J. Physiol. (1987). 393, pp. 123-133 123With 7 text-figuresPrinted in Great Britain

THE EFFECT OF ELECTRICAL STIMULATION OF THESYMPATHETIC CHAIN ON PERIPHERAL LYMPH FLOW IN

THE ANAESTHETIZED SHEEP

BY J. G. MeGEOWN, N. G. McHALE AND K. D. THORNBURY*From the Department of Physiology, The Queen's University of Belfast,

97 Lisburn Road, Belfast BT9 7BL

(Received 5 March 1987)

SUMMARY

1. Pressure fluctuations and lymph flow were measured in cannulated poplitealefferent and metatarsal afferent lymphatics in anaesthetized sheep.

2. Stimulation of the lumbar sympathetic chain at 1, 4 and 10 Hz increased lymphflow and lymphatic contraction frequency. These effects were most marked during10 Hz stimulation where, in some efferent preparations, greater than 5-fold increasesin lymph flow and contraction frequency were observed.

3. Strain-gauge plethysmograph recordings in the lymphatic's drainage areaindicated that during stimulation blood flow was reduced. There was also a slowcontinuous fall in tissue volume throughout the periods of stimulation, presumablydue to a net uptake of fluid by the blood vessels.

4. Intra-arterial infusion of phentolamine at a rate of 10 gg kg-' min- blocked theincreases in lymph flow and contraction frequency.

5. These results suggest that the observed increases in lymph flow were due todirect neurogenic stimulation of lymphatic pumping.

INTRODUCTION

There is histological (Alessandrini, Gerli, Sacchi, Ibba, Pucci & Fruschelli, 1981)and pharmacological evidence (Russel, Zimmerman & Middendorf, 1980; McHale,Roddie & Thornbury, 1980) that lymphatic vessels receive an adrenergic innervation,but so far there have been surprisingly few attempts to investigate the role of thesenerves in vivo. Most previous workers have started with the assumption that lymphis mainly propelled by extrinsic forces (generated outside the lymphatic system) withthe lymphatics offering a variable resistance to flow. Consequently they haveattempted to demonstrate changes in the calibre or tone of the lymphatics duringnerve stimulation, often in the absence of lymph flow measurements (Acevedo, 1943;Rusznyak, Fbldi & Szabo, 1950; Browse, 1968). However, lymphatic vessels in manyspecies including man are spontaneously active, and there is much evidence tosuggest that this represents an important intrinsic mechanism for pumping lymph(Hall, Morris & Woolley, 1965; Campbell & Heath, 1973; Olszewski & Engeset, 1985;McGeown, McHale & Thornbury, 1987 a). In view of the increases in contraction

* To whom correspondence should be sent.

J. a. McGEOWN, N. G. McHALE AND K. D. THORNBURY

frequency observed in mesenteric lymphatics during nerve stimulation (Florey,1927; McHale et al. 1980) it seems possible that the role of the innervation could beto modulate the activity of the intrinsic pump. This is further suggested by the factthat infusions of noradrenaline increase lymph flow and contraction frequency inpopliteal efferent lymphatics of the sheep (McHale & Roddie, 1983). However, directevidence is lacking since there have been no previous attempts to systematicallystudy the effect of sympathetic nerve stimulation on lymph flow. The purpose of thepresent study was to provide such evidence. Here we describe the changes in sheeppopliteal efferent and metatarsal afferent lymph flow and contraction frequencywhich occur in response to lumbar sympathetic chain stimulation. If, as suggestedabove, these vessels receive an adrenergic innervation which is able to increasepumping, one might expect the effect of sympathetic chain stimulation to resemblethat of infusing noradrenaline.A short account of this work has been communicated to the Physiological Society

(McGeown, McHale & Thornbury, 1987b).

