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Is there enhanced lymphatic function in upper body trained females? Dolan, Lianne B. 2006

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IS T H E R E E N H A N C E D L Y M P H A T I C F U N C T I O N I N U P P E R B O D Y T R A I N E D FEMALES?  by  LIANNE BRONAGH DOLAN B . S c , The University o f Western Ontario, 2003  A THESIS SUBMITTED IN P A R T I A L F U L F I L M E N T OF T H E REQUIRMENTS FOR T H E D E G R E E OF M A S T E R OF SCIENCE  in  T H E F A C U L T Y OF G R A D U A T E STUDIES  (Human Kinetics)  T H E UNIVERSITY OF BRITISH C O L U M B I A December 2006 © Lianne Bronagh Dolan  Abstract  Chronic physical activity has been shown to ameliorate various aspects o f human physiology, while specific training can directly influence structural changes.  It remains  unknown i f chronic exercise influences upper extremity lymphatic function in females; thus, the purpose of this cross-sectional study was to compare different exercise stresses on lymphatic function in ten upper body trained females (mean (SD): age= 26.9 (SD 4.4) yrs; ht= 165.0 (SD 11.2) cm; wt= 62.1 (SD 11.8) kg; V 0 = 35.0 (SD 3.2) ml-kg"'-min' ) 1  2  with ten untrained females (age= 31.0 yrs (SD 6.0); ht= 168.1 (SD 6.5); wt= 69.5 (SD 14.7) kg; V 0 2 = 22.2 (SD 4.8) ml-kg" -min" ). 1  1  Participants underwent a maximal upper  body aerobic test on an arm crank ergometer before undergoing three randomly assigned lymphatic stress tests. Lymphoscintigraphy was used to quantify lymphatic function. 99m  T c - a n t i m o n y colloid was injected into the third web space o f each hand followed by 1  minute spot views taken with a y-radiation camera. Axillary acquisitions occurred at 18 (SD 5) and 64 minutes. The maximal stress test required individuals to repeat their initial maximal exercise test and then be imaged every 10 minutes until 60 minutes was reached. The submaximal stress test involved arm cranking for 2.5 minutes at 0.6 W-kg"' followed by 2.5 minutes of rest, repeated for 60 minutes. The final stress test was a 60 minute seated resting session. The amounts cleared at the hand ( A C ) and axillary uptake (Ax) were determined. Four 2 X 3 A N O V A s were used to test for statistical significance between the two groups and three lymphatic stress tests. Only A x post maximal exercise was significantly different between trained and untrained, p=0.009. A l l other measures of lymphatic function between groups were similar. Exercise had a significant impact on lymphatic function: maximal A C was significantly higher at 10 minutes (p=0.000) while submaximal A C was significantly higher at 60 minutes, (p=0.000).  Compared to rest,  exercise A x was significantly greater (p=0.000) but the exercise stress, (maximal or submaximal), A x at 64 minutes did not significantly differ (p=0.426). This study demonstrates no significant difference between upper body trained and untrained females while exercise stress significantly increased A x and A C .  n  Table of Contents  Abstract  ii  Table o f Contents  •  •  iii  List o f Tables  v  List of Figures  y  List o f Abbreviations  '  1. Introduction  1  1.2 Reasoning behind lymphatic research 1.3 Measuring lymphatic function 1.4 Research Problem  ...2 2 4  2. Methods  4 Participants.... Experimental Design Experimental Procedures Imaging Statistical Analysis  4 5 5 6 8  3. Results 3.1 3.2 3.3 3.4  9 Baseline Characteristics Amount Cleared Axillary Uptake Heart Rate and Power Output  4. Discussion 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9  vii 1  1.1 The influence of exercise on lymphatic function  2.1 2.2 2.3 2.4 2.5  i  General Findings Axillary Uptake Clearance from the Hand Untrained vs. Trained Pressure Gradient Respiration Active vs. Passive Transport Mechanisms Adaptations with Training Limitations  • ••  9 ••••9 13 15 16  ;  -16 16 16 17 ••••19 19 ..20 20 21  in  4.10 Future Directions  22  4.11 Conclusion  23  References  24  Appendices 1. Literature Review 2. R a w Data 3. Clinical Research Ethics Board Approval  29 38 46  IV  List o f Tables  Table 1. Participant characteristics.: Table 2. Amount o f  9 9 m  Tc cleared from the hand  Table 3. Axillary Uptake o f  9 9 m  Tc  9 14 14  Table 4. Heart rate and power output  15  Table 5. Individual descriptive data  39  Table 6. Individual data for power out, heart rate and R P E  40  Table 7. Individual data for axillary uptake  41  Table 8. Individual clearance rate equations  42  Table 9. Amount cleared immediately post-maximal exercise  45  v  List o f Figures  Figure 1. Scintigraphs acquired of the axilla region and of the web spaces  3  Figure 2. Clearance o f exercise  10  9 9 m  Tc from the web space of the hand following maximal  Figure 3. Clearance o f " Tc from the web space of the hand during submaximal 99  1  exercise Figure 4. Clearance o f  11 9 9 m  Tc from the web space o f the hand during rest  12  Figure 5. Difference in A C between 30 and 60 minutes  13  Figure 6. Submaximal heart rate vs. A C  15  Figure 7. T w o scintigraphs at initial injection  43  Figure 8. Clearance rates for trained and untrained females  44  vi  List o f Abbreviations  99m j AC  Technetium" ' antimony colloid 99 11  c  .  Amount o f radiopharmaceutical cleared from the web space of the hand in  % Ax BCRL MBq RPE rpm V02max.  Uptake of the radiopharmaceutical in the axillary lymph nodes as a relative % to the initial dose in the webspace Breast Cancer Related Lymphedema Megabecquerel Rating of perceived exertion; where 0 is no effort and 10 is maximal effort Revolutions per minute M a x i m a l rate o f oxygen consumption  vii  1. Introduction The lymphatic system plays a key role in maintaining body homeostasis, yet its role in fluid balance is poorly understood (50). The movement of lymph through the system can be influenced by active and passive mechanisms such as: lymphangion contraction, transmural pressure changes, luminal flow, arteriole pulsations, muscle pump activation, neural input, central venous pressure changes and humoral influences (12).  There are  many known benefits of physical activity; however, whether chronic exercise can improve lymph transport through structural changes remains unknown.  1.1 The influence o f exercise on lymphatic function Exercise has a positive influence on lymph flow (16, 18, 32). Physical activity may stimulate various mechanisms that encourage lymph flow, such as: activating the muscle pump, increasing blood flow, changing various pressures, and augmenting sympathetic nervous activation (22). With exercise-induced increases in arterial blood pressure and vascular surface area in the working muscles, an increase in interstitial pressure occurs, increasing the pressure gradient, which promotes capillaries.  fluid uptake into the lymphatic  With greater lymph formation occurring in the lymphatics, the increase in  fluid causes an increase in wall tension, triggering a stronger contraction in the lymphangions, which increases lymph propulsion (12, 47). There is a continuous drive from the working muscles to aid lymph propulsion by compressing upon the surrounding lymphatics and centrally guiding the lymph towards the ducts to return it to systemic circulation.  Exercise influences lymph flow in both animals and humans. Changes in cardiac output alters lymph flow in sheep (8, 9), while contractions o f different muscle groups have been reported to influence lymphatic clearance rates in humans (16) and in rats (24). Lane et al., (23) studied the acute differences between rest, handgrip exercise, and arm cranking exercise on upper body lymphatic clearance and demonstrated that arm cranking induced the greatest clearance rates (23). It was speculated that the faster clearance rate with dynamic exercise occurred due to increased use o f the muscle pump, and a rise in interstitial pressure.  Male trained runners were shown to have an. overall greater  1  lymphatic clearance rate in their legs than untrained runners, which was attributed to the increase in vascularization in trained individuals. These trained runners had increased resting lymph flow (16), leading to a possibility o f regional differences within the lymphatic system, both functional and in contractile elements (5, 12).  Chronic exercise training leads to various changes in human physiology, but whether adaptations occur within the lymphatic system remains unknown. Due to. improvements in cardiac function with exercise, there is a potential link that the lymph transport mechanisms o f vasomotion and pressure changes could increase the efficiency o f lymph formation and transport, thus improve lymphatic function.  This information leads us to  believe regular exercise programs may have an effect on systemic lymph flow, which begs the question: does chronic exercise result in improved lymphatic function in the upper extremities? '  1.2 Reasoning behind lymphatic research One in nine women is at risk for developing breast cancer and o f those women, up to 50% are at risk for developing breast cancer related lymphedema ( B C R L ) . With the rise in breast cancer diagnosis, combined with the increase in survival rate, there will likely be an increase in the occurrence of lymphedema (44). Unfortunately there is no cure for women diagnosed with this debilitating condition. Understanding the mechanisms that influence lymphatic function may help prevent or treat lymphedema.  