J Appl Physiol 96: 711–718, 2004.
First published October 3, 2003; 10.1152/japplphysiol.00307.2003.
Blockade of the sympathetic nervous system degrades
ligament in a rat MCL model
Kelley W. Dwyer,1,2 Paolo P. Provenzano,1,2 Peter Muir,3 Wilmot B. Valhmu,1 and Ray Vanderby, Jr.1,2
1
Orthopedic Research Laboratories, Department of Orthopedics and Rehabilitation, 2Department
of Biomedical Engineering, and 3Comparative Orthopaedic Research Laboratory, School of
Veterinary Medicine, University of Wisconsin, Madison, Wisconsin 53792
Submitted 25 March 2003; accepted in final form 29 September 2003
(PNS) has three primary functions: 1) relaying sensorimotor information to and from the
central nervous system; 2) controlling local blood flow; and 3)
influencing inflammatory, proliferative, and reparative processes in injured tissue. In the PNS, neurogenic factors, such as
neuropeptides and neurotransmitters, are chemical agents that
mediate these functions (35). These factors can influence
PNS-related actions by modulating immune cell responses,
cytokine response, and local blood flow (5, 35). Although
neurogenic factors are released from stimulated nerve endings,
their actions are not restricted to the point of stimulation (10).
For example, the autonomic neuropeptides vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY) can have angiogenic, vasoregulatory, and cell proliferative actions hours to
days after release from nerve endings (35, 44). In addition,
Ackermann et al. (1) recently reported a role for peripheral
neuropeptides in healing of tendon tissue. The above observations make it likely that these neurogenic factors play an
important role in tissue homeostasis.
Clinically, chemical inhibition of the PNS is used to manage
joint pain. Chemical blockade of the sympathetic nervous
system is often accomplished through the administration of
guanethidine. Guanethidine blocks the release and subsequent
reuptake of norepinephrine (NE) (a major sympathetic neurotransmitter) in patients with osteoarthritis, rheumatoid arthritis,
and reflex sympathetic dystrophy (12, 13, 24, 38, 40). This
treatment, while effective in relieving pain, may have undesirable effects on connective tissues, because it alters the normal
concentration of neurogenic factors.
Growing anatomic and physiological evidence suggest that the
PNS is important to ligament and joint homeostasis. Ligaments,
like many other connective tissues, are innervated with both
unmyelinated afferent (sensory) and efferent (sympathetic) nerves
(2). In normal medial collateral ligament (MCL) tissue, these
nerves are often associated with blood vessels in the epiligament
and contain neuropeptides, including substance P (SP), CGRP,
NPY, and VIP (26). In addition to their association with blood
vessels, efferent fibers can extend into deeper ligament matrix (2).
These efferent nerve fibers in the MCL contain both autonomic
(NPY and VIP) and sensory (SP and CGRP) neuropeptides that
help regulate MCL blood flow (1, 2, 11, 26). Denervation of
peripheral nerves leads to decreased healing of the MCL and
promotes the onset of osteoarthritis (22, 34). Partial injury to the
MCL can lead to increases in vascular volume, a factor that is
largely controlled by ligament innervation (7). During periods of
chronic overuse or disuse, homeostatic changes can be detrimental
to the structural integrity of ligaments. Although the above evidence suggests that peripheral nerves play an essential role in
ligament homeostasis, few studies exist that directly investigate
this role.
Ligament homeostasis and remodeling is a complex process
involving cell responses, cell-matrix interactions, and regulation
through chemical mediators. In soft tissues, such as ligament, the
process of remodeling and degradation is regulated by the production of proteases from fibroblasts (9). Of the many proteases
that are produced by cells, two classes have gained attention for
their ability to degrade collagen: cysteine proteases and matrix
metalloproteinases (MMPs). Within these classes, a number of
proteases have been isolated in rat MCL. MMP-13 has been
thought to play a role in type I collagen degradation in the rat and
Address for reprint requests and other correspondence: R. Vanderby, Jr.,
Dept. of Orthopedics and Rehabilitation, Univ. of Wisconsin, 600 Highland
Ave., Madison, WI 53792 (E-mail: [email protected]).
