1. health and fitness
2. disease

ACL Injury and Reconstruction Impairs Pressure Pain Sensitivity
and Arterial Flow Mediated Dilation
BY
Jeffrey D. Clark
B.S., Central Michigan University, 1998
M.S.P.T, Central Michigan University, 2001
M.B.A., University of Illinois at Chicago, 2006
THESIS
Submitted as partial fulfillment of the requirements
for the degree of Master of Science in Rehabilitation Sciences
in the Graduate College of the
University of Illinois at Chicago, 2013
Chicago, Illinois
Defense Committee:
Shane A Phillips, Chair and Advisor
Carol A Courtney, Advisor, Physical Therapy
Sangeetha Madhavan, Physical Therapy
ACKNOWLEDGEMENTS
I would like to thank my thesis committee--Shane Phillips, Carol Courtney and
Sangeetha Madhavan for their unwavering support and assistance. They provided guidance in all
areas that helped me accomplish my research goals and enjoy myself in the process.
A number of individuals involved with data collection and analysis were extremely
helpful to me during this time, and I would like to thank them as well—
JDC
ii
TABLE OF CONTENTS
CHAPTER
Page
I.
INTRODUCTION……………………………….....................................
A.
Background………………………………………………………..
B.
Main Objective………………………………..............................
C.
Hypothesis…………………………………………………………
D.
Rationale……………………………………................................
1
1
1
1
2
II.
REVIEW OF LITERATURE ACL, OA AND VASCULAR
DYSFUNCTION
A.
Review of Literature on ACL injury and OA
1.
ACL Injury: Prevalence, Cost and OA risk…………..……
2.
OA and Cardiovascular Risk………………………………
3.
Inflammation in ACL injuries and OA – Inflammation
Drives Degeneration……………………………………….
4.
Inflammation in ACL injuries and OA Induces
Nociceptive Sensitization………………………………….
B.
Background of Primary Testing Measures
1.
Arterial Flow Mediated Dilation …………………………
2.
Pressure pain Threshold Testing………………………..
3
4
5
9
11
14
III
METHODS……………………………………………………..................
A.
Study Design and Subjects………………………….....................
B.
Overview of Study Protocol……………………………………….
C.
Pressure Pain Threshold Testing…………....................................
D.
Brachial and Popliteal Artery Flow-Mediated Dilation….............
G.
Statistical Analysis………………………………………………...
15
15
15
16
17
19
IV
RESULTS…………………………………………………………………
A.
Subject Inclusion and Exclusion………………………………….
B.
Demographics……………………………………………………..
C.
Pressure Pain Threshold…………………………………………...
1. PPT at the Hand………………………………………………..
2. Right and Left PPT Comparisons……………………………...
3. PPT at the Tibia ……………………………………………….
4. PPT at the Medial Tibiofemoral Joint…………………………
D.
Brachial Artery Flow Mediated Dilation………………………….
E.
Popliteal Artery Peak Shear Rate………………………………….
F.
Popliteal Artery Flow Mediated Dilation…………………………
G.
Correlation of FMD to PPT……………………………………….
H.
Correlation of Pain to Chronicity…………………………………
20
20
20
22
22
22
23
24
28
29
30
36
39
iii
TABLE OF CONTENTS (continued)
CHAPTER
PAGE
V.
DISCUSSION………………………………………………………........
A.
Subject Characteristics…………….................................................
B.
Pain Comparisons…………………………………………………...
C.
FMD Comparisons………………………………………………..
E.
OA to CVD Connection…………………………………………..
F.
Limitations………………………………………………………..
D.
Conclusions………………………………………………………
40
41
42
45
46
47
49
CITED LITERATURE……………………………………………………..
50
VITA………………………………………………………………………..
61
iv
LIST OF TABLES
TABLE I SUBJECT CHARACTERISTICS ................................................................................. 21
TABLE II PPT AT DOMINANT HAND WEB SPACE ................................................................ 22
TABLE III RIGHT AND LEFT PPT COMPARISON AT KNEE JOINT LINE AND TIBIA...... 23
TABLE IV PRESSURE PAIN THRESHOLD AT MEDIAL TIBIA ............................................ 24
TABLE V PRESSURE PAIN THRESHOLD AT MEDIAL TIBIOFEMORAL JOINT LINE .... 25
TABLE VI BRACHIAL CHARACTERISTICS ........................................................................... 28
TABLE VII POPLITEAL PEAK SHEAR RATE ......................................................................... 29
TABLE VIII POPLITEAL FMD COMPARISONS ...................................................................... 31
TABLE IX POPLITEAL CHARACTERISTICS .......................................................................... 32
v
LIST OF FIGURES
FIGURE 1 PPT AT THE MEDIAL TIBIOFEMORAL JOINT LINE ............................................. 26
FIGURE 2 UNILATERAL PPT COMPARISONS ......................................................................... 27
FIGURE 3 POPLITEAL PEAK SHEAR COMPARISONS .......................................................... 30
FIGURE 4 POPLITEAL FMD CONTROL VS ACL .................................................................... 31
FIGURE 5 RIGHT POPLITEAL FMD COMPARISONS ............................................................. 33
FIGURE 6 LEFT POPLITEAL FMD COMPARISONS ............................................................... 34
FIGURE 7 LEFT POPLITEAL FMD: CONTRALATERAL TO RIGHT SIDE INJURY ........... 35
FIGURE 8 SCATTER PLOT OF POPLITEAL FMD TO MEDIAL JOINT LINE PPT ............... 36
FIGURE 9 SCATTERPLOT OF TIBAL PPT TO POPLITEAL FMD .......................................... 37
FIGURE 10 SCATTER PLOT OF FMD TO PPT IN LEFT LEG OF RIGHT ACL-R .................. 38
FIGURE 11 CORRELATION OF PPT TO CHRONICITY .......................................................... 39
FIGURE 12 THEORETICAL MODEL .......................................................................................... 41
vi
LIST OF ABBREVIATIONS
ACLS
Aerobics Center Longitudinal Study
ACL
Anterior Cruciate Ligament
ACL-R
ACL reconstruction group
BMI
Body mass index
CON
Control Group
CRP
C-reactive protein
CV
Cardiovascular
CVD
Cardiovascular Disease
DBP
Diastolic blood pressure
ECM
Extracellular matrix
ED
Endothelial-dependent
FMD
Flow-mediated dilation
HR
Heart rate
KOS-ADLS
Knee Outcomes Survey – Activity of Daily Living Scale
LDL
Low-density lipoprotein
LLE
Left lower extremity
MET
Metabolic equivalent
MTFJL
Medial tibiofemoral joint line
NO
Nitric oxide
NOS
Nitric oxide synthase
NTG
Nitroglycerin
vii
OA
Osteoarthritis
RA
Rheumatoid Arthritis
ROS
Reactive oxygen species
RLE
Right lower extremity
PPT
Pressure Pain Threshold
SBP
Systolic blood pressure
viii
SUMMARY
A study investigating the pressure pain sensitivity and vascular effects of ACL injury
with subsequent ACL surgical reconstruction was carried out using a non-randomized, crosssectional approach. One investigational visit was conducted on 15 subjects with ACL
reconstructions and 14 subjects without lower extremity injury served as controls. All
participants were between 18-40 years old and free of cardiovascular disease. Information on
demographics, physical activity, medical history, and LE injury was collected for both groups.
Pressure pain threshold with pressure algometry and arterial vascular dilation with
ultrasonography (flow-mediated dilation) were assessed in both the upper and lower extremities.
The ACL injury subjects were found to have lower pressure pain threshold and reduced
FMD as compared to the control group.
ix
I. INTRODUCTION
A.
Background
Anterior cruciate ligament (ACL) injuries are a common problem in the young, athletic
population. Independent of surgical reconstruction, an ACL injury increases the risk for
development of knee osteoarthritis (OA). Historically, people with ACL injuries tend to report
lower levels of physical function and demonstrate altered neuromuscular recruitment patterns
compared to age matched controls. There is a large inflammatory response to the acute injury
and numerous reports of chronic elevation of various inflammatory markers following ACL
injuries. The exact source of this chronic inflammation is unknown. However, its presence may
impair both normal pain processing and vascular function. The relationship between pain,
vascular dysfunction, and inflammation in the knee has not been investigated.
B.
Main Objective
The main objective of this thesis was to determine if there is a relationship between knee
ACL injury and subsequent reconstruction, local and/or remote pressure pain threshold (as a
sign of altered nociceptive processing) and vascular dysfunction, associated with inflammation.
C.