Exercise can  increase lymph flow 2-3 times by altering interstitial pressure and activating the muscle pump (15). Other mechanisms such as transmural pressure, luminal flow, neural input, and humoral influences all affect lymph contractility, which in turn can alter lymph flow (5).  Habitual exercise may influence acute and chronic changes in these factors,  consequently altering the effectiveness o f the pumping action and the pressure-flow relationship (5), potentially improving lymphatic function.  1.3 Measuring lymphatic function Historically, B C R L was monitored by measuring changes in arm circumference or changes in arm volume using a water displacement method. These methods do not relay  2  information on functional changes within the lymphatic system. Lymphoscintigraphy, radiopharmaceutical labeling o f a lymphatic drainage source, is considered the leading technique in primary diagnosis o f lymphedema. It monitors the rate of clearance, or removal of a radiolabeled tracer from an injection site, and is considered one o f the best ways to estimate local tissue lymph flow, characterizing lymphatic function (2, 42). It is considered a highly sensitive, reproducible technique capable o f measuring the transport capacity of the lymphatic vessels (44).  There are a range of techniques used for  lymphoscintigraphy, from the type o f radiopharmaceutical to the location and depth o f the injection. In Canada, Technetium-99m ( indirectly quantify lymphatic flow.  9 9 m  T c ) antimony colloid has been selected to  Antimony colloid is preferentially absorbed by the  lymphatic system (19); the radioisotope is injected subcutaneously into the web spaces and then a gamma camera is used to capture sequential images throughout the testing period, as seen in Figure 1.  Study Name: LYMPH STUDY KH D a t e s Time: 10/17/2008 Manufacturer Model: NumaConneet Icon Vancouver General Hospital Nuclear Medicint^NOWN  Marker Hand  Elbow  Figure. 1 Scintigraphs acquired o f the axilla region and of the web spaces. The lines leaving the two web spaces in the image on the right display lymphatic clearance in the vessels. In the left scintigraph the lines leading up to the axilla region display f l o w in the lymphatic vessels through the arm, accumulating in the lymphatic nodes. The circular image in the top left corner represents a marker designating the right side.  1.4 Research Problem It is acknowledged that exercise increases lymph flow (32), but it still remains unclear i f there is a general improvement in lymphatic function following exercise training. This is an important question that needs to be addressed due to its potential relationship in aiding individuals with B C R L .  If chronic exercise in healthy individuals improves lymphatic  function, then it is logical to implement an exercise program for women who are at risk for B C R L . Ideally, a training study looking at the effect of an upper body exercise program on lymphatic function is preferred, however to explore this concept, we started with a cross-sectional study. Therefore, the purpose of this study was to determine i f upper body trained females have enhanced lymphatic function. The secondary purpose o f this study was to investigate the effect of maximal exercise on lymphatic function.  Hypotheses: •  Upper body trained females w i l l have increased lymphatic clearance rates and axillary uptake in comparison to untrained females.  •  M a x i m a l exercise w i l l induce greater clearance rates and axillary uptake than submaximal exercise.  2. Methods 2.1 Participants Twenty healthy females between the ages o f 20-40 were assigned to two equal groups based on maximal aerobic capacity ( V O 2 max) and upper body training history. Females were considered trained who participated in upper body activities at a competitive level, but were only accepted into the study i f their upper body V O 2 max was greater than 30 ml-kg"--min'' during an arm ergometer maximal exercise test. Females were considered untrained i f they did not participate in regular physical activity (less than twice a week). Exclusion criteria included history of upper extremity axillary surgery, and history of breast cancer or lymphedema. Informed consent was obtained prior to testing and the  4  study procedures were approved by the Clinical Research Ethics Board o f the University of British Columbia.  2.2 Experimental Design A cross-sectional, exploratory, mixed model design was used to compare the effect o f three lymphatic stress protocols on lymphatic function, between both groups at two different time points.  The independent variables were the lymphatic stress protocols  (submaximal, maximal, and rest) and the training status, while the dependent variables, measuring lymphatic function, were the amount o f radiopharmaceutical cleared ( A C ) and the amount of radiopharmaceutical arriving in the axillary region, axillary uptake ( A x ) .  The subjects were randomly designated to treatment order by draw, preventing any sequencing effects from occurring. A minimum of 48 hours separated each test in order to prevent fatigue from biasing data and to allow full decay o f the radioisotope.  2.3 Experimental Procedures Each volunteer was subjected to four different tests on four different days consisting of one  maximal  aerobic  exercise  test  and  three  lymphatic  stress  tests  using  lymphoscintigraphy to quantify lymphatic function. The same technician prepared and administered  subcutaneous  injections  of  the  radiopharmaceutical  Technetium" '  99 11  antimony colloid to each subject for each lymphatic stress test.  Test 1 - Upper body aerobic maximal stress test Day one o f testing consisted of a maximal aerobic stress test on an arm ergometer to determine upper body V O 2  m a x  - Individuals performed the V O 2  m a  x test on a calibrated  Lode arm ergometer (Angio single set model, Netherlands), following a step protocol that started at 10 watts and increased by 10 watts per minute until volitional fatigue. Pulmonary ventilation and expired gas concentrations were measured with a TrueOne 2400 metabolic cart (Parvo Medics Inc, Sandy, U T ) , and values were reported as the average o f 15 second intervals. R p m , heart rate (using a Polar T31 heart rate monitor (Polar Electro Inc, Lake Success, N Y ) ) and rating of perceived exertion (using the Borg  5  scale (4); 0 representing no effort to 10 representing maximal effort), were noted toward the end of every minute.  Test 2- Submaximal intermittent lymphatic stress test After the first static image was acquired, participants began twelve repeated bouts o f arm cranking, on a calibrated Lode ergometer, for 2.5 minutes at a resistance o f 0.6 watts per kilogram o f body weight (Wkg" ) followed by 2.5 minutes of rest. Heart rate was 1  obtained during the last thirty seconds o f each interval bout, and then averaged to give an overall submaximal heart rate.  Test 3- M a x i m a l lymphatic stress test A three to five minute warm up occurred on the same Lode arm ergometer, at 10 watts prior to the initial injection. After the initial injection, the first static image was acquired and then the maximal protocol began. The maximal lymphatic stress test mimicked Test 1, except no ventilatory parameters were measured. Rating of perceived exertion, rpm and heart rate were used to verify i f maximum was reached. A two minute cool down at 10 watts occurred followed by a second static image. The individuals then rested until sixty minutes was reached.  Test 4 - Resting lymphatic stress test The third lymphatic stress protocol was a resting session, where the subjects were asked to remain seated, with their arms resting on a pillow. Heart rate was obtained three times throughout the sixty minute period and averaged to give an overall resting rate.  2.4 Imaging Lymphoscintigraphy occurred at Vancouver General Hospital, in the Department of Nuclear Medicine and Imaging. A total o f 18 M B q of  9 9 m  T c antimony colloid in 0.05 m L  was administered subcutaneously to the third web space o f each hand prior to the start o f the exercise protocol. The radiopharmaceutical purity o f the injected preparation was between 92-96%. Subjects placed both hands prone on a gamma camera equipped^ with a low energy, ultra high resolution collimator (Sopha Medical V i s i o n , Wisconsin, U S A ) .  6  Only the forearms and the hands were in the field of view o f the camera and a single camera head was used.  A n initial one minute static acquisition, with a 20% energy  window centered on a 140 k e V peak, was acquired within one minute o f the injection.  