METHODS

Ewes weighing 40-60 kg were anaesthetized with pentobarbitone (20-30 mg/kg, I.v.) andspontaneously breathed a mixture of 1-3 % halothane in 02 throughout the experiments. 2 ml ofEvan's Blue dye (1 % in 0-9% saline) were injected into the footpad to outline the lymphatics ofthe hindlimb. In each preparation a single lymphatic vessel was cannulated, against the directionof flow, with Polythene tubing (Portex, i.d. 0-4 or 0-5 mm). This was either an afferent in themetatarsal region of an efferent 5-10 cm from the popliteal node.Lymph flow and contraction frequency were measured as described by McHale & Roddie

(1983 a). Each drop of lymph was collected on a small piece of filter paper attached to the lever ofan isometric tension transducer (Statham UC3). Thus the drop was weighed as it formed, afterwhich it fell off the lever resetting the transducer output voltage for the next drop. Lymphaticcontraction frequency was measured by recording side-arm pressure from the lymphatic cannulawith a Statham P23 transducer.

In most experiments tissue volume was estimated using a Vasculab SPG 16 strain-guageplethysmograph. Strain gauges were attached below the fetlock (over the main site drained by thelymphatic) on the same side as the cannulated lymphatic and on the opposite hindlimb which servedas a control. The percentage change in tissue volume was calculated as twice the percentage changein tissue circumference assuming an approximately cylindrical shape (Greenfield, Whitney &Mowbray, 1963). To provide an estimate of blood flow pneumatic cuffs were placed above the hocksand (with the exception of the example shown in Fig. 3) inflated to between 45 and 60 mmHg ina3 s on, 10 s off cycle. This was found to have little effect on the resting rate of lymph flow. Theshort inflation period was chosen because the rate of increase in tissue volume was found to beapproximately linear during this initial phase. Thus the height of the plethysmograph ramp was

proportional to the gradient, allowing changes in blood flow to be demonstrated at the slow chartspeeds suitable for recording lymph flow. The validity of this procedure was tested in a series of fiveexperiments at fast paper speeds where the height was found to be closely correlated with the slope(r in the range 0-94-0-99; P < 0-001 in all cases). Arterial pressure was measured via a cannula inthe carotid artery using a Hewlett-Packard 78200 system. Recordings were made on Gould 4200sand 8600s pen recorders. Access to the left lumbar sympathetic chain was gained through a

paravertebral incision. The chain was then tied and cut and the peripheral end of the cut nerve

enclosed within a fluid electrode similar to that described by Barnes, Bower & Rink (1980). Theelectrode consisted of a plastic tube (2 cm long, diameter 3 mm) containing two platinum rings4 mm apart with the distal ring acting as the cathode. The distal end of the tube was covered witha latex rubber membrane (through which the nerve was threaded) and the proximal end was sealedwith a screw-cap after filling the tube with 0 9 % saline via a side-arm connection. The total volumeof the system was approximately1*5 ml. Square-wave pulses (25 V, nominal; 0-2 ms duration) were

124

SYMPATHETIC NERVES AND LYMPH FLOW

delivered from a Grass S88 stimulator at constant frequencies of 1, 4 or 10 Hz for periods of5-20 min. The femoral artery on the side of the lymphatic to be studied was cannulated via a sidebranch to allow for the administration of phentolamine (Rogitine, Ciba). This was diluted to theappropriate concentration in 09% saline and infused at a rate of 1 ml/min. Where stated,statistical probability values were obtained using Student's paired t test to compare the meanlymph flow during the 5 min stimulation period with that of the preceding 5 min.

RESULTS

Popliteal efferent lymphaticsAll of the preparations studied demonstrated the characteristic pulsatile lymph

flow associated with lymphatic contractions as described previously (McHale &

5 min

I115[ __ Arterial

E_ 751pressure

[p*''LUUIU Change inL/i V FI~l tissue volume

in3 s

<iK /. / / Lymphoutput

0Lymphatic

E Al. [s, . outflowpressu re

1 Hz l10 HZl

Fig. 1. The effect ofstimulating the left sympathetic chain at 1 and 10 Hz for 5 min periods(indicated by arrows). The records are of arterial pressure (top), left hindlimb blood flow(second from top), left popliteal efferent lymph flow (second from bottom) and lymphaticcontraction frequency (bottom).

