The Lymphatic Circulation* Noel G. McHale
School of Biomedical Science, The Queen's University of Belfast, 97 Lisburn Road, Belfast BT9 7BL. The lymphatic system represents a formidable challenge to a potential investigator and one that I am sure I would never have taken up had I had a free choice. Why, then, did I study thetymphatic circulation? Was it because I was inspired by~ the works of William Harvey who, having demonsfi'ated the circulation of blood, was invited~to study the lymphtic circulation'? He declined to do so in the following statement'. "It is useless for you to spur me on to gird myself to any new investigation, seeing that I am now not only ripe in years but also weary.:. R Seems to me indeed that I am entitled to ask for my honourable discharge from duty". Was it that I was keen to take up the guantlet that Harvey was too weary to lift. The answer is "No, it was not!" Was it then that a desire to alleviate human suffering such as that experienced by people afflicted with lymphoedema? And the answer again was "No, it was not!" Was it then the inspiring work by Howard Florey 2 on the lymphatic system which constituted his PhD thesis, published in 1927, the title of which was "Philosophy and Pathology of the circulation of the blood and lymph"? And the answer is "No, it was not, for two reasons: firstly, at that time I had not read his PhD thesis, and secondly, my experience of the lymphatic system up to that point was greatly influenced by a colleague of mine, Denis Mawhinney3, who was sharing a laboratory with me when we were doing our BSc projects. Dennis worked on isolated bovine mesenteric vessels and found that study so difficult that I swore that one thing I would never study, given any choice, was the lymphatic system. However, a year later when I went to discuss my PhD project with my supervisor, Ian Roddie, he offered me a choice of projects. He said I could study lymphatics or nothing! Faced with this "offer that I couldn't refuse" I eudeavoured to look at the lymphatic system in a more positive way. I began to see that here was a system about which so little was known that a whole field that could be opened up if one put in some effort and determination. My project was specifically to establish whether lymph ducts could pump fluid by means of their intrinsic contractions. Much evidence suggested that this was likely. Gasper Aselli4 in 1622 noticed that lacteals became empty after death, implying that they were contractile. Hewson5in 1769 observed directly that lacteals could contract while Heller~ in 1869 described rhythmic contractions of lacteals bat was unable to establish whether or not the contractions were an indirect consequence of gut peristalsis. Flurey 7 in 1927, confirmed that lacteals in the guinea pig could contract spontaneously, and that this activity continued when the vessels were excised from the mesentery. However Florey did not consider that spontaneous eontractity of lymphatics
was universal and he was sceptical about its importance in lymph flow. In his own words: "Under physiological conditions in the cat, therefore, it may be asserted that the forces moving lymph are a v i s a tergo presumably supplied by the blood pressure, squeezing due to gut peristalsis, and a respiratory suction pump mechanism..... There is no evidence that the lacteals in a normal condition farther the passage of lymph by rhythmic contractions4. '' The question with which I was presented at the outset of my studies was: "Do lymphatic vessels contract in such a way that they can propel fluid, and secondly if they are capable of propelling fluid in this way, what is the relevance of this to the function of the lymphatic system"? Pumping in isolated lymphatic vessels. In order to answer the first question, it was thought appropriate to look at isolated vessels first of all, because this removed the complicating effects of extrinsic forces. The vessel chosen was the bovine mesenteric lymph duct since this was large and robust enough to be studied in i~lation. An 8 cm length of duct was dissected free of the mesentery, canndlated at both ends and set up in a horizontal organ bathL The inflow end was connected to a constant pressure reservoir of Krebs solution, while the outflow was connected to a drop counter at the same height. Transmaral pressure could be varied by raising or lowering both reservoir and outflow together. Measurements were made of inflow pressure, outflow pressure and flow. The lymphatic contracted in a very regular, rhythmic fashion. Each contraction resulted in a phasic increase in outflow pressure and caused a series of drops to be expelled through the drop counter. This was followed by a pause until the next contraction expelled a further series of drops. Fig 1 shows the effect of changing transmural pressure from 0 to 21 cm H20. Flow increased with increasing transmural pressure up to a maximum of 8 cm H20 caused flow to decline due to a decrease in stroke volume and despite a continued increase in frequency of contraction.