Hypothesis
It was hypothesized that rupture to the ACL and subsequent reconstruction results in the
activation of numerous chronic pro-inflammatory mechanisms that sensitize both the peripheral
and central nervous system and impair vascular function (flow mediated dilation). Dysfunction
in either the nervous or vascular systems could contribute to the observed accelerated knee
degeneration after ACL injury.
Null hypothesis: No significant difference will exist in pressure pain threshold (PPT) and flow
mediated dilation (FMD) when comparing a healthy population of 25-35 years olds with ACL
reconstructions and a control group matched by age, sex and activity level.
D.
Rationale
The rationale for investigating nociceptive processing and brachial and popliteal artery
FMD in young adults stems from early trends in OA research showing a probable link between
knee injury and the onset of both OA and CV disease. Understanding how knee injury affects
endothelial function and pain processing clinically and physiologically has the potential to
promote the development of new physical or pharmacological treatments and prevent the
functional decline associated with OA.
Since the developmental pathway of vascular dysfunction and nociceptive processing is
complex and demonstrates individual variability, for the purpose of this study, a young subject
sample, absent of cardiovascular disease (CVD) risk factors, age and BMI matched was selected.
Previous work has shown numerous factors can affect vascular FMD response so careful
exclusion criteria were employed to limit these confounding variables.
Prior to reviewing the methods for this study, the background section will review the
potential origins of OA following ACL injury and reconstruction, altered pain processing in
ACL/OA population and the potential direct physiological link between OA and CVD/
endothelial dysfunction.
2
3
II. REVIEW OF ACL AND RELATED LITERATURE
A.
Review of Literature Relating to ACL injury and Osteoarthritis
1.
ACL Injury: Prevalence, Cost and OA risk
The incidence of new ACL injuries in the United States is estimated to be 200,000
annually, with 100,000 ACL reconstructions 1,2 performed each year at an annual cost of $3
billion. 3,4 Current estimates indicate tearing the ACL can ‘age’ the knee by approximately 30
years, leading to OA anywhere from 5-20 years after the injury.5 The highest incidence of ACL
injury is in individuals 15-25 years old who participate in pivoting sports;6 which translates into
subsequent high use demands on an abnormal knee. It has been recommended by the American
Academy of Orthopedic Surgeons that individuals with ACL rupture discuss surgical options
prior to return to high intensity sport, to avoid subsequent intra-articular damage.3 Although it is
clear that ACL reconstruction does not prevent the development of OA,7 the decision to not have
surgery also involves risk. At 10 year follow up in ACL patients who did not undergo
reconstruction, 90% had meniscus damage and 70% had articular lesions;8-10 compared to reports
of meniscal injuries in approximately 50% of initial ACL injuries4. Meniscus injury at or after
the time of ACL injury may be a strong predictor of the development of OA. Neumann et al
reported in a population of 79 subjects with ACL injury, that overall risk of OA was 16%, but
increased to 37% in persons with any meniscal tear and decreased to zero percent in those
without meniscus injury.4
In contrast, Struewer et al 2011, analyzed a population with isolated ACL injuries and no
subsequent meniscal damage, finding at two year follow up 78% had some signs of OA and at
mean 13.5 year follow-up a 95% prevalence of OA signs.11
Presently, the exact link between ACL injury, with or without subsequent reconstruction,
and post-traumatic OA remains a topic of much debate and research. Underlying this debate is
the challenge of identifying the mechanisms which mediate the transition between acute injury
and pain to chronic pain and osteoarthritis. Identifying altered inflammatory and nociceptive
processes following ACL injury may promote understanding of the resultant degenerative
changes.
2.
Osteoarthritis and Cardiovascular Risk
Knee OA affects 27 million Americans and disables approximately 10% of those
affected.12 Although the precise etiology of OA remains unknown, it is considered a
multifactorial condition driven by a combination of local and systemic factors.13 Arthritis and
related conditions, such as OA, cost the U.S. economy nearly $128 billion per year in medical
care and indirect expenses, including lost wages and productivity.14 OA is not classically
considered an inflammatory condition due to the absence of inflammatory cells (ie: neutrophils)
in synovial fluid. Yet, recent research of inflammatory biomarkers shows elevated levels of
cytokines such as interleukin 1, 6, 15 (IL-1, IL-6 and IL-15), tumor necrosis factor- α (TNFα)
and transforming growth factor- β (TGF-β).15-18 Patients with risk factors for atherosclerosis,
including hypertension, obesity, and insulin resistance, also exhibit increased pro-inflammatory
cytokines. These cytokines are known to impair endothelium-dependent vasodilation, an early
4
hallmark of atherosclerosis.19-21 Furthermore, people with OA or rheumatoid arthritis (RA) are
at significantly higher risk of cardiovascular (CV) morbidity and mortality versus the general
population.22-25 In a recent survey study by Golightly et al NFL players with OA are more likely
to report CVD, hypertension, diabetes, and depression than those without.26
The relationships between inflammatory markers, CV risk factor disease states (ie:
obesity, hypertension and insulin resistance19-21 and lower physical function are relatively well
established.27 Previous studies have shown that C-reactive protein (CRP) levels are associated
with age, obesity (BMI) and CVD.28 While the link between OA and CVD remains uncertain,
obesity remains an important factor. Several large, recent reports have not found a relationship
between CRP levels and incidence or progression of OA independent of BMI.29,30
It is postulated that similar to other CVD risk factors, post-traumatic knee OA, increases
circulating pro-inflammatory cytokines that stimulate reactive oxygen species (ROS) generation
leading to impaired nitric oxide (NO) mediated endothelium-dependent dilation of peripheral
blood vessels. In addition, sensitization of nociceptive pathways may be mediated by proinflammatory cytokines, thereby causing pain and subsequent decrease in function. The efferent
secretion of neuropeptides such as Substance P and Calcitonin Gene Related Peptide (CGRP)
from sensory afferents (noxious and non-noxious) may accentuate pain and inflammatory
processes, through a mechanism referred to as neurogenic inflammation. 31
3. Inflammation in ACL injuries and OA – Inflammation Drives Degeneration
Acute increases in cytokine levels after ACL injury are well established and by
increasing inflammation at the knee, may function to promote both degeneration of the joint and
chronic pain. It has been suggested that the two are separate but related processes.32 The
5
degenerative process has been studied in both animal and human models. Within 24 hours after
injury, there is a rapid rise in cytokine levels (IL -6 and 8, tumor necrosis factor and keratin
sulfate) that return in approximately 1 week, to a level comparable to a chronic knee OA group.33
It is unknown, when or if, the inflammatory cytokine levels return to a pre-injury baseline.
Numerous studies have however identified various inflammatory cytokines as biomarkers for
early and late stage knee OA.34,35
Sensory neurons are known afferents, but C-fibers (Group IV) can act efferently and
release neuropeptides (Substance P and CGRP) that serve to increase neurogenic inflammation in
the area.36 This chronic inflammatory environment and sensitization to the nervous system has
the potential to negatively impact normal neuromuscular function, vascular function and articular
cartilage.
Normal articular cartilage is composed of a tight meshwork of collagen fibrils that entrap
proteoglycans. In normal healthy joints, Type II collagen accounts for 90-95% of the collagen in
articular cartilage.37 There are two major classes of proteoglycans: large aggregating
proteoglycans monomers called aggrecans and small proteoglycans including decorin, biglycan
and bifromodulin.37 Aggrecans have large numbers of chondroitin-sulfate and keratansulfate
chains attached to a protein core filament.37 More advanced idiopathic OA has been associated
with increased damage to and loss of type II collagen.38 Unknown mechanisms activate
collagenases and aggrecanases that cleave type II collagen and aggrecans, leading to the
breakdown of these vital articular cartilage building blocks. Nelson et al, in 2006, analyzed
cartilage samples of 28 people undergoing ACL reconstruction and compared this to 21 human
knee samples from autopsy.38 The ACL subjects were divided into two groups, greater than and
less than 1 year since injury. Both groups showed significant increases in denaturation and
6
cleavage of type II collagen compared to controls (collected at autopsy from 21 persons with a
median age of 53 years). The cartilage changes measured in the ACL population are similar to
idiopathic OA and support the theory of an injury-induced degenerative process.38 Another
investigation into type II collagen breakdown reveals urinary (u) CTX II levels (fragments of
type II collagen) are elevated for at least 4 months in humans after ACL reconstruction. 39
Interestingly, after adjusting for BMI, uCTX-II levels were significantly higher at all time points
compared to a control group. 39 This work by Chmielewski also highlights the possibility that
surgical reconstruction is yet another insult to the knee that could further or reinforce the
inflammatory degenerative cycle.
