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The most common histological finding in carpal tunnel syndrome (CTS) is non-inflammatory fibrosis and thickening of the subsynovial connective tissue (SSCT) in the tunnel. While the cause of SSCT fibrosis and the relationship of SSCT fibrosis and CTS are unknown, one hypothesis is that SSCT injury causes fibrosis, and that the fibrosis then leads to CTS. We investigated the sensitivity of the SSCT to injuries. Two types of surgical intervention were performed in a rabbit model: a skin incision with tendon laceration and SSCT stretching sufficient to damage the SSCT, and skin incision alone,. Twelve weeks after surgery, the rabbit carpal tunnel tissues were studied with immunochemistry for TGF-β receptors 1, 2, and 3, collagen III, and collagen VI. All TGF-β receptors were expressed. The percentages of the TGF-β receptors’ expressions were less in the control SSCT fibroblasts than in the fibroblasts from rabbits with surgical interventions. The surgical interventions did not result in any alteration of collagen III expression. However, both surgical interventions resulted in a significant decrease in collagen VI expression compared to the control group. The two surgical interventions achieved similar expression of TGF-β receptors and collagens. Our results provide evidence that the SSCT is sensitive to surgical interventions, even when these are modest. Since SSCT fibrosis is a hallmark of carpal tunnel syndrome, these data also suggest that such fibrosis could result from relatively minor trauma.
Carpal tunnel syndrome (CTS) is the most common compression neuropathy, but in most cases the etiology is idiopathic.1,2 The compression can result from a reduction in size of the carpal tunnel or an increase in the volume of its contents. The most common pathological findings significantly favor the latter.3–8
The subsynovial connective tissue (SSCT) has a unique structure in the carpal tunnel,8–11 loosely connecting the median nerve, flexor tendons, and visceral synovium of the ulnar bursa. Non-inflammatory fibrosis and thickening of the SSCT are commonly identified in CTS patients.3–8,12 While the cause of SSCT fibrosis and its relationship to CTS are unknown, one hypothesis is that SSCT injury causes fibrosis, and that the fibrosis then leads to CTS.
To better understand the role of SSCT fibrosis as an etiologic factor for CTS, a rabbit model was developed.13 In this model, a shear injury of the SSCT is created by surgically stretching the SSCT beyond its elastic limit.14 Rabbits with this injury developed median neuropathy, similar to that seen in human CTS.15
We investigated the effects of this surgical intervention on the synthesis of TGF-β receptors, and collagens III and VI in the SSCT in this model in comparison to a sham procedure, in which the skin was incised but neither the tendon nor SSCT were injured. We hypothesized that the expression of TGF-β receptors and collagen would be affected by the SSCT shear injury, but not by the skin incision.
The experimental protocol was approved by our Institutional Animal Care and Use Committee. 18 New Zealand white rabbits weighing 3.6 to 4.2 kg were used. The rabbits were evenly divided into 2 groups for either skin incision alone or skin incision with tendon laceration and SSCT stretching.13 Nine other rabbits, without any intervention at their wrists, were obtained from other studies in our institution to serve as a normal control group.
Following the induction of anesthesia, both forepaws were scrubbed with povidone-iodine and sterilely draped. A rubber belt was used above the elbow as a tourniquet. For the skin incision with tendon laceration and SSCT stretching intervention (SSCT shear), a volar longitudinal incision (12 mm long) was centered 1 cm proximal to the proximal edge of the wrist cartilage of one randomly selected forepaw, and the flexor digitorum superficialis (FDS) tendon of the middle digit was exposed. After identifying the level of the muscle-tendon junction of the middle digit FDS and marking the flexor carpi ulnaris (FCU) tendon using a 6-0 Prolene (Ethicon, Somerville, NJ) suture to identify the relative position of the middle digit FDS tendon, the middle digit FDS tendon was cut at the muscle-tendon junction, and the distal end of the middle digit FDS was also marked using 6-0 Prolene. Then, a volar longitudinal incision (11 mm long) was made on the 3rd digit centered at the metacarpophalangeal joint level to expose the flexor tendons and proximal annular pulley. Two marks separated by 5 mm were made on the surface of the middle digit FDS tendon in this 2nd incision. Then the 2 tendon marks were approximated and sutured together using 5-0 Ethibond (Ethicon, Somerville, NJ). Thus, the middle digit FDS tendon was distally shifted 5 mm (Fig. 1). The incisions were closed with continuous subcuticular 4-0 Ethibond. For the skin incisions alone group, the distal and proximal contralateral 3rd FDS tendon was similarly exposed, but the tendon was not cut or advanced.