Contribution of intrinsic pumping to lymph flow in the living animal. The above experiments certainly demonstrate that the lymphatic vessel is capable of contracting in isolation, and that these contractions are capable of propelling fluid under conditions where no extrinsic forces could be having any effect. The next question however was: what is the relevance of this to the living animal? In order to investigate this the sheep was chosen as a model. An afferent lymphatic vessel was cannulated in the metatarsal region9. This was connected to a pressure transducer to measure side arm pressure and lymph flow was measured by allowing fluid leaving the cannula to accumulate on the lever of an isometric tension transducer. Each time the lymphatic contracted there was a phasic increase in outflow
"1991 Conway Review Lecture 483
I.J.M.S. August, 1992
484 McHale
lo[L /'. . . . . . . . . . . . . . . %/x ~ A / A
Fmmwcy (omltractions/min)
'I/ OL
~mdko wkJlle Irnl)
/ o-o'~ ~
0.1 0"05
~/~
\o \
o~
0 1"C Flow (rnl/mm)
0"~"
e'l
/
\~
\e
Tran~nural Pr~sum(cmH~) Fig. I - Plotted results of flow, stroke volume and hequez~y of contraction in an experiment where uansmural pressure was increased from 0 to 21 cm H20.
pressure followed by an accumulation of lymph on the transducer lever. This was repeated with each subsequent contraction unttl enouk~@mph had accumulated to form a drop which then fell off.' The drops were of approximately equal size so the slope of the ramp is an index of lymph flow using this technique. One way of determining whether we are looking at an active pump, or an essentially passive process, is to ask the vessel to do some work. This was done by raising the height of the outflow cannula. Between 0 and 30 cm flow remained fairly steady so more work was being doric to drive the same amount of fluid to'a greater height. This was achieved by an increase in frequency of contraction and it is interesting to note that when frequency of contrtaction began to level off (at about 30 cm) flow began to fall off. In other words the lymphatic was working harder by increasing its frequency of contraction, maintaining flow even though there was a decrease in stroke volume.
el) Over Hoof Flow (ul rain-I}
70 60 50
b) Over Metotarsal
~
2on,2off
3O 20 '~
I
t__l
Fig. 2 - Summary of seven experiments in anaesthetized sheep an the effect on flow of intenninem compression (2 see on, 2 see off) was applied either over the hoof or oyer the metatarsal region. (From reference 13 with permission).
Contribution of extrinsic forces to lymph propulsion. One of the reasons why extrinsic forces were thought to be important in the propulsion of lymph is that it has been well known for a very long time that movement increases lymph flow~~ To examine this the metarsal lymphatic was cannulated as before, the animal allowed to recover and measurements were made while the conscious animal was standing in a metabolism caget3. When the animal walked the length of the cage and back again, lymph flow doubled. Observations such as these were, in the past, taken to indicate that contraction of muscles or external compression of lymphatic vessels were largely responsible for lymph propusion. To examine this belief in greater detail the metatarsal lymphatic was cannulated as before and pneumatic cuffs were fitted over both the metatarsal region (so to compress the duct itself) and over the hoof (its drainage hinterland). Fig. 2 shows a summary of seven experiments where intermittent compression at a frequency of 2 seconds on, 2 seconds off, was applied either over the hoof or over the metatarsal region. Compression applied over the hoof dramatically increased lymph flow, but, surprisingly, that applied over the metatarsal region did not significantly increase lymph flow. We took this to mean that the lymphatic was almost empty when the intrinsic pump is working normally. One way of testing this was to control fluid input to the lymphatic and thus ensure that it was always full. External compression could then be expected to greatly enhance fluid propulsion. This was done by cannulating the lymphatic at both inflow and outflow ends and by connecting the inflow end to a constant pressure reservoir of saline. All branches were tied off to make sure that the duct was completely isolated from the rest of the lymphatic system. Now, when the lymphatic was kept filled with fluid, intermittent compression was indeed very effective in promoting fluid propulsion. To determine whether effective compressive forces were encountered during normal walking movements we allowed some of the animals that had been set up as described in previous experiments to recover so that we could measure fluid propulsion by this isolated vessel while the animal was walking round the cage. Under these conditions each time the lymphatic contracted there was a phasic increase in outflow pressure and fluid was added to the lever. However when the animal walked up and down the cage no fluid was added to the lever suggesting that walking provides no effective external compression to aid lymph flow1~under the conditions of this experiment. Role of nerves in the control of lymphflow. The evidence presented so far suggests that lymphatic vessels are capable of spontaneous contractions and that lymph flow can be modulated by changes in intraluminal pressure. What other factors modulate lymph flow? An obvious line of inquiry was to look at the way in which nerves might modulate lymphatic activity since the vessels are known to be innervated Structural evidence suggests that lymphatics have a scant noradreuergic innervation as demonstrated by the Falck technique za,~s. The innervation density is so low
The lymphatic circulation 485
Vol, 161 No. g
~E
~E
(..)~
~g
~:=.
