to be species dependent. The accumulation of fat after tendon detachment is readily apparent in the sheep and humans [4,11,14], and less apparent in the rabbit [1,9,10]. Consistent with these previous studies, we found only a small amount of fat even at 16 weeks post-detachment in the rat (Fig. 4). Fatty accumulation should not be considered a significant factor in the rat model, but the muscles should be analyzed to confirm this assessment for prolonged periods of tendon detachment and following tendon repair. While fatty infiltration is not a significant feature in the rat model and this is different from chronic tears in humans, there are other changes following detachment that are similar to humans. Specifically, there is retraction of the musculotendinous unit, an increase in musculotendinous stiffness [12,151 and an increase in passive tension of the musculotendinous unit [ 12,151. Furthermore, there is a temporary increase in fibrotic tissue and decrease in muscle mass. Therefore, the rat model is useful to investigate many hypotheses related to injury and repair of the rotator cuff, but it may not be an appropriate model for hypotheses related to fatty infiltration. However, the limited amount of fatty infiltration in the rat could lead to the identification of factors which are unique to the rat that prevent fat accumulation in situations in which fat increases normally arise in human patients. We also hypothesized that there would be a shift toward fast fiber types upon tendon detachment. This shift was observed in both the deep and superficial layers of the muscle, and was maintained only in the superficial layers (Table 1). Fiber type distribution is largely
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determined by the activity of the muscle. Without nerve stimulation, and in animals models subjected to denervation, fiber types shift toward fast populations [ 5 , 17,201. While not directly addressed in this study, the results presented here suggest that muscle recruitment ceased for at least four weeks after tendon detachment. However, after this period, motor nerve activity may have returned to the deep layers of the supraspinatus muscle, causing a shift back toward the control fiber type distribution in the deep layers only. This suggests that there was sufficient load on the muscle for the animal to recruit fibers in the deep portions of the supraspinatus. Because this is the only region which contains MHC I/slow fibers, it is most likely the region which is utilized for postural activity [21,22,24]. The fiber type distribution in the remaining regions of the supraspinatus suggests that the superficial region is normally used for high power output activities. Due to tendon detachment, high power output could not be achieved, and it is unlikely that powerful contractions could occur via adhesions to other structures. We examined muscle properties up to 16 weeks after tendon detachment, and it is possible that the fiber type distribution would return to control proportions over longer times. Observations of animal mobility revealed no apparent shifts in gait after one week that were indicative of movement inhibition. It is possible that other synergist muscles which remained intact in the rotator cuff region compensated for the loss of use of the supraspinatus muscle, although this possibility was not addressed in this study. The time course of changes in muscle atrophy in our study is similar to that found in other animal models. These changes reflect those found in the tendon within the same animal model where transient disorganization of the collagen and decreases in mechanical properties of the tendon were detected after tendon detachment [12]. In the rabbit, an increase in atrophy and a reduction in function of the muscle occurred after six weeks of tendon detachment, but muscle atrophy and function partially recovered with time [ 1,9]. The partial recovery of the muscle is thought to be a result of secondary attachment of the supraspinatus muscle to surrounding tissues, with a partial restoration of the contractile activity. The maximum atrophy of the muscle at four weeks post-detachment and the partial return of fiber types to normal found in this study are consistent with the muscle changes after tendon detachment in the rabbit model. However, while the trends in our results are similar to these studies, the reversal of muscle mass and fiber size is more pronounced in the rat model. This dissimilarity may be partially explained by the altered loading environment of the muscle resulting from scar tissue adhesion to the acromion, humerus and other surrounding structures in the rat model. Scar tissue adhesions to the acromion and humerus may play a crucial role in muscle loading following tendon detachment. However,
