INTERACTION EFFECTS OF THE STARTLE RESPONSE AND HORMONAL …

INTERACTION EFFECTS OF THE STARTLE RESPONSE AND

HORMONAL CHANGES ON KNEE STIFFNESS

by

Athena DeAngelis

A thesis submitted to the Faculty of the University of Delaware in partial fulfillment of the requirements for the degree of Master of Science in Exercise Science

Fall 2012

© 2012 Athena DeAngelis All Rights Reserved

INTERACTION EFFECTS OF THE STARTLE RESPONSE AND

HORMONAL CHANGES ON KNEE STIFFNESS

by

Athena DeAngelis

Approved: __________________________________________________ Charles Buz Swanik, Ph.D. Professor in charge of thesis on behalf of Advisory Committee Approved: __________________________________________________ William B. Farquhar, Ph.D. Chair of the Department of Kinesiology and Applied Physiology Approved: __________________________________________________ Kathleen S. Matt, Ph.D. Dean of the College of Health Sciences Approved: __________________________________________________ Charles G. Riordan, Ph.D. Vice Provost for Graduate and Professional Education

iii

ACKNOWLEDGEMENTS

I would like to say a special thank-you to several people who have been a great

support system and have provided me with everything I needed to finish this project.

First and foremost I want to thank all my lab-mates and friends that have helped as

volunteer subjects and assisting in all aspects of performing testing. These special

people include Kathy Liu, Craig Oates, Jen Halterman, Chris Clyde, Peter Braun, and

Dan Tocci. I really want to acknowledge Alan Needle for providing all his expertise,

advice, time and mentorship over the past two years. Finally, I could not do this

without awesome friendships and support that I will treasure forever with Christina

Shields, Rob Hulbert and Matt Astolfi.

The University of Delaware provided me the opportunity to work along side

some of the best researchers including my committee members: Dr. Kaminski, Dr.

Knight and Dr. Royer. I would also like to thank my advisor and chair, Dr. Swanik,

for providing me with guidance throughout this entire thesis process and giving me his

expertise in joint stiffness and neurocognitive function to assist me in completing this

degree.

Finally, I would be nothing without the love and support my family

continuously gives me everyday of my life. Even though they are not experts with my

project, they were always a phone-call away to talk to and always support everything I

was doing in order to earn my degree. Without them, I would not be the person I am

today.

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TABLE OF CONTENTS LIST OF TABLES ........................................................................................................ vi LIST OF FIGURES ...................................................................................................... vii ABSTRACT ................................................................................................................ viii Chapter

1 INTRODUCTION ........................................................................................ 1

2 METHODS ................................................................................................... 5

2.1 Experimental Design .............................................................................. 5 2.2 Participants ............................................................................................. 5 2.3 Instrumentation ....................................................................................... 6 2.3.1 Stiffness and Proprioception Device ............................................. 6 2.3.2 Electromyography ......................................................................... 7 2.4 Procedures .............................................................................................. 8 2.5 Data/Statistical Analysis ......................................................................... 9

3 RESULTS ................................................................................................... 11 3.1 Demographics & Maximum Voluntary Isometric Contraction ............ 11 3.2 Startle Effects on Stiffness and EMG Variables .................................. 11 3.2.1 Stiffness & Startle ........................................................................ 11 3.2.2 EMG & Startle ............................................................................. 11 3.3 Startle Effects on Group Differences ................................................... 13

4 DISCUSSION ............................................................................................. 14 4.1 Stiffness Regulation and the Acoustic Startle ...................................... 14 4.2 Group Effects ........................................................................................ 19 4.3 Limitations ............................................................................................ 21 4.4 Conclusion ............................................................................................ 22

5 LEGEND .................................................................................................... 23

6 LITERATURE REVIEW ........................................................................... 40

v

7 REFERENCES ........................................................................................... 52 Appendix

A: STIFFNESS AND PROPRIOCEPTION DEVICE ........................................... 58 B: PAR-Q FORM ................................................................................................... 61 C: DEMOGRAPHIC AND HEALTH HISTORY QUESTIONNAIRE ................ 62 D: QUADRICEPS & HAMSTRING STRETCHING TECHNIQUE ................... 64 E: IRB APPROVAL LETTER ............................................................................... 65

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LIST OF TABLES

Table 1: Demographics ............................................................................................. 23 Table 2: Short-range (4°) Startle Normalized Stiffness ............................................ 24 Table 3: Total (40°) Startle Normalized Stiffness .................................................... 25 Table 4: Electromechanical Delay (EMD) Values ................................................... 26 Table 5: Peak EMG Values for Startle Passive-Reactive Trials ............................... 27 Table 6: Time to Peak (TTP) Values; Startle Trials ................................................. 29 Table 7: Time to Peak (TTP) Obicularis-Oculi; Startle Trials ................................. 31 Table 8: Onset Values for Passive-Reactive (Startle) Trials .................................... 32 Table 9: Pre-Perturbation (150 prior-0ms) Area Values; Startle Trials ................... 34 Table 10: Post-Perturbation (0-250ms) Area Values; Startle Trials ........................... 36 Table 11: Post-Post Perturbation (250-600ms) Area Values; Startle Trials ............... 38

vii

LIST OF FIGURES

Figure 1: Five Perturbation Techniques and Instructions ......................................... 10

Figure 2: Neuronal Circuit ........................................................................................ 50 Figure A.1: Subject Positioning on the SPAD for testing protocols ............................ 60

viii

ABSTRACT

Context: Growing evidence suggests the nervous system has a significant role in

the high rate of non-contact anterior cruciate ligament injuries. Females are the

most susceptible, but evidence for a hormonal role is conflicting. The startle reflex

is universal across animals and stiffens limb muscles after sudden, unanticipated

events. No studies have investigated how hormonal changes during the menstrual

cycle may interact within the nervous system to alter the startle response and knee

stiffness regulation among males and females. Objective: To assess if reactive

knee joint stiffening strategies are affected differently in males and females during

an acoustic startle. Design: Post-test only with repeated measures. Setting:

University laboratory Patients or Other Participants: 18 males (21.3±2.0 yrs,

82.8±15.8 kg, 179.5±6.9 cm) and 18 females (20.9±2.0 yrs, 61.8±7.6 kg,

164.4±6.8 cm) with no previous knee injury participated in this study. All females

were tested at 2 points in the menstrual cycle to form 3 groups: Males (M),

Female-Follicular (FF), and Female-Ovulation (FO). Interventions: Subjects were

seated on a custom stiffness device that generated a rapid 40° flexion perturbation

to the knee (30° to 70° flexion arc). Subjects remained relaxed prior to the

perturbation, and were instructed to react with maximal extension force as quickly

as possible when the perturbation was sensed. A total of 6 trials were performed,

with an acoustic startle (50ms, 1000Hz, 100dB) applied 100ms prior to the

ix

perturbation on 3 randomly selected trials. Main Outcome Measures: Normalized

knee stiffness (Δtorque/Δposition, Nm/°/kg) was calculated at the short-range (0-

4°) and for the total perturbation (0-40°). The average of the control (CON) and

startle trial (ST) were used for analysis. Repeated-measures analyses of variance

comparing gender (M, FF, FO) and startle condition (CON, ST) were used for

analysis at both short-range and total range. Results: The startle significantly

increased short-range stiffness (F=4.24, p=0.04), and decreased total stiffness

(F=11.25, p<0.001). Pairwise comparisons revealed that at short-range, ST

displayed a significantly greater stiffness of 0.047±0.002Nm/°/kg, (p=0.003)

compared to the CON (0.043±0.003Nm/°/kg). However, for total stiffness, the

startle response caused significantly less total stiffness (0.028±0.002Nm/°/kg) than

the CON (0.037±0.002Nm/°/kg, p=0.004) condition. No significant gender or

menstrual cycle phase differences were observed for stiffness values.

Conclusions: This is the first study to indicate that the startle response can

significantly disrupt the normal knee stiffness regulation strategies that are

required to maintain joint stability, and this effect may occur equally in males and

females. Gender and menstrual cycle phase do not appear to affect knee stiffness

or negatively interact with the startle response. Further studies should explore the

potential role of startle responses in accidents and unintentional non-contact

injuries.

Key Words: anterior cruciate ligament, menstrual cycle, stiffness, acoustic startle

1

Chapter 1

INTRODUCTION

Knee injuries are commonly experienced among the physically active

population, with anterior cruciate ligament (ACL) sprains affecting over 250,000

people per year. (Adachi et al., 2008; J. T. Blackburn et al., 2009; Griffin et al., 2000)

Non-contact ACL (NC-ACL) injuries account for over 70% of all ruptures and likely

result from sudden errors in coordination during abrupt deceleration maneuvers like

landing, or cutting.(Fleming et al., 2003; Kirkendall & Garrett, 2000; Olsen et al.,

2004) Females suffer this injury 2-8 times more often than males, and most

researchers believe intrinsic factors such as knee valgus collapse, anterior tibial sheer

and knee laxity are responsible for the higher incidence. However, these prior

observations may only reflect underlying disruptions in neuromuscular control that

ultimately lead to pathological joint kinematics. (Boden et al., 2000; C.Swanik et al.,

2004; C.Swanik et al., 1999) Moreover, theories for the potential neurophysiological

source of these coordination errors have been limited, especially in males. It is

plausible that two common neurological influences, the startle response and menstrual

cycle, interact to heighten ones risk for NC-ACL injury. (Adachi et al., 2008)

Precise muscle activation strategies can help stress-shield ligamentous

structures from excessive loading by regulating joint stiffness during sport-specific

maneuvers.(Lephart et al., 1992) Both quadriceps and hamstring musculature provide

this capacity of dynamic stabilization at the knee, however, sudden unanticipated

events may interrupt the feed-forward and feedback motor control necessary for

2

optimal temporal/spatial muscle recruitment levels. (Rozzi et al., 1999; C.Swanik et

al., 2004; C.Swanik et al., 1999) Recently, greater attention has been focused on

attributes of the central nervous system (CNS) that may be associated with

unintentional musculoskeletal injuries and the promise of reducing injury prone

behaviors.(C.Swanik et al., 2007; Baumeister et al., 2008, Ribot-Ciscar et al., 2009)

It is well documented that unanticipated events can provoke a universal startle

response within the CNS, which results in a brief, but widespread change in

neuromuscular activity. (Davis, 1984; Koch, 1999) When an unexpected startle

response is provoked, neuromuscular changes to preparatory (feed-forward) and

reactive (feedback) muscle contractions in the extremities can be observed, potentially

altering the stiffness regulation necessary for energy absorption and dynamic joint

stabilization. (LaCroix, 1981; Freeman, 1966; Davis, 1984; Koch, 1999) The most

common startle research model uses an unexpected acoustic stimulus, which during

ordinary physical activities and real-world settings may be synonymous with any

sudden extraneous events, noises, or interpersonal communications. (Leumann et al.,

2001) While much of the previous research has focused on NC-ACL injury

mechanisms in women, the startle response may help explain why males too suffer a

loss of coordination and high incidence of this unintentional musculoskeletal injury.