During the stress tests, sequential images were taken approximately every ten minutes over a period o f sixty minutes, allowing for the calculation of depot clearance rate. Images were not acquired during the maximal protocol, but were acquired prior to the onset o f exercise and then immediately post-maximal exercise and throughout the recovery phase. The clearance rate equation was used to calculate the amount o f radiopharmaceutical cleared from the hand at various time points. T w o , one minute static images were acquired at the designated Test 1 end point time and at sixty-four minutes post-injection to determine the radiopharmaceutical uptake in the axilla relative to the initial activity i n the hand. This image was taken with subjects lying in a supine position. The reliability o f lymphoscintigraphy during intermittent submaximal exercise is r = 0.939, while variability between arms is 30.4% for rest and 14.4% during exercise (20).  The images were processed using Siemens Icon software (Version 8.5, Siemens Medical Systems Inc., Illinois, U S A ) .  The region o f interest (144 pixels) was drawn around the  injection site o f each hand to give the number o f activity counts per hand. corrected for physical decay of  9 9 m  It was  T c using the following equation: A, = A e -  Xt  0  Whereby A = activity after an elapsed time (min), A = activity in original sample, A. = t  0.693/363 m i n physical half-life o f  0  9 9 m  T c , and t = elapsed time from the original image.  The corrected count at each time point was divided by the corrected count measured immediately after injection and was plotted against time. Right and left arm clearance rates are symmetrical during rest (36), and during submaximal exercise (17), accordingly right and left data were averaged to produce one clearance rate per subject. Depot clearance rate from each hand is linear during rest and submaximal exercise; therefore it is expressed as a linear constant (% administered activity min" ). It was unknown how 1  maximal exercise would affect clearance rate, thus clearance rates for dominant and nondominant arms were compared before averaging the two to determine the overall slope.  7  M a x i m a l exercise (with recovery time) percentages  were graphed against time to  determine the line of best fit. A logarithmic slope was determined to represent maximal clearance rate.  Due to the mathematical differences in slopes between maximal,  submaximal and resting clearance rates, the determined clearance rate equations were used to compare the A C in percentages at two selected time points: 10 and 60 minutes.  Region o f interests were drawn around axillary lymph node regions and were corrected for physical decay o f  9 9 n i  T c . The corrected axillary counts were made relative to the  palmar region of interest boxes o f 144 pixels.  There was an anterior and posterior  axillary acquisition taken in the supine position, hence the geometrical mean was determined with the corrected counts then compared to the initial amount o f radioactivity at the injection site which determined percent o f axillary uptake.  A x i l l a r y uptake has  been shown to be equivalent between dominant and non-dominant arms (17) but it was unknown i f that held true for maximal exercise. Thus, A x between dominant and nondominant arms was compared, and i f differences were found, comparisons were made using the dominant arm.  2.5 Statistical Analysis SPSS version 14.0 (SPPSS Inc, Chicago, IL) was used for statistical analysis. For this exploratory study, paired t-tests were run between dominant and non-dominant arms to determine i f A C and A x were equivalent between arms during maximal exercise. Separate 2x3 analyses of variance ( A N O V A s ) were used to determine i f there were significant differences in A C at 10 and at 60 minutes. A s well, separate 2x3 A N O V A s were used for A x at 18 and 64 minutes. A paired t-test was used to determine i f A C and A x at 60 minutes was greater than at 10 minutes. A n alpha level o f 0.05 was used for all tests o f significance. Contrasts were determined and bonferroni adjustments were made for multiple comparisons. Results are given as means (SD) i f not otherwise stated.  8  3. Results 3.1 Baseline Characteristics There was no significant difference in anthropometric data between the trained and untrained females, but there was a significantly higher V O 2 max in the trained group, p= 0.000 (Table 1). A l l subjects except for two in the trained group attained similar maximal values during Test 3 as in Test 1, based on their initial maximal time, power output, heart rate, perceived exertion, and rpms.  Table 1. Participant characteristics (n=10) Age (yrs) Trained .26.9(4.4) Untrained 31.0 (6.0)  Ht (cm) 165.0(11.2) 168.1 (6.5)  Wt (kg) 62.1 (11.8) 69.5(14.7)  VO2 ml-kg''-min"' 35.0(3.2)* • 22.2 (4.8)  BMI (kg/m ) 22.7 (2.9) 24.6 (5.2) 2  V 0 L-mm' . 2.17 (0.4)* 1.48 (0.2) 2  Values are means (SD). * p< 0.05 compared with untrained values.  3.2 Amount Cleared A C , inferring lymph flow, showed equivalence between dominant and non dominant arms in trained and untrained females, p= 0.211. Accordingly, data between extremities was collapsed for further statistical analysis. A C was not significantly different between trained and untrained females at 10 or 60 minutes, p= 0.813 and p= 0.804 respectively, but exercise stress, at both time points, had a significant affect on the amount cleared, p= 0.000 (Table 2). A t 10 minutes, maximal exercise A C was significantly greater than submaximal A C , p=0.000. Approximately seven percent o f the radiopharmaceutical was cleared from the web space immediately post-maximal exercise, but during recovery, the clearance rate slowed generating a logarithmic slope (Fig. 2).  A C between 30 and 60  minutes post-maximal exercise was equivalent to rest A C , 1.95% (SD 0.5) and 1.74% (SD 1.6) respectively, p= 0.52 (Fig. 5). Submaximal and rest stress tests demonstrated a linear clearance rate (Figures 3 & 4 respectively), and at 60 minutes, there was significantly higher A C produced with submaximal exercise than with the other lymphatic stress tests, p= 0.000 (Table 2).  9  Figure 2. Clearance o f T c from the web space o f the hand following maximal exercise of trained females (—) and untrained females (-).  105.00  1 1  12  10.00  8.00  7.00  6.00  g  5.00  • Trained  £  I 5  s  • Untrained 4.00  3.00  2.00  1.00 -  0.00 Maximal  Rest  Submax Intermittent  Figure 5. Difference in A C between 30 and 60 minutes i n trained and untrained females during three modes o f lymphatic stress tests. *p< 0.05 compared to submaximal intermittent exercise.  3.3 Axillary Uptake During each lymphatic stress test A x was measured at two different time points: immediately post maximal exercise at 18 (SD 5) minutes and at 64 minutes. A s expected, there was significantly greater A x at 64 minutes than at 18 minutes within each lymphatic stress test. A x was equivalent at 64 minutes between the dominant and non dominant arm yet there was a significant difference in A x between arms at 18 (SD 5) minutes. p= 0.03.  Immediately post-maximal exercise the trained females had significantly higher A x than untrained, p= 0.009. A t 64 minutes, there was no difference in A x between groups p= 0.531, while there was a significant difference within the lymphatic stresses, p= 0.000 (Table 3). Exercise stresses had higher uptake compared to resting values p= 0.000, but there was no significant difference between maximal and submaximal A x , p= 0.426 (Table 3). 13  Table 2. Amount of  " T c cleared from the hand Maximal Submaximal Rest 10 m i n 60 min 10 m i n 60 m i n 10 m i n 60 m i n Trained 6.41 (SD 1.73) 11.54 (SD 3.10) 3.37(SD2.25) 14.82 (SD 4.28) 0.46 (SD 0.67) 3.17(SD2.09) Untrained 5.97 (SD 1.64) 10.85 (SD 2.95) 3.80 (SD 1.79) 15.72 (SD 4.05) 0.80 (SD 0.84) 3.86 (SD 3.73) T&UT 6.19 (SD 1.66)* 11.19 (SD 2.97)* 3.59 (SD 2.98) 15.27 (SD 4.08) 0.64 (SD 0.76) * "3.51 (SD 2.97) * Values are means (SD) in % o f the initial amount injected at time zero. Due to lack o f significant differences between groups, means were pooled, n= 20 for trained and untrained females, T & U T . * p< 0.05 when compared to submaximal stress 9 9 m  Table 3. A x i l l a r y uptake o f T c Maximal Submaximal Rest 18 ( S D 5) min 64 min 18 (SD 5) m i n 64 min 18(SD5)min 64 min Trained 0.70 (SD 0.30) 1.56 (SD 0.76) 0.61(SD0.82) 1.77 (SD0.78) 0.05 (SD 0.04) 0.71(SD0.86) Untrained 0.28 (SD 0.34) 1.31 (SD 0.76) 0.23 (SD 0.20) 1.62 (SD 1.05) 0.04 (SD 0.08) 0.52 (SD 0.55) T&UT n/a 1.44 (SD 0.75) 0.42 (SD 0.61) 1.69 (SD 0.91) 0.05 (SD 0.06) * 0.61 (SD 0.71) * Values are means (SD) in % relative to the initial amount injected at time zero in the web space. Due to lack of significant differences between groups, means were pooled, n= 20 for trained and untrained females ( T & U T ) except immediately post maximal exercise. * p< 0.05 compared to submaximal stress. w m  3.4 Heart Rate and Power Output Heart rates in the trained females were significantly lower throughout interval and resting protocols, p= 0.000, while maximal heart rate did not significantly differ, p= 0.140 (Table 4). A s expected, maximal wattage was significantly higher in the trained group p= 0.000. but there was no group difference in wattage during the submaximal exercise p= 0.240.  