since the acromion is directly located above the supraspinatus tendon in the rat, unlike in the rabbit, acromial attachments are probably a more significant factor in the rat model. In other studies, prevention of scar tissue adhesions have been achieved by wrapping the end of the tendon with membranes composed of either polyvynilidine fluoride or a dura substitute [4,19]. This methodology can be adopted in the rat model in an effort to prolong the detached state and to test if adhesions to the acromion result in the recovery of muscle mass. The role the acromion plays in the supraspinatus muscle changes may be an important consideration for future studies. The limitations of the rat rotator cuff model have been discussed in a previous publication [12]. However, some limitations that are specific to muscle should be addressed. First, the muscle’s functional changes were not investigated. This is an initial study in which the goal was to characterize the model so that its applicability to the human condition could be assessed. The histologic and geometric changes of the human muscle are partially known which formed the basis for investigating these properties in this study. The findings of the current study imply that the functional capacity of the muscle will be reduced after tendon detachment. However, further study will be required to test this prediction. Second, scar tissue fills the space between the tendon and bone following tendon detachment. While this does not typically occur in human tendon tears, scar tissue adhesions to surrounding tissues do occur in humans like it does in this model. We did find changes that are consistent with the human condition, but the additional adhesions to bone may increase the load transfer between the bone, tendon, and muscle. The consequences of this are unknown, but they may result in a faster and more complete reversal in muscle properties. In order to refine this detachment model, the utilization of anti-fibrosis agents could help to determine if scar formation is responsible for the transient changes in the muscle. Finally, the absence of fat accumulation in the rat signifies a departure of the rat rotator cuff model from tendon tears in humans. These results suggest that endogenous factors exist in rat muscle but not present in human muscle which prevent fat accumulation, and that the rat model presents an experimental platform on which to identify these factors. Lastly, a sham operated control was not used in this study. The surgery by itself, even in the absence of tendon detachment, may result in some small changes in the properties of the muscle. However, it is unlikely that the surgery is responsible for all of the changes observed in the current study, since the sham has been shown to have a negligible effect on the tendon properties of the rat [12]. In summary, the muscle mass results presented in this study suggest that load has returned in some fashion, but the fiber type results suggest that the loading may
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be insufficient to maintain normal activity. The rapid decline in muscle mass after tendon detachment shows that the muscle is a very sensitive indicator of the state of tendon attachment. Future studies will investigate the effects of longer periods of tendon detachment on muscle function, as well as the mechanisms through which these changes in muscle mass occur. Acknowledgments
Study supported in part by grants from the National Institutes of Health, Orthopaedic Research Foundation, and the Muscular Dystrophy Association. The authors thank J. Ellmer and J. Pollock for technical assistance in muscle preparation. References [l] Bjorkenheim JM. Structure and function of the rabbit’s supraspinatus muscle after resection of its tendon. Acta Orthop Scand 1989;60:461-3. [2] Carpenter JE, Thomopoulos S, Soslowsky LJ. Animal models of tendon and ligament injuries for tissue engineering applications. Clin Orthop 1999:S296311. [3] Cofield RH, Parvizi J, Hoffmeyer PJ, et al. Surgical repair of chronic rotator cuff tears. A prospective long-term study. J Bone Joint Surg Am 2001;83-A71-7. [4] Coleman SH, Fealy S, Ehteshami JR, et al. Chronic rotator cuff injury and repair model in sheep. J Bone Joint Surg Am 2003;85A:2391402. [5] Cros N, Tkatchenko AV, Pisani DF, et al. Analysis of altered gene expression in rat soleus muscle atrophied by disuse J Cell Biochem 200 1;83:508- 19. [6] Edwards TB, Boulahia A, Kempf JF, et al. The influmce of rotator cuff disease on the results of shoulder arthroplasty for primary osteoarthritis: results of a multicenter study. J Bone Joint Surg Am 2002;84-A:224@8. [7] Elder GC, McComas AJ. Development of rat muscle during short- and long-term hindlimb suspension. J Appl Physiol 1987;62: 1917-23. [8] Fabis J, Danilewicz M, Omulecka A. Rabbit supraspinatus tendon detachment: effects of size and time after tenotcimy on morphometric changes in the muscle. Acta Orthop Scand 200 1;72:2824. [9] Fabis J, Kordek P, Bogucki A, Mazanowska-Gajdowicz J. Function of the rabbit supraspinatus muscle after large detachment of its tendon: 6-week, 3-month, and 6-month observation. J. Shoulder Elbow Surg. 20009:2114. [lo] Fabis J, Kordek P, Bogucki A, et al. Function of the rabbit supraspinatus muscle after detachment of its tendon from the greater tubercle. Observations up to 6 months. Acta Orthop Scand 1998;69:57W. [ l l ] Fuchs B, Weishaupt D, Zanetti M, et al. Fatty degeneration of the muscles of the rotator CURassessment by computed tomo-
