Investigation of lymph node transplantation as therapy for breast cancer related lymphedema

LEABHARLANN CHOLAISTE NA TRIONOIDE, BAILE ATHA CLIATH TRINITY COLLEGE LIBRARY DUBLIN OUscoil Atha Cliath The University o f Dublin. Terms and Conditions of Use of Digitised Theses from Trinity College Library Dublin. Copyright statement. All material supplied by Trinity College Library is protected by copyright (under the Copyright and Related Rights Act, 2000 as amended) and other relevant Intellectual Property Rights. By accessing and using a Digitised Thesis from Trinity College Library you acknowledge that all Intellectual Property Rights in any Works supplied are the sole and exclusive property of the copyright and/or other I PR holder. Specific copyright holders may not be explicitly identified. Use of materials from other sources within a thesis should not be construed as a claim over them.. A non-exclusive, non-transferable licence is hereby granted to those using or reproducing, in whole or in part, the material for valid purposes, providing the copyright owners are acknowledged using the normal conventions. Where specific permission to use material is required, this is identified and such permission must be sought from the copyright holder or agency cited.. Liability statement. By using a Digitised Thesis, I accept that Trinity College Dublin bears no legal responsibility for the accuracy, legality or comprehensiveness of materials contained within the thesis, and that Trinity College Dublin accepts no liability for indirect, consequential, or incidental, damages or losses arising from use of the thesis for whatever reason. Information located in a thesis may be subject to specific use constraints, details of which may not be explicitly described. It is the responsibility of potential and actual users to be aware of such constraints and to abide by them. By making use of material from a digitised thesis, you accept these copyright and disclaimer provisions. Where it is brought to the attention of Trinity College Library that there may be a breach of copyright or other restraint, it is the policy to withdraw or take down access to a thesis while the issue is being resolved.. Access Agreem ent. By using a Digitised Thesis from Trinity College Library you are bound by the following Terms & Conditions. Please read them carefully.. I have read and I understand the following statement: All material supplied via a Digitised Thesis from Trinity College Library is protected by copyright and other intellectual property rights, and duplication or sale of all or part of any of a thesis is not permitted, except that material may be duplicated by you for your research use or for educational purposes in electronic or print form providing the copyright owners are acknowledged using the normal conventions. You must obtain permission for any other use. Electronic or print copies may not be offered, whether for sale or otherwise to anyone. This copy has been supplied on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgement.. Investigation of Lymph N ode T ransplantation as Therapy for. Breast C ancer Related Lymphedema. by. Dalia T obbia. A thesis sub m itted in conform ity w ith th e req u irem e n ts to r th e degree o f D octor in M edicine. G ra d u a te D e p a rtm en t o f th e Faculty o f H ealth Sciences School o f M edicine. U niversity o f D u b lin T rin ity College. © C opyright by Dalia T o b b ia 2009. Investigation of Lymph N od e Transplantation as Therapy for Breast Cancer related Lymphedema. A thesis su b m itte d in conform ity w ith th e req u irem en ts for th e degree o f D o cto r in M edicine. G ra d u a te D e p a rtm e n t o f the Faculty o f H e a lth Sciences School o f M edicine. U niversity o f D u b lin T rin ity College. D a l j ; ^ T o b b i a - - - - -. ' t r in it y C O L L E G ^. 2 7 JUL 2 0 11 —1. . l i b r a r y DUBLIN ^. H u m a n s have h u n d re d s o f lym ph nodes, collections o f w hich are fo u n d in the. u n d e ra rm s, groin, neck, chest, a n d abdom en. T hey have long been considered 'neutral'. elem ents in term s o f lym ph tra n sp o rt. A n a ssu m p tio n has always been m ade th at the. im p e d im e n t to lym ph tra n s p o rt in post-surgical lym phedem a is due to lym phatic vessel. injury. H ow ever, these vessels are dam aged routinely in surgical procedures, despite this, post-. surgical edem a usually resolves over tim e as th e lym phatics regenerate and c h ro n ic edem a. rarely if ever occurs u n d e r these circum stances. In co n trast, it is clear th a t th e rem oval o f a. lym ph n o d e o r nodes appears to be a prerequisite for lym phedem a dev elo p m en t especially if. this is c o m b in e d w ith radiotherapy. C o nsequently, th e re is reason to believe th a t there are. o th e r factors th a t require co n sid e ratio n . In the first instance th e aim of th e studies reported. here was to develop an anim al m odel o f post-surgical lym phedem a th a t w ould perm it. q u a n tita tio n o f lym phatic tra n s p o rt fu n ctio n after th e rem oval o f a single popliteal lymph. no d e in sheep a n d correlate this w ith edem a form ation. Secondly, we used this m odel to test. w h e th e r th e tra n s p la n ta tio n o f an autologous lyTnph n o d e in to a nodal excision site w ould. restore lym phatic tra n sp o rt fu n c tio n a n d reduce th e m ag n itu d e o f post-surgical lym phedem a.. As a m easure o f lym ph tra n sp o rt, ’"^I-Humaii S erum A lb u m in was injected in to pre-nodal. vessels at 8, 12 or 16 weeks after surgical in te rv en tio n a n d th e plasm a levels o f th e p ro te in . tracer were used to calculate th e tra n s p o rt rate o f th e tracer to blood. (% injected R adioactivity/hr). E dem a was q u a n tifie d from th e circum ferential m easu rem en t. o f th e h in d limbs. C o m p a re d w ith c o n tro l lim bs (17.2 + 0.6, n=7), lym phatic fu n c tio n was. depressed at 8 weeks post n odectom y (10.6 ± 1.5, n=7). A t 12 (14.4 ± 1.0, n=7) and 16 weeks. (13.9 ± 1.0, n=6). Lym phatic fu n c tio n at 8 a n d 12 weeks in limbs subjected to sham surgical. procedures was (16.6 ± 0.7, n=6 a n d 16.1 ± 0.7, n=6) respectively. T h e tra n s p la n ta tio n of. avascular lym ph nodes at sim ilar tim es reduced lym phatic fu n ctio n significantly (12.3 ± 0.5,. n=6 a n d 12.6 ± 0.8, n=6). In c ontrast, w4ien vascularized tran sp lan ts were a ttem p ted ,. lym phatic fu n ctio n was sim ilar to th e c o n tro l groups and significantly greater th a n th a t o f the. avascular group (15.8 ± 0.9, n=9 and 15.7 ± 1.0, n=10). Lymph tra n s p o rt correlated. significantly w ith th e health o f th e tra n sp la n ted nodes (scaled w ith histological analysis).. ■Wlien lym ph nodes were rem oved, all affected lim bs becam e edem atous. T h e vascularized. n o d e tran sp lan ts were associated w ith th e greatest im p ro v em en t in edem a w ith the. m agnitude o f edem a in such lim bs exhibiting significantly lower levels o f edem a th a n non-. treated lim bs. T hese tech n iq u es m ay be helpful in u n d e rsta n d in g th e pathophysiology. associated w ith cancer-related post surgical lym phedem a and may facilitate th e dev elo p m en t. o f new strategies to tre a t o r p rev en t this c o n d itio n .. It is a m iracle th a t curiosity survives form al ed u catio n. Al be r t Ei nstein ( 1 8 7 9 - 1 9 5 5 ). i\^. D e d i c a t i o n. I dedicate this thesis to my d arh n g h u sb a n d , Dr. T h o rste n Sattler, th a n k you for always being. there, constantly encouraging m e a n d giving m e th e freedom to pursue my goals. N o t to. m e n tio n having to e n d u re so m any unsavory d in n ers w hile I was in C a n a d a over the past two. years.. T o my father Dr. Iqdam T obbia for encouraging m e to do my best a n d to a n d my m o th e r. Teresa T obbia w ho has always believed in me.. A c k n o w l e d g m e n t s. I am extrem ely grateful for having th e o p p o rtu n ity to do my research in T o ro n to , a n d all the. effort Dr. Sem ple c o m m itted to o b tain a g ran t for me. For believing in my abilities and. encouraging m e th ro u g h o u t my fellowship, I am grateful.. It has been a great privilege for m e to spend this tim e in professor J o h n s to n ’s lab. I could n o t. im agine a m ore agreeable w orking e n v iro n m e n t or a m ore stim ulating m en to r. H e w ould. com e to th e lab several tim es p e r day to oversee th e ru n n in g experim ents a n d share in th e. successes and fru stratio n s o f everyday lab w ork. His easy-going n a tu re a n d flair for storytelling. m ade even th e m ost d ish e a rte n in g results seem trivial.. Special th an k s to D ia n n a A rm stro n g w ho is u n d o u b ted ly th e q u e e n bee presiding over us,. m aking sure th e Jo h n s to n lab ru n s sm oothly. N o t to m en tio n h e r to u c h o f magic.. I was also truly lucky to have G u rjit N agra, Lena Koh and Amy baker as my lab colleges, w ho. helped to create a m ost friendly e n v iro n m en t.. Sincere thanks to S arah M oore for all th e sheep lifting and late nights in th e lab following. Microsurgery.. Special thanks to A d am Sem ple, w ho d id two consecutive stu d e n t su m m er a tta ch m e n ts at. th e lab and sp en t so m any s u n n y days assisting m e w ith my experim ents.. 1 w ould like to express my g ratitu d e to Professor Reynolds for facilitating th e subm ission o f. this thesis.. Last b u t n o t least, I w ould like to extend special thanks to my sister H ala T o b b ia for all h e r. s u p p o rt d u rin g my stay in C an ad a.. D e c l a r a t i o n. A p art fro m th e exceptions n o ted below, all surgical procedures a n d ex p e rim e n tatio n . c o n ta in ed w ith in this thesis were perform ed by myself. T h e derivation o f all m athem atical. e q u a tio n s were th e w ork o f D r. M ichael Flessner, from th e U niversity o f R ochester.. C o m p u ta tio n a l analysis o f th e d a ta was perform ed by M. Katie from th e D e p a rtm e n t o f. R esearch Design a n d Biostatistics, at S u n nybrook H ealth Sciences C en tre.. Sarah M oore perform ed th e C D 3 A ntibody staining.. T echnical assistance w ith experim ental setup and anim al care d u rin g these studies was. provided by D ian n a A rm stro n g a n d Sarah M oore.. T his thesis has n o t been su b m itted as an exercise for a degree at this or any o th e r University.. I agree th a t th e library may lend o r copy this thesis u p o n request.. T a b l e o f C o n t e n t s. CHAPTER 1: GENERAL INTRO DUCTIO N...................................................................... 1. CHAPTER 2: LYMPHEDEMA DEVELOPMENT AND LYMPHATIC FUNCTION. FOLLOWING LYMPH NODE EXCISION IN SHEEP......................................................23. CHAPTER 3: EXPERIMENTAL ASSESSMENT OF AUTOLOGUS LYMPH NODE. TRANSPLANTATION AS TREATMENT OF POST-SURGICAL LYMPHEDEMA...45. CHAPTER 4: DISCUSSION................................................................................................. 