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
guanethidine; neuropeptide; tissue mechanics; tissue engineering; medial collateral ligament
THE PERIPHERAL NERVOUS SYSTEM
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8750-7587/04 $5.00 Copyright © 2004 the American Physiological Society
711
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Dwyer, Kelley W., Paolo P. Provenzano, Peter Muir, Wilmot B.
Valhmu, and Ray Vanderby, Jr. Blockade of the sympathetic
nervous system degrades ligament in a rat MCL model. J Appl Physiol
96: 711–718, 2004. First published October 3, 2003; 10.1152/
japplphysiol.00307.2003.—We hypothesize that blockade of the sympathetic nervous system degrades ligament. We tested this hypothesis
in a rat medial collateral ligament (MCL) model. Fifteen animals were
treated for 10 days with the sympathetic chemotoxin guanethidine
using osmotic pumps, whereas 15 control rats received pumps containing saline. A reduction in plasma concentrations of norepinephrine
in the guanethidine rats indicated a significant decrease in sympathetic
nerve activity. Vasoactive intestinal peptide and neuropeptide Y were
decreased in MCLs from guanethidine animals, as quantified by
radioimmunoassays. Tissue vascularity was substantially increased in
guanethidine MCLs, whereas mechanical properties were significantly
decreased. Proteases, such as matrix metalloproteinases (MMP) and
cysteine proteases, play a major role in ligament degradation. The
proteases MMP-13, cathepsin K, and tartrate-resistant acid phosphatase (TRAP) have collagenolytic activity and have been shown in rat
ligament tissues. To determine whether the degradation seen in this
study was due to protease activity, we determined the expression of
these enzymes in control and treated MCLs. Real-time quantitative
PCR revealed that guanethidine treatment increased expression of
MMP-13 and cathepsin K mRNAs, although overall expression levels
of MMP-13 and TRAP were relatively low. Histology also identified
increases in TRAP and cathepsin K, but not MMP-13, in guanethidine-treated tissues. Results support our hypothesis that blockade of
the sympathetic nervous system substantially degrades ligament.
712
SYMPATHECTOMY ALTERS LIGAMENT HOMEOSTASIS
METHODS
Surgical procedure. This study was approved by the Institutional
Animal Use and Care Committee and meets the National Institutes of
Health guidelines for animal welfare. Thirty female Wistar rats
(207.4 ⫾ 13 g) were divided into two groups: 1) guanethidine
treatment and 2) saline control. For all groups, anesthesia (isofluorane
0.5–3%) was initiated in an induction chamber and maintained with a
face mask. Into the treatment group (n ⫽ 15 animals), guanethidine
(40 mg䡠kg⫺1 䡠day⫺1) was infused via a subcutaneously implanted
mini-osmotic pump (Alza). Pumps were placed into a subcutaneous
pocket created between the scapulae. Into the control group (n ⫽ 15
animals), a similar volume of sterile saline was infused via the same
method. Rats were observed daily for signs of infection, discomfort,
or unusual behavior. Both groups were euthanized 10 days after
implantation of the pump with intraperitoneal injections of pentobarbitone (150 mg/kg). Throughout the study, only one ligament per
animal was used for a particular experimental test protocol (each
protocol is described below).
NE ELISA. To test the effectiveness of guanethidine on sympathetic
blockade, plasma concentrations of NE (a major sympathetic neurotransmitter) were measured in each rat. Blood samples from each rat
were taken immediately preceding death. Plasma was separated from
whole blood samples by centrifugation. NE was then measured for
each rat by using a NE ELISA kit (IBL-Hamburg). Analysis was
performed following the manufacturer’s instructions. Briefly, ELISA
plates coated in primary antibody were used for the analysis. NE
standards or plasma samples from each rat were added to the wells of
the plates and allowed to incubate at room temperature for 2 h. Plates
were washed, and an enzyme conjugate was applied for 90 min. After
a second wash, amplification reagent was added to the wells, and the
absorbance values were determined for each well. The NE concentration of each sample was determined from a standards curve.