Some research suggests that females may have different reactions to

unanticipated startle events than males. (Hausmann et al., 2000; McCormick &

Teillon, 2001) In response to an auditory stimulus, females have shown faster

responses and higher lower-limb muscle activation. (Kofler et al., 2001) The

3

differences in female startle response have been suggested to result from hormonal

changes. (Braff et al., 2001; Kofler et al., 2001)

The female menstrual cycle and hormone concentration level change is one

remarkable difference between males and females and may be linked to variations in

female neuromuscular control and dynamic stability. (Adachi et al., 2008) Previous

studies tend to focus on ACL characteristics such as laxity and collagen formation;

however, Zazuluk (2006) reported that these results are inconclusive. There is limited

research linking the idea of hormonal alterations to neuromuscular control and joint

stability. Although some debate exists, ACL injuries appear more prevalent during the

ovulation stage of the menstrual cycle, when estrogen is at its peak. (Deie et al., 2002;

Eiling et al., 2007; Park et al., 2009) This hormonal change, along with dysmenorrhea

seen around day 14 of the cycle, may contribute to alterations in sensorimotor

characteristics and athletic performance leading to increased risk of injury. (Adachi et

al., 2008; Eiling et al., 2007) Estrogen levels also cause increased synthesis of the

neurotransmitter dopamine, creating a more responsive effect to the acoustic

startle.(Braff et al., 2001; Jovanovic, 2004) Since estrogen peaks during ovulation, this

would suggest that with an increase of dopamine the reflex would be most affected

during this phase of the cycle. (Jovanovic, 2004) Therefore, changes in hormonal

levels during the menstrual cycle may contribute to differences in both the startle

response and joint stiffness regulations, but these interactions have not been observed.

The purpose of this study was to determine if gender and menstrual cycle

differences occur with an acoustic startle, and whether these alterations influence the

4

quadriceps and hamstring muscle activation strategies responsible for knee joint

stiffness regulation. By combining measures of muscle stiffness and acoustic startle

trials, more information may be found on this potential source for heightened risk of

non-contact ACL injuries. In this study we tested the hypothesis that (1) there is

altered muscle activation and stiffness occurring when the acoustic startle trials occur

compared to the control conditions, (2) males react to the acoustic startle with quicker

muscle activation leading to an increase in stiffness compared to females and (3) the

female menstrual cycle interacts with the startle response leading to a change in

muscle activation and joint stiffness.

5

Chapter 2

METHODS

2.1 Experimental Design

This study utilized a post-test only design with repeated measures. The

independent variables are gender (male and female), menstrual cycle phase (Follicular

(FF), and Ovulation (FO)) and condition (no startle and acoustic startle). The

dependent variables are measurements of short-range and total knee stiffness and

muscle activity (timing and amplitude).

2.2 Participants Eighteen male and eighteen female (tested twice) healthy volunteer

participants within the 18-25 years age range were recruited from the University of

Delaware population. The number of participants is determined through an a priori

power analysis using G*Power v3.1.2 (Heinrich-Heine-Universitat, Dusseldorf) with

parameters set at α = .05, 1- β = 0.80. Prior to testing, participants were provided

university-approved informed consent (UD IRB 231022-1) as well as a Physical

Activity Readiness Questionnaire (PAR-Q) and Demographic and Health History to

determine participation eligibility. If the participant answered, “Yes” to questions

pertaining to pains in the heart or chest, faintness or dizziness, bone or joint problems,

or low back problems; he/she were not included in the study. Participants were tested

on their dominant leg. This is defined by which leg the subject would use to single-leg

jump for distance. (Croce 2004, Russell 2007) Exclusion criteria for all participants

includes: (1) any fractures to the test leg within the previous year, (2) history of injury

6

to the leg within the previous 6 months (3) other knee injuries requiring surgery, (4)

any current bone, muscular, or joint injuries to the hip, knee, and ankle, (5) any

cardiovascular, metabolic or neurological that limits moderate physical activity, (6)

any hearing impairment or complications, and (7) any females taking oral

contraceptive medications or having irregular menstrual cycles.

2.3 Instrumentation 2.3.1 Stiffness and Proprioception Device (SPAD)

Stiffness testing was performed using a custom-built stiffness and

proprioception assessment device (SPAD). The SPAD is a device capable of

providing a perturbation to the knee at a controlled acceleration, deceleration , velocity

and range of motion. The SPAD is a brushless Danaher/Kollmorgen servomotor (B-

404-B-B4) that is fitted into a gearbox (UT018-050, 50:1) and connected to an

amplifier/controller (Copley Xenus driver XSL-12-36-R). The mated servomotor and

gearbox are mounted in a cast aluminum pedestal that is offset from the subject’s

chair. An adaptor arm and torque reaction sensor (Model # T5400, Futek Advanced

Sensor Technology, Irvine, CA) with a 565 N capacity and 1.43 X 105 ft-lb/rad

torsional stiffness is coupled to the gearbox. The signal conditioner digitally displays

torque values and also sends an analog torque signal through an A-D board (NI DAQ

6009, National Instruments, Austin, TX) that can be recorded and displayed in

LabVIEW software. For safety purposes, internal motor settings cannot exceed preset

speeds and there are three emergency stop switches that can disable the motor during

7

testing. The SPAD device is operated using a personal computer with a customized

LabVIEW virtual instrument and motor control software program.

2.3.2 Electromyography (EMG)

Surface electromyography (EMG) were collected from the vastus medialis,

vastus lateralis, medial hamstrings, and lateral hamstrings to determine stiffness

regulation strategies by analyzing the amplitude and timing of the muscular

contractions. There was also one electrooculography (EOG) self-adhesive Ag/AgCl

snap dual electrode (Noraxon USA Inc, Scottsdale, AZ) placed on obicularis oculi to

record EOG activity of the eye blinking and confirm a startle response. Self-adhesive

Ag/AgCl bipolar surface electrodes (Phillips Medical Systems, Andover, MA) were

used to collect EMG data of the leg musculature, and an EMG unit (Bortec AMT-8,

Bortec Biomedical, Calgary, Alberta, Canada) was used to record EMG with a real-

time visual display on the monitor. Electrode placement was identified by bony

landmarks and through palpation of the mid-belly of the contractile component of the

muscle during an isometric contraction. The reference electrode was placed on the

patella. Except for the eye muscle, each electrode is 10mm in diameter and was

placed 25mm apart. Electrode placement site was shaven, abraded, and cleansed with

an alcohol swab (70% ethanol solution) to decrease the impedance from the skin. The

EMG signal was converted from analog to digital data (NI DAQ 6009, National

Instruments, Austin, TX), and then passed to a computer where the raw EMG data was

sampled at 2,400 Hz and further analyzed with LABVIEW software (National

Instruments, Austin, TX). The EMG signal was bandpass filtered at 20-400Hz,

8

rectified, and low-pass filtered at 5Hz to create a linear envelope. EMG is normalized

to maximum voluntary isometric contractions (MVICs) for the muscles of the

quadriceps and hamstrings. All data collected with EMG equipment is used to

determine the timing, sequence, amplitude (area & peak), and pattern of the

quadriceps/hamstrings muscle groups during preparatory and reactive phases of the

stiffness testing.

2.4 Procedures

Participants were asked to report to the Human Performance Lab for a 90-

minute testing procedure. Females were asked to participate twice in the study: once

during the follicular phase and once during ovulation phase of the menstrual cycle.

After providing consent, completing all questionnaires, and satisfying the inclusion

criteria, participants then rode a stationary bike for a 5-minute warm-up followed by 5

minutes of stretching of their quadriceps and hamstrings as instructed with a handout

and verbal direction.

The participant was positioned on the SPAD with the dominant knee flexed to

30 degrees and the trunk positioned at 90 degrees of flexion. The axis of rotation of

the adapter arm attached to the servomotor was in line with the lateral knee joint line.

In order to ensure that all torque responses result from movement at the knee and not

the lower leg, a vacuum splint was secured to the lower leg below the knee to maintain

the ankle in a neutral position. A thigh pad was also used for stability so that

movement occurred only at the knee joint. All electrodes for EMG activity recordings

were attached to the same side determined as the dominant leg. Three maximum

9

voluntary isometric contractions (MVIC) were collected for the quadriceps and

hamstrings to determine an overall average and 30% of their maximum contraction.