64. REFERENCE LIST 79. L i s t o f F i g u r e s. Figure 1-1. Figure 1-2. Sheep popilteal lymph node histology stained with H & E 8. Figure 24. Figure 2-2. Figure 2-3. Figure 2-4. Figure 1-5. Figure 2-6. Figure 34. Figure 3-2. Figure 3-3. Figure 3-4. Figure 3-5. Figure 3-6. Lymphatic vessels highlighted with yellow Microfil® illustrating the extensive branching o f the term inal afferents as they pierce the substance of the lymph n o d e____________________________________________________ 9. Figure 1-3 Fluoroscopy images of the popliteal lymphatic system in sheep___________ 19. Popliteal node highlighted with Evans blue dye intra-operatively and partially. dissected free from its surrounding fat pad____________________________29. Schematic illustrating essential features of the experimental model to. quantify lymphatic fu n c tio n ___________________________________ 36. Lymphedema production after the removal o f the popliteal lymph node 38. Appearance of radioactive album in in plasma over time (quantification. ot lymphatic fu n c tio n )____________________________________________ 40. Averaged lymphatic mass transport rates (quantification of. lymphatic fu n c tio n )______________________________________________ 41. Morphological evidence that some regeneration of lymphatic vessels has. occurred follow'ing popliteal lymph node excision_____________________ 43. Popliteal node tra n s p la n t__________________________________________ 52. Impact of node transplantation on lymphatic fu n c tio n ________________ 57. Relationship between lymphatic function and lymph node health. after tra n sp la n ta tio n _____________________________________________ 59. D evelopment and progress o f edema in the animal groups______________ 63. Relationship between edema and lymphatic f u n c tio n _________________ 65. Fluoroscopic images of the popliteal fossa re g io n ______________________ 67. ix. '-'I-HSA. (% /h r). LV. LN. Vp. Cp(0). Cp(t.). CPM. PBS. H & E. Lis t o f A b b r e v i a t i o n s. lodinated H um an Serum A lbum in. Blood In. T ransport Rate o f the Injected Radioactive Tracer to Blood. Lymphatic Vessel. Lymph Node. A lbum in Elim ination Rate. Plasma Volume. The C oncentration o f Tracer at Time 0. The C oncentration of Tracer at 4 Hours. C ounts per M inute. Phosphate Buffered Saline. Hematoxylin and Eosin Staining. X. CHAPTER 1:. GENERAL INTRODUCTION. 1. CHAPTER 1: GENERAL INTRODUCTION...................................................................... 1. 1.1 Overview___________________________________________________________ 3. 1 .2 Anatomic Design and Special Consideration of the Lymphatic System________ 4. 1.2.1 The Interstitium___________________________________________________ 4. 1.2.2 Formation of Lym ph______________________________________________ 4. 1.2.3 Structure and Function of the Initial Lymphatic Pump___________________ 5. 1.2.4 Function of the Lymphatic Vessel Pump Beyond the Absorbing Lymphatics _6. 1.2.5 Anatomy and Structure of the Lymph N o d e ___________________________ 6. 1.2.6 Flow of Lymph Through Lymph Nodes_______________________________ 9. 1.2.7 Evidence of Lymph Node Concentrating-Diluting Mechanism____________10. 1.2.8 Impact of Lymph Nodes on Lymphatic Pumping Activity________________ 11. 1.2.9 Interaction Between Lymph Nodes and Perinodal Fat Tissue____________ 12. 1.3 Post Surgical Lymphedema Associated with Lymph Node Excision___________ 12. 1.4 Lymphedema, a Problematic Complication of Breast Cancer Therapy________ 14. 1.5 The Psychologic Morbidity of breast Cancer Related Arm Swelling___________15. 1.6 The Concept of Sentinel Lymph Node Sampling_________________________ 16. 1.7 The Role of Lymphangiogenesis in the restoration of Lymphatic Continuity 18. 1.8 Implications of Vascularized Lymph Node Transplantation_________________ 20. 1.9 Wliy Address Issues Related to the Role of Lymph Nodes W ithin the Lymphatic. System and Advance the Field?________________________________________ 20. 1.10 General Hypothesis_________________________________________________22. 1.11 Objectives of the Experimental Studies Outlined in this Thesis____________ 22. 1.1 Overview. T h e lyniphatic circulation is continually responsible for tra n s p o rta tio n o f. extravasated fluid a n d p ro te in from tissue spaces back to th e blood circulation. This. fu n ctio n is frequently d isru p ted w hen lym ph nodes are rem oved as p a rt o f cancer diagnosis. a n d tre a tm e n t. U n fo rtu n ately , th e d evelopm ent o f post-surgical lym phedem a in these. patien ts is n o t u n c o m m o n .. T h e p o p u lar c o n cep tio n is th a t lym phatics are dam aged d u rin g th e excision o f. lym ph nodes and th a t this causes im p e d im e n t to lym ph flow. In this regard, th ere is som e. scientific interest in developing m olecular therapies to en h an ce th e process o f. lym phangiogenesis. How'ever, th ere is reason to believe th a t re-establishm ent o f 'norm al'. fluid dynam ics after tissue injury involves m ore th a n simply th e regeneration of lym phatic. vessels. Is it possible therefore, th a t th e lym ph nodes have im p o rta n t functions in. m ain ta in in g fluid balance in a d d itio n to th e ir m ore recognized im m unological duties?. In light of this, th e goal o f my experim ental studies over th e past two years was to. provide a b e tte r u n d e rsta n d in g o f th e pathophysiology o f th e lym phatic tra n s p o rt system.. S heep were prim arily used for these in vivo experim ental p rep aratio n s, focusing nam ely o n . lym ph clearance in th e n o rm a l situ a tio n and following various surgical interventions,. in cluding th e developm ent a n d progression o f lym phedem a.. 3. Anatomical Design and Special Consideration of the Lymphatic System. 1.2.1 TTie Interstitium. A pproxim ately 9 9 .9 % o f in terstitial fluid, also k n ow n as tissue fluid exists in a gel-. like state (Adair a n d G u y to n , 1985) (Adair, 1985a). T his m eans th a t in n o n-edem atous. tissues less th a n 1% o f th e tissue fluid exists as free fluid available for exchange. T his gel. in te rstitiu m is m ade up o f several types o f proteoglycans a n d collagen. Proteoglycan. filam ents form weak crosslinks w ith each o th er, w ith collagen fibers and p ro te in m olecules. giving rise to th e gel-like consistency o f th e in te rstitiu m . T his m eans th a t tissue fluid does. n o t flow freely in n o rm a l tissues. It is d u e to this feature o f th e in terstitiu m th a t fluid does. n o t collect readily in d e p e n d e n t tissues.. Formation o f Lymt>h. Lym ph is an ultrafiltrate o f blood c o n ta in in g all o f th e co n stitu en ts o f plasm a. Its. fo rm a tio n takes place at th e capillary level according to Starling forces (Starling, 1894;. Starling, 1896) w hich state th a t fluid tran sfer is d e te rm in e d by th e difference betw een. hydrostatic a n d osm otic pressures. T h e transcapillary hydrostatic pressure g rad ie n t causes. fluid m ovem ent from th e capillaries to th e tissue spaces. T his m ovem ent is o pposed by th e. colloid osm otic pressure g rad ie n t w hich ten d s to tra n se r fluid back into th e blo o d capillary.. O n th e arteriolar e n d o f th e blood capillary, th e hydrostatic presure g radient is greater th a n . th e osm otic pressure g rad ie n t leading to a n e t m ovem ent o f fluid in to th e interstitial. spaces. T his is reversed at th e venular side o f th e capillary leading to m o v em en t o f th e . filtered fluid back in to th e blood capillary. H ow ever, in m ost tissues o f th e body th e . hydrostatic pressure g rad ie n t is slightly greater th a n th e colloid osm otic pressure g radient,. 4. causing th e n e t driving pressure directed from th e capillary lu m e n tow ard th e s u rro u n d in g . tissues to be slightly positive a n d thus causing a c o n tin u o u s leakage o f plasm a ultrafiltrate. o u t o f th e capillaries. A small a m o u n t o f p ro te in leaks o u t in to th e tissue space as p a rt o f. this process. T his form s w hat is know n as tissue fluid. T h e re tu rn o f this extravasated fluid. a n d p ro te in back in to th e th e blood circu latio n is an essential physiologic fu n c tio n o f th e. lym phatic system.. S tru ctu re a n d F u n c tio n o f the I n itia l L y m p h a tic P um t). Initial lym phatics possess specialized features, allowing th e active rem oval o f tissue. fluid from the in terstitial space. A djacent endo th elial cells form finger-like ram ifications. created by a series o f valve-like slits in th e vessel wall. Specialized filam ents a n c h o r these. endo th elial cells to th e su rro u n d in g tissue. D ue to this relationship, w hen th e tissues. expand as a result o f fluid accum ulation w ithin th e interstitial spaces, th e lym phatic. capillaries are pulled o p e n a n d autom atically fill w ith tissue fluid (Adair, 1985b). T h e. pressure w ith in these capillaries becom es negative w hen they are o pened, th ere fo re it is. likely th a t the lym phatic p u m p is able to suck fluid directly from th e tissue spaces and. create th e negative tissue interstitial fluid pressures th a t exist in n o n edem atous tissues.. T h e valve flaps are only capable o f o p e n in g inwards, directing th e flow o f lym ph in. a forward d irectio n and preventing backflow. M oreover, en d o th e lia l cells are n o t tightly. b o u n d together, p e rm ittin g large particles such as p ro tein s to pass betw een them .. 