Immunofluorescent evaluation of neuropeptides. Immediately after
death, eight MCLs (from 4 treated, 4 control rats) were exposed and
dissected in toto from the tibial and femoral insertions. Ligaments were
J Appl Physiol • VOL
immediately fixed in 4% formalin for 24 h. Ligaments were washed in
PBS plus 1.0% Tween 20 between incubations. Ligaments were coincubated in a rabbit anti-rat primary antibody for either NPY (1:10,000
dilution) and VIP (1:1,000 dilution) or SP (1:2,000 dilution) and CGRP
(1:2,000 dilution) (all antibodies from Chemicon, Temecula, CA). Each
ligament was then incubated in two goat anti-rabbit fluorescently tagged
(1:50 dilution of 7-amino-4-methylcoumarin-3-acetic acid or rhodamine;
Chemicon) secondary antibodies. Confocal microscopy was used to
examine the sections for fluorescent labeling. Ligaments were viewed in
sagittal planes of focus, and scans were made through the ligament
thickness. Three-dimensional reconstructions were made from individual
confocal images by stacking the sequentially scanned images. Images
were then viewed with a computer and saved digitally.
RIA. Four MCLs were used for quantification of neuropeptide
concentrations via RIA. MCLs were homogenized by using a mortar
and pestle. Homogenate was used to complete standard SP, CGRP,
NPY, and VIP RIA protocols (Phoenix Pharmaceuticals, Belmont,
CA). Briefly, samples were mixed with an antibody specific to one of
the neuropeptides and then incubated with a 125I-traced peptide. The
radioactivity of each sample was measured by using a gamma counter.
Standards for each neuropeptide were included in the kit, and MCL
neuropeptide concentration was determined from a standards curve.
Organ culture. To assess the effect of guanethidine directly on
extracellular matrix (ECM) integrity, organ culture using the MCL
was employed. Bilateral MCLs (n ⫽ 6), including intact femoral and
tibial segments, were aseptically harvested from untreated rats. The
left knee was used as a control tissue, whereas the right knee was
cultured with guanethidine.
During and after harvest, tissues were rinsed in sterile PBS containing penicillin (100 U/ml), streptomycin (100 ␮g/ml), and fungizone (0.25 ␮g/ml). Bone blocks were trimmed to minimize the
amount of bone marrow, which may alter the culture environment.
Tissues were then cultured in DMEM supplemented with 10% fetal
bovine serum, nonessential amino acids (0.1 mM), L-gutamine (4
mM), penicillin (100 U/ml), streptomycin (100 ␮g/ml), and fungizone
(0.25 ␮g/ml). Guanethidine or sterile PBS was added to media for the
treated and control tissues, respectively. The concentration of
guanethidine in media was selected to be the same as the concentration of guanethidine present in the blood during treatment (described
above) using normative rat data for blood volume (5 ml blood/100 g
body wt). Guanethidine was prepared and dissolved in sterile PBS and
added to the media; controls received equal volumes of sterile PBS.
The cultures were maintained in an incubator at 37°C and 5% CO2.
Media were changed every 48 h, at which time tissues were rinsed
with sterile PBS to reduce drug carryover into the fresh media
containing the treatment drug or an equivalent volume of carrier.
Organ culture was maintained for 10 days, after which time the MCLs
were tested mechanically in the same manner as the in vivo treatment
group (described below).
Tissue mechanics. Immediately after death, animal hindlimbs for
biomechanical testing (n ⫽ 8 control, n ⫽ 8 treated MCLs) were
disarticulated at the hip joint and stored at ⫺80°C until testing.
Storage by freezing does not significantly affect the biomechanical
properties of ligament (42). On the day of testing, hindlimbs were
thawed at room temperature, and tissue harvest and testing were
performed using methods similar to those previously published (30).
Each MCL was exposed by carefully dissecting extraneous tissue. The
MCLs, including intact femoral and tibial bone sections, were excised
for ex vivo testing, with care taken not to disturb the ligament
insertion sites. Tissues were kept hydrated in Hanks’ physiological
solution during testing. Ligament thickness and width were measured
optically, and MCL area was estimated with elliptical geometry. Each
femur-MCL-tibia complex was then placed in a custom-designed
tissue bath system. For strain measurements, optical markers were
placed onto the ligament tissue near the insertion sites. A small
preload of 0.1 N was applied to the ligaments to obtain a uniform
starting point. The ligaments were then preconditioned to 1% strain
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has been found in a number of tissues, including uterus, cornea,
bone (31), meniscus, synovium, anterior cruciate ligament, and
MCL (19). Of the cysteine proteases, cathepsin K and an associated lysosomal protease, tartrate-resistant acid phosphatase
(TRAP), also play a role in type I collagen degradation. TRAP,
although not a cysteine protease (but is associated with many
cysteine proteases), has the ability to degrade type I collagen (16)
and was found in canine ligamentous tissue (27). Cathepsin K, a
predominant protease in bone remodeling, has also been found in
rat MCL. There is increasing evidence that cathepsin K plays a
major role in MCL remodeling (27). Cathepsin K is a potent
collagenase and, as such, has a substantial influence on ligament
degradation.