minutes
Fig. 3 - Mean flow, white cell count and white cell output from a popliteal efferent lymphatic in an anaesthetized sheep averaged over 10 min periods. Stimulation of the sympathetic chain at 4 Hz increased flow and white cell output. These effects were blockedbyphentolamine(10 p_g/kg/min). Fromreference21 with permission. that it was questionable if these nerves could have any effect in practice. However when isolated segments of bovine mesenteric lymphatic were stimulated by short duration pulses of electrical field stimulation at 0.5 and 1 Hz 16 frequency of spontaneous contractions and flow increased at both stimulation frequencies. These experiments in themselves do not provide unequivocal evidence of the effects ofantonomic nerves since field stimulation of isolated vessels in an organ bath is very different from stimulating sympathetic nerves in the living animal so it was important to demonstrate the consequences of such stimulation. To do this we examined the effect of stimulation of die sympathetic chain on flow from the popliteal efferent lymphatic in the sheep 17. Lymph flow started to increase from its resting value of 30 I.tl/min within 15 sec of the beginning of stimulation (1 Hz) and averaged 48 I.tl/min during the second half of the stimulus period. There was at the same time a marked decrease in hind limb blood flow and this was accompanied by a slight rise in arterial pressure suggesting that the decrease in blood flow was due to vasoconstriction. The effect on lymph flow of stimulation at 10 Hz was rather more drantatic. At this stimulus frequency lymph fl0w increased more than threefold. Thus, " a time when blood was depressed, lymph flow was
increased and this almost certainly implies that the lymphatic vessels were working harder to expel their contents. In other words the sympathetic nervous system can modulate lymh flow in the living animal. The interesting question raised by these observations is: under what circumstances does this actually happen? What kind of stimuli would cause such a response to be evoked? Bilateral occlusion of carotid arteries had the expected increase in arterial pressure but produced no significant increase in lymph flowTM. Cerebral ischaemia, caused by injection of 2 mls of air into the carotid artery, produced a profound sympathetic response but its effect on lymph flow was not in proportion to that on blood pressure tg. The only circumstance in which lymph flow increased dramatically was when the animal was frightened. We discovered this by accident one day. The animal was startled by someone entering the room unexpectedly whereupon, to our surprise, lymph flow increased more than fourfoldz~ We had always thought in terms of cardiovascular physiology which is why we had looked at the effect of carotid occlusion. Of course an important, perhaps the most important function of the lymphatic system is its immune function. So it was of interest to look at the effects of sympathetic stimulation on lymphocyyte output from the node. The popliteal efferent lymphatic was cannulated and the effect of stimulating the sympathetic chain on both flow and on white cell output was examined 21. Fig. 3 shows a summary of six such experiments. You can see that white cell output increased in response to sympathetic stimulation ~md that this response was blocked by phentolamine. The increase in cell output was a result both of an increase in flow and of an increase in cell count. It could be argued however, that the increase in cell output was due simply to the increased flow of lymph through the node, in other words that cells were simply being washed out as a result of the increased flow. This was tested by massaging the foot from which the lymph was coming. This could be expected to havc the same kind of effect as sympathetic stimulation if increased flow were the sole cause of the increased cell output. That this was the case is shown in Fig. 4. It is clear that, whereas foot massage dramatically increased lymph flow, it did not affect white cell count. Conclusions. The evidence currently available suggests that lymph is propelled mainly by the intrinsic contractions of lymphatic vessels and that these can be modulated by 10 min I--I Drops (16 ~tl) White cell count (per mm 3) White cell output (millions/hour)
~
!
Foot massage [
I
f
UIII[II I [
I I
I~
5000 . 0 A
e--
4 ~ 0
~
t ----.----~D
~ -
Fig. 4 - Flow, white cell count and white cell output from a popliteal dfferent lymphatic in an anaesthetized sheep. Foot massage increased lymph flow but did net increase white cell count.