5. F unction o f the Lymt>hatic Vessel Pum p Beyond the A bsorbing Lymt>hatics. T h e lym phatic p u m p consists o f m ultiple segments separated by one-way valves. referred to as lym phangions, these represent th e basic su b u n it involved in lym ph. p ro pulsion.. Forces th a t com press th e lym ph vessel causing th e p u m ping o f lym ph can e ith e r result from . n o rm a l m ovem ents o f th e body o r from intrinsic contractions o f th e larger collecting ducts. (b o th pre- a n d post-nodal). T hese vessels are equipped w ith sm o o th m uscle cells in their. walls arranged in a circular m a n n e r and vessel contractions occur in c o o rd in a tio n w ith o n e­. way valves th a t p ropel lym ph flow centrally (Benoit et al., 1989; C row e et al., 1997; Elias. a n d Jo h n sto n , 1988; Elias et al., 1990; H argens and Zweifach, 1977; Li et al., 1998;. M cH ale a n d R oddie, 1976).. A n a to m y a n d Structure o f the Lymt>h N ode. Lym ph nodes are small, kidney shaped organs found along th e course o f lym phatic. vessels so th a t lym ph d rain in g back to th e bloodstream passes th ro u g h th em . T hey ten d to. cluster in groups, particularly at locations w here lymph vessels m erge to form larger trunks,. such as in th e axilla. T h e parenchym a o f th e node consists o f an extensive netw ork o f fine. reticu lin fibers w hich provides a loose fram ew ork for th e p o p u latio n s o f lym phocytes th a t. in h a b it th e node. L um ena o f th e afferent and efferent vessels are c o n tin u o u s w ith a. labyrinth o f lym phatic sinuses th ro u g h w hich lymph flows.. T h e o u te r cortex consists o f densly packed lym phoid follicles w here B lym phocytes. are localized, w hereas th e paracortex m ainly holds p opulations o f T lymphocytes. T h e . m edulla co n tain s prim arily B lym phocytes and th eir derivatives. T h e e n tire n o d e is. encapsulated by a dense layer o f connective tissue providing th e m ain stru ctu ral support.. 6. Trabeculae extend from this capsule for a variable distance into the substance o f the node. (Young, 2001).. Blood supply to the node is derived from arteries w hich en ter at the hilum and. b ran ch into the medulla. T he n u m b er o f arteries directly supplying the lymph node of. mammals can range from one to three vessels (H erm an et al., 1969; H erm an et al., 1972;. Sainte-Marie, 1968). However, in some instances there can be as many as ten to twelve. arteries th a t penetrate the node, originating from a delicate arterial anastom otic network. em bedded in the su rro u n d in g fat (H eath an d B randon, 1983).. 7. Figure 1-1 Sheep P opilteal Lym ph N o d e H istology Stained w ith H & E. C - C apsule. CX - C o rtex. P - Paracortical zone. S - Subcapsular sinus. F - Follicle. T - T rabeculae. M - M edulla. M C - M edullary cord. 8. Floiv o f Lymt>h Throush Lym ph Nodes. T h e pre-nodal (afferent) lym ph vessel tra n s p o rtin g p ro te in a n d fluid a b so rb e d from . th e in te rstitial space em p ties in to th e su b c ap su la r sinus o f lym ph nodes. T hese lym phatic. afferents b ra n c h o u t extensively u p o n reach in g th e surface o f th e n o d e giving several. te rm in a l afferents th a t in filtrate in to th e n o d e at m u ltip le locatio n s, p resu m ab ly to help . increase th e surface area over w hich th e lym ph percolates th ro u g h th e n o d e . Lym ph w ith in . th e n o d e m oves first th ro u g h th e tra b e c u la r sinuses a n d th e n in to th e lab y rin th in e . meclullary sinuses. Finally, it is cirained fro m these sinuses by e ffe re n t ly m p h atic vessels. locateci at th e h ilu m o f th e nodes.. Terminal Lym phatics. i. Figure 1-2 Lym phatic Vessels H igh ligh ted w ith Y ellow M icrofil® Illustrating the. Extensive B ranching of the T erm in al A fferents as they P ierce the. Substance of the Lym ph N o d e. 9. E viden ce o f Lymt>h N o d e C o n c e n tr a tin s -D ilu tin s M e c h a n ism. Lymph is alm ost entirely form ed at the lymphatic capillary level, however, protein. c o n cen tratio n o f lym ph can be altered as it flows th ro u g h the lymphatic system. A lthough. lym ph vessels have th e poten tial to alter lymph com position (G uyton et al.,1979; Guyton.,. 1979), m odification w ould appear to take place mainly w ithin the nodes (Renkin, 1979). In fact, one o f the neglected physiological properties o f lymph nodes is th eir ability. to v ent water into th e local vasculature facilitating its removal from the lym phatic system,. due to an extensive netw ork o f blood capillaries th a t are in close contact w ith lymph in. tran sit th ro u g h th e sinuses (Belisle and Sainte-Marie, 1990; Salvador et al., 1992). Since the. colloid osm otic pressure o f prenodal lymph is low in m ost tissues o f the body (i.e. the. pro tein c o n ten t o f lym ph is considerably lower th a n th a t o f blood (Yoffey, 1970)), protein-. free fluid is absorbed from the lymph into the blood capillaries in the nodes to establish an. ecjuilibrium o f Starling forces across the lymph-blood barrier. Remarkably, the protein. co n cen tratio n of lym ph can be increased as m uch as 300-400% after one passage th rough. the node (Adair, 1985c; A dair and G uyton, 1983; A dair and G uyton, 1985; A dair et al.,. 1982). In this sense, the lym ph node acts as a fluid exchange cham ber.. In the studies o f Jila et al., the m easured intra-lymphatic pressures in upstream . popliteal pre-nodal vessels using a servo-null m icropipette system after obstructing flow. dow nstream o f the n o d e, failed to produce the expected rapid rise in pressure w hen lymph. outflow from the netw ork was blocked dow nstream o f the node (post-nodal). This suggests. th a t fluid was lost from the system probably via the m echanism ou tlin ed above (Jila et al.,. 2007).. 10. A lth o u g h this d am p en in g effect caused by th e c o n tin u o u s loss o f w ater in th e nodes. will have finite lim its th a t may be overcom e at hig h er lym ph flow rates, it still represents an. im p o rta n t 'safety m echanism ' to help m ain ta in pressure w ith in th e lym phatic system at a. reasonable level w hen lym ph flow rates are very high o r w hen dow nstream flow is. o bstructed. T his fu n c tio n w ould presum ably be lost w h e n nodes are rem oved in cancer. patients.. I m p a c t o f L y m b h N o d e s on L y m b h a t i c Pumt>ine A c t i v i t y. Lym ph nodes play a vital role w ith in th e lym phatic circulatory system; consequently. th e ir loss may have a m ore significant im pact o n tissue fluid balance th a n is com m only. assum ed.. T h e pressures in pre-nodal vessels can be q u ite high w ith m easurem ents a ro u n d . 22m m H g achieved in rat m esenteric prep aratio n s (Zweifach a n d Prather, 1975) and. 30m m H g in h u m a n popliteal vessels (Olszewski, 1985), facilitating lym ph tra n s p o rt at high. pressures (E isenhoffer et al., 1994). In contrast, post-nodal pressures are considerably lower. m aking it evident th a t th e build-up o f pressure in th e pre-nodal ducts is dissipated in th e. lym ph nodes, w hich appears to anatom ically separate th e lym phatic system in to h igher and. lower pressure areas.. W ith o u t this dissipation o f pressure w ith in th e lym ph nodes, intra-lym phatic. pressures in th e dow nstream vessels are presum ably higher forcing th e m to operate u n d e r. s u b o p tim a l c o n d itio n s. T his w ould m ake th e lym phatic system less effective at tra n sp o rtin g . in terstitial fluid a n d in the cancer p a tie n t, could com prom ise lym ph tra n s p o rt and. c o n trib u te to edem a form ation.. 11. In teraction B etw een L ym ph N odes a n d P erinodal Fat Tissue. Lym ph n o d e collections a ro u n d th e body are em bedded in a p ad o f fatty tissue.. Even in very lean m am m als it is th e last resource o f fat to be depleted (P o n d , 1994). T h e. u n iq u e paracrine interplay betw een lym phoid cells a n d th e adipose tissue th a t envelopes. lym ph nodes seems to play a role in th e local im m u n e response, by regulating th e. m etabolism o f lym phoid cells in th e nodes, m aking it d istinct from o th e r fatty tissue. a ro u n d th e body (Pond a n d M attacks, 1995). T h u s th e local tra n sie n t im m u n e response is. possibly to a significant degree d e p e n d a n t u p o n n u trie n ts supplied by th e ad jacent adipose. tissue (Pond, 2003). In this sense, perhaps th ere is an added ben efit to harvesting th e. p erin o d al fat along w ith th e lym ph n o d e in tran sp lan ts.. Post Surgical Lymphedema Associated with Lymph N ode Excision. In a b ro ad sense lym phedem a is th e d e v elo p m en t o f swelling d u e to a functional. overload o f th e lym phatic system exceeding its drainage capacity. Even w ith m o d ern . advances in research th e precise pathogenesis rem ains elusive.. Lym phedem a associated w ith cancer-related lym ph n o d e dissection has been term ed. ‘th e secret ep idem ic’ (Arm er, 2001). T h e accu m u latio n o f pro tein a n d fluid in th e. in te rstitiu m provides an excellent m ed iu m for th e grow th o f organism s, leading to. recurring b o u ts o f cellulitis and lym phangitis and if left u n tre a te d it can lead to im paired. lim b fu n ctio n , psychosocial problem s and in extrem e cases, m alignant co m plications and. life-threatening infections. W h ile a lym ph tra n s p o rt deficit is recognized to be th e general. cause o f this disorder, we are n o closer to u n d e rsta n d in g why lym phedem a develops in. 12. som e p atients and n o t in oth ers and why th e edem a can be unevenly d istrib u ted in th e arm . w ith som e regions spared com pletely (S tanton et al., 2006).. W lia t is also surprising is th a t this diso rd er does n o t necessarily h a p p e