graphy versus magnetic resonance imaging. J Shoulder Elbow Surg 1999:8:599405. [I21 Gimbel JA, Van Kleunen JP, Mehta S, et al. Supraspinatus tendon organizational and mechanical properties in a chronic rotator cuff tear animal model. J Biomech 200437:73941. [I31 Gore DR, Murray MP, Sepic SB, Gardner GM. Shoulder-muscle strength and range of motion following surgical repair of fullthickness rotator-cuff tears. J Bone Joint Surg Am 1986; 68:26672. [I41 Goutallier D, Postel JM, Bernageau J, et al. Fatty muscle degeneration in cuff ruptures. Pre- and post-operative evaluation by CT scan. Clin Orthop 1994:78-83. Hersche 0, Gerber C. Passive tension in the supraspinatus musculotendinous unit after long-standing rupture of its tendon: a preliminary report. J Shoulder Elbow Surg 1998;7:393-6. Jamali AA, Afshar P, Abrams RA, Lieber RL. Skeletal muscle response to tenotomy. Muscle Nerve 2000;23:85142. Jiang B, Roy RR, Edgerton VR. Expression of a fast fiber enzyme profile in the cat soleus after spinalization. Muscle Nerve 1990;13:103749. Jozsa L, Thoring J, Jarvinen M, et al. Quantitative alterations in intramuscular connective tissue following immobilization: an experimental study in the rat calf muscles. Exp Mol Pathol 1988;49:267-78. Matsumoto F, Uhthoff HK, Trudel G, Loehr JF. Delayed tendon reattachment does not reverse atrophy and fat accumulation of the supraspinatus-an experimental study in rabbits. J Orthop Res 2002;20:357-63. Musacchia XJ, Steffen JM, Fell RD. Disuse atrophy of skeletal muscle: animal models. Exerc Sport Sci Rev 1988;16:61-87. Ohira Y, Jiang B, Roy RR, et al. Rat soleus muscle fiber responses to 14 days of spaceflight and hindlimb suspension. J Appl Physiol 1992;73:51%7S. Ohira Y, Yoshinaga T, Nomura T, et al. Gravitational unloading effects on muscle fiber size, phenotype and myonuclear number. Adv Space Res 2002;30:777-81. Praemer AFS, Rice D. Musculoskeletal conditions in the US. Park Ridge, IL: American Academy of Orthopaedic Surgeons; 1992. Schiaffino S, Reggiani C. Myosin isoforms in mammalian skeletal muscle. J Appl Physiol 1994;77:493-501. Soslowsky LJ, Carpenter JE, DeBano CM, et al. Development and use of an animal model for investigations on rotator cuff disease. J Shoulder Elbow Sum 1996:5:383-92. [26] Soslowsky LJ, Thomopoulos S, Esmail A, et al. Rotator cuff tendinosis in an animal model: role of extrinsic and overuse factors. Ann Biomed Eng 2002;30:105743. [27] Thomopoulos S, Hattersley G, Rosen V, et al. The localized expression of extracellular matrix components in healing tendon insertion sites: an in situ hybridization study. J Orthop Res 2002;20:454-63. [28] Thomopoulos S, Williams GR, Soslowsky LJ. Tendon to bone healing: differences in biomechanical, structural, and compositional properties due to a range of activity levels. J Biomech Eng 2003;125:10613. [29] Tillander B. Franzen L, Norlin R. MMP-I and histologic changes in rotator cuff disease. J Orthop Res 2002;20:1358-64. [30] Yao L, Mehta U. Infraspinatus muscle atrophy: implications? Radiology 2003;226:1614. I
Journal of Ort hopaedic Research
Journal of Orthopaedic Research 23 (2005) 259-265
www.elsevier.com/locate/orthres
Rat supraspinatus muscle atrophy after tendon detachment Elisabeth R. Barton
Jonathan A. Gimbel ’, Gerald R. Williams ‘, Louis J. Soslowsky
’
Department of Anatomy and Cell Biology, School of Denial Medicine, 441A Levy Building, 240 S. 40th Street, University of Peimsylvania. Philadelphia, PA, 19104, USA McKay Orthopaedic Research Laboratory, School of Medicine, University of Pennsylvania, Philadelphia, PA, USA a
Received 29 April 2004; accepted 9 August 2004
Abstract
Rotator cuff tears are one of the most common tendon disorders found in the healthy population. Tendon tears not only affect the biomechanical properties of the tendon, but can also lead to debilitation of the muscles attached to the damaged tendons. The changes that occur in the muscle after tendon detachment are not well understood. A rat rotator cuff model was utilized to determine the time course of changes that occur in the supraspinatus muscle after tendon detachment. It was hypothesized that the lack of load on the supraspinatus muscle would cause a significant decrease in muscle mass and a conversion of muscle fiber properties toward those of fast fiber types. Tendons were detached at the insertion on the humerus without repair. Muscle mass, morphology and fiber properties were measured at one, two, four, eight, and 16 weeks after detachment. Tendon detachment resulted in a rapid loss of muscle mass, an increase in the proportion of fast muscle fibers, and an increase in the fibrotic content of the muscle bed, concomitant with the appearance of adhesions of the tendon 10 surrounding surfaces. At 16 weeks post-detachment, muscle mass and the fiber properties in the deep muscle layers returned to normal levels. However, the fiber shifts observed in the superficial layers persisted throughout the experiment. These results suggest that load returned to the muscle via adhesions to surrounding surfaces, which may be sufficient to reverse changes in muscle mass. 0 2004 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved. Keywords: Supraspinatusmuscle; Chronic; Atrophy; Tendon: Shoulder
Introduction Rotator cuff tears of the shoulder represent a common and challenging orthopaedic problem [23]. While many of these tears are asymptomatic, detachment of the tendon can not only lead to pain, but can also lead to atrophy of the associated muscle, accumulation of fat in the areas normally occupied by muscle, and a subsequent loss in shoulder stability and function [13,15,16]. The reason for these changes may be a combination of * Corresponding author. Tel.: + I 215 573 0887; fax: + I 215 573 2324. E-mail address: [email protected] (E.R. Barton).