Roddie, 1983a). Figure 1 shows a sample record of this activity. The steps on thelymph output record (second trace from bottom) are caused by small amounts oflymph being expelled onto the lever of the tension transducer. Eventually these aresufficient to cause a drop of lymph to fall off the lever, thus resetting the record. Thelymphatic contraction frequency can be estimated from the phasic increasesobserved in the lymphatic outflow pressure (bottom trace). Also shown is arterialpressure (top trace) and hindlimb tissue volume (second from top). Blood flow canbe estimated from the increases in tissue volume during 3 s periods of venousocclusion while changes in vascular volume and fluid exchange may be inferred fromthe slope of the baseline. During the resting period arterial pressure, lymph flow andblood flow were fairly constant. The left sympathetic chain was then stimulated ata frequency of 1 Hz for the 5 min period indicated by the arrows. Lymph flow startedto increase from its resting value of 30 ,td/min within 30 s of the beginning of

125

J. G. McGEOWN, N. S. McHALE AND K. D. THORNBURY

stimulation and averaged 48 #I/min during the second half of the stimulus period. Inthis case contraction frequency did not change much, with most of the increase inflow being accounted for by an increase in the quantity of lymph ejected per stroke(as indicated by the larger steps on the lymph output record). During stimulationthere was a marked reduction in left hindlimb blood flow which was accompanied by

1 Hz 4 Hz 10 Hz

12 m.2 T Tr T.*

8u-Stt >k1|S 9 T T/*AT T T4*'ok~~T X T T A0

* * * * ± *- *i * j. I .

70.

EC50 1-T

> T~TII TTT/ T1IT

10115 15 25 5 15 25 5 15 25

Time (min)

Fig. 2. Summary of the effect of sympathetic chain stimulation on lymph flow (lowerpanel) and contraction frequency (upper panel) in eight popliteal efferent preparations.Flow and contraction frequency were averaged over successive 2-5 min periods in eachpreparation. The circles and vertical bars represent the mean and S.E. of mean, re-spectively, of the eight preparations. The rectangles at the top of the Figure represent 5min periods of stimulation.

a slight rise in arterial pressure thus indicating that the decrease in blood flow wasdue to vasoconstriction. The baseline of the tissue volume record showed an initialrapid decrease followed by a slower continuous decrease. These records are similar totissue volume recordings of the cat hindlimb obtained by Mellander (1960) whoshowed that the initial phase was caused by a decrease in vascular capacitance(venoconstriction) while the slow phase was due to fluid moving from the tissues intothe capillaries. On cessation of the stimulus blood flow and arterial pressure quicklyreturned to control levels while lymph flow dropped slightly below its previousresting value. The right-hand panel of Fig. 1 shows the effect of stimulating the samepreparation at 10 Hz. In this case there was a greater than 3-fold increase in lymphflow associated with an increase in contraction frequency. Mean arterial pressureincreased by approximately 8 mmHg while blood flow was reduced to values belowthe sensitivity of the recording system. As before, there was a continuous decreasein tissue volume throughout the stimulation period. In this preparation, as in themajority of those studied, no visible skeletal muscle twitching or associatedmovement occurred. Thus the increased lymph flow cannot be attributed to

126

SYMPATHETIC NERVES AND LYMPH FLOW 127

5 min

1 %[ _ Change

volumein 1 s

30 ,ul[ , ifLymphoutput

Lymphatic2 cmH2O[ AdAbLiL.JLkL<LJJiWL_,LLhU1k I outflowpressure

4 Hz

Fig. 3. The effect of a longer period of stimulation on the time course of the lymphatic andvascular responses. Shown are left hindlimb blood flow (top record), left efferent lymphflow (middle record) and lymphatic contraction frequency (bottom record). The arrowsindicate a 15 min period during which the left sympathetic chain was stimulated at 4 Hz.