486
sympathetic nerves and by sympathomimetic drugs. It seems unlikely, however, in the light of the results presented here that lymphatic innervation has an important role in cardiovascular pressure and volume regulation since pressor and volume reflexes had so little effect. On the other hand the effect of stress in stimulating lymphatic pumping is a very striking one and might suggest that lymphatic innervation may have a role in the control of lymphocyte circulation. Finally I~would like to thank the Academy for inviting me to give the 1991 Conway lecture and I would like to thank my collaborators: Adair, T. H.; Allen, J. M.; Harty, Helen; Hayashi, A.; IgguldJn, H. L. A.; Johnston, M. G.; McCullough, J. S.; McGeown, J. G.; Roddie, I. C & Thornbury, K. D for their enormous contribution to the work I have just described. I would also like to acknowledge grants from: The Medical Research Council; the Wellcome Trust; DHSS(NI); The British Heart Foundation; The Royal Society'and the Fulbright Commission.
1. 2. 3. 4. 5. 6. 7. 8.
I.LM.S. August. 1992
Mcltale
References Keynes, G. The llfe of WiUiam Harvey. 1961. Clarendon Press, Oxford, p. 312. Florey, H. PhD Thesis, University of Oxford. 1927. Mawhinney, H. J. D. BSc Thesis, The Queen's University of Belfast. 1971. Aselli. De Lacfibus sire [acteis venis. Dissertatin, Mediolani. 1627. Hewson, W. A description of the lymphatic system. Experimental Enquiries Part 11, London, 126, 1774. Heller, A. Uber selbstandige thythmische Contracfionen der Lymphgefasse bei Saugethieren. Centralblt. Med. Wiss. p. 545. Florey, H. Observations on the contractility of lacteals. Part ]L L Physiol 1927: 63, 1-18. McHale, N. G & Roddie, I. C. The effect of transmUral pressure
9.
10.
11.
12.
13. 14.
15.
16.
17.
18. i9. 20. 21.
oil pumping activity in isolated bovine lymphatic vessels. J. Fhysiol 1976: 261, 255-269. McGeown, L G., MclIain, N, G., Roddic, L C & Thooabury, K. D. Peripheral lymphatic responses to outflow pressure in anaesthetisod sheep. J, Physiol 1987: 383, 527-536. Drinker, C. K. and Yoffey, J.M. Lymph flow and lymph pressure. In Lymphatics Lymph and Lymphoid Tisue, Cambridge; Harvard University Press, 1941: 112-145. Courfice, F. C. and Simmoods, W. L Physiological significance of lymph drainage of the serious cavities and lungs. Physiological Reviews 1954: 34, 419-448. White, L C., Field, J. E. and Drinker, C. K. On the protein content and ,1ormal flow of lymph from the foot of the dog. American .Jonmal of Physiology 1933; 103. 34-44-. McGeown, J. G., MeHale, N. G and Thronbury, K. O. The role of extema] compression and movement in lymph propulsion in the sheep hind limb. J. Physinl 1987: 387, 83-93 Todd, G. L. and Bemard, G. R. The sympathetic innervation of the cervical lymph duct of the dog. Anatomical Record 1973: 177,303316. Alessandrini, C., Gerli, R., Sacchi, G., Pucci, A. M. and Fruschelli, C. Cholinergie and adrenergic innervation of mesenterla/ lymph vessels in guinea pig. Lymphology 1981: 14, l-6. McHale, N, G,, Roddie, L C. and Thombury, K. D- Nervous modniation of spontaneous contraetio~as in bovine mesenteric lymphatics. J. Physiof 1980: 309, 461-472. McGeown, J. G., McHale, N. G. & Thombury, K. D The effect of electrical stimulation of the sympathetic chain on popliteal lymph flow in the anaesthetlsed sheep. J. Physiol 1987: 393, 123133, Thombury, K. D. Regulation of lymph flow in the sheep. FhD Thesis 1987. The Queen's University of Belfast, 1987. McHale, N. G and Adair, T. H Reflex modulation of lymphatic pumping in sheep. Circ. Res. 1989: 64, 1165-1171. McHale, N. G and Roddie, I. C, The effect of intravenous adrenaline and noradrenaline infusion on peripheral flow in the sheep. 3. Physiol 1983: 341, 517-526. McHale, N. G. and Thornbury, K. D. Sympathetic stimulation causes increased output of lymphocytes from the popliteal node in anaesthetized sheep. Exp. Physiol. 1990: 847-850.