The parameters of the perturbations for each testing condition remained the

same for each perturbation with a quick acceleration of 1000 deg/sec2 to a velocity of

100o/sec and a flexion arc of 40o going from knee extension to flexion. The various

testing conditions of reactive stiffening included both control trials and acoustic startle

trials. The five perturbation techniques were passive-nonreactive, active-nonreactive,

passive reactive, active reactive and active deactivate. (Figure 1) For the acoustic

startle condition, a high-pitched 100 dB tone, lasting 50ms at a frequency of 1000 Hz

sounded 100ms before perturbation. (Braff et al., 2001) Throughout all conditions, the

knee perturbation was randomly applied within a 10-second time span. Also, the

subjects were wearing headphones for all conditions to mute potential noisy

distractions. Resistance to the perturbation is detected by the torque sensor and

recorded by a computer. Joint stiffness is calculated as the Δ force (Nm) / Δ

displacement (degrees) / body mass (kg). There were six trials of each, and during the

passive reactive trials the acoustic startle was applied three times. We calculated

electromechanical delay (EMD) as the difference of when torque and EMG went

above 5 percent of peak.

2.5 Data/Statistical Analysis

A custom-written software program using LabVIEW was used to analyze

position and torque data, calculate stiffness and analyze all EMG signals. Stiffness

10

values were calculated from the position data at 3° and 40° of the flexion

perturbations. The EMG was averaged over the six trials for each of the five

perturbations. EMG was analyzed from a window of 150ms prior to perturbation and

600ms after the start of perturbation. The normalized stiffness values were obtained

by dividing each volunteer’s stiffness values by their body mass(kg). The significance

for all data is set a priori at p<.05. A repeated-measures ANOVA with one between-

subject factor (group, 3 levels), and one within-subjects factor (condition, 5 levels)

was used primarily to determine the differences in gender (cycle) and knee stiffness

and when the startle is applied. To determine mean statistical differences, pairwise

comparisons were used to assess changes in stiffness while the startle was applied

between males and females, as well as changes in muscle activation strategies post

hoc. This was also used to compare females during the two periods of their menstrual

cycle.

Figure 1: Five Perturbation Techniques and Instructions

Condition Instructions Passive Stiffness (PS) “Remain completely relaxed throughout the entire perturbation”

Active Stiffness (AS) “Push out to [30% MVIC] prior to the move. When you feel the perturbation, hold that amount of contraction without pushing more or less.”

Reactive Stiffness (RS) “Push out to [30% MVIC] prior to the move. When you feel the perturbation, resist it as hard and as fast as possible as if you are stopping your knee from bending.

Passive Reactive Stiffness (PRS)

“Remain completely relaxed prior to move. When you feel the perturbation, resist is as hard and as fast as possible as if you are stopping your knee from bending”

Deactivating Stiffness (DS) “Push out to [30% MVIC] prior to the move. When you feel the perturbation, turn off all your muscles and relax as quickly as possible.”

11

Chapter 3

RESULTS

3.1 Demographics and Maximum Voluntary Isometric Contractions

Demographic data for age, height, mass, and peak MVIC torque are displayed

in Table 1. Females and males ages ranged from 18-25 and were not significantly

different (p=.563). Males displayed significantly greater height, mass, and leg length

compared to female participants (p<.001). Peak torques during MVIC for quadriceps

and hamstrings MVICs also were significantly greater in male participants (p<.05).

3.2 Startle Effects on Stiffness and EMG Variables 3.2.1 Stiffness & Startle Results for normalized short-range (0 to 4°) and total (0 to 40°) stiffness from

PR trials are displayed in Tables 2 & 3, respectively. The startle was observed to

significantly affect both short-range (p=0.037) and total (p<0.001) stiffness. For short-

range stiffness, both the first (FST) startle trial (p=0.003) and the average of the startle

trials (AST) (p=0.005) displayed significantly greater stiffness than control (CON)

trials. However, for total stiffness, FST trials demonstrated significantly less total

stiffness than both CON (p=0.004) and AST (p<0.001) trials.

3.2.2 EMG & Startle EMG variables used for analysis during PR trials include peak EMG (table 5),

TTP for leg musculature (table 6), TTP for obicularis oculi (table 7), onset values

(table 8), areas for EMG activation 250 prior-to (table 9), 250ms following (table 10)

and 250-500ms following (table 11) the perturbation.

12

A significant muscle by condition interaction effect was observed for all

variables (p<.05), with the exception of EMG area 250-500ms following the

perturbation, which was approaching significance (p=.055). The VM displayed

significantly lower peak EMG during FST trials compared to CON and AST trials

(p=0.040 and p=0.017). VM and VL revealed significantly shorter TTP for FST and

AST trials (p<.05) than CON and MH displayed significantly shorter TTP in FST in

relation to CON (p=.046). The VM and MH demonstrated significantly shorter onset

for both FST and AST than CON (p<.05). VM and VL showed a significant increase

in pre-perturbation area with FST and AST compared to CON (p<.05) and LH

presented significant increase in FST trials than CON (p=.037). Post-perturbation area

had a significant decrease for VL in AST trials compared to CON (p<.001).

The muscles also differed from each other within each startle condition.

During CON, FST and AST the quadriceps had significantly increased peak EMG

values than hamstrings (p<.001). CON trials showed a significant delay TTP for the

quadriceps compared to the hamstrings (p<.05). FST and AST had significant longer

TTP in quadriceps than hamstrings (p<.05) with AST also showing slower TTP in

lateral hamstrings than medial hamstrings (p=.048). CON and FST displayed longer

onset of VM compared to VL, MH and LH (p<.001) as well as MH delays to LH

(p<.05). CON also demonstrated decreased onset in VL than MH (p=.026) and AST

had longer onset in VM compared to VL and LH (p=.036 and p=.005). CON showed

increase in VM pre-perturbation activity than VL (p=.041) while FST and AST

displayed decrease in MH activity than VM (p<.05). All trials had an increase in post-

13

perturbation VM activity than the hamstrings (p<.05) with FST and AST also showing

increase in VL than MH (p<.05).

3.3 Startle Effects on Group Differences No significant group differences were observed for stiffness values. No

significant group by trial interaction effects was observed for either stiffness value.

No significant group differences were observed for any EMG variables. A significant

group by muscle interaction was only found for areas 250ms and 250-500ms

following the perturbation (p<.05). For the post-perturbation area, the FF and FO

groups has significantly higher EMG activity in VM compared to LH (p=.008 and

p=.027). Additionally, the FO group showed significantly less activity in MH than

both VM and VL (p<.001 and p=.011). In post-post perturbation area, both female

groups had significantly higher VM and VL activity compared to MH and LH

(p<.001) while ML demonstrated significant less activity in MH than both VM and

VL (p=.003 and p=.004). Furthermore, the VM had significantly decreased activity in

ML than FO (p=.024). No significant group by startle interaction effect for any EMG

variable was observed.

Results for electromechanical delay (EMD) are presented in Table 4. No

significant differences were observed between groups; however, differences were

detected between muscles, p<.001. Post-hoc analysis indicated MH had a significant

higher EMD than VM, VL and LH (p<.001).

14

Chapter 4

DISCUSSION

The purpose of the present study was to determine how stiffness of the knee

joint is affected by an acoustic startle, as well as hormonal differences associated with

gender and the menstrual cycle. Neuromuscular, neurocognitive, and mechanical

differences are thought to play a significant role in the high incidence (70%) of non-

contact joint injury mechanisms (Boden et al., 2000; Fleming et al., 2003), and even

greater factor among females who suffer a disproportionate number (2-8x greater) of

ACL tears compared to males. (Arendt & Dick, 1995; Eiling et al., 2007; Griffin et al.,

2000; C. Swanik et al., 2007) However, few studies have investigated the influence of

extrinsic factors, such as startling events, on the neuromuscular control and joint

stiffness regulation needed for knee stability. The primary finding of this study was

that joint stiffness and muscle activation patterns were significantly affected by an

acoustic startle in both males and females, while differences between groups were not

observed. These findings suggest that startling events may negatively influence the

normal stiffness regulation strategies needed for functional joint stability regardless of

gender, and attenuation of this startle response should be considered in future knee

injury mechanism and prevention studies.

4.1 Stiffness Regulation and the Acoustic Startle

A measure of the resistance provided by a joint to external loading is crucial

for understanding functional stability and injurious mechanisms, as one’s failure to

optimally regulate joint stiffness can lead to excessive loading in capsuloligamentous

15

structures and potential damage. Furthermore, since joint stiffness is largely dependent

on muscle recruitment, including measures of muscle activity can allow greater

understanding of where failures in stiffness regulation may be occurring beyond that

provided by passive connective tissue structures. Muscular contraction can increase

joint stiffness 10-fold, however an optimal level of stiffness is task dependent, and

based on both performance and stability needs (Swanik 1997; Nichols & Houk 1976;

Rack & Westbury, 1974). In order to maintain proper biomechanical alignment the

joint must undergo a controlled, columnar buckling, with the majority of energy being

absorbed and dissipated in the eccentric lengthening of surrounding musculature rather

than capsuloligamentous tissue (Hewett & Torg, 2009). These highly coordinated

muscle activation strategies include pre-programmed preparatory contractions, pre-

planned reflexes and involuntary reactions developed from within the central nervous

system (CNS) and therefore susceptible to strong stimuli and unanticipated events.

The acoustic startle is one example frequently used as a research model to assess the

effects of sensory stimuli on the CNS.

Startle events have been hypothesized to alter neuromuscular control by

disturbing both preparatory and reactive neuronal circuitry at the level of the

brainstem. (Ghez & Krakauer, 1991, Koch, 1999) These planned reflexes can help

explain how people will react to an event when anticipating certain proprioceptive

circumstances during activity (Nieuwenhuijzen et al, 2000). While studies have

investigated the effect of a startle on these neuronal circuits, and involuntary muscular

responses (Yeomans 1995; Koch 1999; Moffit et al. 2012), no research has

16

investigated its potential secondary effect on a measure of joint stability such as

stiffness.