In an animal model, ADAMTS-5 (ADisintegrin and Metalloproteinase with
Thrombospondin Motif) has been identified as an aggrecanase responsible for the rapid onset of
post-traumatic OA40. Malfait et al in 2010, utilized a surgical medial meniscus destabilization
procedure to induce osteoarthritic degeneration and pain (demonstrated by withdrawal to
mechanical stimulus) in wild-type mice as soon as 8 weeks post insult. ADAMTS-5
(aggrecanase-1) null mice were completely protected against degeneration and allodynia,
therefore blocking the expression of aggrecanase prevents the development of OA and related
sensory changes 40, in an animal model.
Additional animal studies, using an ACL injury model, show rapid onset of degenerative
changes in the knee after surgical ACL transection.41 Miller et al found six weeks after ACL
transection in rabbits that synovial hyperplasia, capsular thickening, MCL scarring and bucket
handle tears were observed in all 12 of the skeletally mature 1 year old New Zealand White
rabbits that underwent ACL transection.
7
Animal model studies have demonstrated degenerative changes in both isolated ACL and
meniscus injury models and human observational studies report knee OA will develop in nearly
50% of knees after ACL or meniscal injury.7,42-44 Despite the high prevalence of post-traumatic
knee OA the precise connection between trauma and degenerative changes remains unclear.
Aggrecanase-mediated degeneration of aggrecans is a hallmark feature of OA45. In
subjects with previous knee meniscectomy but without radiographic OA, levels of synovial fluid
aggrecans were weakly and inversely associated with increased loss of joint space over a period
of 7.5 years.46 Although costly and time consuming, western blot analyses is the current gold
standard for measurement of systemic levels of aggrecanase-cleaved aggrecans.47 Clinical tests
of the serum and urine are developing and in the future may serve as excellent biomarkers for
staging of knee OA.45 Byproducts of cartilage degeneration likely serve as a chronic chemical
irritant and may trigger peripheral and central nociceptive processes that perpetuate alterations in
the neuromuscular and vascular systems.
The clinical use of biomarkers remains controversial. In a study sample that included
1235 subjects no association was found between 17 biomarkers measured and radiographic knee
OA.48 Pain was not reported in this study and there was no long-term follow-up, therefore it is
unknown if biomarker levels were associated with pain or the progression of degenerative
changes. Clinically there is a weak correlation between pain and radiographic evidence of
osteoarthritis.49 While knee OA affects an estimated 27 million Americans only 10 million will
seek medical care due to complaints of pain.13 Current MRI based research by Zhang et al 2012
shows a strong link between synovitis and pain, with an odds ratio of 2.4 (p= 0.045) for
“frequent knee pain” when synovitis is present on MRI50. More recent studies have also started
to define the role of central sensitization in knee OA patients who report high levels of clinical
8
pain in the absence of moderate to severe radiographic knee OA.51 Clinical management of knee
OA should improve as researchers develop a better understanding of the relationship between
joint degeneration, inflammation and altered nociceptive processing.
4.
Inflammation in ACL injuries and OA Induces Nociceptive Sensitization
Inflammation can effect nociceptive pathways.31 This can be transient in the case of a
mild injury that resolves, or more long lasting depending on injury severity or a multitude of
factors that can lead to the development of chronic pain. An improved understanding of the local
inflammatory mechanisms and peripheral and centrally mediated nociceptive mechanisms is
necessary to understand the progression from acute injury to chronic pain and OA. Peripheral
sensitization of the nervous system is defined as increased responsiveness and reduced threshold
of nociceptors to stimulation of their receptive fields.52,53 Central sensitization is generally
described as an increased responsiveness of nociceptive neurons in the central nervous system to
normal or subthreshold afferent input leading to hyperalgesia.53 Both peripheral and central
sensitization contribute to the development of hyperalgesia in the ACL injury and knee OA
populations.
Witonski et al in 2004, found elevated levels of the neuropeptide, substance P, in torn
ACLs 1-4 months post injury, indicating the presence of neurogenic inflammation.54 Neurogenic
inflammation is likely one of the key factors in both central nociceptive sensitization and
alterations observed in neuromuscular performance.
9
Neuromuscular impairments in the ACL injury and knee OA populations are abundant.
55,56
Proprioceptive deficits have been identified in both populations and are typically found in
the affected and even the unaffected knee. Functional deficits, such as giving way and gait
limitations, may be associated with diminished proprioceptive acuity56 but limited evidence
exists.
Individuals with prior ACL rupture, complain of pain and ‘giving way’ during functional
activities, even with restored static stability and normal strength of the surrounding musculature
57
. Surgical ACL reconstruction has been shown to improve functional status and quadriceps
activation but not to the level of age matched controls.58 The factors that promote arthritic
changes are complex. In addition to abnormal wear and tear from altered arthrokinematics,
facilitated nociceptive mechanisms may promote pain at the ipsilateral and potentially, the
contralateral knee, due to neurogenic inflammation.59 Heightened nociceptive reflexes, indicating
central sensitization of nociceptive pathways, have been demonstrated in subjects following ACL
rupture, in spite of the fact that all subjects were reportedly pain-free at the time of testing.60,61
Attempts to determine which ACL deficient individuals will be able to cope without
surgical reconstruction have looked at quadriceps and hamstring strength and activation patterns,
extent of joint laxity, proprioception and other sensory changes.62 Courtney et al 2006 found
heightened hamstring activation patterns in response to unexpected platform perturbations,
particularly in the group classified as ‘copers.’63 A later study demonstrated a facilitated flexor
withdrawal reflex in a group of individuals with ACL deficiency, indicative of increased central
nociceptive excitability, and thus suggested that heightened hamstring activation in this
population may be a consequence of this altered pain processing.60 A similar pattern of
heightened flexor withdrawal activation was observed in a knee OA population.64 Similar
10
patterns of heightened flexor withdrawal reflexes have been seen after joint injury in both
animals65 and humans.66,67
B.
Background of Primary Testing Measures
1.
Arterial Flow Mediated Dilation
Brachial artery FMD has been studied extensively in CVD populations and considered a
reliable measurement tool and valid measure of widespread endothelial health.68 Current
research reports, however, show an inconsistent relationship between upper extremity (UE) and
lower extremity (LE) measures. A recent report by Thijssen et al found no correlation between
measures in humans,69 while animal study research correlations do exist.70 Although there is
less published research on LE arterial FMD, research shows an increased likelihood of
atherosclerotic plaque development in the LE compared to the brachial and carotid arteries.71-73
It is possible that the pro-inflammatory environment local to the injury alters endothelial
function. On the other hand, CV risk factors are known to impair endothelial function
systemically. Since, there is a lack of correlation between LE and UE measures, and the impact
of the cellular changes maybe local to the ACL injury; this study will probe FMD in bilateral
popliteal arteries of young and otherwise healthy, lean subjects. In addition, brachial artery FMD
will be used to determine local vs. systemic effect of ACL injury on FMD. The reliability of
popliteal FMD measures has been established.69,74
Abnormal endothelial function marked by reduced dilation to an increase in blood flow
(endothelium dependent FMD) is a well-established early indicator of CVD and is strongly
correlated with the likelihood of future CV events.75 There is an ever growing evidence base
that supports the need to maintain full function of the vascular endothelium in prevention of
11
atherosclerosis. Optimal function of the endothelium likely occurs through release of
endothelial-derived factors such as nitric oxide (NO), which produce anti-proliferative, antiinflammatory, anti-thrombotic, and pain modulating properties, in addition to vasodilation.20,76
The cardiovascular risk associated with OA emphasizes the importance of studying
endothelial dysfunction as a precursory event before CV symptoms arise. Endothelial
dysfunction is an early indicator of blood vessel damage and atherosclerosis, 75,77 and is closely
related to coronary endothelial function. 79 Endothelial function can be assessed through a
reliable, noninvasive method called flow-mediated dilation (FMD), which is an ultrasonic
assessment of FMD in response to occlusion-induced hyperemia. The assessment of endothelial
function through FMD represents endothelium-derived NO availability in humans.79 During the
FMD test, vasodilation occurs following an acute increase in blood flow, typically induced by
circulatory arrest in the arm (supra-systolic cuff occlusion) for a period of 4-5 minutes.