After surgery, a sterile dressing was applied to each paw. An Elizabethan collar was applied for 2 wks to prevent chewing of the incisions. The rabbits were allowed full cage activity. After the wounds had healed (2 to 3 wks post-op), rabbits were allowed 30 mins to 1 hr of exercise outside their cage 2 to 3 times/wk until euthanasia. The exercise was intended to allow the rabbits to perform a full range of motion of the forepaws, and thereby achieve sufficient tendon excursion to affect the SSCT. All rabbits were euthanized at 12 weeks.
After removing the skin, the fore paw was placed in 1% glutaraldehyde and 4% formaldehyde in 0.1 M phosphate buffer, pH 7.2 for 48 hrs for primary fixation. Then, the carpal tunnel tissues were dissected and immersed into 10% formalin for secondary fixation. The fixed tissues were then dehydrated, paraffin embedded, and sectioned at 5 µm thickness. The sections were de-paraffinized with xylene, rehydrated in graded ethanol, and then incubated in 3% H2O2 at room temperature for 10 mins. The sections were blocked with 1.5% normal horse serum for 1 hr and incubated with anti-TGF-β-RI (Santa Cruz sc-398-G, Santa Cruz, CA), TGF-β-RII (Santa Cruz sc-17792), TGF-β-RIII (Santa Cruz sc-6199), collagen III (Acris AF5810, clone III-53, Hiddenhausen, Germany), or collagen VI (Acris AF6210) at 4°C overnight. After extensive washing with PBS, the sections were incubated with the biotinylated secondary antibody and Vectastain Elite ABC reagent (Vector Labs, Burlingame, CA). Visualization was achieved with AEC substrate (Vector Labs). The slides were counterstained with hematoxylin QS (Vector Labs).
Analysis of TGF-β receptors was performed by measuring the percentage of fibroblasts expressing TGF-β receptors. Fibroblasts with positive and negative staining were counted in each sample from the region of SSCT surrounding the middle digit FDS tendon. The total cell count/sample ranged from 33 to 358 (mean of 143). The intensity of collagen III/VI staining was classified as not stained (grade 0), mildly stained (grade 1), moderately stained (grade 2), or intensely stained (grade 3). Three independent observers blinded to the origin of the specimens graded the intensity of immunostaining for the collagen types.
Comparisons of collagen stain intensities, percentage of fibroblasts positively stained for TGF-β receptors among the 3 groups (control, skin incision, and SSCT shear) were analyzed by one-way ANOVA. Student’s t-test was used as a post-hoc test. Significance was set at p<0.05. All analyses were performed with JMP 8 (SAS Institute Inc., Cary, NC).
As in human SSCT, collagen III and VI were identified in the rabbit SSCT (Fig. 2). Collagen III staining had a mean grade of 1.87±0.36 in the control group, 1.81±0.31 in the skin incision group, and 1.82±0.31 in the SSCT shear group (Fig. 3); these differences were not significant. A significant decrease (p < 0.01) occurred in collagen VI staining in both surgical interventions compared to controls (1.79±0.27) (Fig. 4). There was no significant difference in collagen VI staining between the skin incision (0.93±0.36) and SSCT shear (1.16±0.32) groups.
TGF-β receptors 1, 2, and 3 (R1, R2, R3) were detected in the SSCT fibroblasts of all rabbits (Fig. 5). Surgical interventions significantly (p < 0.04) increased the percentage of TGF-β-R1 positive SSCT fibroblasts compared to controls (16.0±8.8%, Fig. 6). The percentages of TGF-β-R1 positive fibroblasts in the SSCT of the skin incision and SSCT shear groups were 42.8±33.8% and 52.2±22.6%, respectively; this difference was not significant.
The surgical interventions increased the percentage of fibroblasts with positive TGF-β-R2 expression compared to controls (Fig. 6), however, significance was not achieved (p = 0.13 and 0.14 for SSCT shear group and skin incision group, respectively). No significant difference was found in TGF-β-R2 staining between the skin incision and SSCT stretching groups.
As with TGF-β-R1, the percentage of fibroblasts with positive TGF-β-R3 staining significantly increased with both surgical interventions (p < 0.03) compared to controls (Fig. 6), but the difference between the two surgical intervention for each TGF-β receptor staining was not significant.
Repetitive, forceful hand or wrist motion, often associated with awkward wrist posture, is a major risk factor for CTS.16–18 Such activity could damage the SSCT from an increased excursion of the flexor tendon that exceeds the tolerance of the SSCT or from a poorly coordinated motion of neighboring digits causing an over stretch of the intervening SSCT.
The intervention of SSCT shear in this study shifted the FDS tendon 5 mm distally, similar to the normal tendon excursion.19 However, this amount of excursion resulted in damage to the SSCT in a rabbit cadaver model,14 and produces fibrosis in vivo.13 These SSCT findings are similar to the SSCT fibrosis seen in clinical cases of CTS.
