We retrospectively reviewed the charts of patients who had had synovial
biopsy specimens sent to our Department of Laboratory Medicine and Pathology
during carpal tunnel release between January 1999 and December 2001. The study
was approved by our institutional review board. The medical history of each
patient was abstracted to identify patients with a clinical diagnosis of
idiopathic carpal tunnel syndrome. Patients with a history of diabetes,
glucose intolerance, thyroid disease, rheumatoid arthritis, osteoarthrosis,
degenerative joint disease, flexor tendinitis, gout, hemodialysis, a body mass
index of >30, sarcoidosis, amyloidosis, peripheral nerve disease, or
traumatic injuries to the ipsilateral arm were excluded, leaving thirty
consecutive patients with presumed idiopathic carpal tunnel syndrome. A total
of fifty-nine charts were reviewed. Of the fifty-nine patients, twenty-one
were excluded because they did not have idiopathic carpal tunnel syndrome, and
eight others were excluded because they were younger (mean age, 35.3 years;
range, twelve to thirty-seven years) than the control population, leaving a
total of thirty patients with idiopathic carpal tunnel syndrome.
There were twelve women and eighteen men with a mean age of 65.3 years
(range, 38.9 to 93.4 years). Five patients had involvement of the left hand;
nine, involvement of the right hand; and sixteen, bilateral involvement. The
tissue was obtained from thirteen left hands and seventeen right hands. The
tissue was obtained from the dominant hand of fifteen patients and from one
patient whose hand dominance had not been recorded. The tissue was the result
of a limited synovectomy in two patients (one endoscopic procedure and one
open release, resulting in 3 and 4 cm3 of tissue, respectively) and
of a synovial biopsy in the rest (resulting in 0.5 to 1.0 cm3 of
tissue). The limited synovectomies were done because, in the opinion of the
surgeon, synovial bulk was impinging on the median nerve, even after release.
The biopsies were performed as part of the routine practice of one surgeon and
to rule out other pathological conditions, such as amyloidosis (tissue from
ten patients was stained with Congo red, all with negative findings), in the
case of other surgeons.
Demographic data, including age, gender, hand dominance, occupation, side
of involvement, and side on which the tissue was obtained, were recorded for
the thirty study patients. The results of the Tinel, Phalen, and carpal tunnel
compression tests, measures of sensibility and strength, and any notation of
thenar atrophy were also sought from the medical records of each patient. The
duration of symptoms (in months) before treatment and the presence of symptoms
such as paresthesias, numbness, and pain in the hand or wrist were also
recorded. Evaluation was aided considerably by employing a standardized hand
evaluation form, in use at our institution since the late 1970s, which
included data fields for all of the above information. Twelve patients had
thenar atrophy and fifteen patients had no thenar atrophy in the hand included
in the study; the presence or absence of thenar atrophy was not documented for
the remainder of the hands. The mean body mass index was 25.8. The mean
duration of symptoms was 28.1 months (range, one to 121.7 months). Nine
patients had had an open carpal tunnel release, and twenty-one had had an
endoscopic carpal tunnel release.
All patients had had diagnostic neurophysiological testing, including
electromyography and nerve conduction studies performed according to the
standards of the American Association of Electrodiagnostic
Medicine16,17,
and the diagnosis of carpal tunnel syndrome was confirmed in each case. The
severity of the carpal tunnel syndrome was determined with use of the Mayo
Clinic scale for neurophysiologic severity of carpal tunnel
syndrome18.
According to these criteria, three patients had mild, fourteen had moderate,
and thirteen had severe carpal tunnel syndrome.
The control group consisted of ten fresh-frozen cadavers from which
subsynovial connective tissue of the ulnar bursa of the carpal tunnel was
obtained. In order to have a close age match with the patient group, we
selected the youngest cadavers available. A total of twenty-six charts were
reviewed to select the cadaver control group. Five subjects were excluded
because a history of carpal tunnel syndrome was noted in the medical record.
Two were excluded because diabetes mellitus was noted in the record and five,
because hypothyroidism was noted (the same exclusion criteria as used in the
patient group). Four were excluded because of old age (mean, 89.5 years;
range, eighty-four to ninety-five years). This left ten cadaver controls
without a history of carpal tunnel syndrome.