lack of muscle activity due to the pain of movement, and a lack of force transmission from the muscle to the bone and the potential shortened state of the muscle fibers due to tendon detachment. Subacromial decompression can be performed to remove pain and tendon repair can be performed to restore a strong connection between the bone, tendon, and muscle. However, the delay between the time of injury and surgical repair is thought to inhibit the healing process, resulting in weak, fatty muscles even after repair. Therefore, a more complete understanding of the muscle changes that occur with time from injury to repair is needed to improve the surgical outcome of chronic rotator cuff tears [3,13]. Significant progress toward understanding the changes in the rotator cuff muscle following tendon
0736-0266/$ - see front matter 0 2004 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.orthres.2004.08.018
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injury has been made utilizing a variety of approaches such as non-invasive CT and MRI measurements in patients [11,30]. In addition to these approaches, several animal models (e.g., rabbit and sheep) have been developed to study the changes in the muscle following supraspinatus tendon detachment [1,4,10,19]. In the rabbit, muscle atrophy increased and muscle function decreased with time after tendon detachment and fatty infiltration of the muscle occurred as early as 4 weeks after tendon detachment [l]. Similar results were found in more comprehensive studies using the same animal model [%lo]. More recently, the effect of a delayed repair on the fatty infiltration of the muscle after tendon repair has been studied. It was found that the supraspinatus muscle did not recover after tendon repair [19] and interestingly, fatty infiltration increased following repair. This suggests that although tendons are intact and load is restored, complete muscle repair is not established. While all models have specific advantages and disadvantages, the anatomy and function of the rat rotator cuff most closely resembles that of the human cuff (other than certain non-human primates). Specifically, the rat has an enclosed arch created by the acromion, coracoid, and clavicle, through which the supraspinatus tendon passes repetitively during shoulder motion [25]. Since the role of the acromion and the enclosed arch is believed to be critical and is at a minimum controversial in the pathogenesis of rotator cuff injuries, an animal model that includes this feature has certain advantages for rotator cuff injury and repair studies. This model has been used previously to address a variety of rotator cuff tendon injury and repair problems [26,28]. The objective of this study was to determine the time course of changes to the supraspinatus muscle in an established rat model of rotator cuff tendon detachment injury [25] to understand the nature of muscle atrophy following tendon detachment and to determine if the potential changes in rat muscle recapitulate those found in muscles from human patients. We hypothesize that tendon detachment would lead to a significant decrease in supraspinatus muscle mass, conversions in fiber properties toward those of fast fiber types, and an increase in the proportion of connective tissue within the muscle bed. Materials and methods Surgical injury model
A total of 24 Sprague-Dawley rats were used in this study. Twenty rats were operated upon bilaterally under general anesthesia to fully detach the supraspinatus tendon at its bony insertion site utilizing a previously described procedure [2,12,27]. Briefly, the supraspinatus was exposed and visualized as it passed under the bony arch created by the acromion, coracoid, and clavicle to its insertion on the greater tuberosity of the proximal humerus. The supraspinatus tendon was separated from adjacent tissues and detached sharply at its insertion on the greater tuberosity using a scalpel blade. The attachments of synergist muscles remained intact during the procedure. A 5-0 proline
suture with long tails was attached to the tendon stump to enable identification of the tendon end at sacrifice. The musculotendinous unit was allowed to freely retract without attempt at repair creating a gap approximately 4mm from its insertion. The overlying deltoid muscle and skin was then closed and the rats were allowed unrestricted cage activity. Rats were sacrificed at one, two, four, eight, and 16 weeks post-detachment (n = 4 each timepoint). The remaining four rats did not undergo tendon detachment, and served as uninjured controls for this study. All animal experiments were approved by the University of Pennsylvania’s Institutional Animal Care and Use Committee. Histological assessment of collagen and,fat inJiltration