The hypothesis of this study is that blockade of the sympathetic nervous system degrades ligament. To test this hypothesis, changes in ligament mechanical properties, ligament vascularity, the presence of neuropeptides, and levels of proteolytic enzymes were examined in a rat MCL model. Results
have importance for a number of reasons. First, because
guanethidine is used in pain management, it is essential to
understand how it affects joint connective tissue. Second,
numerous studies use tissue engineering approaches to regenerate connective tissues but do not consider the role of peripheral nerves or neuropeptides. Last, ligament grafting is commonly performed without consideration of peripheral innervation and neurogenic factors. This study tests our hypothesis and
thus adds insight into the role of sympathetic innervation on all
of the above issues.
SYMPATHECTOMY ALTERS LIGAMENT HOMEOSTASIS
for 10 cycles. After preconditioning, ligaments were pulled to failure
at a rate of ⬃10%/s.
Tissue displacement was obtained by using video dimensional
analysis. The initial ligament length (Lo) was taken to be the length at
preload. The change in distance between optical markers was calculated by analyzing the change in coordinate center of each marker
from stored digital frames by using a custom macro for NIH Image.
Engineering strain (⑀) was measured from displacement data as the
change in length between the stretched length (L) and Lo, divided by
the Lo
ε⫽
L ⫺ Lo
Lo
(1)
Force was measured and displayed on the video screen in synchrony
with the displacement. Engineering stress (␴) was calculated as the
force (F) divided by the initial area (Ao)
␴⫽
(2)
Stress-strain curves were created and fit with the microstructural
model presented by Hurschler and coworkers (21). Elastic moduli
were obtained from this model.
The mechanical properties that were examined in this study were
maximum force, ultimate stress, strain at failure, elastic modulus, and
area. Statistical analysis was performed by using an unpaired t-test
(P ⫽ 0.05) to analyze differences between control and treatment
groups for each mechanical property.
Comparison of wet and dry weights. Ligament hydration was
evaluated by comparing hydrated and dehydrated weights. For conformity, ligaments were hydrated for 2 h in PBS, excess PBS was
removed, and the tissues were then weighed immediately. Ligaments
were weighed on a digital balance (Mettler-Toledo, Columbus, OH) to
an accuracy of 0.00001 g. Each tissue was then dehydrated on glass
coverslips at 60°C for 24 h and weighed a second time.
The difference between wet and dry weights, normalized to the dry
weight, was compared, and group means were evaluated statistically
by using a paired t-test (␣ ⫽ 0.05).
Evaluation of MCL vascularity. To investigate tissue vascularity,
the femoral arteries of six rats (3 treated, 3 controls) were cannulated.
The proximal artery was tied off by using a 4.0 nylon suture. Into the
artery, 4-␮m red fluorescent microspheres (2 ⫻ 107 spheres suspended in 5% albumin solution) were injected as a 3-ml bolus. Five
minutes after injection, the rats were killed. Ligaments were harvested
and fixed overnight in 4% formalin. Tissue was imaged with confocal
microscopy to detect the presence of fluorescent microspheres. Ligaments were viewed in sagittal planes of focus, and scans were made
through the ligament thickness. Stacking the sequentially scanned
images created three-dimensional reconstructions. Images were then
viewed with a computer and saved digitally.
Histological and immunohistochemical evaluation of proteases.
Immediately after death, eight MCLs (from 8 treated, 8 control rats)
were exposed and dissected in toto from the tibial and femoral
insertions. Ligaments were immediately fixed in Zamboni’s fixative
for 3 days at 4°C and then stored overnight in a solution of 30%
sucrose and fixative. Specimens were flash frozen, sectioned (6 ␮m),
and mounted on slides.