n acutely, b u t. can take place m any years following th e initial nodectom y, a n d may be triggered by a m in o r. event such as a laceration o r insect bite in th e affected lim b. T h e rep o rted incidences o f. lym phedem a following m astectom y and axillary n o d e rem oval are fairly significant ranging. betw een 20-40% (A rm er, 2001). T re a tm e n t o p tio n s for these p atients have b e e n m et w ith. som e success b u t rem ain controversial a n d are tar fro m ideal specially w ith e n tre n ch e d ,. c h ro n ic edem a (Badger et al., 2004). M ost individuals are offered som e form o f non-. surgical external com pression therapies. T h e objective o f such in terventions is to 'm ilk' th e. interstitial fluid a n d lym ph th ro u g h th e lym phatic system in o rd er to decongest th e affected. lim b. T h is is achieved by th e application o f com pression garm ents, th e use o f in te rm itte n t. o r sequential pn eu m atic com pression devices and massage therapy. A lth o u g h com pression. therapies do provide a degree ot sym ptom atic im provem ent, they do n o t address th e. underlying p roblem and need to be applied c ontinuously, possibly even o n a daily basis in. m any patients.. Several surgical approaches for lym phedem a therapy have been investigated.. How ever they are n o t com m only available to p atients. These include lym phatic-venous. anastom oses (C am pisi e t al., 2001; K oshim a et al., 2000; Y am am oto et al., 2003),. lym phatic vessel tra n s p la n ta tio n (lym phatic to lym phatic anastom oses) (Baum eister and. Frick, 2003; W eiss et al., 2003), om ental grafts th a t c o n ta in lym phatics (B enoit et al., 2005;. O 'B rie n et al., 1990), im p la n ta tio n o f lym ph n o d e fragm ents (Fu et al., 1998; Pabst and. R o th k o tte r, 1988; R o th k o tte r and Pabst, 1990), and vascularized lym ph n o d e. tra n s p la n ta tio n (Becker et al., 2006; C h e n et al., 1990). It is also interesting to note th a t. 13. c u rre n t th era p eu tic m easures are usually offered only to p atients w ith long-standing,. established edem a. U n d e r such c o n d itio n s th e probability o f com plete reversal o f. sym ptom s seems unlikely n o m a tte r w h at th era p eu tic m easures are applied. T h erfo re, if. n odal therapy was to be applied a t th e tim e o f th e initial surgery or shortly th ereafter and. before th e ch ro n ic sequelae to th e lim b takes place, w ould it have greater p o ten tia l to avert. o r at least result in im proved clinical outcom es?. 1.4 Lymphedema, a Problematic Complication of Breast Cancer Therapy. A m ong th e m u ltitu d e o f late side effects o f breast cancer tre a tm e n t, secondary. u p p er extrem ity lym phedem a is o n e o f th e m ost problem atic and relatively u n d e re stm a te d . com plications. O riginally described by H andley in 1908 (H andley, 1908) and first term ed. as “E lephantasis C h iru rg ic a ” in 1921 by H alsted (H alstead, 1921). M ore th a n a h u n d re d . years since it’s initial description, it rem ains a poorly u n d e rsto o d c o n d itio n th a t has the. p o ten tia l to occuer after any in te rv en tio n affecting lym ph n o d e drainage m echanism .. However u p p e r extrem ity lym phedem a occurs m ost com m only in association w ith breast. cancer (Engel et al., 2003; G eller et al., 2003; M athew et al., 2006; Petrek et al., 2000;. Petrek et al., 2001; van d er V een et al., 2004; Ververs et al., 2001). It is generally viewed as. an inco n seq u en tial c om plication o f breast cancer, and w hen ineffectively m anaged leads to. an increasingly d ebilitating c o n d itio n th a t has no cure (Petrek et al., 2000; Petrek et al.,. 2001).. It is believed th a t th e o p e ra ted lim b has reduced im m u n ity d u e to th e loss o f. lym ph nodes, this in a d d itio n to fibrosis o f tissues leading to stasis o f lym ph w hich. encourages infections a n d secondary inflam m ation. If left u n tre a te d or u n d e rtre a te d can. lead to fu rth e r tissue fibrosis, h a rd e n in g o f skin, lobulations, blistering a n d skin b reakdow n. 14. acting as a vicious cycle w hich ultim ately leads to w orsening o f lym phedem a (King MJ,. 2005).. T h e m o d e rn approach to m an ag em en t o f lym phedem a involves p a tie n t e d u c atio n . a n d p ro m o tin g inform ed self m o n ito rin g o f changes th a t are suggestive o f early disease. onset. Sym ptom s o f heaviness, aching, skin tightness, pain, num bness, weakness or. im paired arm fu n ctio n possibly indicate a pre-clinical phase o f lym phedem a o n se t and. w arran t p ro m p t m edical evaluation (Mayrovitz, 2009; M eneses a n d M cNees, 2007; N ielsen. et al., 2008).. T h e o n set o f lym phedem a evolves at various rates in differen t individuals, th ere can. be a significant delay from th e initial surgical insu lt im til swelling developes. It does n o t. necessarily occur im m ediately post operatively, b u t can h a p p e n up to several years following. th e initial axillary trau m a and often reported to be triggered by a m in o r event (Kosir MA,. 2001; Petrek et al., 2000). hiterestingly 75% of w om en w ho do develop breast cancer. related lym phedem a will have it d u rin g th e first year post operatively a n d 9 0 % by th e th ird . year (P.S. M ortim er, 1996).. 1.5 The Psyhologic Morbidity of Breast Cancer Related Arm Swelling. Lym phedem a is a chronic c o n d itio n involving a bnorm al accum u latio n o f p ro te in . rich fluid in the in terstitial space o f th e affected lim b after lym ph n o d e excision and. rad ia tio n therapy. Breast cancer related lym phedem a is a distressing c o n d itio n affecting one. in five breast cancer survivors. It is increasingly curable a n d as life expectancy im proves for. breast cancer survivors, m ore w om en will be living w ith sym ptom s o f lym phedem a.. T h e im pact of lym phedem a o n quality o f life is rep o rted as being negative (Engel et. al., 2003; M organ et al., 2005) a n d its onset is followed by surprise, em o tio n a l distress,. 15. sorrow a n d depression (A rm er et al., 2004; M organ et a l , 2005; Petrek et al., 2001). For. m any p atients developing lym phedem a post cancer therapy is a c o n s ta n t rem in d e r o f th eir. underlying illness a n d can pose a greater challenge to deal w ith th a n th e cancer itself.. T h e stigm a o f this c o n d itio n a n d th e reaction o f others, o fte n causes patien ts to. develop a p o o r self-image, becom e isolated a n d refrain from th e ir no rm al activities. W h ic h . o ften results in social w ithdraw al a n d d ram a tic life changes (A lim ed et al., 2008; C arter,. 1997; Jo h a n sso n et al., 2003; Passik a n d M cD onald, 1998).. The Concept of Sentinel Lymph N ode SampHng. T h e general tendency in breast cancer surgery over th e years has been a “less is. m o re ” philosophy. Sm aller p o rtio n s o f th e breast are o fte n rem oved in c u rre n t practice and. sen tin el n o d e excision is applied rou tin ely as opposed to taking o u t m any nodes in the. drainage basin o f th e tu m o r.. Axillary lym ph n o d e surgery rem ains an essential aspect o f breast cancer diagnosis. an d tre a tm e n t. A ssessm ent o f th e axilla is req u ired in all cases o f invasive breast cancer.. Lym ph n o d e status assists in staging th e cancer as it provides essential prognostic. in fo rm a tio n w hich helps to guide fu rth e r adjuvant tre a tm e n t such as chem otherapy,. h o rm o n a l therapy o r rad io th erap y a n d rem ove diseased nodes (L uini A, 2005; M orrell et. al., 2005). M etastatic spread to th e axilla occurs in approxim ately 30% o f p atients w ith. breast cancer (C h a n g JC , 2004).. U n til recently full axillary lym ph n o d e dissection was req u ired for m an ag em an t o f. th e axilla. H ow ever this surgery carries a significant risk o f p e rm a n e n t lym phedem a and. increasingly less invasive surgical o p tio n s nam ely axillary n o d e sam pling and se n tin e l lym ph. n o d e biopsy are increasingly em ployed.. 16. T h e first lym ph node(s) stan d in g guard in th e axilla, th a t receives lym phatic. drainage from breast parenchym a and th e tu m o u r is called th e se n tin e l node. T h e tnain. advantage o f sentinel lym ph n o d e biopsy is th a t it reduces th e likelyhood o f developing. lym phedem a by reducing th e n u m b e r o f unnecessary’ axillary lym ph n o d e dissections.. T h e re is also a proven c orrelation betw'een th e n u m b e r o f lym ph nodes rem oved at surgery. a n d th e d evelopm ent of lym phedem a (Engel et al., 2003; G offm an et al., 2004; Q uerci. della Rovere et al., 2003). Early studies proved th a t th e sentinel n o d e was positive in all. b u t few cases th a t had any n o d a l involvem ent o n su b seq u e n t full axillary lym ph n o d e. resection. (Boolbol et al., 2001; Krag et al., 1998). Lym phatic m apping w ith sen tin el node. biopsy has gained wide clinical acceptance a n d has becom e the preferred m eth o d over. axillary lym ph n o d e dissection, increasingly it is used to evaluate axillary status in patients. w ith early stage disease w ith clinically negative nodes (G eller et al., 2003; Kwan et al., 2002;. M ansel et al., 2006; T em ple et al., 2002; W ilke et al., 2006). R ecent literatu re advocates. th a t it accurately stages the axilla while reducing arm m orbidity com m only associated w ith. tra d itio n a l axillary lym ph n o d e staging (B lanchard et al., 2003; Del Bianco et al., 2008;. Kim et al., 2006; S chrenk et al., 2000; V eronesi et al., 2009). H ow ever th e advent o f. se n tin e l lym ph n o d e biopsy will n o t com pletely elim inate the p ro b le m o f postoperative. lym phedem a.. M orbidity following sentinel lym ph n o d e biopsy alone is n o t negligible, b u t still. significantly less fre q u e n t as com pared w ith level I, II a n d com plete axillary lym ph node. dissection (B lanchard et al., 2003; Langer et al., 2007; W ilke et al., 2006). Patients w ith. positive se n tin e l nodes will req u ire full axillary lym ph n o d e dissection o r radiotherapy. (Lyman e t al., 2005; P u ru sh o th a m et al., 2005; V eronesi et al., 2003).. 