The cadavers of six women and four men were included in the control group.
The mean age at the time of death was 75.3 years (range, 67.0 to 82.2 years),
and the mean body mass index was 23.8. The control tissue was obtained from
four left hands and six right hands. Information regarding hand dominance was
not available. Roughly 1 cm3 of synovial tissue was obtained from
each cadaver.
Histological and Immunohistochemical Analysis
For all patients, an initial pathological analysis was performed on frozen
sections at the time of surgery, and all remaining tissue was formalin-fixed
and paraffin-embedded. For the purposes of this study, 5-µm-thick sections
were made from these paraffin blocks. Standard hematoxylin and eosin staining
methods were used. The distribution of collagen types I, II, III, and VI was
investigated with use of monoclonal anti-collagen types I, II, and VI (mouse
IgG1 isotype; Medicorp, Montreal, Quebec, Canada) and monoclonal anti-collagen
type III (mouse IgG1 isotype; BioGenex, San Ramon, California). Anti-human and
TGF-ß RI (rabbit IgG) and TGF-ß RII (rabbit IgG) and TGF-ß RIII
(goat IgG) polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz,
California) were used to stain for TGF-ß receptor subtypes. Biogenex
negative control sera/immunoglobulins were used with each specimen to evaluate
nonspecific staining.
In each staining run, we used paraffin-embedded positive controls. For
collagen type I, this control consisted of a human flexor digitorum profundus
tendon of the long finger in the carpal tunnel from a cadaver; for collagen
type II, human cartilage; for collagen type III, human tissue from a skin
wound closed by secondary intention twenty-one days postoperatively; for
collagen type VI, human skin tissue; and for TGF-ß RI, RII, and RIII,
human skin tissue.
One of the authors, who was not involved in the treatment of any of these
patients, measured the size of twenty randomly chosen transversely or
longitudinally cut collagen fibers on each slide of subsynovial connective
tissue stained with hematoxylin and eosin in the patient and control groups.
The mean size of the collagen fibers was calculated from these
measurements.
Fibroblast density was measured by counting the number of fibroblasts in
five randomly chosen areas on each slide. A light microscope with a ruler,
with the magnification set at 40×, was used to mark the areas by turning
the ruler 360° so that all of the areas were the same size. The microscope
was calibrated with a scale division of 1:0.0235 mm for the 40×
objective. The total surface area in which we counted the number of
fibroblasts was thus 0.0434 mm2.
Three independent observers, blinded to the origin of the specimens, graded
the intensity of immunostaining for the collagen types in both groups. We
randomly chose eight different areas with the magnification set at 20×.
In each of these areas, we classified the intensity of staining as not stained
(grade 0), mildly stained (grade 1), moderately stained (grade 2), or
intensely stained (grade 3).
Analysis of TGF-ß RI staining was done by measuring the percentage of
fibroblasts with TGF-ß RI expression in eight different randomly chosen
areas. The areas were marked by using the total area seen with the microscope
with the 20× objective.
Statistical Methods
The information was entered into a database, and statistical data analysis
was done with the SPSS software program, version 10.0 for Windows (SPSS,
Chicago, Illinois). Two-sample t tests were used to compare continuous
variables between the
groups19. The
proportions of specimens exhibiting the various grades of collagen were
compared between the groups with use of the exact Wilcoxon test for ordered
categorical data20.
The Spearman rank-order
method21 was used
for analysis of correlation among patient age, duration of symptoms, severity
of the carpal tunnel syndrome, and the histological and immunohistochemical
data.
All statistical tests were two-sided, and p values of <0.05 were
considered significant. Results are reported as the mean and standard
deviation unless otherwise indicated.
Histological Analysis
The tissue obtained during surgery included the subsynovial sheet of the
ulnar bursa in all cases. All specimens had initially been given a
pathological diagnosis of noninflammatory fibrous tissue. Compared with the
tissue in the control group, all tissue specimens from the patients showed
vascular proliferation and vascular hypertrophy with intimal thickening.