80, 78
The
hyperemia increases laminar shear forces parallel to the long axis of the vessel, 79-81 which is
transduced through luminal mechanoreceptors to the endothelial cell80. The increase in arterial
diameter, as a consequence of reactive hyperemia, is compared to the baseline diameter and is
expressed as % FMD. However, it is arguable whether NO is the only mediator of endothelium
dependent vasodilation82, since the mechanism of vasodilation depends on vessel type, its size
and how FMD is induced.83,84 Impairment of endothelial function is apparent in many
cardiovascular diseases such as hypertension, stroke, coronary heart disease, and
atherosclerosis.85
Atherosclerosis research trends have moved away from the classic view of passive
cholesterol storage and toward the current view of active, inflammatory-driven metabolism in the
arterial walls.86 In a sample of 329 myocardial infarctions only 19% occurred in coronary
12
arteries with greater than 75% stenosis while nearly 50% occurred in arteries with less than 50%
stenosis.87 This work shows that coronary angiography has a limited ability to predict the site of
subsequent myocardial infarction. CRP, a biomarker of systemic inflammation, is targeted in
many CVD studies and is linked to elevated risk of CV events. Data from the Physician Health
Study (n=550) shows that men in the highest quartile of CRP levels have triple the relative risk
of a CV event of those in the lowest quartile.88 Human physiology investigations have
confirmed that CRP is primarily produced in the liver in response to IL-6.89 The FMD response
is impaired by inflammatory cytokines such as CRP and is considered an early prognostic
indicator of cardiovascular disease.
Proteases (such as ADAMTs) play a critical role in the synthesis and degradation of the
extracellular matrix (ECM) of the intimal wall. Versican is a proteoglycan in the arterial wall that
holds low density lipoprotein (LDL) in place. Inflammation drives a shift in the balance of the
ECM. Salter et al in 2010 reports inflammation induced activity of ADAMTs contributes to
plaque instability, increased risk of rupture and thrombosis90. Inflammation induced activity of
the ADAMT’s can initiate degradation of articular cartilage and increase vascular instability.
Nitric Oxide (NO) plays a role in both pain perception and endothelial function.(reviewed
in Mackenzie et al76). The challenge with creating the link between knee OA and atherosclerosis
comes down to the lack of established optimal levels of NO.91 NO in the proper amount is an
essential component of a diverse range of physiological processes.76 However, NO production
in excess, as a component elevated inflammation, will lead to the reaction of NO with superoxide
free-radicals, resulting in short lived oxidant species such as peroxynitrite, which is a potent
inducer of cellular death and blocks nitric oxide synthase (NOS) enzyme activity.92
pathway may represent a link between early OA and vascular dysfunction.
13
This
2.
Pressure Pain Threshold Testing
According to the International Association for the Study of Pain, pressure algometry is
the most commonly used quantitative technique to assess tenderness in myofascial tissues and
joints. 52 A reduction in pressure pain thresholds or increased pain ratings at numerous sites
indicate widespread hyperalgesia. Exact mechanisms of widespread hyperalgesia are complex
and not fully understood but the presence of both peripheral and central sensitization are
commonly discussed components. Hyperalgesia of deep somatic tissues has been demonstrated
in patients with knee OA, using PPT.93,94 Nociceptors in deep somatic tissue, such as joint and
muscle, show pronounced sensitization to mechanical stimuli in contrast to cutaneous
nociceptors.93 Numerous studies have shown decreased PPT in acute and chronic
musculoskeletal conditions both local95,96 to the source and remotely.97,98 In the present study,
PPT at the involved knee represented local hyperalgesia, while measures at the involved side
tibia, contralateral lower extremity and hand are a sign of central sensitization.
14
III. METHODS
A.
Study design and Subjects
This was a non-randomized prospective, cross-sectional study. A convenience sample of
men and women (n=29) were recruited from an urban university setting. A total of 15 individuals
with ACL reconstruction (ACL-R) (male, n=3; female, n=12), defined as those who had under
gone surgical ACL reconstruction more than 6 months prior to participation in the study.
Fourteen healthy control subjects, (CON) (male=4; female = 10) defined as individuals with no
history of lower extremity, participated. Exclusion criteria were as follows: current use of
vasoactive medications, history of diabetes, hypertension, pregnancy, tobacco use in the past 6
months, illicit drug use, cardiovascular disease or events, thyroid disease, pituitary tumor, a
genetic disease causing disability, gout and a body mass index (BMI) ≥30 kg/m2. The study was
approved by the Office of Protection of Research Subjects and Institutional Review Board (IRB)
at the University of Illinois at Chicago.
B.
Overview of Study Protocol
All data was collected in the Outpatient Care Center at the University Of
Illinois Hospital & Health Sciences System Chicago. All subjects fasted (8 hours) and abstained
from exercise (8 hours) before testing. Subjects completed medical history questionnaires,
including specifics on injuries. Average physical activity in the participants was assessed by a
metabolic activity questionnaire for the last two months [Aerobics Center Longitudinal Study
(ACLS)]. Previous studies validate the ACLS questionnaire for associations between physical
activity levels and health, as well as fitness.99,100 Reported activity was later computed to
determine total metabolic equivalent (MET) hrs/week.101 Lower extremity self-reported
15
functional level was assess with the Knee Outcomes Survey-Activities of Activity of Daily
Living Scale (KOS-ADLS). The KOS is one of the most commonly used self-report functional
measures. Its reliability and validity are well established in both the OA and ACL patient
population.102-104 A lower extremity physical exam was completed by a single investigator (JDC;
a physical therapist with 11 years of clinical experience), to screen for the presence of knee
ligament or meniscus injury and other major lower extremity range of motion or alignment
abnormalities. Next, PPT testing (see section C below) was completed using pressure algometry
at five sites: 1) thumb web space of dominant hand, 2) medial tibiofemoral joint line of bilateral
knees, 3) mid shaft tibia bilaterally. Lastly, the ultrasound protocol for flow-mediated dilation
(FMD) was completed to the right brachial and bilateral popliteal arteries.
C.
Pressure Pain Threshold Testing
PPT testing was completed with a Wagner Pain Test FPX algometer (Wagner
Instruments, Greenwich CT), utilizing 1cm2 applicator. Pressure was applied at a rate 50kPa/sec
and subjects were clearly instructed to signal their “first pain” or the moment the pressure
became painful. Four measures were taken at each site (30 seconds rest between measures) and
the averages of the final three measures were used in the analysis.105 PPT as a quantitative
measure of hyperalgesia in musculoskeletal conditions has been found to be reliable in numerous
conditions and body regions.96,106,107 and ICC’s are reported to range from 0.83-0.91 and have
been reported in the lower and upper extremities of control and knee OA sample populations.108
16
D.
Brachial and Popliteal Artery Flow-Mediated Dilation
Due to the lack of correlation in human research between LE and UE measures and the
relative youth and health of the subjects in this investigation, bilateral LE popliteal FMD will be
assessed in addition to the right brachial artery FMD. In order to control for other variables that
are known to affect FMD results, all subjects were tested in the early morning (approximately
8am), in the same room with consistent climate control, after fasting at least 8 hours from food,
caffeine and vitamins. Subjects also abstained from vigorous exercise for at least 12 hours prior
to testing and all females were tested 7-10 days after the first day of their most recent menstrual
cycle. Prior to FMD and blood pressure measurement, subjects were placed supine on a plinth
for at least 20 minutes in the same temperature controlled room, while physical exam and PPT
testing was completed. Ultrasound imaging was conducted using M-Turbo ultrasound (Sonosite;
Seattle, WA). Imaging of the brachial artery was performed in a longitudinal plane, at
approximately 5 cm proximal to the antecubital fossa of the right arm, abducted approximately
80° from the body, with the forearm supinated. The ultrasound probe (11 MHz) was positioned
at a 60° insonation angle to visualize the anterior and posterior lumen-intima interfaces to
measure diameter or central flow velocity (pulsed Doppler). Baseline images and blood pressure
readings of the opposite arm with an automated sphygmomanometer were recorded. After
baseline ultrasound imaging, Doppler readings of peak flow and average flow were performed
for at least 5 seconds. A blood pressure cuff was placed on the forearm, distal to the antecubital
fossa on the right arm being imaged and inflated 60 mmHg above baseline systolic blood
pressure (SBP) for 5 minutes. Once the cuff was released, blood pressure and HR measurements
in the opposite arm were taken, along with Doppler readings of the first 10 seconds after cuff
17
release. The brachial artery was then imaged continuously to capture 30 seconds, 1 min, 2 min,
and 3 min post cuff release.