1 7. 1.7 The Role of Lymphangiogenesis in the Restoration of Lymphatic. C ontinuity. It is generally accepted th a t dam age to th e lymphatics is involved in the. pathogenesis o f lym phedem a. T his has led to th e a ssum ption th a t stim ulating new vessel. grow th is a n a p p ro p riate th era p eu tic m easure. Several groups are trying to achieve this by. applying selected agents directly to affected tissues (Szuba et al., 2002) or by in tro d u c in g the. m olecules th ro u g h gene th erapy approaches (Saaristo et al., 2002; Y oon et al., 2003). W liile. lym phangiogenesis has b e e n researched for m any years w hat is generally n o t appreciated is. th e fact th a t it is a very vigorous process (W itte, 2001; Yoffey, 1970). Indeed, once. dam aged, th e rem arkable regenerative capacity o f lym phatics and th eir ability to develop. collateral pathways a ro u n d th e site o f o b stru c tio n soon bridge th e gap (H adam itzky and. Pabst, 2008; Filler, 1985). Also an im p o rta n t c o m p o n e n t o f th e lymph tra n sp o rt system is. th e ability o f pre- a n d post-nodal lym phatics to c o n tra ct and p u m p lym ph. From th e studies. o f Ikom i (Ikom i et al., 2006), it w ould seem th a t th e regenerating lym phatic vessels re­. acquire a contractile p h e n o ty p e as early as fo u r weeks after nodal excision.. 18. Pre-nodal v e s s e ls . R egenerating":. : fymphatlcM at^i th e nodectbrny. (t-nodal fsiels .. Figure 1.3 Fluoroscopic Images of the PopUteal Lymphatic System in Sheep. A - Sheep p ophteal lym phatic region in th e n o rm a l/c o n tro l situ a tio n . B ' Illustration o f regenerating lym phatics at 16 weeks post n odectom y. 19. Implications of Vascularized Lymph N od e Transplantation. In th e co n te x t o f secondary lym phedem a, th e physiological role o f lym ph n o d e s in. tissue fluid balance has generally been ignored. It is com m only assum ed th a t rem oval o f th e . n o d e merely in te rru p ts th e flow o f lym ph betw een th e cu t ends o f th e pre- and post-nodal. vessels.. However, th ere are several reasons to suspect th a t th e re-in tro d u ctio n o f an. autologous vascularized lym ph n o d e at th e excision site w ould have a beneficial effect. First,. th e tra n s p o rt o f p ro te in , solutes and w ater from th e in te rstitiu m to the b lo o d strea m . involves an integrated netw ork o f lym phatic vessels a n d lym ph nodes. T h e rep lacem en t o f a. n o d e could help to restore th e correct physiological relationships betw een pre- a n d post-. nodal lym phatic vessels. Second, in c o m b in a tio n w ith th e ap p ro p riate cellular a n d . m olecular cues, th e lym ph n o d e may provide structural o rie n ta tio n d u rin g . lym phangiogenesis a n d facilitate th e re-alignm ent o f pre- and post-nodal vessels. T h ird , o n e. c a n n o t d isc o u n t th e possibility o f im m unological benefits as well. In a d d itio n , lym ph nodes. also act as filters a n d presum ably provide som e im p e d im e n t to th e m etastatic d istrib u tio n . o f cancer cells.. W hy Address Issues Related to the Role of Lymph Nodes W ithin the. Lymphatic System and Advance the Field?. Lym phedem a has puzzled th e b iom edical c o m m u n ity for m any years. T h ere are still. m any unad d ressed issues regarding th e path o g en ic m echanism s responsible for this. d iso rd e r and its tre a tm e n t co n tin u es to pose a challenge for h e a lth care professionals.. In d eed , th e exact n a tu re o f th e surgically in d u ced deficit rem ains to be defined. W liile. various m ouse m odels have proven valuable in elucidating th e m olecular factors th a t. 20. c o n tro l lym phatic vessel grow th, due to th e ir small size they are less suitable for analysis and. q u a n tita tio n o f th e physiological param eters th a t govern lym ph flow. In this regard, there. w ould seem to be a need for th e d evelopm ent o f anim al m odels o f lym phedem a th a t. p e rm it th e evaluation o f lym phatic fu n ctio n a n d allow assessm ent o f th e practical. effectiveness o f surgical or o th e r form s o f therapy.. W ith this in m ind, lym phatic vessels in sheep may provide u n iq u e o p p o rtu n itie s. since th e collecting ducts are relatively large a n d can be m an ip u la te d individually.. In this respect, th e establishm ent o f a new ap p ro a c h to tre a tm e n t lies in th e b o u n d s. o f fu rth e r u n d e rsta n d in g th e pathophysiology o f post-surgical lym phedem a.. 21. General Hypothesis. It is th e absence o f lym ph nodes after excision for cancer th a t shifts th e balance. tow ards a p red isp o sitio n for lym phedem a a n d n o t necessarily an inability to regenerate new . lym phatic vessels. T h erefore, tra n s p la n ta tio n o f a lym ph n o d e in to th e excision site will. have th e p o ten tia l to im prove tissue drainage com pared w ith no treatm en t.. Objectives of the Experimental Studies O utlined in this Thesis. T o fu rth e r expand o u r u n d e rs ta n d in g o f lym phatic system fu n ctio n in th e n orm al. situ atio n a n d following surgical m an ip u la tio n , th e m a in goal o f this thesis is to deal w ith. the following experim ental objectives:. 1) T o develop a m odel o f lym phedem a based o n th e rem oval o f a single lym ph node. in sheep.. 2) T o q u antify lym phatic tra n s p o rt over tim e a n d correlate this w ith edem a fo rm atio n . in th e lym phedem a m odel.. 3) Assess w h e th e r th e tra n s p la n ta tio n o f an autologous lym ph n o d e will reduce or. p revent th e developm ent o f lym phedem a.. CHAPTER 2:. LYMPHEDEMA DEVELOPMENT AND LYMPHATIC. FUNCTION FOLLOWING LYMPH NODE EXCISION IN. SHEEP. 23. CHAPTER 2: LYMPHEDEMA DEVELOPMENT AND LYMPHATIC FUNCTION. FOLLOWING LYMPH NODE EXCISION IN SHEEP.................................................... 23. 2.1 Introduction_______________________________________________________2 5. 2.2 Development of the Experimental Model Used in O ur Inestigations_______ 2 6. 2.3 Materials and M ethods_____________________________________________ 2 8. 2.3.1 Animals__________________________________________________ 2 8. 2.3.2 Surgery____________________________________________________28. 2.3.3 Assessment of Edema________________________________________ 30. 2.3.4 Fluoroscopy_______________________________________________ 30. 2.3.5 Immunohistochemistry______________________________________31. 2.3.6 Quantitative Assessment of Protein Transport to Plasma__________ 32. 2.3.7 Statistical Analysis__________________________________________ 35. 2.4 Results___________________________________________________________ 37. 2.4.1 Development of Lymphedema________________________________ 37. 2.4.2 Assessment of Lymphatic Function____________________________ 39. 2.4.3 Fluoroscopy and Immunohistochemistry________________________42. 2.5 Conclusion________________________________________________________ 44. 24. Introduction. Lym phatic vessels rapidly regenerate following lym ph n o d e excision in an a tte m p t. to bridge th e resu ltan t gap a n d re-establish continuity. However, very little is kno w n ab o u t. th e physiologic properties o f these newly form ed vessels and how th e tra n s p o rt capabilities. o f regenerating ducts are integrated functionally in to a lym phatic netw ork th a t includes. lym ph nodes as well as pre- a n d post-nodal collectors.. T h e studies co n ta in ed w ith in this ch ap ter were designed to achieve th e first two. experim ental objectives o f developing an anim al m odel th a t w ould p e rm it q u a n tita tio n o f. lym phedem a a n d lym phatic fu n ctio n after lym ph n o d e excision.. 25. D evelopm ent of the Experimental M odel U sed in Our Investigations. In th e first instance a study using 40 sheep was perform ed to exam ine. lym phangiogenesis at 6, 12 a n d 16 weeks following surgical o b stru c tio n o f postnodal. lym phatic vessels in th e popliteal, prescapular, a n d m esentaric lym ph systems (Jila et al.,. 2007). It was observed in this instance th a t th e in te rru p tio n to lym ph flow th ro u g h th e. popliteal lym phatic vessels resulted in th e g en e ra tio n o f a fine netw ork o f lym phatic vessels. th a t helped to re-establish lym ph flow across th e site o f o b stru ctio n , despite this fact a. significant resistance to lym ph flow was detected at all tim e points. Lym ph nodes were n o t. excised in these experim ents and th e ir presence appeared to lessen th e im pact o f post nod al. lym phatic o b stru c tio n , presum ably by facilitating ex traction o f w ater th ro u g h th e nod al. vasculature. T h e c onclusions o f this study inspired fu rth e r investigation o f th e physiologic. properties o f newly regenerated lym phatic vessels a n d how they are integrated to fu n ctio n . as ch annels o f lym ph tra n s p o rt over tim e in th e presence or absence o f lym ph nodes or. no d e tran sp lan ts. Several dissections were perform ed initially to ascertain th e ideal lym ph. n o d e system for this study. W e fo u n d th e popliteal lym ph node to be easily accessible and. m icro-dissections revealed reasonably sized vessels piercing th e peri-nodal fat pad to supply. blood to this n o d e. T h e m ain pedicle was derived from a b ra n c h o f e ith e r th e m edial. circum flex fem oral artery o r th e caudal fem oral artery, th e form er was m ore consistent. (arteries betw een 1.0-3.0 m m in diam eter). T h e m ain vein d rain in g th e n o d e was a b ran c h . o f th e lateral sa p h en o u s vein (betw een 3.0-5.0 m m in diam eter). T h e laparotom ies we. perfo rm ed o n sheep revealed q u ite large m esenteric lym ph nodes w ith reasonably sized. vascular pedicles, a n d we c onsidered harvesting these nodes for use as free transplants,. however this idea was a b a n d o n e d as we felt th a t in a d d itio n to th e intra-operative risk o f. 26. bowel p e rfo ra tio n , th e recovery period after a successful