Our results found an increase in short-range stiffness following the startle, but

then a decrease in total stiffness, which encompasses and larger, functional range of

motion. Limited research is available with the startle and biomechanics, but a study

by Nieuwenhuijzen et al showed decreases in leg musculature range-of-motion during

a gait analysis (2000) and Moffit et al displayed changes in neuromuscular activation

with an applied startle (2012). Short-range stiffness is typically associated with the

passive components of the joint, a reverse pivot of bound actin-myosin cross-bridges,

as well as the series and parallel elastic components of the muscle. (Sinkjaer et al.,

1988, Crago et al, 1976; Nichols & Houk 1976; Houk et al, 1967) Our startle response

was purposely initiated in a brief period of time before the external knee perturbation,

in an attempt to replicate a potential unanticipated sensory disturbance just prior to

joint loading, which may occur during physical activity. The higher short-range

stiffness likely reflects a disturbance in neuromuscular control as we found earlier

quadriceps muscle activation during these trials. Quadriceps activation is important for

maintaining knee stability because this group is among the primary antigravity

muscles (Bellew, 2002). However, when contracting earlier that anticipated during

the joint range of motion, the quadriceps may contribute to increased anterior shear

forces and a knee hyperextension moment. This combination would potentially place

the knee in a more vulnerable position for ACL injury (Rozzi et al, 1999; Griffin et al,

2000).

17

In addition to altered short-range stiffness, our results also supported

decreased stiffness for the entire 40-degree perturbation. The total-stiffness is largely

dependent on the ability to regulate the reverse cross-bridge cycling of an eccentric

contraction as the knee is forced into flexion (Sinkjaer et al, 1998). Our results showed

that the startle caused an initial, early heightening of joint stiffness that then was

attenuated throughout the remainder of the perturbation; a potentially detrimental

combination for avoiding knee injury. Previous studies have also linked alterations in

muscle stiffness to knee injury. (Swanik 2007; Blackburn et al 2009; Boden et al

2000) The inability to sufficiently stiffen the knee, as one would normally accomplish

through muscle activation, could expose capsuloligamentous structures to excessive

loads that would otherwise be absorbed through controlled muscle lengthening.

When considering these muscle activation patterns, nearly all variables were

affected by the acoustic startle. While the quadriceps muscles were observed to have

greater EMG activity than the hamstring muscles throughout all trials, the startle

prolonged the onset and time-to-peak (TTP) activity of the vastus medialis muscle.

Furthermore, the startling event before the perturbation caused an early increase in

EMG activity but decreased muscle activity when compared to control trials during

and after the perturbation (Tables 10 & 11). We hypothesize that this overall

decreased EMG activity, in conjunction with our findings of decreased total joint

stiffness, may be related to an “uncontrolled buckling” observed in a jump landing

where absorption of ground reaction forces occurs with the joint in a more vulnerable

position. (Boden et al., 2000; Fleming et al., 2003; Hewett & Torg, 2009) If an

18

unanticipated event occurs just prior to joint loading, a startle reflex may be initiated.

The involuntary startle causes an early quadriceps activation and knee extension

moment, appearing to us as heightened short-range stiffness. Under real-world

conditions such as landing or cutting, this effect may cause the knee joint to move

towards a hyper-extended position, which is associated with ACL tears. Furthermore,

as the startle response subsides, less muscle activity was observed and subsequently

less total stiffness of the joint resulted. The level of total joint stiffness that resulted

from the startle event was less than under normal conditions, suggesting that the

optimal stiffness regulation strategy for dynamic stability was significantly disrupted.

The inability to maintain normal stiffness regulation throughout the total range of

motion could result in uncontrolled columnar buckling during functional activities

(Hewett & Torg, 2009).

For further analysis, we compared the average of 3 applied startles to the initial

startle. Previous research has suggested that following the initial startle, the effect of

subsequent startles would be diminished and therefore may make differences more

difficult to detect. (Blumenthal et al., 2005) Our results supported this finding as the

greatest differences in joint stiffness were observed in the initial trial, and several

EMG differences were observed as well (Tables 3, 5–8). The first startle had

significantly decreased total stiffness when compared to the control and average trials.

A decreased peak EMG was observed during initial trials as well as increased time-to-

peak and decreased onset times. This demonstrates that there is an attenuation of the

startle for both stiffness and muscle activation after the initial startle is introduced. It

19

is unclear how long the attenuation of a startle response may last, but these changes do

show promise for future research into prevention and rehabilitation approaches that

can minimize the potentially disruptive neuromuscular effects of sudden unanticipated

events.

4.2 Group Effects

No group differences were observed for any stiffness or EMG measures.

Throughout the literature, females are observed to tear their ACL more frequently than

males.(Huston & Wojtys, 1996; Rozzi et al., 1999) However, the reasons behind this

common phenomenon are inconclusive, often attributing differences to

neuromuscular, strength, stiffness and the female menstrual cycle characteristics.

Moreover, little attention has been directed toward explaining why males also suffer

NC-ACL injuries. (Wojtys et al., 1998; Blackburn et al., 2009; Griffin et al., 2000; C.

Swanik et al., 2007) The menstrual cycle tends to show the most hormonal changes

during the ovulation phase with some data showing increases in laxity and stiffness.

(Van Lunen et al., 2003; Wojtys et al., 1998) This study did not support the claim that

the menstrual cycle causes significant changes in neuromuscular control or stiffness.

Some theories also blame quadriceps dominance in females, leading to knee joint

hyperextension and improper mechanics; however, our study observed that all groups

recruited these muscles similarly. Previous studies tend to show a decrease in stiffness

in females compared to males, which leads to a decrease in joint stabilization and

increases in ACL injuries, however, many of these studies do not account for

normalized body weight between genders as our study did. (Blackburn et al., 2009;

20

Granata et al., 2002) Unlike other stiffness studies comparing genders, our study did

not use dynamic tasks such as jumping rather than a controlled seated dynamic

movement in order to minimize other joint axial loads and to concentrate specifically

on the knee joint.

Males tend to have greater strength, a more balanced muscle contribution,

greater muscle mass, and higher muscle fiber recruitment than females. (Blackburn et

al., 2009; Wojtys et al., 2002) In our study, females showed a decrease in MVIC for

quadriceps and hamstrings than males, supporting that females have less over-all

quadriceps and hamstring strength. The reason for females having a higher injury rate

is often contributed to the neuromuscular control strategy and their lack of control

during dynamic tasks (Rozzi et al., 1999, Griffin et al., 2000; C. Swanik et al., 2007)

Currently there is conflicting evidence, however, supporting the claims of why males

and females both suffer from a higher incidence of non-contact ACL injury. In fact,

the mechanism explaining why males also suffer from non-contact injuries has not

been offered. This study showed similar activation strategies with no significant

difference between groups. Although, from the results, female groups did have data

that was trending towards slower TTP, lower peak EMG, and slower muscle onsets. A

larger subject pool would provide more data to see if this effect continues to lead

towards significance. A group-muscle significant difference was seen with both

female groups following the 40-degree perturbation where the VM had higher EMG

activity compared to the other three muscles measured during the first startle. This

could lead towards a quad-dominance effect following startling perturbation

21

maneuvers, and this dominance could support the incidence of higher injuries. It is

possible that our startle response main effect size was large enough to conceal

potential interaction effects between groups. However, more research, with larger

subject pools is needed to provide additional evidence on whether there could be

group and gender differences with a startle event.

4.3 Limitations

One of the largest limitations to this study was that the subject pool was small

and larger groups could potentially lead to overall group differences. In future studies,

more participants are warranted to have a better understanding of how gender groups

could have altered stiffness and muscle activation patterns. In this study we measured

body weight (kg) to normalize stiffness values, however, it may be prudent to use lean

body mass due to the variations in body types that were included in this study. Lean

body mass is a more accurate portrayal of a person’s body composition. Another

limitation was that females faced the acoustic startle in more than one sitting, which

could lead to some suspicion of a habituation effect; however, prior research shows

that there is a minimal learning effect when a startle stimulus is tested on two separate

days making this limitation unlikely. The females also self-reported the accuracy of

their menstrual cycle, which is difficult to assess how honest these reports were for the

precision of our study. In future studies, ovulation kits and hormonal level testing can

improve the accuracy of the menstrual cycle timeline, which may give a better

understanding of how the 28-day cycle can alter muscle stiffness and activation.

22

4.4 Conclusion

The results from this study indicate that startle events can significantly alter

neuromuscular control and stiffness regulation strategies at the knee joint. Regardless

of gender, this event caused alterations in preparatory and reactive muscle recruitment

strategies necessary for dynamic restraint and joint stability. Overall, the first startle

caused the greatest significant differences in the measures investigated. This suggests

a single, uniquely timed startling event could expose both males and females to a

failure of the dynamic restraint mechanism and non-contact related ACL injuries.

More-over, startle events could be used in prevention and rehabilitation exercises,

however, from this study’s results, proper care should be taken due to the significant

disruption in neuromuscular control that the startle is found to have on unanticipated

perturbation maneuvers.