School of Biomedical Science, The Queen's University of Belfast, 97 Lisburn Road, Belfast BT9 7BL. The lymphatic system represents a formidable challenge to a potential investigator and one that I am sure I would never have taken up had I had a free choice. Why, then, did I study thetymphatic circulation? Was it because I was inspired by~ the works of William Harvey who, having demonsfi'ated the circulation of blood, was invited~to study the lymphtic circulation'? He declined to do so in the following statement'. "It is useless for you to spur me on to gird myself to any new investigation, seeing that I am now not only ripe in years but also weary.:. R Seems to me indeed that I am entitled to ask for my honourable discharge from duty". Was it that I was keen to take up the guantlet that Harvey was too weary to lift. The answer is "No, it was not!" Was it then that a desire to alleviate human suffering such as that experienced by people afflicted with lymphoedema? And the answer again was "No, it was not!" Was it then the inspiring work by Howard Florey 2 on the lymphatic system which constituted his PhD thesis, published in 1927, the title of which was "Philosophy and Pathology of the circulation of the blood and lymph"? And the answer is "No, it was not, for two reasons: firstly, at that time I had not read his PhD thesis, and secondly, my experience of the lymphatic system up to that point was greatly influenced by a colleague of mine, Denis Mawhinney3, who was sharing a laboratory with me when we were doing our BSc projects. Dennis worked on isolated bovine mesenteric vessels and found that study so difficult that I swore that one thing I would never study, given any choice, was the lymphatic system. However, a year later when I went to discuss my PhD project with my supervisor, Ian Roddie, he offered me a choice of projects. He said I could study lymphatics or nothing! Faced with this "offer that I couldn't refuse" I eudeavoured to look at the lymphatic system in a more positive way. I began to see that here was a system about which so little was known that a whole field that could be opened up if one put in some effort and determination. My project was specifically to establish whether lymph ducts could pump fluid by means of their intrinsic contractions. Much evidence suggested that this was likely. Gasper Aselli4 in 1622 noticed that lacteals became empty after death, implying that they were contractile. Hewson5in 1769 observed directly that lacteals could contract while Heller~ in 1869 described rhythmic contractions of lacteals bat was unable to establish whether or not the contractions were an indirect consequence of gut peristalsis. Flurey 7 in 1927, confirmed that lacteals in the guinea pig could contract spontaneously, and that this activity continued when the vessels were excised from the mesentery. However Florey did not consider that spontaneous eontractity of lymphatics
was universal and he was sceptical about its importance in lymph flow. In his own words: "Under physiological conditions in the cat, therefore, it may be asserted that the forces moving lymph are a v i s a tergo presumably supplied by the blood pressure, squeezing due to gut peristalsis, and a respiratory suction pump mechanism..... There is no evidence that the lacteals in a normal condition farther the passage of lymph by rhythmic contractions4. '' The question with which I was presented at the outset of my studies was: "Do lymphatic vessels contract in such a way that they can propel fluid, and secondly if they are capable of propelling fluid in this way, what is the relevance of this to the function of the lymphatic system"? Pumping in isolated lymphatic vessels. In order to answer the first question, it was thought appropriate to look at isolated vessels first of all, because this removed the complicating effects of extrinsic forces. The vessel chosen was the bovine mesenteric lymph duct since this was large and robust enough to be studied in i~lation. An 8 cm length of duct was dissected free of the mesentery, canndlated at both ends and set up in a horizontal organ bathL The inflow end was connected to a constant pressure reservoir of Krebs solution, while the outflow was connected to a drop counter at the same height. Transmaral pressure could be varied by raising or lowering both reservoir and outflow together. Measurements were made of inflow pressure, outflow pressure and flow. The lymphatic contracted in a very regular, rhythmic fashion. Each contraction resulted in a phasic increase in outflow pressure and caused a series of drops to be expelled through the drop counter. This was followed by a pause until the next contraction expelled a further series of drops. Fig 1 shows the effect of changing transmural pressure from 0 to 21 cm H20. Flow increased with increasing transmural pressure up to a maximum of 8 cm H20 caused flow to decline due to a decrease in stroke volume and despite a continued increase in frequency of contraction.