laparotom y w ould be longer a n d . m ore com plicated for th e anim als.. Q u a n tita tiv e assessm ent of lym phatic function; th e ability to tra n sp o rt a k n o w n . mass o f radiolabeled A lb u m in to plasm a provides a q u a n tita tiv e m easure o f th e lym ph. tra n s p o rt effectiveness o f a given lym phatic netw ork. U n d e r n orm al c o n d itio n s, m olecules. w ith m olecu lar weights >6000 KDa injected in to th e lim b lym phatic vessel are alm ost. com pletely recovered in th e thoracic d u c t lym ph (A ukland a n d Reed, 1993). T h u s we. p la n n e d to inject radioactive p ro te in tracer in to o n e o f th e pre-nodal popliteal vessels a n d . m easure it’s rate o f tra n s p o rt to plasm a. T his m eth o d o r variations th e re o f has been used. by th e J o h n s to n lab to assess lym phatic drainage from th e su b arach n o id c o m p a rtm e n t o f. th e b rain (B oulton et al., 1997; B o u lto n et al., 1999) a n d from th e pericardial space. (B oulanger et al., 1999). Initially th e above m eth o d o f assessing lym ph tra n s p o rt rate was. tested o n eleven c o n tro l sheep to assess if consistent tra n s p o rt rates could be o b ta in e d and. if this was a rep ro d u cib le m odel for o u r experim ental purpose. In th re e o f th e eleven. anim als tested, th e experim ents failed d u e to technical difficulty related to c a n n u la tio n o f. th e pre-nodal lym phatic vessels. T h e o th e r eight anim als show ed fairly co n sisten t lym ph. mass tra n s p o rt readings. T h e n we applied th e same co n c ep t after lym ph n o d e excision to. com pare w ith th e controls.. 27. Materials and Methods. A n im a b. A to tal o f 41 random ly bred m ale a n d fem ale D orset sheep (30.3 ± 1.1 kg) were. used in this investigation. A nim als were given free access to food a n d w ater for an. o bservation p eriod o f o n e week preceding surgery. All experim ents o u tlin e d in this paper. were approved by the ethics com m ittee at S u n n y b ro o k H e a lth Sciences C en tre and. co n fo rm ed to th e guidelines set by th e C a n a d ia n C o u n c il o n A nim al C are and th e. A nim als for Research A ct o f O n ta rio .. Sursery. Sheep are fasted 24 ho u rs prio r to anesthesia. T h e anim als are anesthetized initially. w ith 20m l l.V. injection o f so d iu m p e n to th a l. Subsequently, 2.0-3.5% Isotluorane is. delivered th ro u g h an endo trach eal tu b e via a M oduflex, D ispom ed m achine w ith Hallowell. resp irato r for surgical m aintenance. T h e surgical site is shaved a n d w ashed w ith alcohol. an d b e ta d in e th e n draped w ith sterile sheets. Sterile Evans blue dye (1% in saline) is. injected subcutaneously just proxim al to th e h o o f to e n h a n ce visualization o f th e lymph. n o d e a n d th e pre- and post-nodal lym phatic vessels.. A vertical skin incision (approxim ately 8-lOcm long) is m ade over th e lateral aspect. o f th e p o p liteal region. T h e popliteal fossa is trian g u lar in shape and can be found by. retracting th e biceps fem oris m uscle caudally at th e level o f th e stifle jo in t. A t its distal. angle, a single popliteal lym ph n o d e lies e m b e d d e d in a pad o f fat. O n c e th e node is. exposed as illustrated in figure 2.1 th e pre- and post-nodal lym phatic vessels are tied off. w ith a silk su tu re and th e n o d e is excised. H em ostasis is en su red p rio r to closure o f th e. 28. surgical site. T h e anim als are re tu r n e d to th e ir h o ld in g p e n s after recovery fro m th e . a n e sth e tic . S u b c u ta n e o u s o p io id analgesic is given p o st operatively for p a in m an a g e m e n t.. A n tib io tic (D up lo cillin ) is given in tra m u sc u la rly o n e day b efore surgery a n d again tw o days. p o st operatively.. Pre-nodal VesselPost-nodal. Vessel. Popliteal L ym ph. Figure 2.1 Popliteal N od e Highlighted W ith Evans Blue Dye Intra-operatively. and Partially Dissected Free From its Surrounding Fat Pad. 29. Assessment o f E dem a. T h e h in d legs are shaved closely and leg circum ference m easurem ents are taken at a. p o in t 10cm distal from th e hock (tarsus). This landm ark is highlighted pre-operatively w ith. a skin m arker. U sing a b lan k piece o f umbilical tape, circum ference m easurem ents are. taken daily in th e first week post-surgery and once a week after th a t u ntil the animals are. sacrificed. T h e lim b circum ferences are divided by the original (pre-surgical value) and. expressed as percentage change over time.. T o com pare th e edem a outcom es in the various groups, the percentage change in. lim b circum ference is p lo tted against time. Between the h o o f an d hock joints the sheep. limb is fairly unifo rm in shape and diam eter, elim inating the need for a circumference. m easurem ent at m ore th a n one point. Similarly, the forelimbs o f 21 animals were shaved. an d m easured th ro u g h o u t the experim ents as controls at a p o in t 10cm distal to the hock. joint.. Fluoroscotrj. Imaging was perform ed on four sheep at eight weeks (one animal, two limbs), 12. weeks (one anim al, two limbs) an d 16 weeks post surgery (one animal, two limbs), in. ad d itio n to one co n tro l anim al (two limbs).. Evans blue dye (1% in saline) is injected subcutaneously to highlight the lymphatics. prio r to can n u latio n o f an upstream popliteal pre-nodal vessel w ith a 26G angiocatheter.. A n x-ray co n trast m edium (l-3m l, Lipiodol, EZ-EM C anada, Therepex) is injected th ro u g h . the cannula an d a m obile fluoroscopy system is used (BV Pulsera, Philips) to visualize the. lym phatic vessels aro u n d the popliteal region.. 30. Im m u n o h isto c h e m istry. In te n anim als th e grow th o f new lym phatic vessels was assessed at th e nodal. excision site (lym phangiogenesis). Briefly, tw’o m ethods w ere used to identify th e lym phatic. vessels. First, CFDA-SE (5(6)-CFDA SE [5'(and'6)'Carboxyfluorescein diacetate succinim idyl. ester] M olecular Probes # C '1 1 5 7 ) is injected in to an u p stream pre-nodal lym phatic. This. m olecule diffuses passively in to cells and rem ains non-fluorescent u n til its acetate groups. are cleaved by intracellular esterases (it fluoresces green). T his provided unequivocal. iden tificatio n o f th e lym phatics since th e dye is in tro d u c ed directly in to th e vascular lum en.. As well, th e sections are co-stained wnth antibodies to th e lym phatic en d o th e lia l receptor. to r h yaluronan (LYVE-1) (tagged w ith Cy3'red).. Tissue blocks from th e popliteal region are rem oved surgically. T h e sam ples are. th e n em b ed d ed in Tissue-Tek O .C .T c o m p o u n d , placed in a base m old a n d frozen. im m ediately at. - 80°C . Sections (7 |im thick) are cut using a Leica cryostat (m odel Leica CM 3050S'3-1'1). a n d placed o n glass m icroscope slides. T h e slides are washed 5 tim es in p h osphate. buffered saline (PBS-O.l M) a n d blocked for o n e h o u r at ro o m tem p e ra tu re w ith 10% goat. serum in p h o sp h a te buffered saline (PBS). A fter w ashing w ith PBS, th e sections are. in cu b ated overnight at 4 °C w ith 1:50 or 1:100 d ilu tio n s o f rabbit, an ti-h u m an LYVE-1. prim ary an tibody (Research D iagnostics Inc.). T h e next day, th e sections are w ashed w ith. PBS and incubated w ith 1:100 dilu tio n s o f goat, anti-rabbit IgO an tib o d y tagged w ith Cy3. (Jackson Im m u n o Research). In controls th e prim ary an tib o d y to LYVE-1 was om itted.. Finally, th e slides are m o u n te d in a q u ap o ly m o u n t and cover slipped.. Im m unofluorescence m icroscopy is perform ed w ith a Zeiss Axiovert lOOM laser. scanning confocal m icroscope. T h e argon a n d h e liu m /n e o n lasers are set to w avelengths of. 31. 488 and 5 4 3 n m for excitation o f CFDA-SE a n d Cy3 respectively. W lie n b o th approaches. are co m bined in th e same tissue sam ple, th e lym phatic vessels appear yellow-orange.. 2 . 2.6 Q u a n tita tive Assessment of Protein Transport to Plasma. T h e m ain role o f th e lym phatic vessel is to absorb extravasated vascular derived. p ro te in from th e in terstitial spaces a n d re tu rn it to th e venous circulation. T herefore, the. ability to tra n s p o rt a kno w n mass o f radiolabeled a lb u m in to plasm a provides a qu an titativ e. m easure o f th e lym ph tra n s p o rt effectiveness o f a given lym phatic netw ork. A schem atic. illustrating the features o f th e experim ental design is provided in Figure 2.2. Q uantitative. studies were perform ed at 8, 12 and 16 weeks following popliteal n odectom y as well as in a. node-intact group.. T h e anim als are fasted and anesthesia is in duced as previously described. T h e h in d . lim bs are shaved and a final circum ference m ea su rem e n t is m ade at th e form erly explained. landm ark. A heparinized neckline is inserted in to th e jugular vein and secured for. collecting blood sam ples th ro u g h o u t th e experim ent. Evans blue dye (1% in saline) is. injected u n d e r th e skin to highlight th e lym phatics. W lie n injected subcutaneously, th e dye. b in d s to p ro te in a n d readily enters th e absorbing lym phatics. It is d istrib u ted rapidly. th ro u g h th e lym phatic n etw ork a n d o utlines th e collecting vessels clearly. A n incision is. m ade th ro u g h th e skin and subcutaneous tissues over th e lower lateral aspect o f th e h in d . lim b, extending distally from th e h o o f to expose several pre-nodal lym phatic vessels in close. proxim ity to th e lateral sap h en o u s vein. W ith th e aid o f m agnifying C arl Zeiss loupes. (3,5x400) a single vessel is dissected free from th e su rro u n d in g tissues a n d cannulated w ith . a 26G angiocatheter. T his is secured w ith 4-0 silk ties a n d a clam p applied distally to. p rev en t backflow.. 