The same protocol was utilized for FMD measurements in the right and left popliteal
arteries. The blood pressure cuff remained on the left upper arm, the subject turned into a right,
semi-prone side lying position and a pillow was placed between the lowers limbs and the knees
were flexed to a comfortable position of approximately 30 degrees of knee flexion. Longitudinal
imaging of the popliteal artery occurred approximately 5cm proximal to the center of popliteal
fossa. The proximal edge of the occlusion cuff was placed just distal the popliteal fossa.
Images were digitally recorded using Brachial Imager (Medical Imaging, Iowa City,
Iowa, USA) and analyzed as previously described80. 450 frames (7.5 frames per second for 10
seconds) were captured, digitized, and analyzed from the M-line (border between intima and
media of brachial artery) of the same location of blood vessel using visible landmarks through
edge detection software. Approximately 75 frames were analyzed for each baseline and time
point measurement through an average of artery diameters over the entire R-R interval.
Electrocardiogram (ECG) gating was not performed for all subjects during ultrasound analysis.
Previous research demonstrated that when comparing FMD and NTG-induced dilation analyses
through QRS gating or an average of brachial diameters over the entire R-R interval, a strong
agreement was found between both methods for FMD and NTG-induced dilation, with
measurements based on average diameter not reducing accuracy.109 Percent FMD was calculated
using the averaged minimum mean brachial artery diameter at baseline compared with the largest
mean values obtained after release of the forearm occlusion. The peak flow velocity was
observed from 5 seconds of baseline diameter Doppler readings and 10 seconds of post-cuff
release Doppler readings were recorded for shear rate calculations. Shear rate was calculated as
18
blood velocity (cm/s) divided by vessel diameter (cm).110 Baseline arterial diameter measures
were used shear rate calculations.
G.
Statistical Analysis
All data are reported as mean± SE, with P <.05 as significant unless otherwise noted.
The Shapiro-Wilk test for normality was completed as this test is appropriate for sample sizes of
less than 50. The Kruskal-Wallis Test (non-parametric ANOVA) was completed to assess for
significant difference between in FMD and PPT of subgroups within and between the ACL-R
and the control groups. (ie: right and left knees, involved and un-involved knees within the ACLR group to right and left knees within the control group). Independent samples t-tests
(parametric data) and Mann Whitney U tests (non-parametric data) were employed to compare
differences in subject characteristics between groups. Post-hoc between group comparisons were
completed with the Mann-Whitney U test following the Kruskal-Wallis for the FMD and PPT
data. PPT testing results are reported in N/cm2 and dilation in dose response data is expressed as
a percentage change from the resting/baseline diameter to the maximal diameter. For correlation
analyses, Pearson product-moment was used for parametric variables, and Spearman rho for
nonparametric variables. Effect sizes were also calculated when possible. Analyses were run
with IBM SPSS Statistics software (Version 20.0, SPSS Inc., Chicago, Illinois, USA). Plotting
was completed in SigmaPlot Version 12.3 (San Jose, CA, USA)
19
IV. RESULTS
A. Subject Inclusion and Exclusion
27 subjects were tested and in a subset of 22 individuals, FMD testing was performed.
One subject was entirely excluded from both PPT and FMD analysis since she was not in the
fasting state and her PPT 2SD’s above the mean. Another subject was also excluded from the
control group analysis due to a remote history of several lower extremity injuries and PPT results
nearly 2 SD’s below the mean.
B. Subject Characteristics
Age, gender, BMI, HR, SBP and DBP (mmHg) were not significantly different between
control and ACL reconstruction (ACL-R) groups (TABLE I). The ACL-R subjects did report a
higher level of activity level (MET Hours/wk) on the ACLS questionnaire (z= -2.115, U= 47.5,
p= 0.034 with an effect size of r= 0.407) but a lower functional level on the KOS-ADLS (z= 3.456, u=21, p= 0.001 with an effect size of r= 0.665). The worst pain on NPRS for the ACL-R
group was significantly higher than the CON group (z= -2.259, u= 53.5, p= 0.024 with an effect
size of r= 0.435).
Using Cohen’s criteria the above effect sizes are considered medium and
large (0.1= small effect, 0.3= medium effect, 0.5 = large effect). 111
20
Table I SUBJECT CHARACTERISTICS
Control
ACL
N
14
15
Age
Subjects
Total (n=29)
26.6±3.25
N=14
10 female, 4 male
N=13
9 female, 4 male
26.9±1.29
N=15
10 female, 5 male
N=14
9 female, 5 male
0.402‡
0
-
Pain included
p-value
-
Total ACL knees
Side of ACL
Reconstruction
Included
FMD included
Subjects
Popliteals (R/L)
Brachials
0
20
RLE only: 5
LLE only: 3
BLE:6
N=10
N=20
N=10
N=11
N=21
N=11
-
HR
59.8±1.99
61.5±2.04
0.563†
SBP
109.9±3.26
114.1±2.21
.109‡
DBP
70.1±2.01
70.7±1.82
0.830†
BMI
ACLS
(METhours/wk)
22.27±0.34
23.12±0.81
0.467‡
31.42±3.44
46.34±5.35
0.034‡
KOS-ADLS
Avg Pain
(NPRS)
Worst Pain
(NPRS)
Chronicity
(years since
surgery)
98.08±1.1
90.80±1.82
Mean: 0.2±0.15
Mean: 0.36±0.2
Median: 0
Median: 0
Mean: 0.31±0.31
Mean: 1.57±0.52
Median: 0
Median: 0.5
ACL Group Only
Mean: 7.79 ±0.88 (median: 8.04)
Range: 1.25-13.9
†: T-test
‡: Mann-Whitney U Test
21
-
0.001‡
0.432
0.345
0.049†
0.02‡
C. Pressure Pain Threshold
1. PPT at the Hand
PPT results did not differ between groups at the dominant hand 1st and 2nd digit (thumb)
web space; control: 26.04N±1.64, ACL-R: 27.99±3.38, p=0.617. (Table II)
Table II PPT AT DOMINANT HAND WEB SPACE
PPT Dominant Web Space
ACL-R Group
Con Group
(Mean ± SE)
(Mean + SE)
p value
26.04N±1.64
27.99±3.38
p=0.617
2. Right and Left Leg PPT Comparisons
Comparisons between the right and left limbs of the control group and the ACL-R groups
at the medial tibiofemoral joint line and tibia showed no significant differences (Table III).
Therefore the right and left LE measures were combined as indicated into involved and
uninvolved limbs based on the side of the ACL reconstruction.
22
Table III RIGHT AND LEFT PPT COMPARISON AT KNEE JOINT LINE AND TIBIA
Right
Left
(Mean ± SE)
(Mean + SE)
p value
Joint Line CON
41.86 ± 4.66
40.17 ± 4.74
0.803†
Joint Line ACL-R
32.07 ± 3.004
27.0 1± 2.93
Median
29.16
23.2
Tibia CON
39.80 ± 4.98
41.82 ± 4.77
Median
30.8
40.13
Tibia ACL-R
31.35 ± 3.25
30.68 ± 3.91
Median
29.0
32.0
0.129‡
0.608‡
0.806‡
†T-test
‡Mann-Whitney U test
3. PPT at the Tibia
PPT measures at the tibia (control, ACL-R all, ACL-R involved) were compared with the
Kruskal-Wallis test (non-parametric), due to result of robust tests for equality of means (BrownForsythe p=<0.05) during initial attempts to compare via ANOVA. . The result of the KruskalWallis revealed X2 =4.438, df=2, p=0.109. Post hoc Mann-Whitney U testing of the differences
observed in PPT’s measures at the tibia show a strong trend toward lower PPT in the ACL-R
group. PPT results from the control tibia: median= 36.2 to the ACL-R all group: median= 30.14
(z= -1.833, u= 207.5, p= 0.067). (Table IV) Trending but non-significant differences were also
observed when the involved tibias were compared to the control group (p=0.082) (Table IV).
23
Table IV PRESSURE PAIN THRESHOLD AT MEDIAL TIBIA
PPT Grouping
Tibia CON vs.ACL-R all
Tibia CON vs. ACL-R Inv
CON
Median
36.2 N
36.2 N
ACL
Median
30.14 N
29.57 N
Z
U
Sig.