23

Chapter 5

LEGEND

Table 1: Demographics

Female (N=18) Male (N=18) p-value Age (yrs) 20.89 ± 1.97 21.28 ± 2.02 0.563 Weight (kg) 61.77 ± 7.63 82.75 ± 15.84 <.001† Height (cm) 164.41 ± 6.79 179.49 ± 6.86 <.001† Leg Length (cm) 40.34 ± 1.88 43.82 ± 2.02 <.001† QMVIC (Nm) 740.61 ± 230.70 1023.20 ± 272.42 .002* HMVIC (Nm) 647.20 ± 198.02 1075.81 ± 258.99 .002* *Significance at p<.05, †Significance at p<.001; QMVIC-Quadriceps maximum voluntary contraction, HMVIC – hamstrings maximum voluntary contraction

24

Table 2: Short-range (4°) Startle Normalized Stiffness (Nm/o/Kg) Independent

Variable

Mean ± SD F-Value

DF

p-value

Pairwise comparison

p-value Group Males

Females-FF Females-FO

.047±.002

.048±.005

.044±.004

.506

2

.606

Trials

Control First Average

.043±.003

.047±.002

.050±.003

4.236

1.198

.037*

C-F .003* C-A .005*

Group, Trials

Control

M FF FO

.046±.011

.042±.012

.040±.008

.461

2.395

.668

First

M FF FO

.047±.012

.049±.014

.045±.011 Average

M FF FO

.049±.018

.053±.028

.047±.024 *Significance at p<.05, M-Male, FF-Female Follicular, FO-Female Ovulation; C-Control Trials, A-Average of 3 startle trials, F-First startle trial

25

Table 3: Total (40°) Startle Normalized Stiffness (Nm/o/Kg) Independent

Variable

Mean ± SD F-Value

DF

p-value

Pairwise comparison

p-value Group Males

Females-FF Females-FO

.034±.006

.032±.005

.036±.006

.373

2

.691

Trials


.037±.002

.028±.002

.038±.003

11.248

1.385

.000†

C-F .004* F-A .000*

Group, Trials Control

M FF FO

.038±.016

.035±.012

.038±.013

.126

2.770

.934

First

M FF FO

.027±.022

.027±.021

.030±.020 Average

M FF FO

.038±.012

.035±.016

.041±.017 †Significance at p<.001; M-Male, FF-Female Follicular, FO-Female Ovulation; C-Control Trials, A-Average of 3 Startle Trials, F-First Startle Trial

26

Table 4: Electromechanical Delay (EMD) Values Independent

Variable

Mean ± SD (ms)

F-Value

DF

p-value Pairwise

comparison p-value

Group

Males Female-FF Female-FO

73.142 ± 7.705 73.013 ± 8.367 68.671 ± 8.550

2.315

2

.109

Muscles

VM VL MH LH

67.909 ± 4.548 66.003 ± 3.120 83.218 ± 2.083 69.306 ± 4.072

15.450

3

.000†

VM-MH .000†

VL-MH .000† MH-LH .000†

Group, Muscle

VM

M 69.738 ± 11.974 FF 71.258 ± 17.172 FO 62.732 ± 15.118

.721

6

.633

VL

M 65.448 ± 14.112 FF 69.444 ± 12.800 FO 63.117 ± 17.131

MH

M 83.472 ± 15.929 FF 85.162 ± 10.337 FO 81.019 ± 10.626

LH M 73.912 ± 15.853 FF 66.188 ± 15.301 FO 67.816 ± 16.244

†Significance at p<.001, M-Male, FF-Female Follicular, FO-Female Ovulation; VM-Vastus Medialis, VL-Vastus Lateralis, MH-Medial Hamstrings, LH-Lateral Hamstrings

27

Table 5: Peak EMG Values for Startle Passive-Reactive Trials Independent

Variable

Mean ± SD (% Max)

F-Value

DF

p-value Pairwise

comparison p-value

Group

Male Female FF Female FO

37.836 ± 17.032 40.536 ± 24.840 41.455 ± 25.768

.231

2

.795

Muscles

VM VL MH LH

62.769 ± 7.825 58.910 ± 9.267 15.806 ± 2.435 22.285 ± 4.560

55.404

1.722

.000†

VM-MH .000†

VM-LH .000† VL-MH .000† VL-LH .000†

Trials


42.557 ± 24.964 36.317 ± 19.204 40.954 ± 23.747

6.290

1.342

.009*

C-F .013* F-A .037*

Muscles, Trial

VM

C 68.967±3.804 F 54.820±3.906 A 64.521±7.626

4.349

2.106

.015*

C-F .017* F-A .040*

C-F .025*

C, F, A VM-MH

.000† VM-LH .000† VL-MH .000† VL-LH .000†

VL

C 62.510±9.994 F 53.367±6.483 A 60.853±11.419

MH

C 15.997±2.729 F 15.489±2.811 A 15.931±2.854

LH

C 22.755±4.031 F 21.592±6.137 A 22.509±5.305

Muscles, Group

VM

M 57.660±6.566 FF 62.988±8.558 FO 67.659±7.312

1.254

3.443

.297

VL

M 48.611±2.666 FF 64.580±9.328 FO 63.539±6.298

MH

M 17.493±2.411 FF 17.288±9.476 FO 12.636±4.490

LH

M 27.581±3.014 FF 17.287±8.514 FO 21.988±4.740

Control

M 40.412±21.686 FF 44.538±30.529

28

Trials, Group

FO 42.721±29.521 .537

2.684

.639

First M 35.528±16.346 FF 35.471±21.826 FO 37.951±24.562

Average

M 37.569±17.610 FF 41.598±28.232 FO 43.698±30.571

†Significance at p<.001, *Significance at p<.05; M-Male, FF-Female Follicular, FO-Female Ovulation; VM-Vastus Medialis, VL-Vastus Lateralis, MH-Medial Hamstrings, LH-Lateral Hamstrings

29

Table 6: Time to Peak (TTP) Values; Startle Trials Independent

Variable Mean ± SD (s) F-Value DF p-value Pairwise

comparison p-value

Group


.367±.069

.398±.079

.375±.067

.215

2

.808

Muscles

VM VL MH LH

.419±.057

.438±.052

.308±.047

.356±.048

12.717

2.345

.000†

VM-MH.000† VL-MH .000† VL-LH .015*

Trials


.426±.074

.351±.063

.363±.056

10.762

2

.000†

C-A .002* C-F .001†

Muscles, Trial

VM

C .487±.025 F .367±.016 A .402±.023

2.466

4.337

.043*

C-A .002* C-F .000†

C-A .003* C-F .050*

C-F .046*

C VM-MH .001† VM-LH .003* VL-MH .002* VL-LH .006*

F VM-MH .000† VL-MH .001†

A VM-MH .023* VL-MH .000† MH-LH .048*

VL

C .496±.033 F .412±.020 A .405±.036

MH

C .357±.025 F .267±.018 A .301±.039

LH

C .365±.045 F .357±.066 A .345±.053

Muscles, Group

VM

M .426±.059 FF .432±.072 FO .398±.055

1.202

4.690

.316

VL

M .416±.047 FF .441±.071 FO .456±.045

M .299±.044

30

MH

FF .303±.073 FO .324±.031

LH

M .327±.022 FF .417±.014 FO .323±.019

Trials, Group

Control

M .412±.076 FF .458±.067 FO .409±.088

.333

4

.855

First M .342±.075 FF .360±.075 FO.350±.056

Average

M .347±.046 FF.377±.074 FO .366±.058

†Significance at p<.001, *Significance at p<.05; M-Male, FF-Female Follicular, FO-Female Ovulation; VM-Vastus Medialis, VL-Vastus Lateralis, MH-Medial Hamstrings, LH-Lateral Hamstrings; C-Control Trials, A-Average of 3 startle trials, F-First startle trial

31

Table 7: Time to Peak (TTP) Obicularis-Oculi; Startle Trials Independent


comparison p-value

Group


.771±.341 1.110±.480 .995±.227

.642

2

.530

Trials


.685±.142 1.268±.297 .923±.348

4.007

1.452

.034*

Trials, Group

Control

M .521±.813 FF .762±.832 FO .773±.990

1.091

2.905

.357

First

M 1.160±1.719 FF 1.657±1.871 FO 1.226±1.622

Average

M .632±.787 FF .911±1.009 FO .987±1.054

*Significance at p<.05, M-Male, FF-Female Follicular, FO-Female Ovulation; C-Control Trials, A-Average of 3 startle trials, F-First startle trial

32

Table 8: Onset Values for Passive-Reactive (Startle) Trials Independent


comparison p-value

Group


.094±.040

.068±.049

.076±.028

1.738

2

.186

Muscles

VM VL MH LH

.131±.031

.063±.016

.081±.027

.043±.023

27.450

2.517

.000†

VM-VL .000† VM-MH.000† VM-LH .000† MH-LH .006*

Trials


.097±.024

.063±.005

.080±.005

10.348

1.419

.001†

C-A .013* C-F .002* F-A .040*

Muscles, Trial

VM

C .166±.024 F .102±.005 A .124±.005

4.161

3.775

.004*

C-A .001† C-F .000†

C-A .024* C-F .006*

C VM-all .000† VL-MH .026* MH-LH .000†

F VM-all .000† MH-LH .027*

A VM-VL .036* VM-LH .005*

VL

C .070±.023 F .053±0.00 A .068±.017

MH

C .105±.024 F .058±.018 A .079±.020

LH

C .045±.023 F .038±.032 A .048±.023

Muscles, Group

VM

M .139±.045 FF .134±.032 FO .119±.022

1.114

5.035

.356

VL

M .079±.023 FF .055±.003 FO .056±.003

MH

M .101±.028 FF .068±.033 FO .073±.010

M .058±.008

33

LH FF .015±.012 FO .058±.010

Trials, Group

Control

M .120±.052 FF .087±.064 FO .082±.042

2.838

1.002

.394

First M .070±.023 FF .050±.044 FO .068±.019

Average

M .093±.030 FF .067±.043 FO .079±.027


34

Table 9: Pre-Perturbation (150 prior-0ms) Area Values; Startle Trials Independent