Immunohistochemical labels were applied by using a diaminobenaldehyde immunohistology kit for rodent tissue (Innogenex, San
Ramon, CA). Slides were labeled for MMP-13 (collagenase 3),
MMP-1, and cathepsin K after the manufacturer’s instructions. Histological evaluation of TRAP was performed following previously
published methods (3, 15). Briefly, slides were incubated for 2 h in
equal parts of two buffer solutions: naphthol AS-BI phosphate buffer
and hexazonium pararosaniline. Slides were evaluated by using light
microscopy.
J Appl Physiol • VOL
Real-time quantitative PCR. Primer sets for cathepsin K, rat collagenase (MMP-13), TRAP, and GAPDH have been developed and
tested for specificity in our laboratory. The primer sets were either
derived from previously published reports [rat collagenase (25, 39)],
or designed from sequences available from GenBank (GAPDH, cathepsin K, and TRAP: GenBank accession numbers are AF106860,
AF010306, and M76110, respectively) using the PrimerSelect module
of the Lasergene 5.01 software (DNAStar, Madison, WI). Primer
sequences are as follows: MMP-13: forward: 5⬘-AAA GAA CAT
GGT GAC TTC TAC C-3⬘, reverse: 5⬘-ACT GGA TTC CTT GAA
CGT C-3⬘, length: 283 bp; cathepsin K: forward: 5⬘-TGC GAC CGT
GAT AAT GTG AAC C-3⬘, reverse: 5⬘-ATG GGC TGG CTG GCT
TGA ATC-3⬘, length: 205 bp; TRAP: forward: 5⬘-CGC CAG AAC
CGT GCA GAT TAT G-3⬘, reverse: 5⬘-AAG ATG GCC ACG GTG
ATG TTC G-3⬘, length: 297 bp; and GAPDH: forward: 5⬘-GAC TGT
GGA TGG CCC CTC TG-3⬘, reverse: 5⬘-CGC CTG CTT CAC CAC
CTT CT-3⬘, length: 239 bp.
For total RNA isolation, the tissues (n ⫽ 4 control, n ⫽ 4 treated)
were rinsed, after sterile harvesting, in DNase-, RNase-, and proteasefree PBS to remove blood or other contaminants from the tissue and
placed in RNAlater solution (Ambion, Austin, TX). Tissues were
pooled to ensure adequate measurable RNA yield. RNA isolation was
performed by using methods similar to those utilized by Reno and
coworkers (32), which combine the TRIzol method with the column
fractionalization steps of the RNeasy Total RNA kit (Qiagen, Valencia, CA). Methods primarily followed the manufacturer’s instructions
for the respective steps. For tissue homogenization, MCLs were
frozen with liquid nitrogen, placed in a liquid nitrogen-cooled Braun
Mikro-Dismembrator vessel (B. Braun Biotech International, Allentown, PA), and reduced to powder. After total RNA isolation, yield
and purity of RNA were quantified by spectrophotometric measurement at 260, 280, and 325 nm (UltraSpec 3000 Spectrophotometer,
Pharmacia Biotech, Cambridge, UK). RNA was reverse transcribed
into cDNA (20 ␮l total volume) by combining a mixture of 5 ␮l total
RNA, 1 ␮l oligo(dT)15 (250 ng/␮l), and 5 ␮l RNase free water, with
9 ␮l of First Strand Synthesis buffer (Life Technologies) containing
40 units of RNase inhibitor (Ambion), 500 ␮M dNTP mix, 10 mM
dithiothreitol, and 200 units of Superscript II reverse transcriptase.
Quantitative PCR (QPCR) standards for the genes indicated above
were prepared from purified PCR products of the target sequences.