Table 4. Heart rate and Power Output (n=T 0) H R rest H R submax H R max  P O max  P O submax  37.0(7.1) 129 (22)* 58 (6)* 103 (14)* 180 (9) Trained 40.3 (7.5) 76 (7) 79 (9) 123 (11) Untrained 173 (2) Values are means (SD) in beats per minute for heart rate (HR), and watts for power output (PO). Results are presented between trained and untrained during maximal, submaximal and resting protocols. * p < 0.05 compared with untrained values.  In Figure 6, average heart rates for each 10 minute interval during submaximal stress were calculated for the trained and untrained groups and plotted against A C for each time point. The generated trend demonstrates that the trained group required a lower, steady heart rate to clear the equivalent amount as the untrained group.  160 140 120 100 CO  •e CO CO  X  80 60 40 20 0 5  10  15  20  A C % at 10, 20, 30, 40, 50, 60 min  Figure 6. Submaximal heart rate vs. A C . Average heart rate (bpm) was calculated for each time point at 10, 20, 30, 40, 50 and 60 minutes for trained (—) and untrained females (-) and graphed against A C o f T c as a percent. 9 9 m  15  4. Discussion 4.1 General Findings This exploratory study provides new information concerning the differences in lymphatic function between upper body trained and untrained females. Further, this study compares different exercise stresses on axillary uptake and the amount o f radiopharmaceutical cleared in the upper limb o f healthy females.  The principal findings o f this study are: 1) N o significant difference was found between groups except A x immediately postmaximal exercise; 2) M a x i m a l A C was significantly greater at 10 minutes, whereas submaximal A C was significantly higher at 60 minutes; and 3) M a x i m a l and submaximal A x did not differ.  Our results confirm other findings that exercise increases clearance rates, inferring lymph flow, due to exercise induced activation o f lymph transport mechanisms enhancing lymph propulsion (15, 16, 23). We also demonstrated an increase in axillary uptake o f  9 9 m  Tc  during exercise, an alternate measurement to infer lymph flow in the upper limb.  4.2 Axillary Uptake It remains unknown what influences retention time of a radiopharmaceutical within the lymphatic nodes and what physiological changes promote its return into the lymphatic vessels. With the inability to monitor the exact amount of radiopharmaceutical entering, staying and exiting a lymph node, we are unable to state i f axillary uptake, at these time points, is an accurate view o f total lymph transport within the upper limb. However, axillary acquisitions give an estimation of lymph flow from the hand and its movement through the upper limb to the axilla region. With this estimation, this study demonstrates the importance o f exercise in promoting lymph flow in the upper limbs.  4.3 Clearance from the Hand High variability occurred within the resting data (see F i g 4), which is not unexpected considering previous results found in other studies (20). The initial injection of the  16  radiopharmaceutical into the web space may cause high variability due to the immediate change in resting fluid homeostasis triggering an increase in lymph formation and flow, to prevent this instant edema (49). This natural response o f increasing flow may differ between individuals, thus producing excessive variability within the resting values, promoting the concept o f using an exercise stress for greater consistency to investigate lymphatic function. Another reason for the variability could be attributed to the lack of compliance of the subjects to stay still. Previous studies have shown that slight changes in body posture can alter resting lymph flow (10, 49). Cooke et al. demonstrated that a slight 15 degree change in supine body position altered resting lymph flow 104±36ul/h to 341±126pl/h (10).  from  During the resting period, the majority o f women  altered their posture so they could go to the bathroom, make a phone call or they simply forgot to stay still.  4.4 Untrained vs; Trained Baseline physical characteristics o f the two groups were not significantly different in age, weight or height, while physiological differences in  VC>2ma.\  were shown (Table 1).  Females were defined as upper body trained who participated in upper body, sports such as swimming, rock climbing, rowing or outrigger paddling. The subjects in this study participated in their sport at least four times a week with a minimum o f 3 years (up to 18 years) o f experience and all used other modes o f cross training such as running and weight lifting. The untrained females did not participate in regular exercise, but monthly activity consisted of a yoga class or casual walks.  Contrary to the hypotheses, lymphatic function was similar between groups through all stresses, except immediately post-maximal exercise.  Specifically, A x for the trained  group was significantly higher than that of the untrained subjects' A x immediately postmaximal exercise. A viable explanation is the time difference and the total amount o f work completed during the maximal condition when comparing the two groups. Trained individuals exercised approximately four minutes longer with a power output o f approximately forty extra watts; meanwhile, maximal heart rate was not significantly different between the groups. Even with greater work attained by the trained, A x was  17  similar between the trained and untrained group at the 64 minute mark, which introduces the possibility o f a maximal flow limitation.  The trained females had a lower resting heart rate, and a lower submaximal heart rate even though the average power output required was equivalent between the two groups (see table 4). These results imply that the trained required less cardiac effort to clear an equal amount o f radiopharmaceutical from the hands and transport an equal amount to the axilla region than the untrained (Fig. 6). The lower heart rate in the trained, yet equivalent A C and A x between groups insinuates the possibility o f an effective lymph transport mechanism such as enhanced arteriole pulsation in the trained. Magnetic resonance imaging has recently demonstrated vascular remodeling in both peripheral and central conduit arteries in upper body trained athletes (38). Enhanced diameters in the brachial arteries have been observed with training (29), and chronic changes were found in the vascular system: greater pulse pressure and stroke volume with a decreased heart rate (38). Perhaps trained individuals transport the same amount of lymph with a lower heart rate due to this increased pulse pressure, and greater alterations in artery and arteriole diameters, enhancing the lymphatic transport mechanism of vasomotion. This interpretation encourages the concept of greater efficiency within the lymphatic system in trained females.  Only one other study has looked at differences in training status in humans and lymph flow (16).  The lower extremity lymphatic clearance rates in four runners and four  untrained males were compared. N o differences were found in clearance rates during 10 minute exercise bouts or during the 65 minute recovery phase between each exercise condition.  When the three recovery phases were combined, the overall resting mean  lymphatic clearance rate in the trained was significantly higher than in the untrained, (0.06% (SD 05) and 0.03% (SD 0.03) respectively, p= 0.008). Our study had a greater sample size, and for upper body trained and untrained females, the resting clearance rates in the upper limb were 0.06% S D (0.03) and 0.06% (SD 0.07) respectively, but no significant difference  was found during the one hour resting protocol.  Possible  explanations may be due to differences in adaptation within the lymphatic system based  18  on gravitational stresses, such that vessels within the lower limbs may show greater changes than in the upper limbs. Another explanation is the difference in the injection site. The trained runners received injections into their vastus lateralis, an area most likely to have enhanced vascularization with lower body training (16). Ideally, to find a difference between trained and untrained females, an injection into an area of high capillary density within the arm muscles would be ideal, but in this study injections occurred in the web space o f the hand.  4.5 Pressure Gradient There are multiple mechanisms that affect the movement o f lymph, one being alterations in the various pressure gradients (12) at the capillary level or within the lymphatic vessel. H i g h central venous pressure is directly linked to lymphatic outflow pressure which is linked to an attenuation in lymph flow velocity (49); however, whether there is a linear decrease in lymph flow with increased outflow pressure in surrounding lymph vessels remains to be clarified (35). Thus, any change in pressure at the initial lymphatics, within the collector vessels, or at the final destination o f the right lymphatic or the thoracic duct, has potential to alter lymph flow. If the thoracic duct pressure is high, the lymphatic vessels leading into the duct w i l l be competing not only against gravitational forces, but against a higher pressure gradient within its own system, possibly slowing overall lymph flow.  