(2 tail)
-1.83 207.5 0.067
-1.74 181.5 0.082
Effect
Size
0.262
0.257
N
49
46
4. PPT at the Medial Tibofemoral Joint
The Kruskal-Wallis test with an independent groups design to determine more specific
changes in pain sensitivity either locally or regionally was completed at the medial knee. The
different cases (or independent groups) were as follows: CON all, ACL-R all, ACL-R involved
knees, ACL-R contralateral knees, CON R LE, CON LLE, ACL-R RLE, ACL-R LLE. Results
revealed statistically significant difference in PPT values across the 8 different groups X2=
18.584, p=0.010. The CON group had a median PPT of 37.79 N while the ACL all and ACL
involved group medians were 26.48N and 27.63N respectively. Post hoc analysis with Mann
Whitney U test to determine difference between pairs of groups revealed significant difference
between all control knees (median: 37.79) and all knees in the ACL-R group (median: 26.48) (z=
-2.545, u=217, p=0.011 with an effect size of r = 0.346). Significant difference was also found
when only the involved medial knee PPT (median: 27.63), was compared to the control group
(z=.2.016, u= 169, p=0.044 with an effect size of 0.297). A Bonferroni adjustment of the alpha
value (0.05/2= 0.025), to control for a Type I error in the presence of multiple post-hoc
comparisons, confirms the only significant finding was the comparison of whole group median
scores (ACL-R vs Control: p=0.011 with effect size of r=0.346)(Figure I). A post-hoc PPT
24
comparison of the LLE CON group to the LLE of the ACL-R group also reveals a statistically
significant difference between the CON median: 36.27, ACL-R median: 23.2 (z= -2.329, u: 43,
p=0.02, effect size of r=0.45). This was the largest effect size observed in all the comparisons.
Further investigation of the LLE shows the lowest PPT mean when measures taken from the left
medial knee of a unilateral R LE injury (mean = 25.186±3.27; median 21.6) (z=-2.021, u= 64,
p=0.043, effect size r=0.476). There are 5 observations in this LLE (contralateral to R LE
injury) uninvolved grouping. (Figure II) Only 3 observations occurred in the R LE
(contralateral to LLE injury) and the PPT data was as follows: 17.6N, 58.67N and 20.8N. Due
to the low number of observations and variability in this data, this comparison was not
completed.
Table V PRESSURE PAIN THRESHOLD AT MEDIAL TIBIOFEMORAL JOINT LINE
PPT Grouping
ACL
Median
ACL-R all vs CON all*
26.48 N
ACL-R Inv vs CON all*
27.63 N
LLE ACL-R vs. LLE CON*
23.2 N
RLE ACL-R vs. R LE CON
29.16 N
*Mann-Whitney U Test p=<.05
CON
Median
37.79 N
37.79 N
36.27 N
38.7 N
25
Z
-2.54
-2.01
-2.32
-1.31
U
217
169
43
64
Sig.
(2
tail)
0.011
0.044
0.02
0.19
Effect
Size
0.346
0.297
0.448
0.252
N
54
46
27
27
Figure 1 PPT AT THE MEDIAL TIBIOFEMORAL JOINT LINE
50
PPT (N)
40
30
20
10
0
Control
ACL
Mann-Whitney U Test p=0.011
Mann Whitney U test of PPT measures from all CON knees and all ACL-R knees at the medial
tibiofemoral joint line revealed statistically significant difference (p=0.011).
26
Figure 2 UNILATERAL PPT COMPARISONS
50
PPT (N)
40
30
20
10
0
CON RLE CON LLE
ACL RLE
ACL LLE ACL LLE Univ
Control LLE vs ACL LLE p=0.02; Control LLE vs. ACL LLE uninvolved= 0.043
Significant difference was also determined when only the injured knees were compared
to the control group. The LLE of the CON group also demonstrated different pain sensitivity
than that of the ACL-R group (p=0.02).
27
D. Brachial Artery Flow Mediated Dilation
Comparison of between group differences at the right brachial artery showed a similar
FMD response of 9.07±2.26% in the CON group and 8.27±1.32 in the ACL group with p=0.755.
Peak shear response and FMD normalized to peak shear response were also similar between the
ACL-R and the Control group. (Table VI)
Table VI BRACHIAL CHARACTERISTICS
ACL-R Group
Con Group
Brachial Artery (right) FMD %
(Mean ± SE)
8.27±1.32
(Mean + SE)
9.07±2.26
p value
0.869
Peak Shear Rate
394.93±39.65
417.70±38.53
0.686
Normalized (FMD%/Shear)
0.0256±.008
0.0206±.004
0.806
28
E. Popliteal Artery Peak Shear Rate
Initial investigation of the popliteal FMD data revealed statistically significant
differences in the peak shear response between the right and left limbs of control group p=0.012
and trending differences in the ACL reconstruction group, p=0.068 (Table VII) (Figure 3).
These differences introduce error into combined group comparisons that average FMD results
from both right and left limbs. However, despite limb differences, combined average peak shear
rates from both limbs of the CON and ACL-R group did not differ significantly, 121.31 ±9.37 vs.
119.74±12.18, respectively with p=0.877.
Table VII POPLITEAL PEAK SHEAR RATE
Peak shear rate, -sec ± SE
N
CON all
121.31 ±9.37
16
CON R LE
143.01 ±12.06
9
CON LLE
100.1±9.19
8
ACL all
119.74±12.18
17
CON all vs ACL all: p=0.877‡
ACL R LE
145.16 ±26.12
8
ACL R LE vs LLE: p=0.068‡
ACL LLE
100.83±13.07
9
ACL involved
128.27 ±14.376
12
ACL contralateral
101.99 ±18.576
6
Grouping
‡: Mann-Whitney U Test
29
p value
CON R LE vs. LLE p= 0.012‡
Figure 3 POPLITEAL PEAK SHEAR COMPARISONS
180
160
Peak Shear Rate
140
120
100
80
60
40
20
0
CON R LE CON L LE ACL R LE ACL L LE
CON all
ACL all
Group and Limb Side
Control R LE vs. LLE p=0.012; ACL R LE vs LLE p=0.068; Control All vs. ACL all p=0.877
F.
Popliteal Artery Flow Mediated Dilation
The popliteal FMD response was different between the two groups. (Table VIII)
Mann-Whitney U test revealed median FMD of the control group at 5.785% compared to 3.93%
in the ACL-R group (z=-2.254, u= 109, p = 0.024, with effect size r = 0.361).( Figure 4)
Comparison of the control (median: 5.785%) to the ACL-R involved only limb (4.525%) FMD
30
failed to reach significance (z = -1.481, u = 87, p = 0.138). (Table VIII) Therefore the FMD %
in the involved limbs was slightly higher than in the uninvolved.
Table VIII POPLITEAL FMD COMPARISONS
FMD CON vs ACL all
CON
Median
5.785
Z
ACL
Median
3.94
-2.254
FMD CON vs. ACL Inv
5.785
4.525
FMD Grouping
-1.481
U
109
Sig.
(2 tail)
0.024
Effect
Size
0.361
87
0.138
0.262
Figure 4 POPLITEAL FMD CONTROL VS ACL
7
6
% FMD
5
4
3
2
1
0
Control all
ACL all
ACL Inv
Control all vs. ACL all: p=0.024; Control all vs. ACL Inv: p=0.138
31
N
CON:18
ACL: 21
CON: 18
ACL: 14
Same side limb comparisons between the CON and ACL-R groups were then completed.
Statistical significant difference was not achieved when the right popliteal FMD from the control
group 6.73% ±0.88 (n=10 ) was compared to the ACL-R group 4.65% ±0.77 (n=8), p=0.102.
This comparison included three FMD’s from subjects with B LE ACL-R, when the three FMD’s
from subjects with B LE injuries were excluded mean FMD did decrease in the ACL-R group
but the difference did not reach statistical significance. (ACL-R group, R LE only: 3.9% ± 1.05
compared to the control group: 6.73±0.88 , p=0.076.) (Figure 5) FMD normalized to peak
shear rate did not differ between the ACL-R and the CON groups in the right limb, p=0.418. Left
limb popliteal FMD comparisons between the CON and the ACL-R groups also did not reach
significance differences, 5.18% ± 0.63 to 4.54 ± 0.76 respectively, p=0.554. (Table IX and
Figure 6). FMD normalized to peak shear rate also did not differ in LLE, p=0.412. (Table IX)
Table IX POPLITEAL CHARACTERISTICS
Right Popliteal Artery FMD %
Peak Shear Rate
Normalized (FMD%/Shear)
Left Popliteal Artery FMD%
Peak Shear Rate
Normalized (FMD%/Shear)
ACL-R Group
CON Group
p value
4.65 ± 0.766
6.73 ± 0.877
0.102
145.16 ±26.12
143.01 ± 12.06
0.0356 ± 0.0035
.0418 ±.0056
0.418
4.53 ± 0.896
5.18 ± 0.625
0.554
100.83±13.07
100.1±9.19
0.0385± 0.0068
0.0439±.0027
32
0.412
Figure 5 RIGHT POPLITEAL FMD COMPARISONS
8
% FMD
6
4
2
0
Right Control
Right ACL-R all Right ACL-R only
Right Control vs All R ACL p=0.102; R Control vs. R unilateral solo: p=0.076
R ACL-R all= 5 RLE ACL only, 3 BLE ACL
33
Figure 6 LEFT POPLITEAL FMD COMPARISONS
7
6
% FMD
5
4
3
2
1
0
Control LLE
ACL LLE
Control LLE vs. ACL LLE p=0.554
The final comparison that revealed a statistical significant difference was the popliteal
FMD from LLE of the control group compared to a subset of the LLE of the ACL-R group that
was contralateral to a unilateral R LE ACL reconstruction. FMD was 5.18%±0.63 in the control
versus 2.76%±0.81 in the contralateral LLE of the ACL-R groups, independent samples t-test
p=0.037. (Figure 7) There were a total of seven popliteal FMD results contralateral to a
unilateral injury, five of this seven are from the left LE, the two R LE FMD’s were excluded
from the above comparison due to previously mentioned limb differences in peak shear
responses.