At sacrifice, the supraspinatus tendon and muscle were exposed and removed for histological analysis. The wet weight of each muscle was measured, after which the muscles were surrounded in embedding medium (Tissue-Tek, Torrance, CA) and rapidly frozen in liquid nitrogen cooled isopentane. Muscles were stored at -80°C for subsequent analysis. Frozen cross-sections (10pmthick) were taken from the proximal, mid-belly and distal portions of the supraspinatus muscle. Masson’s trichrome was utilized to assess the presence of collagen infiltration and fibrosis. Oil red 0 staining was utilized to assess the presence of fat infiltration. Microscopy was performed on a Leitz DMR microscope (Leica Microsystems, Bannockburn, IL). Image acquisition and analysis was carried out using a MicroMAX digital camera system (Princeton Instruments, Inc., Trenton, NJ) and imaging software (OpenLab, Improvision, UK). Six high powered fields were analyzed per muscle cross-section to determine the proportion of collagen or fat infiltration in the muscles. For collagen content, high powered fields were acquired away from the central tendon, which could potentially obscure differences. These images were then quantitatively analyzed to determine the area of fibrotic tissue per high powered field. The analysis was performed by applying a consistent threshold to each image and calculating the area of fibrotic tissue using the above imaging software (OpenLab, Improvision, UK). Immunohistochernistry Fiber size analysis. Immunostaining with polyclonal Laminin antibody (Neomarkers, Fremont, CA) was utilized to measure muscle fiber size distribution. Muscle cryosections (10pm thick) were incubated in 5%) bovine serum albumin (BSA) in PBS for 20min. and then incubated in the same solution plus anti-laminin (1:200) overnight at 4°C. After several washes in PBS, the sections were incubated in Rhodarnine-conjugated anti-rabbit (1:200) (Jackson lmmunoresearch Laboratories, Inc., West Grove, PA) in 5%BSA for 1 h a t room temperature. After several more washes in PBS, sections were mounted in aqueous mounting medium containing DAPI (Vector Laboratories, Burlingame, CA) to counterstain the nuclei. Stained sections were visualized on an epifluorescent microscope, as described above. For each muscle cross-section, six high power fields (HPF) were acquired for analysis. The number of fibers per HPF was counted to determine if there is a significant change in fiber size. For example, if the fibers decrease in size, then the number per HPF will increase. Fiber t.vpe composition. Immunohistochemistry was also used to determine myosin heavy chain composition [24]. Primary antibody dilutions were as follows: Type I myosin (BA-FS), 150; Type IIa myosin (SC-71), l:lO; Type IIb myosin (BF-F3), 1:3; Embryonic myosin (BF45). 150.FITC-conjugated, goat anti-mouse IgM antibodies and donkey anti-mouse IgG (H + L) (Jackson lmmunoresearch Laboratories, Inc., West Grove, PA) were used as secondary antibodies. Microscopy was performed on an epifluorescent microscope, as described above. Preliminary experiments revealed an asymmetric fiber type distribution across the supraspinatus muscle. Therefore, the fiber type compositions in the superficial and deep layers were analyzed independently, with images of five high powered fields acquired for each region. Statistical analysis
Between group differences were compared using an ANOVA followed by Fisher’s post-hoc test. Statistical significance was set at p < 0.05.
.
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Results
m,
All of the animals tolerated the surgery well. The animals initially favored their arms and shoulders for the first week after surgery, but appeared normal after that time. Gross inspection of the specimens at the time of dissection revealed that scar was present throughout the rotator cuff region. The musculotendinous units were retracted to the glenoid (approximately 4 mm) and scar filled the space between the bony insertion site and the tendon stump, Scar tissue adhesions to the acromion, infraspinatus, and subscapularis were also apparent. Less scarring was observed with time but the scar remained throughout all time points studied. Despite the scar tissue in all injured specimens, the end of the tendon stump could easily be identified by the marking suture attached during detachment surgery. Muscles exhibited a statistically significant loss in mass initially following tenotomy. By one week after tendon detachment the mass was 13% below that of uninjured controls (Fig. 1). Muscle mass remained decreased by this amount until four weeks. Consistent with the loss in muscle mass, fiber size was also significantly reduced at these time points, resulting in an increase in the number of fibers per HPF (Fig. 2). However, by eight weeks post-detachment, muscle mass returned to near-control levels, and this trend continued until 16 weeks after tendon detachment. In fact, by 16 weeks, muscle mass and fiber size were not significantly different from muscles isolated from age- and weight-matched control animals. There was no evidence of muscle repair as indicated by the lack of central nuclei in the fibers (data not shown). Therefore, rapid but transient muscle atrophy was observed in association with surgical detachment of the supraspinatus tendon.
800,
*
T
1
*
*
11 2
1
4
1-L16
Tim post teootomy (weeks)
Fig. 1. Mass of supraspinatus muscle after tendon detachment. Significant loss of muscle mass was observed by one week after tendon detachment. Mass remained at this level until four weeks after detachment, after which muscle mass returned to control values. Data (mean f SD) represents n = 4 muscles for each condition.
*
26 1
*
150.
100
-
50-
0, 0
I
2
4
8
16
Time post teootomy (weeks)
Fig. 2. Muscle fiber size analysis after tendon detachment. The number of fibers per high powered field (HPF) was utilized as an index of fiber size. A decrease in fiber size results in an increase in the number of fibers per HPF. Fiber size remained depressed until four weeks after tendon detachment. By sixteen weeks, there was no significant difference in the fiber size compared to images from untreated control muscles. Data (mean f SD) represents n = 4 muscles for each condition.