It is possible that lymphatic outflow pressure reaches an upper limit due to a  leveling off o f central venous pressure, causing a maximal flow rate within the lymphatic system. Other human and animal studies infer the idea of a maximal flow rate, such that after the initial spike in clearance rate at the onset o f exercise, a plateau in clearance rate is demonstrated, even with further increases in intensity (8, 15).  4.6 Respiration Respiration has been shown to have variable influence on lymph flow in different animals due to changes in pleural pressure, affecting thoracic duct flow, consequently altering the pressure gradient throughout the lymphatic vessels (6, 35). To try to maintain a similar breathing pattern between subjects, a consistent rpm was used, but ventilation was not measured during the lymphoscintigraphy tests. A t maximal exercise, rpms were  19  85 (SD3) and during interval exercise, rpm was held at 70. Other than avoiding an entrainment response with breathing, a secondary reason for consistency with rpms was to ensure no variation in contractile rhythm. Muscle lengthening allows for lymphatic filling,  and the duration of the lymphatic refilling period within the vessels influences  lymphatic flow (25). Thus, muscle movement was kept as constant as possible, so when the arm muscle lengthened lymphatic filling stayed consistent.  4.7 Active vs. Passive Transport Mechanisms Gashev and colleagues have demonstrated  a decrease in contractile frequency and  amplitude within the lymphangion when there is an increase in lymph flow (13). The proposed explanation suggests that the lymphatic system attempts to conserve metabolic energy by creating a balance between the active and passive transport forces (12). L y m p h flow is dominantly influenced by the lymphangion at rest, but with an instant increase in edema, due to exercise or an injection, the passive pumps such as the suctioning o f the lungs, arteriole pulsations, pressure changes and the muscle pump become the dominant forces behind lymph transport (14). This balance between lymph transport mechanisms could limit lymph flow.  4.8 Adaptations with Training The trained required less cardiac effort (a lower heart rate) than the untrained to attain an equivalent amount of A C and A x , which suggests that alterations in blood flow mechanics and vascular adaptations occurred.  It has been documented that upper body trained individuals have increased capillary density of greater than 500 capillaries/mm in the upper limbs depending on the sport and 2  training level (7). This increase in capillary density and accompanying blood flow (1, 46), may increase potential fluid leakage into the interstitial space, causing the lymphatic system to adapt to the rising fluid demand to prevent edema. A potential adaptation by the lymphatic system to this chronic stress o f increased fluid may be an augmentation in lymphatic vessel development. Upper body trained athletes may have a greater number o f lymphatic vessels, compensating for the increased number of vascular capillaries.  20  If there is an upper limit for maximal flow, the increase in lymphatic vasculature may not change the rate o f lymph flow. But, i f no chronic increase in lymph flow is possible, perhaps an increase in lymphatic vasculature w i l l inadvertently produce a safety net to prevent a decline in lymphatic function. If damage occurs to the lymphatic system (as in B C R L ) , trained individuals may compensate through the development rerouting  mechanisms  with their  extra  number  of  lymphatic  o f potential  vessels,  avoiding  compromised lymphatic function.  4.9 Limitations A s a cross-sectional study, our major limitation was the inability to control for type, .intensity, duration and frequency o f training. A s well, our definition of trained using a VChmax value may have been flawed because it is well documented that V 0 2  m a x  is also  influenced by genetics. To get an accurate measurement o f the effect o f upper body exercise's influence on lymphatic function, a training study needs to occur.  One o f the potential limitations to this study was changing the lymphoscintigraphy protocol from two injections into the second and fourth web spaces to one injection into the third web space o f the hand. This study demonstrated similar clearance rate values to that o f previous studies using two injections (21). Resting values had a clearance rate o f -0.06% while intermittent submaximal exercise produced a clearance rate of -0.22% compared with two injection induced clearance rates of -0.08%) and -0.24% (20) respectively. This finding w i l l allow future studies using lymphoscintigraphy to decrease the number  o f injections per hand, potentially increasing compliance rate of the  volunteers.  A s previously discussed, understanding lymph node retention mechanisms needs to occur. The use o f axillary acquisitions is required because clearance rates do not differ from the hands in women with compromised lymphatic function in the upper limb (20) showing a lack o f sensitivity to defining lymphatic function efficiency. Axillary uptake in this study only gives an estimation o f the lymph movement occurring within the upper  21  limb, but perhaps i f multiple images were taken, time o f first appearance may be a more sensitive measurement.  Our choice o f submaximal intermittent exercise did not elicit equivalent workload intensities between groups.  The wattage was calculated based on weight; calculating a  load based on a percentage of VOnnax, or on equivalent cardiac output would be the next step to obtain more accurate comparisons in training differences.  Further, hormonal fluctuations were not controlled even though it is acknowledged that changes in hormone levels can affect various other physiological systems in the body.  We estimated our sample size using the averages in intensity differences (21), producing a sample size requirement of 10 in each group. Using differences in clearance rate slopes produced from this study, a sample size of 2321 may have detected differences between groups during rest. A sample size o f 60 during submaximal exercise and a sample size of 119 at the end o f maximal exercise were calculated to detect differences.  The injection location may be a limitation when trying to determine differences between groups. The amount o f change in capillary density in the hand due to upper body training may be less than that found in the arm muscles. A n injection into the forearm or upper arm instead o f into the web spaces may be a more viable method to determine i f there are differences with training.  4.10 Future Directions This  cross-sectional  study  investigated  lymphatic function in women who  were  previously trained and physically healthy. A training study using sedentary subjects is the next step. This training study would involve a change i n injection location, as well as investigating various exercise intensities to verify i f there is a maximal flow limitation in healthy females. If a limitation is found, then a prospective training study in women with compromised lymphatic function would follow. If their lymphatic function cannot reach the defined upper limit o f lymph  flow,  perhaps training could induce rerouting  22  mechanisms, and dissipate the effect o f impaired lymph transport. The type o f training, intensity, frequency and the duration that could generate alterations in lymphatic vasculature remains unknown, but measurements of not only clearance rate, but axillary uptake are necessary.  4.11 Conclusion In summary, the results demonstrate that lymphatic function is similar between trained and untrained females. This introduces the concept of a maximal flow limitation within the lymphatic system in healthy females. O n further examination this study proposes that lymphatic adaptations occurred in the trained females due to a decrease in cardiac requirement needed to produce equivalent lymphatic clearance rates and similar axillary uptake values.  23  References: 1.  Ahlborg G and Jensen-Urstad M . A r m blood flow at rest and during arm exercise. 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Lymphoscintigraphy to evaluate the effects o f upper body dynamic exercise and handgrip exercise on radiopharmaceutical clearance from the hands o f healthy females. Lymphatic Research and Biology 3: 16-24. ,2005.  24.  Mazzoni M C , Skalak T C , and Schmid-Schonbein GW. Effects o f skeletal muscle fiber deformation on lymphatic volumes. American Journal of Physiology 259: H1860-1868, 1990.  25  25.  26.  McGeown JG, McHale NG, and Thornbury KD. The role o f external compression and movement i n lymph propulsion in the sheep hind limb. of Physiology 387: 83-93, 1987.  Journal  McHale NG and Adair T H . Reflex modulation o f lymphatic pumping i n sheep. Circulation  Research  64: 1165-1171, 1989.  27.  McHale NG and Roddie IC. The effect o f intravenous adrenaline and noradrenaline infusion o f peripheral lymph flow in the sheep. Journal of Physiology 341: 517-526, 1983.  28.  