Contribution of intrinsic pumping to lymph flow in the living animal. The above experiments certainly demonstrate that the lymphatic vessel is capable of contracting in isolation, and that these contractions are capable of propelling fluid under conditions where no extrinsic forces could be having any effect. The next question however was: what is the relevance of this to the living animal? In order to investigate this the sheep was chosen as a model. An afferent lymphatic vessel was cannulated in the metatarsal region9. This was connected to a pressure transducer to measure side arm pressure and lymph flow was measured by allowing fluid leaving the cannula to accumulate on the lever of an isometric tension transducer. Each time the lymphatic contracted there was a phasic increase in outflow
"1991 Conway Review Lecture 483
I.J.M.S. August, 1992
484 McHale
lo[L /'. . . . . . . . . . . . . . . %/x ~ A / A
Fmmwcy (omltractions/min)
'I/ OL
~mdko wkJlle Irnl)
/ o-o'~ ~
0.1 0"05
~/~
\o \
o~
0 1"C Flow (rnl/mm)
0"~"
e'l
/
\~
\e
Tran~nural Pr~sum(cmH~) Fig. I - Plotted results of flow, stroke volume and hequez~y of contraction in an experiment where uansmural pressure was increased from 0 to 21 cm H20.
pressure followed by an accumulation of lymph on the transducer lever. This was repeated with each subsequent contraction unttl enouk~@mph had accumulated to form a drop which then fell off.' The drops were of approximately equal size so the slope of the ramp is an index of lymph flow using this technique. One way of determining whether we are looking at an active pump, or an essentially passive process, is to ask the vessel to do some work. This was done by raising the height of the outflow cannula. Between 0 and 30 cm flow remained fairly steady so more work was being doric to drive the same amount of fluid to'a greater height. This was achieved by an increase in frequency of contraction and it is interesting to note that when frequency of contrtaction began to level off (at about 30 cm) flow began to fall off. In other words the lymphatic was working harder by increasing its frequency of contraction, maintaining flow even though there was a decrease in stroke volume.
el) Over Hoof Flow (ul rain-I}
70 60 50
b) Over Metotarsal
~
2on,2off
3O 20 '~
I
t__l
Fig. 2 - Summary of seven experiments in anaesthetized sheep an the effect on flow of intenninem compression (2 see on, 2 see off) was applied either over the hoof or oyer the metatarsal region. (From reference 13 with permission).
Contribution of extrinsic forces to lymph propulsion. One of the reasons why extrinsic forces were thought to be important in the propulsion of lymph is that it has been well known for a very long time that movement increases lymph flow~~ To examine this the metarsal lymphatic was cannulated as before, the animal allowed to recover and measurements were made while the conscious animal was standing in a metabolism caget3. When the animal walked the length of the cage and back again, lymph flow doubled. Observations such as these were, in the past, taken to indicate that contraction of muscles or external compression of lymphatic vessels were largely responsible for lymph propusion. To examine this belief in greater detail the metatarsal lymphatic was cannulated as before and pneumatic cuffs were fitted over both the metatarsal region (so to compress the duct itself) and over the hoof (its drainage hinterland). Fig. 2 shows a summary of seven experiments where intermittent compression at a frequency of 2 seconds on, 2 seconds off, was applied either over the hoof or over the metatarsal region. Compression applied over the hoof dramatically increased lymph flow, but, surprisingly, that applied over the metatarsal region did not significantly increase lymph flow. We took this to mean that the lymphatic was almost empty when the intrinsic pump is working normally. One way of testing this was to control fluid input to the lymphatic and thus ensure that it was always full. External compression could then be expected to greatly enhance fluid propulsion. This was done by cannulating the lymphatic at both inflow and outflow ends and by connecting the inflow end to a constant pressure reservoir of saline. All branches were tied off to make sure that the duct was completely isolated from the rest of the lymphatic system. Now, when the lymphatic was kept filled with fluid, intermittent compression was indeed very effective in promoting fluid propulsion. To determine whether effective compressive forces were encountered during normal walking movements we allowed some of the animals that had been set up as described in previous experiments to recover so that we could measure fluid propulsion by this isolated vessel while the animal was walking round the cage. Under these conditions each time the lymphatic contracted there was a phasic increase in outflow pressure and fluid was added to the lever. However when the animal walked up and down the cage no fluid was added to the lever suggesting that walking provides no effective external compression to aid lymph flow1~under the conditions of this experiment. Role of nerves in the control of lymphflow. The evidence presented so far suggests that lymphatic vessels are capable of spontaneous contractions and that lymph flow can be modulated by changes in intraluminal pressure. What other factors modulate lymph flow? An obvious line of inquiry was to look at the way in which nerves might modulate lymphatic activity since the vessels are known to be innervated Structural evidence suggests that lymphatics have a scant noradreuergic innervation as demonstrated by the Falck technique za,~s. The innervation density is so low
The lymphatic circulation 485
Vol, 161 No. g
~E
~E
(..)~
~g
~:=.