The mean fibroblast count in the measured area (0.0434 mm2) was
51.9 ± 16.3 in the specimens from the patients with carpal tunnel
syndrome and 31.3 ± 9.3 in the control specimens. The mean fibroblast
density was 1195 ± 374/mm2 in the specimens from the
patients with idiopathic carpal tunnel syndrome and 721 ±
215/mm2 in the control group (p < 0.001).
The mean size of the collagen fibers in the subsynovial connective tissue
was estimated to be 6.59 ± 3.43 µm in the specimens from the
patients with carpal tunnel syndrome and 3.80 ± 1.51 µm in the
control specimens (p < 0.001) (Figs.
2-A and
2-B).
Collagen Typing
No specific collagen type-I staining, except for immunostaining localized
within a few small blood vessels (Fig.
3-A), was seen in the subsynovial connective tissue from either
the patients or the controls. There was also no specific collagen type-II
staining of the subsynovial connective tissue from either group.
The results of the collagen type-III staining showed a mean grade of 1.41
± 1.04 in the patient group, which was significantly higher than the
grade in the control group (0.35 ± 0.62) (p < 0.001). One (3%) of
the patients had grade-0 staining; eighteen (60%), grade-1; seven (23%),
grade-2; and four (13%), grade-3. In the control group, eight specimens had
grade-0 staining and two had grade-1. Collagen type III was randomly present
within the tissue and was not concentrated around vessels.
Figure 3-B shows the
immunohistochemical staining for collagen type III in a flexor tendon within
the subsynovial connective tissue of one of our patients at 4×
magnification. The subsynovial connective tissue in this image shows specific
staining for collagen type III in the area under the tendon.
The results of the collagen type-VI staining showed a mean grade of 2.16
± 0.83 in the patient group compared with 2.52 ± 0.67 in the
control group (p = 0.213). There were five patients (17%) with grade-1
staining, fifteen (50%) with grade-2, and ten (33%) with grade-3. In the
control group, there was one specimen with grade-1 staining, three with
grade-2, and six with grade-3. Figure
3-C shows immunohistochemical staining for collagen type VI in a
human flexor tendon within the subsynovial connective tissue of one of our
patients at 4× magnification. The subsynovial connective tissue in this
image shows specific staining for collagen type VI as marked by the red color.
Immunohistochemical staining for collagen type VI showed strong mural staining
of small and intermediate-size muscular arteries within the adventitial and
internal elastic lamina and was also seen throughout the subsynovial
connective tissue.
TGF-ß
The results of the TGF-ß RI staining showed expression in the
endothelial cells and in the fibroblasts of all specimens of subsynovial
connective tissue from the patients and the controls; however, there was a
markedly higher number of fibroblasts with expression of TGF-ß RI in the
patient group than in the control group. The mean percentage of TGF-ß RI
expression in eight randomly chosen areas in specimens from the patients was
62.7% ± 20.9%, whereas it was 19.5% ± 15.0% in the control group
(p < 0.001). Figure 4 shows
immunohistochemical staining of TGF-ß RI within the endothelial cells and
fibroblasts in subsynovial connective tissue from a patient.
TGF-ß RII was seen in the endothelial cells, endovascular smooth
muscle, and a few fibroblasts of the subsynovial connective tissue from the
patients, but it was seen only within the endothelial cells of the control
tissue. TGF-ß RIII was found in the endothelial cells within the
subsynovial connective tissue of both the patients with carpal tunnel syndrome
and the controls. We found it too difficult to use a grading system for the
analysis of TGF-ß RII and TGF-ß RIII. Our positive control tissue
showed staining for TGF-ß RI and RIII within the epidermis. We found
staining for TGF-ß RII of the basement membrane of the skin.
Association of Pathological Findings with Severity of Carpal Tunnel
Syndrome
With the numbers available, we found no correlation between the severity of
the carpal tunnel syndrome and the duration of symptoms, fibroblast density,
collagen size, or grades of collagen types I, II, III, and VI. There was also
no correlation between the duration of symptoms and the fibroblast density,
collagen size, or grades of collagen types I, II, III, and VI or between age
(patient or control) and fibroblast density, collagen size, or grades of
collagen types III and VI.