McHale NG and Roddie IC. The effects o f catecholamines on pumping activity in isolated bovine mesenteric lymphatics. Journal of Physiology 338: 527-536, 1983.  29.  Naylor L H , O'Driscoll G, Fitzsimons M , Arnolda L F , and Green DJ. Effects of training resumption on conduit arterial diameter in elite rowers. Medicine and Science  30.  in Sports and Exercise  38: 86-92, 2006.  O'Brodovich H and Coates G. Effect o f exercise on lung lymph flow in unanesthetized sheep with increased pulmonary vascular permeability. American Review of Respiratory  Disease  134: 862-866, 1986.  31.  O'Brodovich H and Coates G. Effect o f isoproterenol or exercise on pulmonary lymph flow and hemodynamics. Journal of Applied Physiology 60: 38-44, 1986.  32.  O'Mahony S, Rose SL, Chilvers AJ, Ballinger JR, Solanki CK, Barber RW, Mortimer PS, Purushotham AD, and Peters A M . Finding an optimal method for imaging lymphatic vessels o f the upper limb. 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European Journal of Nuclear  Medicine  and Molecular  Imaging  30: 657-661, 2003.  37.  Pain SJ and Purushotham AD. Lymphoedema following surgery for breast cancer. British Journal of Surgery 87: 1128-1141, 2000.  38.  Petersen SE, Wiesmann F, Hudsmith L E , Robson MD, Francis J M , Selvanayagam JB, Neubauer S, and Channon K M . Functional and Structural Vascular Remodeling in Elite Rowers Assessed by Cardiovascular Magnetic Resonance. Journal  of the American  College  of Cardiology  48: 790, 2006.  .39.  Petrek JA, Senie RT, Peters M , and Rosen PP. Lymphedema in a cohort o f breast carcinoma survivors 20 years after diagnosis. Cancer 92: 1368-1377, 2001.  40.  Stanton AW, Holroyd B, Mortimer PS, and Levick JR. Comparison o f microvascular filtration in human arms with and without postmastectomy oedema. Experimental  41.  42.  84: 405-419, 1999.  Stanton AW, Svensson WE, Mellor RH, Peters A M , Levick JR, and Mortimer PS. Differences i n lymph drainage between swollen and non-swollen regions i n arms with breast-cancer-related lymphoedema.[comment]. Clinical Science 101: 131-140, 2001. Swartz MA. The physiology of the lymphatic system. Advanced Reviews  43.  Physiology  Drug  Delivery  50: 3-20, 2001.  Szuba A, Shin WS, Strauss HW, and Rockson S. The third circulation: Radionuclide lymphoscintigraphy in the evaluation o f lymphedema. Journal Nuclear  Medicine  of  44:43-57,2003.  44.  Tomczak H , Nyka W, and Lass P. Lymphoedema: lymphoscintigraphy versus other diagnostic techniques—a clinician's point o f view. Nuclear Medicine Review 8:37-43,2005.  45.  Valic Z, Buckwalter JB, and Clifford PS. Muscle blood flow response to contraction: influence o f venous pressure. Journal of Applied Physiology 98: 7276,2005.  46.  Volianitis S, Yoshiga CC, Nissen P, and Secher NH. Effect o f fitness on arm vascular and metabolic responses to upper body exercise. 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Microcirculation 12: 141-150, 2005.  28  A P P E N D I X I: L I T E R A T U R E R E V I E W  Anatomy and Physiology: The lymphatic vascular transport system is composed of: lymph, lymphatic capillaries, pre-collector vessels, collector vessels, lymphatic trunks, lymphatic ducts and lymph nodes. L y m p h is a clear fluid made up o f water, macromolecules, dying cells and other waste products from the interstitial fluid (3). The non-contractile lymphatic capillaries, or the initial lymphatic vessels, consist solely o f endothelium and have anchoring filaments attached at right angles. Under increased interstitial stress, the'filaments exert a radial tension on the lymphatic vessel, expanding the luminal volume and creating new gaps for fluid entrance (42). This expansion drops the luminal pressure,, favoring the pressure gradient, driving the interstitial fluid through the openings between  the  filaments, forming lymph. The lymph then drains from the capillaries into the precollector vessels within the dermis. Towards the lower aspect of the dermis, the precollector vessels develop valves and a layer o f smooth muscle. The functional units within the muscular lymphatic vessels are called lymphangions, and are separated by the lymphatic valves (5). The main function of the numerous pre-collector vessels is to propel the lymph from mid to lower dermis to the larger collector vessels. The collector vessels transport the lymph through the lymph nodes to the lymphatic trunks, where they return the lymph into the bloodstream via one o f the two lymphatic ducts: the large thoracic duct or the right lymphatic duct.  Each day approximately 16-18 L o f interstitial fluid develops and 2-4 liters are drained back into the lymphatic system, maintaining the necessary homeostatic balance (3). Active pumps, passive pumps and valves are used to form and move this newly formed lymph against gravitational forces, a hydrostatic pressure gradient  and a protein  concentration gradient (5). Active pumps, or intrinsic forces are coordinated contractions of the lymphangions with the lymphatic valves (12). Passive pumps, or extrinsic driving forces, consist o f lymph formation, arterial pulsations, changes i n venous pressures, skeletal muscle contractions, and respiration (12).  The lymphatic muscle plays an important role in lymphatic function because it influences the generation and regulation of lymph flow (5). Lymphatic smooth muscle produces  30  phasic and tonic contractions of its vessels to modulate lymphatic flow (47).  Tonic  contractions change the diameter o f the lymphatic vessels, which alters lymph outflow resistance, therefore manipulating lymph flow (5). Phasic contractions, which are similar to those seen in cardiomyocytes, are brief yet intense, and effectively open and close lymphatic valves, providing energy to produce lymph flow (5).  Chemically, acetylcholine inhibition, (3-adrenoreceptor  activation, and humoral agents  inhibit lymphatic contractions by increasing c G M Y and c A M P production (5) while nitric oxide can reduce the action potentials  in the pacemaker  muscle, reducing  contractions (43). The intravenous administration of adrenaline and noradrenaline has been shown to increase the force and frequency o f lymphatic contractions in popliteal, prefemoral  and mesenteric  lymphatic vessels  (27).  Too much administration o f  noradrenaline depresses lymphatic flow, perhaps due to the decrease in stroke volume with the increase in contractile frequency (28).  Filtration forces are responsible for maintaining the equilibrium o f fluid  exchange  between the circulatory and the lymphatic systems. Hydrostatic pressure is based on the pressure-volume relationship, whereas colloid osmotic pressure is based on the changing protein concentration within the interstitial fluid (2).  Hydrostatic and osmotic colloid  pressures are forces that influence the transmural pressure, which can influence the pacemaking activity within the lymphangions, resulting in a change in lymph flow (12). A rise in transmural pressure causes distension of the lymphatic wall, this may increase the number and the force of lymphatic muscular contractions, which in turn enhances flow (12, 47). A rise in lymph flow can provoke the pacemaking potential o f the smooth muscle cells by increasing their spontaneous transient depolarization, while a reduction in lymph flow decreases the activity o f the spontaneous transient depolariztions (47). These depolariztions can occur in the absence o f innervation or endofhelieum, thus provide possible evidence o f a myogenic effect (47). Although contrary to this lymphatic myogenic hypothesis, too much luminal pressure can diminish the lymphatic pumping action and when there is no distension due to a lack of transmural pressure, the  31'  lymphatics still contract (47).  This' suggests that distension of the lymphatic wall is  influential, not mandatory, in altering the pacemaker activity in the lymphatics (12).  Research has demonstrated  that increases in flow cause a decrease in contractile  frequency and amplitude (14). This may be due to an overriding regulatory factor in the lymphatic system, and at high levels o f lymph formation, the passive lymph pump becomes a greater force than the active lymph pump. The flow inhibition response may occur to save metabolic energy by decreasing the contractions and outflow resistance because other forces are providing a strong enough drive to propel the lymph (14).  Respiratory movements, muscle contractions and arterial pulsations are considered secondary mechanisms that influence lymphatic dynamics over the intrinsic contractions of the lymphatics (33). However, there is a building opinion that the muscle pump makes a strong contribution to the increase in lymphatic flow (16, 23).  The lymphatics  transport fluid centrally against a hydrostatic pressure gradient and against a protein concentration gradient (47); hence, mechanisms which aid in altering pressure gradients, can help improve lymph flow. When the muscle is stretched, the cross-sectional area o f the muscle fibers decreases and radially pulls on the adjoining connective tissue, this in turn pulls open the initial lymphatics (24). This opening lowers the pressure within the lymphatic vessel, allowing it to fill with interstitial fluid. When the muscle shortens to its minimal length, the contractile tissues compress the surrounding lymphatic vessels to propel the lymph forward (16). The contraction and elongation o f the muscles drive the fluid along the lymphatic network, demonstrating a key relationship between exercising skeletal muscle and lymphatic flow.  