minutes
Fig. 3 - Mean flow, white cell count and white cell output from a popliteal efferent lymphatic in an anaesthetized sheep averaged over 10 min periods. Stimulation of the sympathetic chain at 4 Hz increased flow and white cell output. These effects were blockedbyphentolamine(10 p_g/kg/min). Fromreference21 with permission. that it was questionable if these nerves could have any effect in practice. However when isolated segments of bovine mesenteric lymphatic were stimulated by short duration pulses of electrical field stimulation at 0.5 and 1 Hz 16 frequency of spontaneous contractions and flow increased at both stimulation frequencies. These experiments in themselves do not provide unequivocal evidence of the effects ofantonomic nerves since field stimulation of isolated vessels in an organ bath is very different from stimulating sympathetic nerves in the living animal so it was important to demonstrate the consequences of such stimulation. To do this we examined the effect of stimulation of die sympathetic chain on flow from the popliteal efferent lymphatic in the sheep 17. Lymph flow started to increase from its resting value of 30 I.tl/min within 15 sec of the beginning of stimulation (1 Hz) and averaged 48 I.tl/min during the second half of the stimulus period. There was at the same time a marked decrease in hind limb blood flow and this was accompanied by a slight rise in arterial pressure suggesting that the decrease in blood flow was due to vasoconstriction. The effect on lymph flow of stimulation at 10 Hz was rather more drantatic. At this stimulus frequency lymph fl0w increased more than threefold. Thus, " a time when blood was depressed, lymph flow was
increased and this almost certainly implies that the lymphatic vessels were working harder to expel their contents. In other words the sympathetic nervous system can modulate lymh flow in the living animal. The interesting question raised by these observations is: under what circumstances does this actually happen? What kind of stimuli would cause such a response to be evoked? Bilateral occlusion of carotid arteries had the expected increase in arterial pressure but produced no significant increase in lymph flowTM. Cerebral ischaemia, caused by injection of 2 mls of air into the carotid artery, produced a profound sympathetic response but its effect on lymph flow was not in proportion to that on blood pressure tg. The only circumstance in which lymph flow increased dramatically was when the animal was frightened. We discovered this by accident one day. The animal was startled by someone entering the room unexpectedly whereupon, to our surprise, lymph flow increased more than fourfoldz~ We had always thought in terms of cardiovascular physiology which is why we had looked at the effect of carotid occlusion. Of course an important, perhaps the most important function of the lymphatic system is its immune function. So it was of interest to look at the effects of sympathetic stimulation on lymphocyyte output from the node. The popliteal efferent lymphatic was cannulated and the effect of stimulating the sympathetic chain on both flow and on white cell output was examined 21. Fig. 3 shows a summary of six such experiments. You can see that white cell output increased in response to sympathetic stimulation ~md that this response was blocked by phentolamine. The increase in cell output was a result both of an increase in flow and of an increase in cell count. It could be argued however, that the increase in cell output was due simply to the increased flow of lymph through the node, in other words that cells were simply being washed out as a result of the increased flow. This was tested by massaging the foot from which the lymph was coming. This could be expected to havc the same kind of effect as sympathetic stimulation if increased flow were the sole cause of the increased cell output. That this was the case is shown in Fig. 4. It is clear that, whereas foot massage dramatically increased lymph flow, it did not affect white cell count. Conclusions. The evidence currently available suggests that lymph is propelled mainly by the intrinsic contractions of lymphatic vessels and that these can be modulated by 10 min I--I Drops (16 ~tl) White cell count (per mm 3) White cell output (millions/hour)
~
!
Foot massage [
I
f
UIII[II I [
I I
I~
5000 . 0 A
e--
4 ~ 0
~
t ----.----~D
~ -
Fig. 4 - Flow, white cell count and white cell output from a popliteal dfferent lymphatic in an anaesthetized sheep. Foot massage increased lymph flow but did net increase white cell count.