With prolonged periods of disuse, muscle can exhibit an increase in fibrotic tissue [5,18]. Cryosections were stained with Masson's trichrome to assess whether this occurred after tendon detachment. Intermuscular collagen could be observed in muscle sections throughout the period of tendon detachment (Fig. 3A). The proportion of fibrotic tissue in the muscles was significantly higher than controls at one, two, and four weeks post-detachment (Fig. 3B). At eight and 16 weeks post-detachment, there was no significant quantifiable difference in collagen content between control and detached muscles, even though fibrosis could be observed in these samples. In human rotator cuff injuries, there is a significant increase in fatty infiltration of the muscles [5]. Therefore, proportion of fat in the rat supraspinatus muscle was assessed after tendon detachment. Oil red 0 staining revealed a slight increase in the appearance of fat in the muscles from the eight and 16 week post-detachment group (Fig. 4), although the presence was not significantly different than control muscles. Lastly, muscle fiber type has been shown to shift toward faster fiber types when there is a lack of muscle activity [17,20,22]. For the control specimens, the deep layers contained a mixture of slow (MHC I) and fast oxidative (MHC IIalx) and fast glycolytic (MHC IIb) fibers, whereas the superficial layer was predominantly fast glycolytic fibers, and there were no slow fibers (Fig. 5, Table 1). In the superficial layer, there was a significant shift toward the fastest fiber type by two weeks after tendon detachment. Specifically, there was an increase in MHC IIb fibers, a comparable loss in the IIa/x population, and no appearance of MHC I fibers. The shift in fiber type was maintained for the entire length of the study. In the deep layers, there was also
E. R. Barton et al. I Journal of Orthopaedic Reseurch 23 (2005) 259-265
262
I
1
Fig. 4. Assessment of fat infiltration after tendon detachment by Oil Red 0 staining. Intermuscular fat was detected in cross-sections of supraspinatus muscles after eight weeks detachment (panel B, black arrow) compared to control muscles (panel A), which had no apparent intermuscular fat. Scale bar, 100pm.
T
0
16
2
Time post tenotomy (weeks) Fig. 3. Assessment of collagen content after tendon detachment. (A) Representative images of muscles stained with Masson’s trichrome from control (left panel) and eight weeks (right panel) after tendon detachment showed increases in fibrosis (white arrows) between the muscle fibers. Scale bar, 100pm. (B) Image analysis of collagen content. The region of positive collagen staining in a high powered field (HPF) was utilized to determine collagen content. At one, two and four weeks after tendon detachment, there was a significant increase in collagen content compared to untreated control muscles. Data (mean f SD) represents n = 4 muscles for each condition.
a shift toward fast fiber types, which was detectable at two weeks after tenotomy. Specifically, a loss in MHC I fibers was reflected in an increase in the MHC IIa/x population. By 4 weeks, there was also an increase in the proportion of MHC IIb fibers and a further decrease in the MHC I fibers. However, the shift reversed between four and eight weeks after tenotomy, and by 16
Superfkial Layer MHC IIb
MHC I
weeks, the fiber type distribution in the deep layer was not significantly different than that found in control muscles. Therefore, tendon detachment resulted in a persistent shift toward fast fiber types in the superficial regions of the supraspinatus muscle, and a transient shift in the fiber type distribution in the deep layers of the detached muscle.
Discussion This study determined the histological modifications to the rat supraspinatus muscle that occur with time following tendon detachment. We hypothesized that tendon detachment would result in a decrease in muscle mass. Our results support this hypothesis in that the supraspinatus muscle underwent a rapid and statistically significant decrease in muscle mass and fiber size. However, the loss of muscle mass was not persistent, and by 16 weeks after detachment, muscle mass and fiber size returned to control levels. This transient loss in muscle mass suggests that there was a return of load to the muscle. Even though the muscle was in a significantly shortened state due to the separation of the tendon from its original insertion site, scar tissue adhesions to the
Deep Layer MHC IIb
MHC I
Fig. 5. Immunohistochemistry of myosin heavy chain (MHC) isoforms in the superficial (left panels) and deep (right panels) layers of the rat supraspinatus muscle. The distribution of MHC isoforms differed in the regions within the muscle, such that MHC 1 fibers were found only in the deep layers of the supraspinatus muscle. An antibody directed at laminin was used to outline the muscle fibers (in red). and antibodies which recognized specific MHC isoforms identified muscle fibers which were MHC IIA, Ilalx, and I (in green).