Variable Mean ± SD (%) F-

Value DF p-value Pairwise

comparison p-value

Group


1.407±.353 1.639±.389 1.726±.497

1.311

2

.280

Muscles

VM VL MH LH

1.954±.474 1.459±.431 1.302±.199 1.648±.275

3.198

3

.026*

VM-MH .034*

Trials


1.392±.357 1.707±.467 1.673±.413

11.522

1.719

.000†

C-A .001† C-F .000†

Muscles, Trial

VM

C 1.636±.392 F 2.131±.579 A 2.095±.427

5.370

2.787

.002*

C-A .031* C-F .001†

C-A .000† C-F .000†

C-F .037* C

VM-VL .041* A

VM-MH .020* F

VM-MH .022*

VL

C 1.087±.291 F 1.660±.476 A 1.629±.346

MH

C 1.264±.204 F 1.342±.249 A 1.300±.231

LH

C 1.578±.342 F 1.697±.265 A 1.668±.321

Muscles, Group

VM

M 1.437±.211 FF 2.189±.210 FO 2.235±.457

1.652

6

.138

VL

M 1.085±.273 FF 1.509±.344 FO 1.782±.423

MH

M 1.214±.007 FF 1.549±.056 FO 1.143±.025

LH

M 1.892±.045 FF 1.307±.086

35

FO 1.744±.120 Trials, Group

Control

M 1.273±.467 FF 1.455±.368 FO 1.448±.294

1.341

3.437

.266

First M 1.456±.290 FF 1.689±.370 FO 1.977±.641

Average

M 1.493±.345 FF 1.772±.457 FO 1.754±.482


36

Table 10: Post-Perturbation (0-250ms) Area Values; Startle Trials Independent

Variable Mean ± SD (%) F-Value DF p-value Pairwise

comparison p-value

Group


4.252±.078 4.711±1.482 4.879±1.819

.958

2

.392

Muscles

VM VL MH LH

6.125±1.106 5.078±1.055 3.234±.602 4.020±.872

9.218

3

.000†

VM-MH.000† VM-LH .005* VL-MH .017*

Trials


4.379±1.188 4.571±1.431 4.892±1.648

4.219

1.511

.028*

C-A .003*

Muscles, Trial

VM

C 5.844±.847 F 5.968±1.408 A 6.562±1.301

3.217

3.015

.025*

C-A .000† C

VM-MH .000†

VM-LH .025* F

VM-MH .000†

VM-LH .000† VL-MH .002*

A VM-MH

.002* VM-LH .026* VL-MH .031*

VL

C 4.508±.361 F 5.064±1.431 A 5.662±1.129

MH

C 3.219±.668 F 3.262±.732 A 3.222±.683

LH

C 3.946±.963 F 3.991±.856 A 4.123±1.163

Muscles, Group

M

VM 4.771±.461 VL 3.999±.488 MH 3.256±.066 LH 4.983±.289

2.214

5.876

.047*

VM-LH .008*

FF

VM 6.538±.573 VL 5.388±.853 MH 3.914±.103 LH 3.006±.043

VM 7.066±.536

37

FO VL 5.847±.842 MH 2.532±.036 LH 4.072±.126

VM-MH.000† VM-LH .027* VL-MH .011*

Trials, Group

Control

M 4.341±.782 FF 4.296±1.225 FO 4.501±1.742

1.591

3.023

.199

First

M 3.901±.715 FF 4.844±1.622 FO 4.969±1.879

Average

M 4.514±.910 FF 4.995±1.886 FO 5.168±2.294


38

Table 11: Post-Post Perturbation (250-600ms) Area Values; Startle Trials Independent

Variable Mean ± SD (%) F-Value DF p-value Pairwise

comparison p-value

Group


6.765±2.655 7.367±4.134 7.894±4.861

1.057

2

.357

Muscles

VM VL MH LH

11.134±1.533 10.676±2.038 2.950±.484 4.607±1.047

55.329

2.309

.000†

VM-MH.000† VM-LH

.000† VL-MH

.000† VL-LH .000†

MH-LH .032*

Trials


7.450±4.052 6.769±4.353 7.806±4.535

1.309

4.029

.039*

F-A .008*

Muscles, Trial

VM

C 11.500±1.316 F 9.935±1.439 A 11.969±1.484

1.770

3.140

.055

VL

C 10.753±1.914 F 9.803±2.232 A 11.474±2.420

MH

C 2.964±.571 F 2.844±.514 A 3.041±.563

LH

C 4.585±1.058 F 4.496±1.311 A 4.740±1.225

Muscles, Group

M

VM 9.714±1.220 VL 8.242±.823 MH 3.197±.219 LH 5.905±.168

2.452

4.618

.043*

VM-

MH.003* VL-MH

.004*

VM-MH.000† VM-LH

.000† VL-MH

FF

VM 11.195±.737 VL 11.402±.797 MH 3.325±.034 LH 3.546±.176

FO

VM 12.494±1.253 VL 12.386±1.108 MH 2.328±.056

39

LH 4.369±.142 .000† VL-LH .000†

VM-MH.000† VM-LH

.000† VL-MH

.000† VL-LH .000†

VM M-FO .024*


40

Chapter 6

LITERATURE REVIEW

Incidental, non-contact anterior cruciate ligament (ACL) sprains affect

primarily young (aged 15-25) athletic populations and are continuing to be a very

common knee injury. (Griffin et al., 2006; Wojtys et al., 1998) Approximately

100,000 ACL reconstruction surgeries are performed annually in the United States

with an estimated cost of $17,000 to $25,000 per case for surgical and rehabilitation

interventions.(Grindstaff, 2006) Additionally, an average of 6 months is lost from

normal activity when surgery is performed to repair the ACL. Recent research shows

osteoarthritis after 10 years in 905 surgical and nonsurgical ACL cases as well as

alterations in joint loading, which also causes complications with this injury. (Holm et

al., 2010) Generally, ACL sprains affect young women more often than men, however

conclusive studies do not exist to explain the underlying mechanisms that may be

causing this unintentional injury. (Griffin et al., 2000; Griffin et al., 2006) Because

over 70% of ACL tears are a non-contact injury, which means there was no player-on-

player contact, this infers that errors in neuromuscular control led to the injurious

biomechanics during routine functional tasks such as jump landings running and

cutting maneuvers. (Boden et al., 2000; Fleming et al., 2003; Kirkendall & Garrett,

2000; Olsen et al., 2004) It is imperative to continue research towards understanding

the mechanisms behind unintentional non-contact injury, such that prevention

programs can be implemented.

41

Joint Stabilization

The knee joint relies on both static and dynamic restraint to maintain stability.

(Lephart et al., 1992, Freeman, 1966, Johansson, 1991, C.Swanik et al., 1999)

Stiffness properties can measure the muscle tension that assists with joint stability.

Knee joint stability is influenced by multiple factors including capsulo-ligamentous

structures, muscle forces, and proprioceptive influences from static and dynamic

restraints. (C.Swanik et al., 1999) Static restraints include ligaments, bones and the

capsule that control and limit joint motion. Dynamic restraints are comprised of the

musculotendinous units, which help produce and absorb forces in response to changes

in joint loads. When static and dynamic restraints work together, they can help guide

joint kinematics and absorb loads during the high velocity movements of physical

activity. Generally, when dynamic stabilization is compromised, an increase in the

accessory motion of tibial translation can increase the risk of ACL injury. As a static

stabilizer, the ACL provides 86% of restraint against anterior tibial translation, and

when surrounding musculature do not help resist this motion, stresses beyond the

ACLs strain limit can cause tissue failure. (Butler et al., 1980) In some cases the loss

of an ACL does not result in a functionally unstable joint because quadriceps and

hamstring musculature act in synergy with the remaining static restraints, allowing

some patients to cope with the loss of the ligament. These “copers” rely on proper

muscle activation to provide dynamic stabilization. (Eastlack & Snyder-Mackler,

1999)

Dynamic stability can be described as the ability to prepare for and react to

42

unanticipated joint loads throughout activity. (Williams et al., 2001) Muscle fibers

contracting at optimal force, length and velocity relationships help maintain this type

of stability, with the quadriceps and hamstrings serving as the primary dynamic

stabilizers of the knee joint. (Branch et al., 1989; C.Swanik et al., 2004; C.Swanik et

al., 1999) Through co-contraction, both muscle groups create a state of joint force

equilibrium to help reduce the amount of anterior translation placed upon the ACL,

which if left unrestrained leads to ligament ruptures. (Li et al., 1999) Prior research

has shown that aggressive quadriceps loading increases the anterior tibial translation,

which places greater stresses on the ACL and may lead to injury.(Kirkendall &

Garrett, 2000) The quadriceps are the primary anti-gravity muscles that absorb loads

during sudden knee flexion during tasks such as landing and cutting.(Snyder-Mackler,

2007) The hamstring muscles contractions may often protect against ligament injury

as they provide a posterior tibial shear force limiting loads placed on the ACL.

(MacWilliams et al., 1999) Some females demonstrate earlier recruitment of the

quadriceps than the hamstrings delaying the hamstring reactions to anterior stress on

the ACL.(Hurd et al., 2006; Huston & Wojtys, 1996) Delayed recruitment of muscle

fibers can affect the timing of force production and mechanical restraint necessary for

joint stability.

Electromechanical delay (EMD) is defined as the time between onset of

electrical muscle activity and force production or joint motion. (Granata et al., 2000;

Moore et al., 2002) When perturbations are introduced, males tend to have a shorter

EMD compared to females (Bell & Jacobs, 1986), which for women can delay muscle

43

stiffening and place them in a more vulnerable state for injury. Pre-motor planning

and pre-activation of the muscles can affect mechanoreceptors and have been shown

to affect reactions to sudden perturbations. (Dietz et al., 1981) Rozzi et al (1999)

showed females had increased joint laxity and EMD when reacting to joint motion.

Precise pre-contractions of knee musculature can increase the joint stiffness because it

allows for a quicker force production. (Draganich et al., 1989) Stiffness properties can

measure the muscle tension that assists with joint stability.

Muscle stiffness is defined as the muscle’s ability to resist changes in length

(change in force/change in length). (Latash, 1993) Joint stiffness from the energy

exchange between static and dynamic restraints is drastically enhanced by voluntary

contractions, reflexive contractions, descending corticospinal drive, and muscle tone.