5 minPhentolamine

I 105 _ ArterialE 60[ _ pressure

30,11/JIIII Il/ll7X///r <Lymphoutput

25cmH2O[ J d ~ j U 4 zI~zl& l.Mls~l~iplQ~C LymphaticI ~~ 1~~fI~i~~ '~~USJ~~5~IU U~~ ~ ~ aa~ outflow

pressure

10 Hz 1t Hz

Fig. 4. The effect of stimulating the sympathetic chain at 10 Hz on arterial pressure (toprecord), popliteal efferent lymph flow (middle record) and lymphatic contraction fre-quency (bottom record), both before and 15 min after the beginning of phentolamineinfusion to the femoral artery at a rate of 10 Fsg kg-' min-. Phentolamine was presentthroughout the period indicated by the arrow at the top of the Figure.

movement and is unlikely to be related to external compression of the lymphaticvessels by skeletal muscles. Curiously, even when skeletal muscle contraction wasallowed to occur (e.g. if non-insulated electrodes were used) there appeared to be verylittle difference in the lymph flow response.The effect of stimulating the sympathetic chain for 5 min periods at frequencies of

1, 4 and 10 Hz was examined in eight preparations. The order was varied frompreparation to preparation and at least 10 min was allowed for recovery betweenstimulation periods. These results are summarized in Fig. 2. In each experimentlymph flow and contraction frequency were averaged over successive 2-5 min periods.

J. G. McGEOWN, N. G. McHALE AND K. D. THORNBURY

The filled circles represent the mean values of the eight preparations over each 2-5min block, while the vertical bars represent 1 S.E. of mean. The Figure shows thatlymph flow increased from 21+6 (mean+s.E. of mean) to 26+7 /tl/min at 1 Hz,from 22 + 7 to 40+134I/min at 4 Hz and from 19+8 to 50+22jd/min at 10 Hz.The increases during stimulation at 4 and 10 Hz were statistically significant

10 Hz 10 Hz

> 16

E 80 T

C

C T T~ ~ T

E400 -1 10a:1

0 .0 .-0'~~ ~~~~Tm

C0C. 4-

100-

80 1/-

0. T

±A r20 11111 ,jL i -*=

5 15 25 5 15 25Time (min)

Fig. 5. Summary of the effect of intra-arterial infusion of phentolamine (10/,Igkg-1min') on the popliteal efferent response to 5 min periods of sympathetic chain stimulationat 10 Hz. The method of presentation is similar to Fig. 2. Filled circles indicate the meansof five preparations which received a first stimulus before (left-hand panel), and a secondduring, phentolamine infusion (right-hand panel). Open circles represent the means of fivecontrol preparations where the stimulus was applied in the absence of blocker on bothoccasions. Vertical bars represent + S.E. of mean.

(P < 0 05). Contraction frequency increased from 5-9 + 1-4 to 6-5 + 1-3 beats/min at1 Hz, from 6-8 + 1-5 to 8-1 + 1-7 beats/min at 4 Hz and from 5-2 + 1-5 to 9-1 + 1-7beats/min at 10 Hz. Thus contraction frequency did not keep pace with lymph flow,implying that an increase in the mean volume of lymph ejected per stroke was partlyresponsible for the increased lymph flow. Indeed this could be clearly seen to be thecase in some of the individual records where the size of the steps on the lymph outputramps became larger during stimulation.The reductions in blood flow during stimulation were estimated from the heights of

the plethysmograph ramps. These were expressed as a percentage of the maximumvalue recorded during the 5 min period before stimulation in each case. On the left-hand side, blood flow decreased to 19+99% at 1 Hz, 8± 6% at 4 Hz and 3+ 3 % at

128

SYMPATHETIC NERVES AND LYMPH FLOW

10 Hz (n = 8) while on the right (where the chain was not stimulated) blood flowsometimes increased or decreased slightly but the mean values were not significantlydifferent from control (99+ 6% at 1 Hz, 100+ 8% at 4 Hz and 95+ 8% at 10 Hz,n = 5). The rate of the slow phase of decline in baseline tissue volume duringstimulation was 0-1+0-02%/min at 1 Hz, 0-16 +0-06%/min at 4Hz and0-22 + 0-1 %/min at 10 Hz (n = 8).