32. Saline (lOOpL) is th e n injected slowly over 30 seconds to check for any leaks at th e. c a n n u la tio n site a n d to observe th e flushing o f th e Evans blue dye in th e lym phatic vessel.. T his process ensures th e correct placem ent o f th e cannula. R adiolabeled h u m a n serum . alb u m in ('"^I-HSA, 2mg in a 200pL volum e) is injected in to a pre-nodal lym phatic vessel. over a 60 second period w ith a 250pL H a m ilto n syringe a n d flushed w ith lOOpL o f saline. over 30 seconds. T h e can n u la is th e n capped to p revent backflow. Swabs are tak e n from . a ro u n d th e tip o f th e can n u la as well as th e injection site a n d later analyzed in th e gam m a. c o u n te r to indicate w h eth er o r n o t th ere was a leak or spill o f th e radioactive tracer.. Blood samples are taken from th e neckline to m o n ito r th e recovery o f radioactivity. in th e blood over a period o f to u r hours. Sam ples are taken at tim e zero, 15m in, 3 0 m in . a n d th e n every 3 0 m in up to four hours. T h e anim al is sacrificed at th e e n d of th e. ex p erim ent w ith 20m l o f E uthanyl adm inistered intravenously.. T he co n cen tratio n s o f th e radioactive p ro te in tracer in plasm a (C P M /m l) are. divided by th e a m o u n t injected to arrive at percen t in je c te d /m l, w hich is p lo tted over tim e.. However, once a p ro te in tracer enters blood, it re-filters back in to th e various tissues o f th e. body a n d hence, tracer recoveries are inherently u n d erestim ated . A n alb u m in e lim in a tio n . rate (K̂ p̂) been defined in previous experim ents (B oulton et al., 1997) a n d is used in a. mass balance e q u a tio n (eq u atio n 1) to reflect m ore accurately th e ability o f th e lym phatic. system to re tu rn p ro te in to blood. T h e mass balance e q u a tio n o u tlin e d below is used to. estim ate a single averaged mass tra n s p o rt rate:. g k P ( ' / exp ‘f ) - ‘̂ / ’ (o)] (^ e x p > P ) ex p (A ^ex p '/)-l. 33. Bin (blood in) = th e mass tra n sp o rt rate ( C P M /h o u r) is averaged fro m tim e zero to tim e t,. (d u ra tio n o f experim ent) w hich in o u r case was 4 ho u rs. T h e values for Bĵ derived from . e q u a tio n (1) are divided by th e to tal radioactivity injected to give % in je c te d /h r. Cp(t,) = th e . c o n c e n tra tio n o f th e tracer at 4 hours; Cp(0) = th e c o n c e n tra tio n o f tracer at tim e zero;. = is th e coefficient o f e lim in a tio n o f tracer from plasm a. Since o u r previous experience. indicated th a t this coefficient did n o t differ significantly betw een anim als o f various ages. a n d weights, a n average value derived from 41 anim als used in previous studies (Boulanger. et al., 1999; B o u lto n M, 1998a; B o u lto n et al., 1997; B oulton M , 1998b). Since th e volum e. o f d istrib u tio n o f th e tracer (plasma volum e, Vp) w ould differ betw een anim als, we adjusted. th e plasm a recoveries to reflect this. Based o n data derived in previous studies from o u r. group (B oulanger et al., 1999; B oulton M, 1998a; B oulton et al., 1997; B o u lto n M, 1998b),. we plo tted th e plasm a volum es derived from 41 anim als against th e ir weights. W e used a. regression analysis of these data (eq u atio n 2) to calculate a plasm a volum e in each sheep. based o n th e following equation:. y = 2 1 .77x + 649.68 ( 2). 34. 2 . 2.7 S ta tis tic a l Analysis. All data is expressed as th e m ean ± SEM. T h e data was analyzed w ith a one-way. analysis o f variance A N O V A followed by contrasts back to baseline using a one-sided. D u n n e tt’s t-test. W e in te rp re te d P < 0.05 as significant.. 35. M onitor recovery in Plasma. Surgically rem ove ̂ N ode V -. Post-nodal (efferent) lymphatic. Popliteal Lym ph N ode. Pre-nodal (afferent) lymphatics. C ircumference m easurem ent. Exclusive popliteal drainage basin. Figure 2.2 Schem atic Illustrating Essential Features of the Experim ental M odel to. Q uantify Lym phatic F unction. 36. Results. D e v e lo p m e n t o f L ym phedem a. T h e rem oval o f a single popliteal lym ph n o d e resulted in edem a fo rm a tio n in th e. low er h in d limbs o f all anim als. A n exam ple is illustrated in Figure 2.3A. Figure 2.3B. displays th e averaged data. A t day o n e after surgery th e average p e rc e n t increase in leg. circum ference was 19.5% , at day two 29.1% a n d at day th re e 33.8% (the peak o f th e. response). T h e edem a declined over tim e b u t in m ost anim als did n o t subside com pletely. a n d rem ain ed elevated u n til th e sheep were sacrificed (up to 16 weeks post surgery). In. th re e sheep, th e edem a subsided com pletely before the e n d p o in t o f th e study. All o f the. c o n tro l forelim bs w hich were m easured show ed no increase in circum ference. All anim als. were am bulatory during th e course o f th e experim ents.. 37. — N o d e ex c ise d C ontrol Lim b. Picture taken 1 day after n o d e rem oved. B. 0 ) o c p 0 ) 0 ). <4— O ) p c. ro o x :o O D ) 0 ). — I. 40 3 8 - 4 6 4 - 181 8 - 2 8. # lim bs. 30. 20. 10. 0 20 4 0 60 80 100 120. Time (days). Figure 2.3 L ym phedem a P ro d u ced A fter the R em oval of the P opliteal Lym ph N ode.. (A) Example o f edema development following lymph node excision in. com parison w ith a control limb.. (B) Q uantification o f edema after lymph node excision expressed as percentage. change from pre-surgical levels. The numerals at the top of the figure illustrate. the num ber o f limbs th a t were assessed at various times following nodectomy.. 38. 2.3.2 Assessment of Lymphatic Function. W lie n '"^I'HSA was injected in to an u pstream pre-nodal popliteal lym phatic vessel. in co n tro l lim bs, it en tered plasm a w ith peak c o n c en tra tio n s being achieved approxim ately. o n e h o u r after injection (Figure 2.4). M easurem ent o f plasm a levels at 8, 12 and 16 weeks. following lym ph n o d e excision revealed lower blo o d co n cen tratio n s.. Figure 2.5 illustrates th e averaged tracer mass tra n s p o rt rates over four h o u rs. calculated from e q u a tio n o n e (Bin). A one-w’ay A N O V A revealed th a t th e groups were. significantly different. T he values obtain ed at 8 (10.6 ± 1.5), 12 (14.4 ± 1.0) and 16 weeks. post'surgery (13.9 ± 1.0) were less th a n th a t n o ted for th e intact lim bs (17.2 + 0.6) b u t only. th e data at 8 weeks reached statistical significance w ith th e D u n n e tt’s t-test.. T hese data suggested th a t pre- and post-nodal lym phatic vessels had regenerated to a. considerable extent, since th e en try o f tracer in to plasm a show ed th a t som e lym ph. c o n tin u ity had been re-established. Indeed, at 12 a n d 16 weeks after surgery, lym phatic. fu n ctio n had retu rn ed to approxim ately 80% o f th e level observed in co n tro l lim bs. A t 8. weeks post-surgery, only a b o u t 6 0 % o f lym ph tra n s p o rt had been restored.. 39. P er. ce nt. In. je ct. ed. d o. se /m. l 0.080. 0.070 ^ C ciitrol. 0.060. 0.050 12 î êel < - 16 wee.. 0.040 ^ 8 v/eek. 0.030. 0.020 0.010. 0.000 180 24030 " 6 0 120. Time. Figure 2.4 Appearance of Radioactive A lb u m in in Plasma Over T im e. (Q uantification of Lymphatic Function). Radioactive alb u m in was injected in to a pre-nodal lym phatic vessel and th e. c o n c e n tra tio n o f th e tracer m o n ito re d in plasm a for four ho u rs after injection. in th e four groups o f anim als; c o n tro l g roup (n=7) and 8 (n=7), 12 (n=7) and. 16 weeks (n=6) post-surgery.. 40. Figure 2.5 Averaged Lymphatic Mass Transport Rates (Q uantification of Lymphatic. Function). E quations 1 and 2 were used to calculate an average mass tra n sp o rt rate for. each group o f anim als. C o n tro ls (n=7), 8 week (n=7), 12 week (n=7) a n d 16. weeks (n=6). A one-way A N O V A revealed th a t th e groups were significantly. different. T he values o b tain e d at 8, 12 and 16 weeks post-surgery were less. th a n th a t n o ted for the in ta c t lim bs b u t only th e d a ta at 8 weeks reached. statistical significance w ith th e D u n n e tt’s t-test (m arked w ith asterisk in. Figure).. 41. Fluoroscopy and Im m unohistochem istry. Fluoroscopic analysis at 8, 12 a n d 16 weeks after n o d a l excision co nfirm ed th a t. som e degree o f fluid c o n tin u ity had b e e n established betw een th e pre-nodal a n d post-nodal. lym phatic vessels in th e absence o f th e lym ph node. T h e co n tra st agent injected into o n e o f. th e pre-nodal lym phatics in th e lower lim b could be observed in th e relatively large d u c t. th a t w ould norm ally collect lym ph from th e popliteal n o d e (post-nodal vessel) (example. illustrated in Figure 2.6A). Sm all lym phatic vessels were observed in th e n o d a l excision. area. T h e possibility th a t these vessels were newly form ed lym phatics a tte m p tin g to bridge. th e gap left by th e absent n o d e was s u p p o rte d by im m unohistochem istry.. W e observed co-localization o f LYVE-1 (red) a n d CFDA-SE (green) indicating th a t. lym phatic vessels were present at th e area vacated by th e popliteal lym ph node. A n example. is provided in Figure 2.6B. T h e newly form ed lym phatics observed at 12 weeks p o st surgery. were variable in size b u t at th e u p p e r en d , were a b o u t 4 0 |im in diam eter. T hese vessels were. th e co n d u its th a t connected th e originally placed pre- and post-nodal lym phatics and. c o n trib u te d to th e resto ratio n o f lym ph tra n s p o rt th a t was observed in th e tracer and. fluoroscopic studies.. 42. Figure 2.6 Morphological Evidence that Some Regeneration of Lymphatic Vessels. has Occurred Following Popliteal Lymph N od e Excision. (A) Fluoroscopy p e rfo rm e d at 12 weeks after lym ph n o d e rem oval illu stratin g . som e degree o f c o n tin u ity b etw een th e lym phatic vessels th a t w ere originally. pre- a n d p o st-nodal to th e p o p litea l lym ph n o d e .. (B) C o n fo c a l m icroscopy image o f reg en erated p ost-nodal lym phatics in th e . n o d a l excision site. T issue sections were p re p a re d as d escribed in th e . M aterials a n d M e th o d s. For in iequivocal id e n tific a tio n o f th ese vessels, a. non-specific cell dye C FD A -SF (green) was infuseci in to an u p stre a m pre-. n o d a l lym phatic a n d a t th e sam e tim e, th e tissues w ere s ta in e d w ith . a n tib o d ie s to th e lym phatic e n d o th e lia l m a rk e r LYVE-1 (red). T h e sta in e d . sections a p p e are d yellow-orange in d ic a tin g co-existence o f th e tw o stains. A t. 12 weeks, a n irreg u lar n e tw o rk o f sm all in te rc o n n e c tin g lym phatics c o u ld be. o bser\'ed in th e area originally occu p ied by th e p o p litea l lym ph n o d e . W e. p resu m e th a t all vessels in th is image are newly fo rm e d in resp o n se to th e . rem oval o f th e noele.. 