34
Figure 7 LEFT POPLITEAL FMD: CONTRALATERAL TO RIGHT SIDE INJURY
7
6
% FMD
5
4
3
2
1
0
Left Control
Left Contralateral
Left Control vs. Left Contralateral to Right side ACL: p=0.037
35
G.
Correlation of FMD to PPT
Spearman’s rho result determined no relationship between involved knee PPT at the
medial joint line of ACL-R subjects compared to popliteal FMD of the same knees r = -0.038,
n=21, p=0.871. (Figure 8) Pearson’s Correlation coefficient r = -0.213, n=12, p= 0.465 also
revealed no significant relationship, between PPT measures at the involved tibia and same side
popliteal FMD. (Figure 9) Pearson Correlation of PPT and FMD in the LLE uninvolved leg to
the right unilateral injury returned an r =0.827,n=5 p =0.084. (Figure 10) This correlation
reveals a strong possible connection of PPT and FMD in the lower limb contralateral to a
unilateral injury.
Figure 8 SCATTER PLOT OF POPLITEAL FMD TO MEDIAL JOINT LINE PPT
36
Figure 9 SCATTERPLOT OF TIBAL PPT TO POPLITEAL FMD
37
Figure 10 SCATTER PLOT OF FMD TO PPT IN LEFT LEG OF RIGHT ACL-R
38
H. Correlation of Pain and Chronicity
Pearson correlation coefficient r = .157, n= 18, p = 0.520, revealed no significant
relationship between involved knee PPT and chronicity in years since injury. (Figure 11)
Figure 11 CORRELATION OF PPT TO CHRONICITY
39
V. DISCUSSION
To our knowledge this is the first study to report decreased pressure pain sensitivity and
arterial flow mediated dilation chronically after ACL reconstruction. The combined results of
differences in PPT at the knee and percent FMD in the popliteal artery of the ACL reconstruction
group versus the control reached statistical significance. Based on current results and
background research we propose a cyclical mechanism, initiated by joint injury and
inflammation, that activates intrinsic cellular degradation mechanisms (aggrecanases), which
release bioactive aggrecan fragments that propagate the inflammation cycle. (Figure 12)
Animal model research shows antigenic t-cell (immune system) responses to cartilage fragments
(proteoglycan aggrecan epitopes);112 supporting the plausibility of the theory, that once activated,
the aggrecanase-mediated joint degradation is cyclical. This cycle creates an interesting
dilemma for an individual after ACL injury as ligamentous instability is associated with high risk
of subsequent injury (especially to the meniscus), while surgical reconstruction introduces
another nociceptive and inflammatory insult to the knee and neuromuscular systems.
Chronic
hyperalgesia in the knee effects normal neuromuscular control61 and vascular impairments may
contribute locally to degenerative changes113 and relate, chronically, to cardiovascular disease26.
40
Figure 12 THEORETICAL MODEL
JOINT INJURY, CHRONIC PAIN AND VASCULAR DYSFUNCTION
A. Subject Characteristics
The mean difference in worst pain between the two groups was 1.2 points on the 0-10
NPRS scale (ACL-R group: 1.57 and CON: 0.31). With a reported MCID of two points on the
NPRS114, this difference does not reach clinical significance. 114114 However, more importantly
the ACL-R group did report a median score on the KOS-ADLS 8 points lower than the control
41
group, 92 vs. 100, respectively. The MCID for the KOS-ADLS has been reported as 8-10
points103,104. Therefore, the KOS-ADLS difference between groups, especially in combination
with the NPRS scores, is noteworthy and represents low- grade impairment in pain and function
chronically after ACL reconstruction.
The groups differed further in their exercise levels with the ACL reconstruction group
reporting a median difference of 13 MET hours/week of activity. With an average weight of the
study participants of 65.5 kg this means that he ACL reconstruction group is expending
approximately 850 calories more each week than the control group. This finding has potential
significance when considering the cardiovascular and all-cause mortality risk associated with
sedentary lifestyle. 115 Exercise has been shown to have anti-inflammatory effects in humans. In
a group of 45 sedentary males, 12 weeks of moderate intensity exercise significantly decreased
IL-6 levels116. The exact mechanism remains unknown however some theorize that exercise
drives the release of myogenic IL-6 which displays anti-inflammatory properties that counteract
the pro-inflammatory properties of cytogenic IL-6117. Furthermore, aerobic exercise has a
cumulative large effect size of 0.52 for pain relief in knee OA populations in a systematic review
of 13 randomized controlled trials118,119. This specifically relates to the hypothesis of the current
study by providing a mechanism for a protective effect to both hyperalgesia and vascular
function. Future study samples that match activity levels in the groups would control for the
possible protective effect of exercise.
B. Pain Comparisons
Summative comparisons of the two groups show hyperalgesia in ACL reconstruction
group which corresponds to this group’s report of both higher NPRS (worst pain levels) and
42
lower self- report functional levels (KOS-ADLS). Differences in pain are robust locally, at the
medial knee, but are also trending distally at the tibia. No difference was noted at remote
locations (thumb web space).
Statistically significant differences in hyperalgesia were found at the medial knee of the
ACL-R group compared to the controls. This is in accordance with numerous previous studies in
the knee OA populations 93,94,120,121, however to our knowledge; this is the first reporting of such
a finding in a chronic ACL reconstruction population. Based on knee OA studies it was
hypothesized that in this relatively young sample we would find hyperalgesia isolated only to the
involved knee. However, the tibial measures were also lower but not significant (p=0.067) in the
ACL reconstruction group; tibial mean PPT of 31N vs. 40N in the reconstruction vs. control
group respectively. Although this difference was not statistically significant, PPT in the ACL
reconstruction group was nearly 25% less than the controls and a larger change in hyperalgesia at
distal sites than expected. Numerous previous studies have reported hyperalgesia (lower PPT) in
chronic painful conditions such as osteoarthritis, whiplash, and lateral epicondylalgia.122-124
However the higher NPRS level and lower PPT in this ACL-R sample, a group that would be
expected to fully recover from surgery is a novel finding. This chronic low-grade hyperalgesia,
especially at the tibial sites, is a sign of central sensitization and has the potential to further
sensitize nociceptive pathways and promote the spreading of hyperalgesia. The spreading of
hyperalgesia has been attributed to an increased responsiveness of spinal cord dorsal and ventral
horn neurons outside of their original receptive field (ie: leg and thigh sites responding to knee
joint stimulation).125-127
Experimental nociceptive stimulus induces neurogenic inflammation and activates the
release of neuropeptides responsible for hyperalgesia both locally and in the corresponding
43
contralateral limb.59,128-130 Nociceptive stimulus alone may be sufficient in some individuals to
produce chronic changes in pain perception. Research has shown, that phenotypic changes to
low threshold A-β fibers occur, leading to expression of neuropeptides only observed in highthreshold nociceptive fibers (A ∆ and C).31,53 131 This phenotypic change to the A-β fibers may
then serve as a foundation for non-noxious stimulus to further sensitize human pain perception,
in the absence of subsequent injury or noxious exposure.