There was a significant positive correlation between patient age and the
severity of the carpal tunnel syndrome (correlation coefficient, 0.591; p <
0.01). There was also a significant association between the presence of
TGF-ß RI and collagen size (p < 0.001), fibroblast count (p <
0.001), fibroblast density (p < 0.001), and the presence of collagen type
III (p < 0.05).
We hypothesized that, if there were an insult to the subsynovial connective
tissue in patients with carpal tunnel syndrome, there should be some
histological and immunohistochemical evidence of it, and that these findings
would not be present in unaffected individuals. This injury could be
mechanical, as postulated by
Guimberteau11, or
ischemia-reperfusion, as suggested by Freeland et
al.22. The vascular
proliferation and vascular hypertrophy with intimal thickening, the
significant increase in fibroblast density and collagen fibril size, and the
presence of collagen type III in the subsynovial connective tissue in the
specimens from our patients with idiopathic carpal tunnel syndrome all support
this hypothesis of injury.
The histological findings in the tissue from the patients compared with
those in the control tissue support the findings of previous histological
analyses of carpal tunnel
syndrome8,10,23-29.
The difference between our study and others is the analysis of collagen type,
fiber size, and TGF-ß. The distribution of the different collagen types
provides information about the structure of the subsynovial connective tissue
and, perhaps, about the pathophysiology of carpal tunnel syndrome. For
example, as collagen type III is characteristic of an injury response and is
weaker than collagen type
I30, it could
predispose to a vicious cycle of further injury.
Collagen type I is the major component of tendon, but there was no positive
staining for collagen type I in the subsynovial connective tissue from either
the patients or the controls. Instead, collagen type VI seems to be a major
component of the subsynovial connective tissue. This is a new and potentially
important finding as, again, collagen type VI does not have the strength of
collagen type I and could be more susceptible to injury.
Additional immunohistochemical analysis should be performed to identify
other components of the subsynovial connective tissue. Collagen types V and XI
are also fibrillar
collagens31 and
might be present in the subsynovial connective tissue. There was an increase
in collagen fiber size in our patients relative to the size in the control
group. These changes could affect the material properties or permeability of
the synovium, as has been suggested by Freeland et
al.22. Those
changes may, in turn, contribute to the elevation in carpal tunnel pressure
seen in patients with carpal tunnel syndrome.
Fibroblasts play important roles in granulation tissue formation, wound
contraction, matrix synthesis, wound repair, and scar
formation32-34.
Our data confirmed that the fibroblasts express TGF-ß receptor isoforms
and thus would be responsive to TGF-ß signaling within the subsynovial
connective tissue. The finding of greater amounts of TGF-ß RI in the
fibroblasts within the noninflammatory subsynovial connective tissue of the
patients than in the control tissue supports our hypothesis that there is a
wound-healing process in the subsynovial connective tissue of these
patients35-42.
The increase in TGF-ß RI expression in the fibroblasts within the
subsynovial connective tissue in our patient group also supports our
hypothesis that this process leads to scarring and fibrosis and may thus play
a role in the etiology of carpal tunnel syndrome.
The strength of this study lies in the systematic histological analysis of
the subsynovial connective tissue from individuals with and without carpal
tunnel syndrome. The weaknesses are those inherent in any retrospective study.
Specifically, the synovial tissue that was available from patients with carpal
tunnel syndrome was not collected routinely or randomly but from specific
patients (thirty of more than 1000 treated with carpal tunnel release at our
institution over the three-year study period) in whom a synovectomy was
performed for reasons other than to treat a comorbid condition, such as
rheumatoid arthritis. The specimens that were available to us may therefore
not be representative. In addition, we did not have complete histories on the
activity of these patients; thus, we cannot make any comment about whether the
activity level of the patients with carpal tunnel syndrome was in any way
different from that of the control individuals.
In conclusion, we found evidence of histological and immunohistochemical
changes in the subsynovial connective tissue of patients with carpal tunnel
syndrome that were different from those seen in individuals of similar age but
without carpal tunnel syndrome. The findings were similar to those seen after
injury in other tissues and therefore are consistent with the hypothesis that
the subsynovial connective tissue had sustained some sort of injury. The exact
nature of this injury and its relationship, if any, to carpal tunnel syndrome
remain to be elucidated.