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263
Table 1 Myosin heavy chain composition in the supraspinatus muscle Time (weeks) Control 1 2 4 8 16
Deep MHC (YO)
Superficial MHC (Yn) IIb
IIdx
I
IIb
IIalx
68.6 f 0.7 72.5 f 5.2 82.9 ? 5.0' 78.7 f 3.0' 82.7 f 2.1' 86.9 f 1.1'
31.3 f 0.7 27.5 f 5.1 17.1 f 5.0' 21.3 f 3.0' 17.3 f 2.1' 13.1 f 1.1'
0 0 0 0 0 0
22.6 f 3.3 35.1 f 7.2 28.1 f. 3.6 42.5 f 2.7' 36.8 f. 3.4' 21.6 f 3.9
58.6 f 52.2 f 58.9 f 49.0 f 45.5 f 61.9 f
I 4.3 5.4 3.2 3.5' 6.1' 4.7
18.7? 1.0 12.7? 3.2' 13.0 k 2.0' 8.5 ? 1.7' 17.7 f 3.7 20.2 f 3.0
Data represents mean k SD for six high powered fields in each superficial and deep layer from n = 4 muscles per timepoint. p < 0.05 compared to control.
acromion, humerus, and other surrounding structures may have resulted in a return of load to the muscle and may have borne sufficient load to allow the muscle to regain mass. A similar timecourse of histological and functional changes have been observed in the tendon in this detachment model [ 121. While other potential mechanisms exist that could lead to transient effects of tendon detachment to muscle (and to the tendon), the high correlation between muscle and tendon remodeling and the scar formation leads us to conclude that the most likely basis for our observations are due to the return of load to the muscle-tendon complex via scar adhesions. The accumulation of fibrotic tissue and fat in the supraspinatus muscle is a problem that plagues individuals with rotator cuff injuries [6,11,14,23,29]. We hypothesized that there would be an increase in collagen and fat with time post-detachment. Histological evidence of increased collagen content was observed in the supraspinatus muscle by one week post-detachment, and began to decrease by eight weeks after tenotomy (Fig. 3). Although not addressed in this study, an increase in fibrotic tissue in muscle has been shown to be associated with a decrease in muscle function [7,20,21] and is thought to increase the passive tension of the musculotendinous unit after prolonged tendon rupture [15]. An increase in collagen can also have important implications to muscle function which may adversely affect the post-operative healing of the muscle. In other rodent models of muscle atrophy, such as hindlimb unloading, there is little significant collagen accumulation until after load has been returned 10 the muscle or if the muscle is stretched, which suggests that fibrosis can occur when muscles experience acute changes increases in load or length [181. It is also possible that the increase in collagen content at one week post-detachment is triggered by the onset of adhesion formation to adjacent structures, thereby changing load on the muscle. While not examined in this study, it is possible that further increases in collagen might be found after tendons are reattached, and the muscle is subjected to normal length and load. Future studies using this model are needed to determine whether collagen content increases after tendon repair.
In chronic tendon tears in humans, fat accumulation can occur within the muscle and can remain even after repair [11,14]. Despite a small increase in fat, there was not a significant amount of fat accumulation in the rat as in the human, nor was its appearance uniform across samples. No quantification of fat was pursued because changes on this scale are not detectable by such measures. The lack of significant fat accumulation is not surprising based on other unloading studies in the rat, where there was no fat accumulation in rat muscles even after 6 months of unloading [7]. Fat accumulation within muscle appears to be species dependent. The accumulation of fat after tendon detachment is readily apparent in the sheep and humans [4,11,14], and less apparent in the rabbit [1,9,10]. Consistent with these previous studies, we found only a small amount of fat even at 16 weeks post-detachment in the rat (Fig. 4). Fatty accumulation should not be considered a significant factor in the rat model, but the muscles should be analyzed to confirm this assessment for prolonged periods of tendon detachment and following tendon repair. While fatty infiltration is not a significant feature in the rat model and this is different from chronic tears in humans, there are other changes following detachment that are similar to humans. Specifically, there is retraction of the musculotendinous unit, an increase in musculotendinous stiffness [12,151 and an increase in passive tension of the musculotendinous unit [ 12,151. Furthermore, there is a temporary increase in fibrotic tissue and decrease in muscle mass. Therefore, the rat model is useful to investigate many hypotheses related to injury and repair of the rotator cuff, but it may not be an appropriate model for hypotheses related to fatty infiltration. However, the limited amount of fatty infiltration in the rat could lead to the identification of factors which are unique to the rat that prevent fat accumulation in situations in which fat increases normally arise in human patients. We also hypothesized that there would be a shift toward fast fiber types upon tendon detachment. This shift was observed in both the deep and superficial layers of the muscle, and was maintained only in the superficial layers (Table 1). Fiber type distribution is largely