(Sinkjaer et al., 1988, C.Swanik et al., 2004) Additionally, joint stiffness may be

altered by gender differences including different muscle activation patterns, joint

biomechanics, and hormonal changes associated with the menstrual cycle; as well as

extrinsic influences such as, cognitive processes, and external stimuli. (Hausmann et

al., 2000; McCormick & Teillon, 2001; C.Swanik et al., 2007; Wojtys et al., 1998) All

of these factors can potentially cause variations in joint stiffening and neuromuscular

control, leading to an increased risk of injury to the knee ligaments. Blackburn et al

(2009) has shown that males have increases in muscle stiffness, and these have

corresponded to decreases in EMD. Females also showed a decline in force

production, which may limit the ability to maintain joint equilibrium reducing overall

joint stability. However, Hinsey et al (2010) did not find gender differences in

44

stiffness when normalizing for body size and strength. If muscle stiffness is increased,

this can help maximize joint stabilization at the knee and help decrease injuries seen

within the joint. (Blackburn et al., 2009)

Neuromuscular Control

Neuromuscular control is the coordinated ability of the nervous and

musculoskeletal systems to control movements, prepare for and react to joint

perturbations. (Freeman, 1966, Lacroix, 1981, Williams et al., 2001) Unfortunately,

sport competition presents many unanticipated events that may alter the nervous and

musculoskeletal systems. Neuromuscular control incorporates proprioception, visual

influences, and vestibular feedback with cortical and spinal motor commands. (Ghez

& Krakauer, 1991; Rozzi et al., 1999; C. Swanik et al., 2007) Mechanoreceptors

embedded in muscles, joint capsules, and ligaments play a large role as sensory organs

contributing to neuromuscular control. (Johannsson, 1991) These receptors send

afferent signals to the central nervous system in response to tension, compression,

loads and other forms of mechanical deformation.(Prochazka et al., 1989) Feedback

and feed-forward motor control processes counter most unexpected changes that may

affect joint stability. (Dietz et al., 1981; Lacroix, 1981; Rozzi et al., 1999; Williams et

al., 2001) Feed-forward systems work by the pre-activation of muscles in anticipation

of changes in joint movements and loads. (Ghez & Krakauer, 1991) Feedback

systems work to provide reactive muscle activity during unanticipated tasks and

impact dynamic restraint. (C. Swanik et al., 1999; Ghez & Krakauer, 1991)

Musculotendinous mechanoreceptors include Golgi tendon organs and muscle

45

spindles. (Gordon & Ghez, 1991) Golgi tendon organs are generally found within the

musculotendinous junction of muscles and react to changes in muscle tension or force,

providing the nervous system with specific force-feedback. (Houk & Henneman,

1967; Houk & Rymer, 1981; Nichols & Houk, 1976) Muscle spindles are sensitive to

changes in muscle length and velocity and have modifiable sensitivity through the

fusimotor system. (Gordon & Ghez, 1991; Matthews, 1981) Primarily, they increase

firing in response to increases in muscle length and changes in rate of muscle

lengthening. (Gordon & Ghez, 1991; Hulliger, 1984) Their sensitivity is set in advance

depending on the specific anticipated task. Muscle stiffness is affected by the force

and length feedback properties of Golgi tendon organs and muscle spindles. (Houk &

Rymer, 1981; Nichols & Houk, 1976) If joint perturbations and outside stimuli

coincide with unforeseen events like movements to the limb or acoustic noises, the

feed-forward and feedback processes that enable dynamic stabilization and stiffness

regulation may be compromised. Some research has suggested that females may be

more prone to knee injury because of these influences.(Griffin 2000)

Gender Factors

Females are at 2-8 times greater risk of ACL injury than their male counterparts.

(Arendt & Dick, 1995; Eiling et al., 2007; Park et al., 2009) Recent research has

shown biomechanical, neuromuscular and cognitive characteristic differences

suggesting potential reasons why females may be more susceptible to this injury.

(Griffin et al., 2000; C. Swanik et al., 2007) Decreases in joint proprioception, delays

in muscle reflexes, increases in knee laxity and increases in quadriceps dominance all

46

heighten the risk for ACL injuries and are seen more in females than males.(Huston &

Wojtys, 1996; Rozzi et al., 1999)

Blackburn et al (2009) performed a study comparing gender-based

characteristics that may contribute to stiffness differences. An increase in

musculotendinous stiffness leads to changes in muscle timing and magnitude of force.

(Myer et al., 2005; Rozzi et al., 1999) Decreased values of force production and

stiffness seen in females may contribute to the greater risk of ACL injury because the

neuromechanical properties needed to activate the muscle do not transpire as quickly

in females than males. A key finding of this study showed greater stiffness values

correlated with the cross-sectional area of the muscles suggesting males will have

increased stiffness. (Blackburn et al., 2009) However, there is limitation to

Blackburn’s study as stiffness was extrapolated from oscillating perturbation, rather

than using true measured values of stiffness. Males demonstrated a heightened

musculotendinous stiffness allowing for increases in joint stability. (Blackburn et al.,

2009) Since females generally have less muscle mass than males, females show

decreases in stiffness and delays in producing force, which could cause an increase in

injuries. (Blackburn et al., 2009; Wojtys et al., 2002) Due to a relatively quicker force

production of the hamstrings in males, the amount of anterior tibial translation would

therefore be limited and provide better stability to the knee joint. Hormonal effects

have been linked to differences seen between males and females caused primarily by

the menstrual cycle.

The 28-day female menstrual cycle is one of the greatest differences between

47

genders. (Van Lunen et al., 2003; Wojtys et al., 1998) Estrogen is the leading hormone

that shows changes throughout the menstrual cycle and is thought to contribute to the

increase of female ACL injury. (Deie et al., 2002; Eiling et al., 2007; Adachi et al.,

2008) Estrogen peaks during the ovulation days: 10-14(Wojtys et al., 1998), and is

found to affect decreases in soft tissue strength, increases in laxity, decreases in

muscle function and the central nervous system. (Sciore et al., 1998; Shikata et al.,

1979) Another hormone that has gained increasing attention is relaxin, not found in

males, which also has receptors on the ACL and may be influenced by estrogen.

(Dragoo et al., 2003; Sherwood et al., 1993) Several studies have found laxity to peak

during the ovulation period of the female cycle. (Deie et al., 2002; Eiling et al., 2007;

Park et al., 2009) There is an increase in injury rate during the ovulation stage found

as well. (Wojtys et al., 1998) However, Zazulak et al (2006) produced a systematic

review showing six of the nine studies examined did not show a laxity and cycle

correlation.

Effects on motor skills from estrogen increases are influenced by menstrual

symptoms, which cause decreases in neuromuscular joint protection. (Wojtys et al.,

1998) Some of these suggested motor effects include fine motor dexterity skills,

diminished muscular protection to the joint and decreases in musculotendinous

stiffness. (Eiling et al., 2007) Neural effects caused by menstrual cramps and

discomfort, usually peaking during ovulation, has also been thought to add to high

incidence of ACL injury in women. (Eiling et al., 2007) It has been suggested that

these premenstrual symptoms causing discomforts and pain contribute to alterations in

48

athletic performance and neuromuscular control, which may contribute to non-contact

injury. (Arendt & Dick, 1995; Eiling et al., 2007) Dysfunctions to the females during

the cycle have been studied previously showing changes, however, the results are not

conclusive. This study will look into how an acoustic startle disruption will affect

female muscular activity and joint stiffness during the menstrual cycle changes.

Startle Response

Disruptions from acoustic startles such as whistles and crowd noise may also

alter the processes for muscle stiffness and neuromuscular control regulations in

protecting the joint.(Koch, 1999) A universal startle response is provoked during these

unexpected events, and neuromuscular control changes can be observed through

alterations in preparatory (feed-forward) and reactive (feedback) muscle contractions,

potentially altering the stiffness regulation necessary for dynamic joint stabilization.

(Ghez & Krakauer, 1991) The startle reflex usually begins by a contraction of the

facial muscles in response to sudden intense visual, tactile or auditory stimuli, with the

eye-blink being the first seen response controlled by the orbicularis oculi: a striated

sphincter muscle encircling the orbit that closes the eye during a blink. (Blumenthal et

al., 2005; Braff et al., 2001; Koch, 1999; Landis & Hunt, 1939) The acoustic startle is

primarily used in the laboratory setting to elicit a response.(Favaron et al., 2010)

Blumenthal et al (2005) reported that the best way to record EMG activity of the

orbicularis oculi and record the first activity after a startle is to place electrodes below

the lower eyelid. Collecting the response from this location shows the first central

nervous system response to the startle from the facial musculature. An acoustic

49

startle causes a response to an outside stimulus where the body has a parasympathetic

response and is described as a startle reflex through a motor response. (Favaron et al.,

2010; Nieuwenhuijzen et al., 2000) The acoustic startle is influenced by stimulus

intensity, interval, other motor behavior and genetic differences. (Davis, 1970)

Habituation is shown in a reduction of response from an acoustic startle meaning that

the first introduction to the stimulus generally creates the greatest response compared

to trials without the stimulus.(Davis, 1984; Koch, 1999) Males are shown to have a

greater pre-pulse inhibition (PPI) response to the startle compared to females. (Braff

et al., 2001; Swerdlow et al., 1993b) Swerdlow et al suggest that the hormonal

changes between males and females cause variability in the response to outside

stimuli. PPI limits the reaction to a strong stimulus, such as loud acoustic noise. This

allows the nervous system to adjust when this occurs and the muscles functionally

adapt to the outside stimuli. (Braff et al., 2001; Swerdlow et al., 1993b)

A neuronal circuit made of three sets of synapses in the lower brainstem

mediates the startle response. (Davis et al., 1982) These three synapses are started with

spiral ganglion cells in the cochlea, followed by the cochlear root neurons synapsing

onto the pontine reticular nucleus (PnC) and the PnC neurons synapsing on spinal and

cranial motor neurons. (Davis et al., 1982) This circuit is highly controlled through

the role of PnC. (Koch et al., 1992) Although the pathway ends with cranial motor

neurons, parallel pathways can also effect extremities including the knee

joint.