486
sympathetic nerves and by sympathomimetic drugs. It seems unlikely, however, in the light of the results presented here that lymphatic innervation has an important role in cardiovascular pressure and volume regulation since pressor and volume reflexes had so little effect. On the other hand the effect of stress in stimulating lymphatic pumping is a very striking one and might suggest that lymphatic innervation may have a role in the control of lymphocyte circulation. Finally I~would like to thank the Academy for inviting me to give the 1991 Conway lecture and I would like to thank my collaborators: Adair, T. H.; Allen, J. M.; Harty, Helen; Hayashi, A.; IgguldJn, H. L. A.; Johnston, M. G.; McCullough, J. S.; McGeown, J. G.; Roddie, I. C & Thornbury, K. D for their enormous contribution to the work I have just described. I would also like to acknowledge grants from: The Medical Research Council; the Wellcome Trust; DHSS(NI); The British Heart Foundation; The Royal Society'and the Fulbright Commission.
1. 2. 3. 4. 5. 6. 7. 8.
I.LM.S. August. 1992
Mcltale
References Keynes, G. The llfe of WiUiam Harvey. 1961. Clarendon Press, Oxford, p. 312. Florey, H. PhD Thesis, University of Oxford. 1927. Mawhinney, H. J. D. BSc Thesis, The Queen's University of Belfast. 1971. Aselli. De Lacfibus sire [acteis venis. Dissertatin, Mediolani. 1627. Hewson, W. A description of the lymphatic system. Experimental Enquiries Part 11, London, 126, 1774. Heller, A. Uber selbstandige thythmische Contracfionen der Lymphgefasse bei Saugethieren. Centralblt. Med. Wiss. p. 545. Florey, H. Observations on the contractility of lacteals. Part ]L L Physiol 1927: 63, 1-18. McHale, N. G & Roddie, I. C. The effect of transmUral pressure
9.
10.
11.
12.
13. 14.
15.
16.
17.
18. i9. 20. 21.
oil pumping activity in isolated bovine lymphatic vessels. J. Fhysiol 1976: 261, 255-269. McGeown, L G., MclIain, N, G., Roddic, L C & Thooabury, K. D. Peripheral lymphatic responses to outflow pressure in anaesthetisod sheep. J, Physiol 1987: 383, 527-536. Drinker, C. K. and Yoffey, J.M. Lymph flow and lymph pressure. In Lymphatics Lymph and Lymphoid Tisue, Cambridge; Harvard University Press, 1941: 112-145. Courfice, F. C. and Simmoods, W. L Physiological significance of lymph drainage of the serious cavities and lungs. Physiological Reviews 1954: 34, 419-448. White, L C., Field, J. E. and Drinker, C. K. On the protein content and ,1ormal flow of lymph from the foot of the dog. American .Jonmal of Physiology 1933; 103. 34-44-. McGeown, J. G., MeHale, N. G and Thronbury, K. O. The role of extema] compression and movement in lymph propulsion in the sheep hind limb. J. Physinl 1987: 387, 83-93 Todd, G. L. and Bemard, G. R. The sympathetic innervation of the cervical lymph duct of the dog. Anatomical Record 1973: 177,303316. Alessandrini, C., Gerli, R., Sacchi, G., Pucci, A. M. and Fruschelli, C. Cholinergie and adrenergic innervation of mesenterla/ lymph vessels in guinea pig. Lymphology 1981: 14, l-6. McHale, N, G,, Roddie, L C. and Thombury, K. D- Nervous modniation of spontaneous contraetio~as in bovine mesenteric lymphatics. J. Physiof 1980: 309, 461-472. McGeown, J. G., McHale, N. G. & Thombury, K. D The effect of electrical stimulation of the sympathetic chain on popliteal lymph flow in the anaesthetlsed sheep. J. Physiol 1987: 393, 123133, Thombury, K. D. Regulation of lymph flow in the sheep. FhD Thesis 1987. The Queen's University of Belfast, 1987. McHale, N. G and Adair, T. H Reflex modulation of lymphatic pumping in sheep. Circ. Res. 1989: 64, 1165-1171. McHale, N. G and Roddie, I. C, The effect of intravenous adrenaline and noradrenaline infusion on peripheral flow in the sheep. 3. Physiol 1983: 341, 517-526. McHale, N. G. and Thornbury, K. D. Sympathetic stimulation causes increased output of lymphocytes from the popliteal node in anaesthetized sheep. Exp. Physiol. 1990: 847-850.