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determined by the activity of the muscle. Without nerve stimulation, and in animals models subjected to denervation, fiber types shift toward fast populations [ 5 , 17,201. While not directly addressed in this study, the results presented here suggest that muscle recruitment ceased for at least four weeks after tendon detachment. However, after this period, motor nerve activity may have returned to the deep layers of the supraspinatus muscle, causing a shift back toward the control fiber type distribution in the deep layers only. This suggests that there was sufficient load on the muscle for the animal to recruit fibers in the deep portions of the supraspinatus. Because this is the only region which contains MHC I/slow fibers, it is most likely the region which is utilized for postural activity [21,22,24]. The fiber type distribution in the remaining regions of the supraspinatus suggests that the superficial region is normally used for high power output activities. Due to tendon detachment, high power output could not be achieved, and it is unlikely that powerful contractions could occur via adhesions to other structures. We examined muscle properties up to 16 weeks after tendon detachment, and it is possible that the fiber type distribution would return to control proportions over longer times. Observations of animal mobility revealed no apparent shifts in gait after one week that were indicative of movement inhibition. It is possible that other synergist muscles which remained intact in the rotator cuff region compensated for the loss of use of the supraspinatus muscle, although this possibility was not addressed in this study. The time course of changes in muscle atrophy in our study is similar to that found in other animal models. These changes reflect those found in the tendon within the same animal model where transient disorganization of the collagen and decreases in mechanical properties of the tendon were detected after tendon detachment [12]. In the rabbit, an increase in atrophy and a reduction in function of the muscle occurred after six weeks of tendon detachment, but muscle atrophy and function partially recovered with time [ 1,9]. The partial recovery of the muscle is thought to be a result of secondary attachment of the supraspinatus muscle to surrounding tissues, with a partial restoration of the contractile activity. The maximum atrophy of the muscle at four weeks post-detachment and the partial return of fiber types to normal found in this study are consistent with the muscle changes after tendon detachment in the rabbit model. However, while the trends in our results are similar to these studies, the reversal of muscle mass and fiber size is more pronounced in the rat model. This dissimilarity may be partially explained by the altered loading environment of the muscle resulting from scar tissue adhesion to the acromion, humerus and other surrounding structures in the rat model. Scar tissue adhesions to the acromion and humerus may play a crucial role in muscle loading following tendon detachment. However,
since the acromion is directly located above the supraspinatus tendon in the rat, unlike in the rabbit, acromial attachments are probably a more significant factor in the rat model. In other studies, prevention of scar tissue adhesions have been achieved by wrapping the end of the tendon with membranes composed of either polyvynilidine fluoride or a dura substitute [4,19]. This methodology can be adopted in the rat model in an effort to prolong the detached state and to test if adhesions to the acromion result in the recovery of muscle mass. The role the acromion plays in the supraspinatus muscle changes may be an important consideration for future studies. The limitations of the rat rotator cuff model have been discussed in a previous publication [12]. However, some limitations that are specific to muscle should be addressed. First, the muscle’s functional changes were not investigated. This is an initial study in which the goal was to characterize the model so that its applicability to the human condition could be assessed. The histologic and geometric changes of the human muscle are partially known which formed the basis for investigating these properties in this study. The findings of the current study imply that the functional capacity of the muscle will be reduced after tendon detachment. However, further study will be required to test this prediction. Second, scar tissue fills the space between the tendon and bone following tendon detachment. While this does not typically occur in human tendon tears, scar tissue adhesions to surrounding tissues do occur in humans like it does in this model. We did find changes that are consistent with the human condition, but the additional adhesions to bone may increase the load transfer between the bone, tendon, and muscle. The consequences of this are unknown, but they may result in a faster and more complete reversal in muscle properties. In order to refine this detachment model, the utilization of anti-fibrosis agents could help to determine if scar formation is responsible for the transient changes in the muscle. Finally, the absence of fat accumulation in the rat signifies a departure of the rat rotator cuff model from tendon tears in humans. These results suggest that endogenous factors exist in rat muscle but not present in human muscle which prevent fat accumulation, and that the rat model presents an experimental platform on which to identify these factors. Lastly, a sham operated control was not used in this study. The surgery by itself, even in the absence of tendon detachment, may result in some small changes in the properties of the muscle. However, it is unlikely that the surgery is responsible for all of the changes observed in the current study, since the sham has been shown to have a negligible effect on the tendon properties of the rat [12]. In summary, the muscle mass results presented in this study suggest that load has returned in some fashion, but the fiber type results suggest that the loading may
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E. R. Burron rt ul. I Journal of Orthopuediic Reseurcli 23 (200s) 259-265
be insufficient to maintain normal activity. The rapid decline in muscle mass after tendon detachment shows that the muscle is a very sensitive indicator of the state of tendon attachment. Future studies will investigate the effects of longer periods of tendon detachment on muscle function, as well as the mechanisms through which these changes in muscle mass occur. Acknowledgments
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