50

Figure 2: Neuronal Circuit (http://www.acoustics.org/press/138th/davis.htm)

As a motor response to stimuli, the startle elicits a flexor and extensor reaction,

(Davis, 1984; Landis & Hunt, 1939) and was reported to show similar results in a

study by Nieuwenhuijzen et al (2000). These flexor and extensor co-activation

reactions were seen during the early/mid-stance of gait in the leg musculature while a

startle was applied and caused a decrease in range of motion. This study was

performed to develop a better understanding of how a startle could disrupt functional

movements. Nieuwenhuijzen et al found that the startle affected locomotion through

co-contraction and showed increases in muscle activation strategies. Unanticipated

events such as startles have shown to alter functions in previous literature. (Braff et

al., 2001; Swerdlow et al., 1993b) In studies focused on neuropsychiatric disordered

patients, the startle is showing impairment to sensorimotor and cognitive reaction due

to its effect on PPI. When the PPI is inhibited, this can cause delays in functional tasks

and muscle activation creating possible deficits in stabilization. (Braff et al., 2001)

The relationship between auditory stimulus and changes to joint stability specifically

51

at the knee is not highlighted in current literature which could add further knowledge

to the ACL injury occurrences in sport.

Summary

ACL injuries affect many athletes every year, but more females than males.

Quadriceps and hamstring stiffness contributes to muscle firing and activation

differently in males and females. Decreased stiffness values are often seen as a

contributing factor to ACL injuries, which can be preventable with better dynamic

stabilization. The female menstrual cycle has continuing estrogen hormonal fluxes

during the 28-day period, which shows alterations in neuromuscular control and joint

stability. When these processes are altered, the risk of injury increases in the female

population. The acoustic startle response can also disrupt muscular stiffness, dynamic

joint stability, and may interact with the menstrual cycle to cause variations in

neuromuscular control. Studying gender differences combined with a startle stimulus

may lead to further insight to causes of ACL injury.

52

Chapter 7

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APPENDIX A

Stiffness and Proprioception Assessment Device

Stiffness testing will be performed using a custom-built stiffness and

proprioception assessment device (SPAD) (Appendix 2). The SPAD is a brushless

Danaher/Kollmorgen servomotor (B-404-B-B4) that is fitted into a gearbox (UT018-

050, 50:1) that is connected to an amplifier/controller (Copley Xenus driver XSL-12-

36-R). The amplifier (input: 230 VAC, 3PH, output: 18 FLA, 50/60 Hz) is mounted

~4ft above the ground and connected to the motor with a 12-foot long feedback cable

(Model # CEF-RO- 006-900, Pacific Scientific, Rockford, IL) and also connected to a

personal computer through a Kvasar CAN cable. The controller receives a three-

phase, 240-volt, 30-amp enclosed I-T-E switch power supply through a power cable

(Model # CEP-A6-006-904, Pacific Scientific, Rockford, IL). The mated servomotor

and gearbox are mounted in a cast aluminum pedestal that is offset from the subject’s

chair. An adaptor arm and torque reaction sensor (Model # T5400, Futek Advanced

Sensor Technology, Irvine, CA) with a 565 N capacity and 1.43 X 105 ft-lb/rad

torsional stiffness is coupled to the gearbox. Signals from the torque reaction sensor

pass through a conditioner (Model # D502, Futek Advanced Sensor Technology,

Irvine, CA) at 60 Hz and have a 0 to 10 Vdc analog output range. The signal

conditioner digitally displays torque values and also sends an analog torque signal

through a BNC box so that is can be recorded and displayed in LabVIEW software.

For safety purposes, internal motor settings cannot exceed preset speeds and there are

three emergency stop switches that can disable the motor during testing. The operator

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and test participant will each hold an emergency stop button. When depressed, the

motor is disabled. Also, if the adaptor arm moves within the last 5% of the

participant’s end range of motion a proximity sensor (#S4602896, Turck Inc. USA,

Minneapolis, MN) will disable the motor. When the motor is disabled, the operator

must re-start the power supply to continue tests. The LabVIEW motor control

software also has “soft limits” which will disable the motor amplifier if motion

exceeds individualized motion limits. The SPAD is also fitted mechanical stops to

limit motion through an adjustable range and brass screws in the adapter arm flange

connected to the gear box which will fail under excessive torque. The SPAD device is

operated using a personal computer with a customized LabVIEW virtual instrument

and motor control software program.

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Figure A.1: Subject Positioning on the SPAD for testing protocols

Motor Amplifier

Torque Sensor

Motor & Gear Box

Subject Emergency Stop Switch

Proximity (stop) Sensors & Mechanical Stops

Adapter Arm

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APPENDIX B

PAR-Q Form

1. Has your doctor ever said that you have a heart condition and that you should only do physical activity recommended by a doctor?

________ NO _______ YES 2. Do you frequently feel pain in your chest when you do physical activity? ________ NO ________YES 3. Do you often feel faint or have spells of severe dizziness? ________ NO ________ YES 4. Has a doctor ever said your blood pressure was too high ________ NO ________ YES 5. Has your doctor told you that you have a bone or joint problem such as arthritis

that could be aggravated or made worse by a change in your physical activity? ________ NO ________ YES 6. Do you know of any other reason why you should not do physical activity? ________ NO _________ YES 7. Are you over the age of 65 and not accustomed to vigorous exercise? ________ NO _________ YES If you answer YES to one or more of these questions please answer the following questions: 8. Have you recently consulted your physician about increasing your physical activity

and/or participating in a fitness evaluation? ________ NO _________ YES 9. If you answered “NO” to question 8, will you agree to consult your physician prior

to increasing your physical activity and/or participating in a fitness evaluation? ________ NO _________ YES Signature of PARTICIPANT _____________________ Date ________________ Signature of Investigator __________________________ Date ________________

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APPENDIX C

Demographic and Health History Questionnaire

Participant ID: ______________ Date: ___________ Gender: M F Cycle: L F N/A Age: _______ Sport: _______ PLEASE ANSWER THE FOLLOWING CONDITIONS TO THE BEST OF YOUR ABILITY. 1. Are you currently unable to participate in lower extremity activities due to injury? NO___ YES___ (if so, explain)_______________________________ 2. Have you injured either knee within the last 6 months? NO___ YES___ (if so, explain)_______________________________ 3. Have you injured either ankle within the last 6 months? NO___ YES___ (if so, explain)_______________________________ 4. If yes to any of the above, please answer: Have you ever seen a doctor for your injury (s)? NO___ YES___ (if so, explain)_______________________________ 5. Do you have any other muscle or skeletal disorders that the researcher should be aware of? NO___ YES___ (if so, explain)_______________________________ 6. Are you presently taking any medications? (including prescribed and over the counter) NO___ YES___ (if so, explain)_______________________________ 7. Have you ever had any type of surgery that may prevent your participation in this study? NO___ YES___ (if so, explain)_______________________________ 8. Do you have any hearing deficits? NO___ YES___ (if so, explain)______________________________ 9. Do you have a history of neurological disorders that may prevent your participation in this study? NO___

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YES___ (if so, explain)_______________________________ 10. Have you ingested any caffeine in the past 12 hours? NO___ YES___ (if so, explain)_______________________________ 11. Have you taken any over-‐the-‐counter medications in the past 12 hours? NO___ YES___ (if so, explain)_______________________________ 12. FEMALES: Have you had a regular menstrual cycle (lasting 26-‐32 days) during the past 6 months? NO____ YES____ Please indicate any medical and/or health concerns you may have which have not been addressed by the previous items on this form. If there are any questions please feel free to contact one of the investigators at the following: Athena DeAngelis, BS, ATC 541 South College Avenue, University of Delaware Newark, DE, 19716 (856)-‐816-‐7286 -‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐ Signature of participant Date -‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐ Signature of investigator Date

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APPENDIX D

Quadriceps & Hamstring Stretching Techniques QUADRICEPS:

Stand and touch the wall for support. Bend your heel towards your back and pull your ankle and forefoot to your rear end. Hold for 30 seconds and repeat 3 times.

HAMSTRINGS:

Stand and slightly bend your knees. Bend at the waist and reach towards your toes or floor and hold for 30 seconds. Repeat 3 times.

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APPENDIX E

IRB Approval Letter

RESEARCH OFFICE 210 Hullihen Hall University of Delaware

Newark, Delaware 19716-1551 Ph: 302/831-2136 Fax: 302/831-2828

DATE: May 6, 2011 TO: Athena DeAngelis FROM: University of Delaware IRB STUDY TITLE: [231022-1] The Effects of an Acoustic Startle and Hormonal Changes on Knee Joint Stiffness SUBMISSION TYPE: New Project ACTION: APPROVED (E. Peloso, W. Farquhar) APPROVAL DATE: May 6, 2011 EXPIRATION DATE: May 5, 2012 REVIEW TYPE: Expedited Review REVIEW CATEGORY: Expedited review category # 4 Thank you for your submission of New Project materials for this research study. The University of Delaware IRB has APPROVED your submission. This approval is based on an appropriate risk/benefit ratio and a study design wherein the risks have been minimized. All research must be conducted in accordance with this approved submission. This submission has received Expedited Review based on the applicable federal regulation. Please remember that informed consent is a process beginning with a description of the study and insurance of participant understanding followed by a signed consent form. Informed consent must continue throughout the study via a dialogue between the researcher and research participant. Federal regulations require each participant receive a copy of the signed consent document. Please note that any revision to previously approved materials must be approved by this office prior to initiation. Please use the appropriate revision forms for this procedure. All SERIOUS and UNEXPECTED adverse events must be reported to this office. Please use the appropriate adverse event forms for this procedure. All sponsor reporting requirements should also be followed. Please report all NON-COMPLIANCE issues or COMPLAINTS regarding this study to this office. Please note that all research records must be retained for a minimum of three years. Based on the risks, this project requires Continuing Review by this office on an annual basis. Please use the appropriate renewal forms for this procedure. If you have any questions, please contact Jody-Lynn Berg at (302) 831-1119 or [email protected]. Please include your study title and reference number in all correspondence with this office.

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