From spectrophotometric quantitation of the PCR products, the number of copies per microliter of each standard was calculated, and
10-fold serial dilutions (ranging from 1 ⫻ 109 to 10 copies/␮l) were
prepared. The housekeeping gene GAPDH was used as internal
control. Real-time QPCR was performed by using a Bio-Rad iCycler
iQ real-time PCR system (Bio-Rad, Hercules, CA). All reactions were
carried out in a total volume of 20 ␮l containing 1⫻ Platinum QPCR
Supermix (Life Technologies), 10 nM fluorescein, 200 nM forward
primer, 200 nM reverse primer, 0.25⫻ SYBR green, 5 ␮l template,
and 3.95 ␮l diethyl pyrocarbonate-treated H2O. PCR cycling consisted of initial denaturation at 95°C for 3 min and up to 50 cycles of
95°C denaturation for 15 s, 55 or 60°C annealing (depending on
template) for 30 s, and 72°C extension for 30 s. During each cycle,
optical data were collected during the annealing and extension steps.
Each gene was run in triplicate. The copy numbers of the respective
cDNAs in the samples were determined relative to the standard
curves. The cDNA copy number of the target gene was normalized to
the copy number of GAPDH.
RESULTS
Surgical outcomes. All guanethidine-treated rats developed
ptosis (an indication of sympathetic inhibition) by the second
day of treatment, and ptosis persisted throughout the 10-day
period. No other gross changes were observed in vivo. No
abnormalities in movement, body weight, or behavior were
observed in either the treated or control groups. On tissue
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F
Ao
713
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SYMPATHECTOMY ALTERS LIGAMENT HOMEOSTASIS
Fig. 1. After 10 days of treatment, plasma concentrations of norepinephrine
(NE) were measured in control and guanethidine-treated rats. Values are
means ⫾ SD. Guanethidine-treated rats had significantly lower (P ⫽ 0.04)
concentrations of NE compared with controls.
DISCUSSION
PNS inhibition degrades ligament. Results of this study
support the hypothesis that inhibition of the sympathetic nerves
Fig. 2. After 10 days of treatment, ligaments
were harvested and labeled for the sympathetic neuropeptide Y (NPY) and vasoactive
intestinal peptide (VIP). Presence of NPY
(blue) and VIP (red) in rat medial collateral
ligaments (MCLs) is shown (note: green
color indicates tissue autofluorescence).
Guanethidine-treated animals showed depletion of VIP. Neuropeptides have an important vasoregulatory role in connective tissues. NPY and NE have vasoconstrictive
actions and play a predominant role in regulating blood flow, whereas VIP plays an
important role in fine-tuning blood flow
through connective tissues.
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harvest, no morphological differences were macroscopically
observed between control and treated MCL tissue.
Neurotransmitter and neuropeptide concentrations. Plasma
concentrations of NE in the treated group were significantly
lower than those in control animals (0.1717 vs. 6.272 ng/ml;
P ⫽ 0.04; Fig. 1), indicating significant inhibition of the
sympathetic nervous system. Immunofluorescent labeling of
neuropeptides revealed a disappearance of VIP from ligament
tissue after guanethidine treatment, whereas NPY, SP, and
CGRP remained unchanged (Fig. 2), indicating an impairment
to sympathetic and parasympathetic neuropeptides but not
neuropeptides from sensory nerves. RIA data confirmed the
absence of VIP from guanethidine-treated MCLs compared
with controls (0.28 vs. 164.42 pg/100 ␮l; P ⬍ 0.001) and
showed greatly decreased concentrations of NPY in treated
MCLs (1.65 vs. 14.67 pg/100 ␮l; P ⬍ 0.001). SP concentrations were also increased in guanethidine-treated MCLs
(50.173 vs. 1.97 pg/100 ␮l; P ⫽ 0.03); however, CGRP
concentration remained unchanged (3.36 vs. 1.58 pg/100 ␮l).
MCL mechanics. Results from organ culture experiments
performed to examine the direct effect of guanethidine on
ECM structural integrity revealed no changes in ultimate
stress, area, or force. Ultimate stress values were nearly identical between contralateral ligaments [71.01 ⫾ 19.20 MPa
(control) vs. 70.29 ⫾ 19.15 MPa (guanethidine); P ⫽ 0.96].
Ultimate stress in guanethidine-treated animals was significantly decreased (Fig. 3A; P ⫽ 0.0006), as was the strain at
failure (Fig. 3B; P ⫽ 0.031) and elastic modulus (Fig. 3C; P ⫽
0.0003) compared with MCLs from saline-receiving control
animals. A significant increase in MCL area was also present in
treated tissues. The mean ultimate tensile force also decreased
in treated tissues, but it was not statistically significant (P ⫽
0.76). This increase in area is due (at least in part) to an
increase in fluid content in the ligament, as supported by a
statistically significant increase in the wet weight (P ⫽ 0.042).