43. 2.4 C onclusion. Lymphedema was initiated by the removal o f a single lymph node and a. quantitative m ethod was developed to assess lym phatic fu n ctio n over time. These. techniques may be helpful in u nderstanding the pathophysiology associated w ith. lym phedem a and may facilitate the developm ent o f new strategies to treat or prevent this. condition.. 44. CHAPTER 3;. EXPERIMENTAL ASSESSMENT OF AUTOLOGOUS LYMPH. NODE TRANSPLANTATION AS TREATMENT OF POST-. SURGICAL LYMPHEDEMA. 45. CHAPTER 3: EXPERIMENTAL ASSESMENT OF AUTOLOGUS LYMPH NODE. TRANSPLANTATION AS TREATMENT OF POST-SURGICAL LYMPHEDEMA _45. 3.1 In tro d u c tio n __________________________________________________________47. 3.2 Materials and M eth o d s________________________________________________ 4 8. 3.2.1 A n im a ls ________________________________________________________4 8. 3.2.3 A natom ic Dissections_____________________________________________ 48. 3.2.3 Surgical P ro ced u res______________________________________________ 49. 3.2.4 Assessment of Edem a_____________________________________________ 53. 3.2.5 Fluoroscopy_____________________________________________________ 54. 3.2.6 Histologic Assessment of Lymph N odes_____________________________ 54. 3.2.7 Q uantitative Assessment o f Lymphatic F un ctio n ______________________ 55. 3.2.8 Statistical A n aly sis_______________________________________________ 55. 3.3 R e s u lts ______________________________________________________________ 56. 3.3.1 Lymphatic F unction______________________________________________ 56. 3.3.2 C orrelation of Lymphatic Function with Health of Lymph N o d e s 58. 3.3.3 E d e m a _________________________________________________________ 61. 3.3.4 Fluoroscopy S tudies______________________________________________ 66. 3.4 C o n clu sio n s__________________________________________________________69. 46. Introduction. Lym phedem a in breast cancer p atients represents a distressing co nsequence of. axillary n o d e resection. Since th e actual cause o f lym phedem a is still being d ebated, th e. m ost a p p ro p riate target for new th era p eu tic approaches is n o t entirely apparent.. Follow ing th e d evelopm ent o f a sheep m odel th a t perm its q u a n tita tio n o f edem a. and lym phatic fu n ctio n after th e rem oval o f a single po p liteal n o d e in sheep, th e objective. o f this c h a p te r is to exam ine th e im pact o f autologous vascularized and non-vascularized. lym ph n o d e tra n sp la n ta tio n o n lym phatic fu n ctio n a n d edem a fo rm atio n im m ediately. following th e rem oval o f th e popliteal node.. 47. Materials and Methods. Animals. A total o f 50 random ly bred m ale a n d fem ale D orset sheep (30.6 ± 0 . 7 kg) were. used in th e experim ents presented in this chapter. A n a d d itio n a l 10 sheep were used solely. for anatom y dissections. All experim ents o u tlin e d in this pap er were approved by th e ethics. com m ittee at S u n n y b ro o k H ealth Sciences C e n tre and c onform ed to th e guidelines set by. the C a n a d ia n C o u n c il o n A nim al C are a n d th e A nim als for R esearch A ct o f O n ta rio .. Anatomy Dissections. A total o f te n sheep were used for an ato m y studies o f th e popliteal lym ph node,. particularly focusing o n its blood supply. S heep are fasted and anesthetized as described in. earlier sections. Evans blue dye (1% in saline) is injected u n d e r th e skin close to th e hoof.. T his colors th e d rain in g lym phatics o f th e lim b up to th e popliteal no d e. Also in several. sheep, yellow M ic ro fil® was used. T h e best results were o b tain e d using a p rep a ra tio n th a t. was m ore dilu te th a n th a t reco m m en d ed in th e p ro d u c t literature. F or every 2.5m l o f. yellow M icrofil® 2m l o f d ilu e n t is a dded a n d th e m aterial catalyzed w ith 10% (of total. volum e) o f th e curing agent. T h e fem oral artery is catheterized a n d 10 o r 20m l o f th e. M ic ro fil® p rep a ra tio n is infused in to th e vasculature.. 48. Polymerized M icrofil highlights even th e tiniest vessels p e n e tratin g th e no d e. T h e. popliteal lym ph n o d e w ith its su rro u n d in g fat was carefully dissected w ith th e aid o f eith er. C arl Zeiss Loupes (3,5x400) o r a larger Surgical M icroscope C a rl Zeiss (O PM I l-FC). T h e. arterial supply to the n o d e seems to com e m ainly from th e m edial circum flex fem oral artery. (0.1'0.3m m ) a n d to a lesser degree from th e caudal fem oral artery (0.1-0.2m m ). U p o n . enterin g th e fat pad, these vessels b ran c h o u t to form an extensive arterial netw ork, w hich. in tu rn gives rise to m ultiple sm aller vessels responsible for n u rtu rin g th e node. In only one. o f th e dissections, th e m ain arterial b ran c h was fo u n d to e n te r th e n o d e directly. T h e m ain. d rain in g vein consistently is th e lateral saphenous vein (0.3'0.5m m ).. It was thus decided th a t th e n o d e needed be harvested w ith th e popliteal fat intact. to b e tte r th e chances o f preserving th e delicate blood supply to th e node.. Surgical Procedures. N o d e c to m y F ollo w ed by A v a s c u la r N o d e G r a f t In sertion. Sheep are fasted 24 h ours prio r to anesthesia. T h e anim als are anesthetized initially. by I.V. injection o f so d iu m p e n to th a l. Subsequently, 2.0-3.5% isofluorane is delivered. th ro u g h an endo trach eal tube via a M oduflex, D ispom ed m achine w ith Hallow ell. respirator for m aintenance. T h e surgical site is shaved a n d w ashed w ith alcohol and. b e ta d in e th e n draped w'ith sterile sheets.. A vertical skin incision (approxim ately 8-10cm long) is m ade over th e lateral aspect. o f th e popliteal region. T h e popliteal fossa is triangular in shape a n d is foim d by retracting. th e biceps fem oris m uscle caudally at th e level ot th e stifle joint. A t it’s distal angle, a single. p opliteal lym ph n o d e lies em bedded in a pad o f fat.. 49. A small o p e n in g is m ade in th e popliteal fat a n d th e node is carefu.ly dissected o u t.. T h e pre- a n d post-nodal lym phatic vessels are tied o ff w ith a silk su tu re and th e n o d e is. carefully excised. O n c e th e nodes from b o th h in d lim bs are rem oved, each is th e n placed. in to th e c o n tra lateral popliteal fossa th ro u g h th e w indow open in g created in th e fat as a. free graft w ith o u t vascular reco n n e c tio n . T his o p e n in g is th e n su tu re d to ensure th e n o d e is. n o t displaced. H em ostasis is perform ed as needed a n d th e skin is closed.. N o d ecto m y Followed by L ym ph N ode Transt>lant by M icrovascu lar A n astom osis. S heep are fasted, anesthesia is induced a n d th e popliteal fossa is exposed as. described above. T h e lym ph n o d e is harvested tog eth er w ith the popliteal tat pad intact, to. preserve th e delicate n u trie n t vessels supplying th e lym ph node. T h e popliteal fossa is. exposed by retracting th e biceps fem oris m uscle caudally at the level o f th e stifle joint. T h e. tra n s p la n t tissue is carefully dissected off th e su rro u n d in g muscles w ith the use o f C arl. Zeiss Loupes (3,5x 400) in a distal to proxim al d ire c tio n (starting distal to and dissecting. tow ards th e vascular pedicle). C are is tak e n n o t to h a rm th e tibial a n d com m on pero n eal. nerves w hich lie ju st m edial to th e popliteal fat. T h e vascular pedicle form ed by th e lateral. sap h en o u s vein a n d th e m edial circum flex fem oral artery w hich lie in the superior-m edial. angle o f th e popliteal fossa are identified. G re a t care is taken to dissect the pedicle free. from th e s u rro u n d in g tissues before ligation and clipping o f th e vessels. O nce th e free. tra n s p la n t is harvested it is covered w
Show more

New documents

This paper proposes an unsupervised label propagation algorithm Un-LP for word clustering which uses multi-exemplars to represent a cluster.. Experiments on a synthetic 2D dataset show

While Open IE presents a promising direction for ID, thanks to its robustness and scalability across domains, we argue that it currently lacks representation power in two major aspects:

We present an automated approach that is based on the hypothesis that if two sentences are a found in the same cluster of news articles and b contain temporal expressions that reference

Columns denote odds ratio OR, sample size n, method, factor for r2VIM, total number of SNPs, number of causal SNPs, number of clumps based on causal SNPs, number of SNPs in clumps based

In Second Joint Conference on Lexical and Computational Semantics *SEM, Volume 2: Proceedings of the Seventh International Workshop on Semantic Evaluation SemEval 2013, pages 1–9,

With this in mind, it is nonetheless worth reminding ourselves that his contributions included information theory, fiducial probability and inference, discriminant function, likelihood,

We ran experiments with three systems; the jointly learned entity filtering-relation extraction approach using imitation learning henceforth 2stage, the one-stage classification

This paper describes a two-stage process for stochastic generation of email, in which the first stage structures the emails according to sender style and topic structure high-level

I am especially grateful to the following for commenting on an earlier draft: Donal Nevin and Ken Whitaker the only two persons still serving on the Council of the Institute from its

!"#$%&'#*'+,-./' Abstract We present a novel algorithm for inducing Combinatory Categorial Grammars from dependency treebanks, along with initial experiments showing that it can be used