We chose to study collagens III and VI and TGF-β receptor 1 because they have been identified in human SSCT and change when comparing normal SSCT and the SSCT of individuals with CTS.8,10 A significant increase in TGF-β receptor 1 expression occurs in the fibroblasts of CTS patients compared with unaffected individuals, and collagen III is significantly more abundant in CTS patients than in controls. In addition, collagen VI is abundant in the SSCT of both CTS patients and unaffected individuals.
TGF-β plays a vital role in mediating extracellular matrix deposition and remodeling, which occur in tissue repair and fibrosis.20,21 TGF-β signals through TGF-β receptors, which are essential for the biological activity of TGF-β.22–25 Three major receptors (TGF-β-R1, TGF-β-R2, and TGF-β-R3) form part of the larger TGF-β superfamily of receptors. R1 and R2 are activated following engagement by members of the TGF-β superfamily of ligands. R3 promotes TGF-β ligand binding to the signaling receptors, such as R1 and R2.
TGF-β receptors are increased in wound healing and fibrosis.26,27 R1, R2, and R3 protein levels at the repair site were up-regulated in transected and repaired rabbit flexor tendons.28 R1 and R2 receptors returned to normal level by day 56 postop, but R3 production remained high. In contrast to the flexor tendon, the surgical interventions in our study resulted in significant increases in the R1 and R3 staining in the rabbit SSCT 12 wks after surgery, suggesting either a longer healing process than in the flexor tendon or, as we hypothesized, an ongoing process of injury and healing due to repetitive injury of the SSCT following an initial insult.
The percentage of fibroblasts with positive R1 staining in the SSCT of control rabbits (16.0±8.8%) is similar to that detected in normal human SSCT (19.5±15.0%).8 Both surgical interventions resulted in a significant increase in the percentage of fibroblasts with R1 staining in the rabbit SSCT, to 42.8±33.8% and 52.2±22.6%, respectively. These are less, though, than the rate of positive R1 staining of 62.7±20.9% seen in the SSCT of patients with CTS.8
Collagen III expression changed in a time-dependent fashion during the highly regulated process of wound healing.29,30 Collagen III characteristically constitutes the early matrix in wound repair. Collagen III expression is elevated in the SSCT of CTS patients.8 In contrast to the clinical situation, the surgical interventions in our study did not increase collagen III expression, possibly due to timing: at 3 mos, the injury may be sufficiently healed so collagen III synthesis is no longer elevated.
Collagen VI is a structural component within interstitial connective tissues31,32 and serves as an important mediator for cell adhesion,33 cell survival,34 cell proliferation,35 the activation and activity of matrix-metalloproteinases,36 and tissue remodeling.32,34,36,37 Collagen VI also plays a significant contributory role in the complex mechanisms of disordered matrix protein disposition leading to arthrofibrosis.38 Collagen VI expression is also regulated in a time-dependent fashion after injury.39–41 Expression increases during wound healing, reaching its maximum 2 wks after insult. In the late phase of healing, a reduction of collagen VI is observed. Collagen VI is normally present in human SSCT.8,10 In our study, a significant decrease of collagen VI staining was found. Again, with a single time point it is impossible to be certain that collagen VI levels were different at earlier times; but certainly this alteration in collagen VI staining suggests an effect on normal SSCT structure.
Two surgical interventions were performed in our study. The FDS tendon was shifted 5 mm distally in the SSCT shear intervention. The 2nd intervention, skin incision only, was envisioned to be a sham intervention. However, contrary to our hypothesis, both surgical groups appeared to have a similar impact on expression of fibrosis-associated proteins in the SSCT, suggesting that this structure can be quite sensitive to even minor trauma.
The importance of this study lies in the finding of the sensitivity of SSCT to surgical interventions. This lends credence to the hypothesis that relatively minor trauma might be responsible for the SSCT fibrosis seen typically in clinical CTS cases. However, this study has several weaknesses. Specifically, only one time point was investigated. More meaningful knowledge could be achieved if more time points were studied. But we were reluctant to sacrifice large numbers of animals without knowing if some lasting effect from the experimental intervention was possible. We now plan to do both shorter and longer term interventions. Also, a limited number of proteins were investigated, although they included important proteins altered in the SSCT of CTS patients.8,42,43
We conclude that SSCT is sensitive to surgical interventions, even those which do not directly occur in the carpal tunnel. These findings may be useful in future studies of the etiology of CTS.
This study was funded by a grant from NIH (NIAMS AR 49823).
None of the authors have any financial conflicts of interest.