MCL vascularity. Analysis of tissue vascularity using fluorescent microspheres qualitatively revealed that guanethidine
treatment increased vascularity compared with that in control
animals (Fig. 4). Substantial and consistent differences in
microsphere deposition were easily detectable between treated
and control animals.
Proteolytic enzymes. The MCLs from the guanethidinetreated animals labeled positively for cathepsin K and TRAP
(Fig. 5). Cathepsin K labels were observed near the peripheral
portions of the ECM and within the ligament matrix. TRAP
staining was seen throughout the ligament, with the heaviest
staining seen in the ligament matrix. No ligaments (except the
positive control slides) had positive staining for MMP-1 or
MMP-13 (results not shown). Real-time QPCR produced consistent results and showed increases in cathepsin K, MMP-13,
and TRAP (Fig. 6). Increases in the expression of cathepsin K
(58.04 ⫾ 8.08 vs. 31.97 ⫾ 9.40 copies/1,000 copies GAPDH)
and MMP-13 (6.91 ⫾ 2.98 vs. 0.271 ⫾ 0.04 copies/1,000
copies GAPDH) were seen in the treated tissues compared with
control tissues. In both the untreated and treated tissues, the
overall expression of TRAP was very small.
SYMPATHECTOMY ALTERS LIGAMENT HOMEOSTASIS
degrades ligament. Data reveal that guanethidine does not
directly affect structural integrity (via tissue culture experiments) but that chemically induced sympathectomy does. Reduced plasma concentrations of NE as well as ptosis in the
treated group indicate that guanethidine was effective in blocking the sympathetic nervous system. Although histological
ligament sections only showed changes in VIP immunoreactivity, RIA data from the guanethidine-treated rats showed
decreases in both NPY and VIP, indicating that levels of
autonomic neuropeptides were altered. SP concentrations were
also increased in guanethidine-treated MCLs, indicating that
blockade of efferent nerve fibers may alter concentrations of
afferent neuropeptides as well. In vivo administration of
guanethidine decreased ultimate stress, decreased elastic modulus, decreased strain at failure, and increased cross-sectional
area. The mean ultimate force decreased after 10 days of in
vivo guanethidine treatment, but the change was not statistically significant. The increased area is likely the result of
increased tissue hydration, as exhibited by a significant increase in the MCL wet weight. Increased hydration may be due
to changes in vessel permeability (as mediated by neuropeptides). Alternatively, changes in ECM components, such as
proteoglycans, which are known to affect tissue water content
as well as stabilize collagen ECM organization, could account
for the increased hydration. Strain at failure was significantly
altered, implying some alterations to ECM organization and
integrity. Further studies are necessary to elucidate details of
the changes and the mechanisms.
PNS influences MCL physiology. Tissue vascularity was
significantly higher in MCLs from guanethidine-treated animals, which is consistent with previous reports (6, 26, 35). The
increased vascularity was most likely the result of altered
concentrations of neurotransmitters and neuropeptides. Our
results in rats agree with rabbit experiments (26) in which the
authors report that altering levels of neuropeptides and NE
alters MCL blood flow. Others (7) report that partial tears to
both the MCL and anterior cruciate ligament increase ligament
blood volume, which also may be mediated by the PNS.
Guanethidine-treated rats increased their expression of proteolytic enzymes (MMP-13 and cathepsin K) within MCL
tissue. Expression of TRAP was low in control and treated
MCLs. Histology confirmed the presence of cathepsin K and
showed the presence of TRAP in guanethidine-treated MCLs.
The presence of these enzymes may have altered the MCL
mechanical properties in the treated animals. Remodeling and
Fig. 4. After 10 days of treatment, red fluorescent-labeled microspheres were injected
into the femoral artery. Yellow arrows locate
some of the microspheres in each image.
Guanethidine-treated rats had a qualitatively
higher presence of microspheres located
within their MCLs. This result indicates increased vascularity in the MCL after chemical sympathectomy with guanethidine.