Lymphatic vessels are adjacent to the arterioles in skeletal muscle, potentially making arteriolar pulsations and vasomotion mechanisms for lymph formation (24). Arteriolar constriction and dilation may provoke the movement of interstitial fluid toward the lymphatic capillaries, while the changing diameter may be responsible for the opening and closing o f lymphatics, causing lymph formation and flow (24).  32  The contractions o f the lymphatic system have been compared to the contractions o f the heart over those in the vascular system (5).  muscular  Perhaps, with the  development similarities between cardiomyocytes and lymphatic muscle cells (5), there could  be  similar  beneficial exercise  effects  on lymphangions, as  there are  on  cardiomyocytes (11, 48).  Exercise and lymphatic function: Various studies have used exercise stress to investigate the functional components o f the lymphatic system. In sheep, a rise in cardiac output with short term exercise increased lymph flow due to. the recruitment of previously unperfused microvascular beds (30, 31). Exercise time in these studies averaged approximately 40 minutes, thus lymphatics may not have reached a steady state condition (30, 31). This led to another study by Coates et al., (8) which looked at a longer bout o f exercise and it's effect on lung and hindlimb lymph flow. Twelve sheep were trained on a treadmill for 2-4 weeks prior to onset o f testing: A s Havas showed in humans (15), the sheep had a 5 fold increase in lymph flow at the onset o f exercise, but then with continued exercise, it tapered off to 130% above baseline values. The authors believe the marked increase resulted from the rise in tissue pressure, and the gradual reduction (but still above baseline values) may be contributed to the reduced volume in the compartment drained by the lymph.  With exercise, it was  noted that the combination o f both increased vascular surface area and hydrostatic pressure aided in the steady state increase in lymph flow. The exercise induced a drop in systemic  vascular  resistance  secondary  to  the  recruitment  and  dilation o f  the  microvascular beds in the exercising muscles, therefore increasing microvascular surface area for fluid exchange (8).  In male  humans,  lymphatic clearance  rate increased  while walking  (34), while  performing specified muscular contractions (16) and during prolonged running (15). Based on the concept o f increased arterial pulsations and muscle fiber deformations, Havas et al., (16) looked at the removal rate o f injected albumin from the gastrocnemius and tibialis anterior muscles.  Compared to resting values, greater clearance rates were  observed during isometric flexion, isometric extension and concentric contractions. The  greatest clearance rates were seen in isometric extension and concentric contractions, proposing that the most efficient phase in lymph propulsion occurs when the muscle shortens to its minimal length. This study also looked at the difference between resting clearance rates in trained and untrained runners. The trained runners had greater resting clearance rates due to potential adaptations o f exercise induced increases in vascularity in skeletal muscle and perhaps enhanced adaptations within the lymphatic system (16).  Trained runners, undergoing prolonged running, had similar effects (15) to those found in sheep (8). There was a 5 fold increase in lymphatic clearance rate at the onset o f exercise, and then it dropped to twice the rate o f resting levels (15). This rapid increase was proposed to be associated with an overshoot in capillary filtration due to an increase in blood delivery and a rise in intramuscular osmotic pressure, secondary to muscle contractions. This effect may be due to the inability o f the lymphatic system to match fluid accumulation demand, however, other studies have shown that the lymphatic system responds rapidly to the exercise stress and limits the accumulation of interstitial fluid in the muscles (16). This study demonstrates the capability of the lymphatic system to handle the stress o f prolonged exercise, however the muscle contractions, as well as the arteriole pulsations, remained constant throughout the exercise, nonetheless the estimated lymphatic flow did not.  Lymphatic clearance rates during isometric handgrip exercise have been compared with dynamic arm cranking exercise in healthy females (23). A r m cranking produced higher clearance rates (-28% per min), while handgrip exercise produced comparable results to the resting control clearance rates (-18% per min, -14% per min respectively). The dynamic exercise was thought to increase the lymphatic clearance rates through multiple changes in lymphatic transport mechanisms.  Exercise increased the subjects cardiac  output, inducing vasodilation in the working muscles which augmented muscle blood flow. This rise in muscle blood flow promoted capillary filtration, which increased interstitial pressure, leading to greater lymph formation and lymph flow (23).  34  Central and peripheral changes with chronic exercise include increased capillary density, increased sympathetic tone, and increased muscular adaptations, which are mechanisms linked to improving lymphatic function (46).  Sympathetic nervous system activity  increases with dynamic exercise which may enhance the frequency and force o f smooth muscle contraction in larger contractile lymph vessels promoting lymph propulsion (23, 27). M c H a l e and Adair suggest that the reflex activation o f the sympathetic nervous system could promote lymph movement due to a rise in circulating catecholamines (26), while V a l i c and colleagues have demonstrated that vasodilation plays a greater a role in lymph propulsion than previously thought (45). If upper body trained individuals have greater upper body sympathetic tone, increased capillary beds within the trained muscles and increased metabolic activities (46), the rise in vasodilation and vascular conductance may increase lymph flow in these individuals.  Imaging Techniques Lymphoscintigraphy is a highly reproducible method used to quantify lymphatic function (44). A n injected amount o f a radiopharmaceutical is monitored through a gamma camera, and quantitative and qualitative data can be observed. The radiation dose received is low and is less invasive compared to the past technique o f lymphography which involved the cannulation o f lymphatic vessels (44).  Unlike the old method,  lymphoscintigraphy cannot directly measure lymph flow, because the total volume o f lymph remains uncertain, but one can infer lymph flow by the clearance rate of the radioisotope from the injection site (41). There is no standardized procedure  for  lymphoscintigraphy, and variations include the selected radiotracer, the site of injection (32), whether an exercise stress is used and different acquisition times (43, 44).  Because there is the possibility o f a disturbance in homeostasis with the injection, due to increases in blood flow and swelling (42), there has been questionable results in the first 20 minutes after the injection (16). To encourage the disappearance o f impurities and provoke uptake into the lymphatic capillaries after injection, patients are encouraged to massage the site o f injection (43) or in one study, subjects walked for 10 min, then rested for 15 minutes before imaging began (15).  35  Sites for injection vary from intradermal, intermuscular, subdermal to locations.  subcutaneous  Intradermal injections produce rapid visualization o f the dermal lymphatics,  where subdermal and subcutaneous injections seem to have a slower uptake into the lymphatics (18). The dermis has a higher concentration o f lymphatics, which provides greater surface area for lymphatic uptake (32). One study looked at various radiotracers and their site of injection, and showed that intradermal injections produced the clearest images o f lymphatic vessels under 30 minutes, whereas subcutaneous injections took longer for a quality image o f the lymphatics to be produced (32). In general, both limbs are injected, with two injections per limb (43). Injections vary between the webspaces of the hands, to the dorsum o f the foot, to areas around the styloid process or at the malleolous i n the lower extremity (18) .  Technetium-99m sulphur colloid is the most commonly used radiotracer because it is inexpensive with a high safety profile. Imaging is always performed on a high-resolution collimater (44) and should be recorded with a 20% window centered on the 140-keV photopeak of  9 9 m  T c using a scan speed o f lOcm/min (43).  