In several experiments the time course of the vascular and lymphatic responseswere compared when the stimulus was applied for periods of 15-20 min. Figure 3shows an example. During the control period lymph flow and blood flow were fairlyconstant. The sympathetic chain was then stimulated at a frequency of 4 Hz for15 min. Blood flow fell almost immediately after the beginning of stimulation, whilelymph flow and contraction frequency began to increase approximately 1 min later.Lymph flow reached a peak after about 5 min and then declined so that after 10 minit had returned to control level again. The vascular response, however, was wellmaintained throughout the entire period of stimulation.The effect of a-blockade. Figure 4 shows a typical record of the effect of the a-

antagonist phentolamine on the lymphatic responses to 10 Hz. Before phentolamine,5 min of stimulation at 10 Hz increased lymph flow from 15 to 75 ,tl/min, anapproximate 500% increase while arterial pressure rose from 94/60 to 99/65mmHg. After recovery, phentolamine was infused into the left femoral artery at arate of 10 jug kg-' min-. Fifteen minutes later arterial pressure had fallen to 60/38 mmHg while lymph flow decreased to 6 jsl/min. When the 10 Hz stimulation wasrepeated lymph flow increased to 10 #sl/min, an increase of less than 70%, whilearterial pressure showed little change. This protocol was repeated in five experiments,the results of which are represented by the filled circles in Fig. 5. The method ofsummary is similar to that used in Fig. 3, with the circles and vertical barsrepresenting the mean values and S.E. of mean respectively, during successive 2-5min periods. The left-hand panel (filled circles) shows the effect of stimulating the fivepreparations before the addition of phentolamine. This had the effect of increasinglymph flow from 28+ 6,l/min to a peak of 56+ 9 jcl/min during stimulation(P < 0-05). Frequency increased from 7.3 + 0-9 to 10-2 + 2-2 beats/min. The right-hand panel shows the effect of repeating the stimulus 15 min after the beginning ofphentolamine infusion. Basal flow had now fallen to 20 + 6 #Il/min and duringstimulation this rose slightly to 24+ 8 ,ul/min (not significant). Contraction frequencyincreased slightly from 6-2 + 0-6 to 7-5 + 1 beats/min. To exclude the possibility thatthe decreased reponses after phentolamine could have been due to factors other thana-blockade, such as desensitization or deterioration of the preparations, the sameprotocol was followed in the absence of blocker in five control animals. The meanlymph flow values of these are represented by the open circles in the lower panel ofFig. 5. In this case the lymph flow increased significantly (P < 005) during bothstimulus periods, although the peak lymph flow was slightly lower on the secondoccasion. The open circles in the upper panel of Fig. 5 represent the mean frequencycounts of four of the five preparations (the 5th was rejected because of difficulty incounting contractions due to a high resting frequency). Frequency increased from8-6 + 1-8 to 13-7 + 2-7 beats/min during the first period of stimulation and from8-6 + 2-4 to 12-9 + 2-7 beats/min during the second period.

PHY 393

129

130 J. G. McGEOWN, N. G. McHALE AND K. D. THORNBURY

2 minChange

111*in tissuevolume

L ~~~~~~in3s

--Jr _ A output

r 1111111 III fl ~~~~~~~~~~Lymphatic5cmH2Ol I L outflow

10 Hz

Fig. 6. The effect of stimulating the left sympathetic chain on left hindlimb blood flow(top), left metatarsal afferent lymph flow (middle) and lymphatic contraction frequency(bottom).

10 Hz

oX 6[ ~ Z II

&i, 4-o2 T

20

20 100~~~~

55 10 15 20

Time (min)Fig. 7. Summary of the effect of sympathetic chain stimulation on lymph flow (lowerpanel) and contraction frequency (upper panel) in six metatarsal afferent preparations.The method of presentation is similar to Fig. 2.

Metatarsal afferent lymphaticsThe effect of stimulation at 10 Hz was also examined in six afferent preparations.