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Fig. 3. After 10 days of treatment, mechanical properties of rat MCLs were
measured in control and guanethidine-treated rats. A: guanethidine-treated rats had
significantly lower ultimate stress compared with controls (P ⫽ 0.0006). B:
guanethidine-treated rats had significantly lower strain (P ⫽ 0.031) compared with
controls, which may indicate that the ligament matrix organization was disrupted
after sympathectomy. C: guanethidine-treated rats also had significantly lower
elastic moduli compared with controls (P ⫽ 0.0003). Values are means ⫾ SD.
715
716
SYMPATHECTOMY ALTERS LIGAMENT HOMEOSTASIS
degradation of connective tissues depend greatly on the action
of proteolytic enzymes, including MMPs and cysteine proteinases (such as cathepsins). MMPs have been implicated in
remodeling the ECM of a number of orthopedic tissues, including ligament, cartilage, and bone (18, 19, 28, 37, 41).
Fig. 6. Real-time quantitative PCR analysis of TRAP, matrix metalloproteinases (MMP)-13, and cathepsin K mRNA levels in control and guanethidinetreated animals. Tissues from treated had ⬃26- and 1.8-fold increases in
MMP-13 and cathepsin K mRNA levels, respectively, over control tissues.
Each point represents the mean ⫾ SD of triplicate determinations.
J Appl Physiol • VOL
MMP-13 has enzymatic specificity for type I collagen and,
therefore, may contribute to matrix degradation. Cysteine proteases also degrade collagen as well as other ECM components. Cathepsin K plays a substantial role in bone remodeling
and also has specificity for type I collagen (4, 8, 14, 23, 33, 36,
43). Cathepsin K has been found in synovial fibroblasts and
multinucleated giant cells, suggesting a role for the enzyme in
rheumatoid arthritis and in response to calcified tendonitis in
human rotator cuff tendon (20, 29). TRAP, which may play a
collagenolytic role in calcified tissue, is also present in canine
cruciate ligaments after a rupture (16, 27). TRAP is capable of
generating reactive oxygen species that can target and cleave
type I collagen (16). Hence, the presence of TRAP and cathepsin K in guanethidine-treated ligaments suggests that the collagenolytic activity of these enzymes contributes to the observed changes in mechanical behavior and morphology. The
association herein between collagenolytic enzymes and the
PNS suggests that neurogenic factors are important in chronic
ligament and tendon problems, such as disuse or overuse
(16, 27).
Limitations. Limitations of this study must be considered
when interpreting the results. First, although guanethidine has
been shown to inhibit sympathetic nerves, its specific effect on
neuropeptides has not been quantified. A decrease in NPY and
VIP was apparent in this study, but many other neuropeptides
require further study. It should be noted that VIP is localized to
a number of nerve fiber types, including sympathetic and
parasympathetic fibers. Therefore, guanethidine affects autonomic nerves, but not necessarily sympathetic nerves exclusively. We have observed biomechanical and catabolic changes
after chemical sympathectomy but have only correlated them
with NPY and VIP. The specific mechanisms of the observed
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Fig. 5. After 10 days of treatment, histological sections of rat MCLs were labeled for
collagenolytic enzymes. Guanethidine-treated
animals had an increase in proteolytic enzymes
(indicated by arrows) compared with controls.
Tartrate-resistant acid phosphatase (TRAP; B)
and cathepsin K (D) were observed in guanethidine-treated MCLs but not in control MCLs (A
and C, respectively). Both TRAP and cathepsin
K have potent collagenolytic activity and may
contribute to tissue degradation in guanethidine-treated ligaments.
SYMPATHECTOMY ALTERS LIGAMENT HOMEOSTASIS
ACKNOWLEDGMENTS
Special thanks are due to Zhengling Hao for assistance with animal
treatments and immunohistochemical preparations, to Adriana AlejandroOsorio for assistance with organ culture preparation, and to Kal Watson and
Dr. Robert Conhaim for technical assistance with vascularity experiments.
GRANTS
This study was partially funded by National Aeronautics and Space Administration Grant NAG9-1152, National Science Foundation (NSF) Grant
CMS-9907977, and a NSF graduate research fellowship (to K. W. Dwyer).
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