Size is an important factor when determining uptake and retention in the lymphatics. Molecules smaller than lOnm are preferentially absorbed into the circulatory system, whereas molecules between lOnm and lOOnm are ideal for lymphatic uptake (42). The larger the size, the greater preference to lymphatic uptake, yet the rate of uptake is slower (42).  Breast Cancer Related Lymphedema ( B C R L ) : The etiology and pathophysiology of breast cancer related lymphedema is multi-factorial and requires greater understanding (37).  Breast cancer treatment frequently involves  surgical mastectomy, chemotherapy and radiation in order to increase survival rates following diagnosis. However, these treatments have strong associations with  the  development o f lymphedema of the upper extremity (40). Other risk factors  are  pathological nodal status, obesity and tumor stage (39). There is a lack o f evidence-based  36  medical information on the management  of lymphedema; furthermore, there is no  standardized test to evaluate the functional aspect o f the. lymphatic system in women with B C R L (20).- Understanding the mechanisms that influence lymph flow in the upper extremities may help to prevent or alleviate post-mastectomy lymphedema in breast cancer survivors.  With the combined technique o f lymphoscintigraphy and potential  lymphatic training adaptations  (as previously discussed), the concept o f rerouting  mechanisms and increased efficiency in lymph transport may help women suffering from BCRL.  37  A P P E N D I X 2- R A W D A T A  Table 5. Individual descriptive data  Trained CC AS WW Kl SHu Shi LG JD AW KP Untrained JM CM AL SHa LH MM RL JH MMc SB  Dominant arm  Elbow nodes  1.81 2.77 1.62 1.78 2.72 2.22 2.48 2.55 2.09 1.70  R R R R R R R R R R  N R N B R L B N R N  1.46 1.56 1.28 1.42 1.55 1.46 1.60 1.50 1.79 1.22  . R R R R R R R R L R  R L L B B B B N B L  Age years  Ht cm  Wt kg  BMI kg-m"  V02 ml-kg- min "  V02 L-min  33 29 35 28 26 22 24 26 23 23  156.5 180.8 150.7 151.7 176.0 161.4 155.3 174.4 175.0 167.8  59.8 75.8 46.8 49.7 85.0 65.6 60.8 64.9 62.0 50.6  24.4 23.2 20.6 21.6 27.4 25.2 25.2 21.3 20.2 18.0  30.3 36.5 34.6 35.8 32.0 33.8 40.7 39.3 33.8 33.6  26 25 32 38 25 33 35 22 35 39  165.3 160.9 164.0 159.0 164.8 177.0 173.0 178.2 171.3 167.7  63.6 55.6 50.0 85.3 52.2 61.6 91.6 76.8 79.5 79.0  23.3 21.5 18.6 33.7 19.2 19.7 30.6 24.2 27.1 28.1  22.9 28.1 25.5 16.7 29.8 23.7 17.5 19.6 22.5 15.4  2  1  R= Right A r m , L= Left arm, B = Both arms, N = None.  1  Table 6. Individual Data for Power Output, Heart Rate and R P E Trained CC AS WW Kl SHu Shi LG JD AW KP Untrained JM CM AL SHa LH MM RL JH MMc SB  TimeMI  POM1  HR M1  Time M2  PO M2  HR M2  PO SM  HR SM  HR Rest  RPE M1  RPE M2  10 10 10 10 9.5 10 10 10 10 10  10 10 10 10 10 10 10 10 10 10  11.20 17.20 11.25 11.25 17.20 13.20 14.00 14.00 10.75 10.00  110 170 110 110 170 130 140 140 110 100  189 185 184 177 202 181 186 175 190 171  10.00 16.00 12.00 10.00 15.00 11.00 11.00 14.00 11.00 9.00  100 160 110 100 150 110 110 140 110 100  187 181 184 180 192 173 165 174 192 176  36 45 28 30 51 39 36 39 37 30  123 95 120 93 124 100 91 89 104 96.4  62 44 58 60 52 66 60 52 62 60  8.00 8.25 7.00 7.20 9.00 7.20 8.00 7.50 9.75 7.00  80 80 70 70 90 80 90 80 100 80  174 178 167 167 181 178 188 173 178 150  8.00 8.00 6.00 7.00 8.00 7.00 8.00 8.00 8.00 7.00  80 80 60 70 90 80 80 80 80 90  172 178 161 162 179 182 188 177 184 151  38 33 30 50 31 37 50 45 45 44  130 115 118 143 111 132 119 134 119 111  78 88 77 69 75 67 84 64 78 78  10 10 9 10 10 10 10 • 10 10 10  10 10 10 10 10 10 10 10 10 10  Time = min, P O = wattage, H R = beats-min" . M l = maximal test 1; M 2 = maximal lymphatic stress test; S M = submaximal intermittent protocol at 0.6W-kg~'; R P E = Rating o f perceived exertion at end o f maximal test (0 = no effort to 10 = maximal effort).  o  Table 7; Individual data for axillary uptake  Trained AS CC SHU WW SHI Kl LG JD AW KP Untrained AL JM CM SH MM LH RL MMc SB JH  time  Maximal R-18  L-18  R-64  L-64  R-18  Submax L-18  R-64  L-64  R-18  Rest L-18  R-64  L-64  22 17 25 23 25 25 19 25 15 15  0.48 0.62 0.70 0.37 0.61 0.83 0.68 1.14 1.23 0.34  0.97 0.10 0.23 0.10 0.19 0.18 0.24 1.19 0.99 0.12  1.23 2.13 1.39 1.17 1.41 1.63 1.82 1.81 . 3.10 1.87  1.78 0.86 0.57 0.55 0.83 0.47 1.11 2.16 3.89 1.43  0.62 2.90 0.53 0.14 0.45 0.38 0.59 0.10 0.30 0.20  0.34 0.22 0.93 0.11 0.45 0.05 0.20 0.14 0.93 0.01  1.74 4.52 0.94 1.09 1.67 0.71 1.96 1.01 1.62 2.98  1.44 1.42 2.76 1.72 1.55 0.54 1.66 0.48 4.29 1.3  0.09 0.04 0.12 0.02 0.09 0.02 0.04 0.03 0.02 0.02  0.1 0.04 0.08 0.01 0.04 0.02 0.04 0.01 0.01 0.08  0.20 0.8.9 1.00 0.12 0.82 0.29 0.27 0.02 1.62 0.28  0.35 0.26 0.55 0.21 0.68 0.05 0.16 0.07 4.29 2.00 .  13 14 17 21 13 14 15 14 13 14  0.13 0.11 0.09 0.47 0.03 0.05 0.81 0.31 0.18 0.93  0.02 0.07 0.08 0.15 0.08 0.04 0.5 0.01 0.04 0.08  2.81 2.92 0.81 1.29 0.12 0.63 1.23 0.74 1.82 3.41  1.45 1.59 0.91 0.43 1.01 0.51 1.13 0.34 1.43 1.6  0.09 0.13 0.58 0.32 0.03 0.03 0.48 0.29 0.35 0.28  0.28 0.12 0.10 0.03 0.03 0.03 0.11 0.02 0.07 0.14  4.45 2.40 3.01 1.39 0.17 0.62 1.52 0.58 1.66 2.00  3.4 2.1 1.4 0.5 1.0 0.6 0.7 0.7 2.3 1.8  0.03 0.02 0.03 0.00 0.02 0.01 0.03 0.29 0.02 0.27  0.03 0.04 0.10 0.00 0.02 0.03 0.03 0.02 0.02 0.02  2.21 0.17 0.13 0.16 0.05 0.01 1.37 0.58 0.11 1.72  0.52 0.16 1.27 0.13 0.42 0.02 0.69 0.65 0.08 0.97  A x , in % relative to initial amount injected at time zero, acquired at 18 (SD 5) and 64 minutes during maximal, submaximal and resting lymphatic stress tests for the right (R), and left (L) sides.  Table 8. Individual clearance rate equations Maximal C R Intermittent Submaximal C R Trained KP y = -3.326 L n ( x ) + 100.78 R = .90 -0.243x + 100.43 R = .98 SHi y = R = .96 -0.186x+ 100.55 R = .98 -2.328 L n ( x ) + 100.01 y = CC -2.224 Ln(x) + 99.97 R = .96 -0.210x +97.05 R = .86 SHu y = -2.727 L n ( x ) + 100.47 R = .92 -0.274x + 98.83 R = .96 y = LG -2.104 L n ( x ) + 100.17 R = .99 -0.225x + 99.52 R = .98 JD -2.885 L n ( x ) + 100.41 R = .97 -0.104x +99.85 R = .97 y = WW y = R = .91 -1.859 L n ( x ) + 100.00 -0.161x+ 101.03. R = .94 AW y = -4.394 L n ( x ) + 100.48 R = .98 -0.281x4-98.53 R = .95 KI -3.451 L n (x) +99.73 R = .98 -0.203x + 96.50 R = .77 y = y = AS -3.332 L n ( x ) +99.83 R = .98 -0.195x+ 101.00 R = .94 Untrained RL -3.689 L n (x) + 99.63 R = .97 R = .87 -0.268x +98.10 y = JM -2.757 L n (x) + 100.85 R = .93 -0.250x+ 100.49 R = .98 y= CM y = -1.687 L n (x) + 100.22 R = .82 -0.267x + 99.57 R = .96 AL y = -2.719 L n (x)+ 100.12 R = .95 -0.283x +98.72 R = .94 SHa y = -2.488 L n (x) + 99.64 R = .91 -0.229x +97.21 R = .84 MM y = -2.330 L n ( x ) + 100.54 R = .96 -0.167x + 99.11 R = .93 MMc -2.943 L n (x) + 100.41 R = .88 -0.194x + 97.03 R = .84 y = LH -1.450 L n ( x ) +99.64 R = .88 R = .98 -0.146x + 100.31 y = SB -3.443 L n (x) + 100.94 R = .92 -0.315x +95.79 R = .86 y = JH y = -3.490 L n (x) + 100.48 R = 97 -0.268x + 99.44 R = .98 Equations and R values for maximal, intermittent and resting clearance rates, y = the amount 2  to  2  2  2  2  2  2  2  2  2  2  2  2  2  2  2  2  2  2  2  2  2  2  2  2  2  2  2  2  2  2  2  2  2  2  2  2  2  2  2  2  Resting C R -0.095x +98.799 -0.082x+100.92 -0.020x+ 100.51 -0.105X+ 100.4 -0.020x + 99.78 -0.064x+ 100.48 -0.043x+100.48 -0.040x+ 101.42 -0.060x + 99.83 -0.037x+ 100.27  R R R R R R R R R R  2  2  2  2  2  2  2  2  2  2  = .77 = . 86 = .36 = .92 = .20 = .87 = .63 = .30 = .75 = .82  -0.183x+ 101.49 R = .85 -0.010x +99.98 R = .23 -0.042x + 99.55 R = .55 -0.125x+ 100.74 R = .96 -0.046x+ 101.25 R = .46 R = .01 -0.001x +98.79 -0.036x +98.86 R = .48 0.041x +99.74 R = .39 -0.023x +99.307 R = .38 -0.150x +98.855 R = .87 cleared (%) for x time (min). 2  2  2  2  2  2  2  2  2  2  Figure 7. Two scintigraphs at initial injection. These scintigraphs visually demonstrate what occurs i f the T c is injected into the circulatory system, preventing any further testing. 9 9 m  43  a  102  Trained  100  Untrained y = -2.7Ln(x) + 100.25  y = -2.86Ln(x) + 100.19  98  1  Irn o  96 94 92 90 88 86  40  20 T  i  m  60 e  80  (min)  101  Trained  y = -0.4748x+ 100.47  100  Untrained  y = -0.5833x+ 100.58  99 "D CD CD  O  98 97 96 95 94 93 92 10  15  20  Time (min)  Trained  v = -0.2 l x + 99.329  Untrained.  20  40 Time (min)  60  -0.24x +98.577  Fig 8. Clearance rates for trained and untrained females. Data was averaged and represents clearance rates for trained and untrained females during maximal exercise over 60 minutes in (a), while (b) represents maximal clearance rates for trained and untrained females excluding the recovery period post maximal exercise. The clearance rate during submaximal exercise is represented in (c) over 60 minutes.  44  Table 9. Amount cleared immediately post-maximal exercise. AC Untrained Time AC Trained Time  JM 4.47 11 CM 11 4.41 AL 10 6.05 SHu SH 10 7.90 WW MM 10 3.68 Shi LH 12 4.62 Kl JH 16 8.92 LG RL 11 10.79 JD SB 16 7.26 AW MMc 8.40 KP 17 Amount Cleared (AC), as a % relative to the initial injection, of Tc from the web spaces of the hand. Time (min) represents the time of the hand acquisition immediately post-maximal exercise. AS  cc  19 13 20 19 13 15 . 16 19 16 19  10.85 5.41 6.72 5.47 6.88 10.2 5.13 7.02 10.46 7.36  A P P E N D I X 3 - C R E B ' s C E R T I F I C A T E OF A P P R O V A L  


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