Chronic inflammation can negatively affect neural tissue. Witonski et al in 2004, found
elevated levels of the neuropeptide, substance P, in torn ACLs 1-4 months post injury, indicating
the presence of neurogenic inflammation.54 Following acute ACL injury, nociceptive pathways
become sensitized.60
A type II collagen biomarker study found an association between
biomarkers, pain and function in an ACL reconstruction population during monthly testing for 4
months. The authors reported a positive correlation of uCTX-II levels with pain and a negative
correlation to function.39
Classic injury and healing models support the resolution of inflammation and
hyperalgesia at some point after injury; however this does not appear to occur in many
individuals after ACL injury and reconstruction. Therefore some combination of inflammatory
mechanisms and nociceptive pathway change is likely occurring. Further testing is necessary to
determine if intrinsic cellular degradation mechanisms (ie: induction of aggrecanases) in humans
play the vital role they do in animal model research. 40 As an observational study, it is
impossible to determine the exact mechanism of the hyperalgesia observed, however several
plausible explanations exist.
44
C. FMD Comparisons
Peak shear response between the R LE and LLE of the CON group was significantly
different and nearly reached significant in the ACL-R group; revealing a consistent difference in
the peak shear response and resultant FMD in the right and left legs. Most importantly whole
group shear stress between ACL-R and CON did not differ significantly (p= 0.840) but FMD
response between groups did (p=0.024). Due to the reported high sensitivity of the popliteal
arteries to shear stress132 consideration of this variable is of the utmost importance. If mean
peak shear rates between groups were different then whole group comparisons would not be
possible. Therefore whole group differences need to be interpreted carefully, while intra-limb
comparisons control for the differences observed in peak shear rates.
Summative comparison of the two groups reveal impaired FMD in the ACL-R group as a
whole (p=0.024). More specific comparison of involved knee FMD (popliteal artery local to
injury) to the controls weakens this difference (p=0.138). (Table VIII and Figure 4)
Interestingly, comparison of popliteal FMD of the control groups LLE to the LLE of the ACL
reconstruction group contralateral to a R LE only injury revealed significant difference
(p=0.013). Additionally, there is the possibility of relationship between FMD and PPT with
Pearson product-moment correlation coefficient r=0.827, n= 5, p =0.084. (Figure 10) This
reduced arterial function, in the limb contralateral to the injured side, is contrary to our initial
hypothesis that the pain and arterial dysfunction would be most significant local to the
inflammatory source (injured knee).
45
Neurogenic inflammation (NI) triggered both by chronic nociceptive input and elevated
inflammatory cytokines local/ipsilateral to a unilateral ACL reconstruction is the most plausible
explanation. NI as a well-known vasodilator128, may be minimizing the local inflammatory
mediated vascular impairment through the stimulation of dorsal root and axonal reflexes. 129,130
NI, through spinal circuitry, can induce vascular change in the contralateral, uninvolved
limb.59,129 Kelly et al reported changes in vascular dilation but not permeability (which would
lead to edema) in an experimental NI model. The vasodilation observed by Kellyet al, was
slightly greater ipsilateral vs. contralateral but the difference was not significant.59 The
contralateral changes observed in the present study likely represent complex interactions of local
inflammatory factors and nociceptive driven neurovascular changes. The role of the autonomic
nervous system, specifically sympathetic stimulation to the lower extremities, should also be
considered. Sympathetic stimulation is a known vasoconstrictor133, while neurogenic
inflammation is a known vasodilator. If previous injury and surgery and current hyperalgesia
stimulates a low-grade sympathetic response, this vasoconstrictive response would affect the
bilateral LE’s but be overpowered ipsilateral to the injury.
Exercise could possibly play a role in minimizing both the arterial dysfunction and
hyperalgesia. Exercise has been shown to have anti-nociceptive effects on OA pain.118,119
Others have proposed exercise either stimulates a biochemical anti-inflammatory effect116,117 or
the local shear stimulus to the arterial endothelium improves NO bioavailability.134
Unfortunately the methodology of the current study does not allow for the determination of the
exact physiological mechanisms at play.
46
D. OA to CVD Connection
Surprisingly, in a large sample of nearly 3600 people, OA in any finger joint predicted
cardiovascular deaths in men (relative risk: 1.42, 95% CI 1.05 to 1.92)22. Others have also
questioned if OA, especially subchondral bone sclerosis and cyst formation is an atheromatous
vascular disease.113 In a cross-sectional observation, Kurunlahti et al, reported a significant
association between atheromatous lesions in the abdominal aorta and low back pain, as
determined by presence of atherosclerotic lesions on CT scans in a low back pain population
compared to age matched controls.135 MRI investigation in a knee OA population revealed
increased popliteal artery wall thickness (a measure of metabolic disease) in a knee OA
population versus an age matched control136. The evidence continues to build supporting a
direct physiological connection between OA and cardiovascular disease, beyond just similar comorbid conditions in a similar cohort. Future research is recommended that builds on the current
project with larger sample sizes, more extensive investigation of inflammatory cytokines, serum
or urine analysis of type II cartilage byproducts and a more detailed analysis of arterial function.
E.
Limitations
Subjects with ACL injury and subsequent construction were used; therefore the
changes observed may not be only a result of the ACL injury but may also be related to the
surgical reconstruction procedure. The ACL reconstruction sample was of greater convenience
and reports show increased incidence of post-traumatic knee OA after ACL injury independent
of surgical reconstruction.
47
Significant findings of pain and arterial dysfunction in the left leg contralateral to a
right unilateral ACL reconstruction, was limited by only five observations. Unfortunately there
were only 3 subjects in this study with isolated left ACL reconstructions and FMD and PPT data
from the right LE of these subjects was very limited. PPT data was low for 2 of the 3 subjects,
but FMD results were only available for 2 of the subjects, therefore no formal analysis was
completed. The outcomes of the left limb contralateral to the right injury in the present study
would have been strengthened if a similar occurrence was observed in the right limb of those
with left unilateral injury and reconstruction.
Differences in peak shear response in the right vs. left popliteal FMD limited
combined group comparisons. Baseline artery diameter was slightly greater in the left popliteal
arteries but did not reach significance (p=0.3-0.5). The larger resting diameter of the left
popliteal artery would decrease peak shear rates. Differences in arterial size and hyperemic
response could be due to both limb dominance and distal muscle mass. However 20 of the 21
subjects who completed FMD testing were R hand dominant (leg dominance was not assessed)
and leg muscle mass was also not measured.
Finally, the majority of data was analyzed with non-parametric tests which are less
likely to find significant differences between groups. Future investigations should include larger
sample sizes to improve ability to complete contralateral comparisons in both lower extremities
and obtain more normally distributed data that allows for comparisons with parametric tests.
48
F.
Conclusions
Hyperalgesia and impaired FMD are both present in a group of individuals
chronically after ACL reconstructive surgery compared to an age matched healthy control group.
This is a novel finding in human research and as developed can improve the understanding of
post-traumatic knee OA and a possible link to cardiovascular disease. Significant hyperalgesia
was observed local and contralateral to the ACL reconstruction. A trending pattern of
hyperalgesia was also observed at tibial measurement sites. FMD comparisons between groups
reached statistical significance, while, interestingly the greatest impairment in FMD occurred in
the popliteal artery contralateral to a unilateral ACL reconstruction. This contralateral arterial
impairment likely represents an imbalance between local inflammatory factors and spinally
mediated reflexes effecting smooth muscle control. Future research is recommended to expand
on the current project with larger sample sizes, more extensive investigation of inflammatory
cytokines, serum or urine analysis of type II cartilage byproducts and a more detailed analysis of
arterial function.
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60
VITA
NAME:
Jeffrey Donald Clark
EDUCATION:
B.S., Central Michigan University, 1998
M.S.P.T, Central Michigan University, 2001
M.B.A., University of Illinois at Chicago, 2006
TEACHING:
Department of Physical Therapy, University of Illinois as Chicago,
2003-2012
PROFFESIONAL
MEMBERSHIP:
ABSTRACTS:
American Physical Therapy Association
American Academy of Orthopaedic Manual Physical Therapy
Immediate changes in quantitative sensory testing following
lumbar manipulation. Poster Presentation: AAOMPT 2010
Conference, San Antonio, Texas
Clark JD, Rhon D and Courtney CA. Acute knee injury to chronic
pain: clinical/neurophysiological features of osteoarthritic
progression. Platform Presentation (Focused Symposia). IFOMPT
2012 Conference, Quebec City, Canada
PUBLICATIONS:
Courtney CA, Clark JD, Duncombe AM, and O’Hearn MA.
Clinical presentation and manual therapy for lower quadrant
musculoskeletal conditions. Journal of Manual & Manipulative
Therapy, Volume 19, Number 4, 2011 , pp. 212-222(11)
61