In the example shown in Fig. 6, lymph flow and contraction frequency almostdoubled during the first 2 min of stimulation, thus demonstrating a responsequalitatively similar to that seen in efferents. However, the afferent effect was

SYMPATHETIC NERVES AND LYMPH FLOW

usually of a relatively short duration and as this example shows, flow often returnedto near the control value before the end of the 5 min stimulation period. A summaryof the effect of stimulating the six preparations for 5 min is presented in Fig. 7.Lymph flow increased from 10-7+ 2,I/min before to a peak of 16-3+ 3-6 #I/minduring the first 2-5 min stimulation. Mean lymph flow during the 5 min of stimulationwas significantly (P < 0-05) greater than that of the previous 5 min.

In four preparations the effect of stimulating at 10 Hz for 20 min was studied. Inall cases lymph flow increased initially and then declined continuously until, by theend of the stimulus period, it had fallen to less than 50% of control.

DISCUSSION

In the present study stimulation of the sympathetic chain increased lymph flowand lymphatic contraction frequency in sheep hindlimb lymphatics. The mechanismof this response is not clear but the results would appear to be consistent with a directneurogenic stimulation of lymphatic pumping since in many respects the lymphaticresponses are similar to those observed in isolated vessels. Isolated segments ofbovine mesenteric lymphatic, cannulated at both ends, will contract spontaneouslyand pump fluid from an inflow reservoir to an outflow at the same height (hencemoving fluid in the absence of a perfusion gradient). Field stimulation (McHale et al.1980) or addition of noradrenaline (McHale & Roddie, 1983 b) nearly alwaysincreased the contraction frequency of these vessels and this was sometimesaccompanied by an increase in the quantity of fluid pumped. Intravenous infusionsof noradrenaline also increased flow and contraction frequency in a doublycannulated preparation in anaesthetized sheep (McHale & Thornbury, 1986). Sincethe sole source of fluid supply to this preparation was from the inflow reservoir itfollows that the increased flow must have resulted from an increase in pumping.However, in the present experiments input to the lymphatic was not controlled inthis way, thus making it difficult to exclude the possibility that the increase in lymphflow was secondary to an increased rate of lymph formation in part of thelymphatics's catchment area. Nevertheless, at all of the stimulus frequencies tested,lymph flow increased despite a marked degree of vasoconstriction in the regionbelow the fetlock, which supplies over 80% of the lymph draining, via the metatarsalafferents, to the popliteal efferent (J. G. McGeown, N. G. McHale & K. D.Thornbury, unpublished observations). It may be deduced from the slow changes inthe baseline of the tissue volume records that the vasoconstriction was accompaniedby uptake of fluid by the capillaries (see Figs 1 and 2), a factor which would usuallybe expected to reduce lymph formation. No account was taken, however, of thepossibility that outward shifts of fluid could have occurred in more proximalcapillaries such as those in skeletal muscle or the lymph node. Skeletal muscle seemsan unlikely source of extra lymph since capillary filtration in this tissue is usuallyreduced during sympathetic stimulation (Mellander, 1960) although exceptionally,at high stimulus frequencies (> 20 Hz), the reverse is true (McLeod & Cobbold,1979). Activation of cholinergic vasodilator nerves to skeletal muscle would also failto provide an adequate explanation of the above results since, although acetylcholineproduces parallel increases in lymph flow and blood flow in the cat hindlimb (Lewis

5-2

131

J. C. McGEOWN, N. C. McHALE AND K. D. THORNBURY

& Winsey, 1970), the responses in the present study were blocked by the a-adrenergicantagonist phentolamine (Figs 4 and 5).Much less is known about the way in which the lymph node influences the volume

and composition of the efferent lymph. Two groups of workers (Adair, Moffatt,Paulsen & Guyton, 1982; Knox & Pflug, 1983) have demonstrated that a significantamount of fluid may be exchanged between the lymph and blood across the richnodal vasculature. The factors which control this process are poorly understood atpresent thus leaving open the possibility that altered haemodynamics at this sitemight have accounted for some of the increase in efferent lymph flow. However,sympathetic stimulation also increased lymph flow and contraction frequency inafferent vessels (Figs 6 and 7) thus demonstrating that the response can occurindependently of any effect at the node.The time courses of the lymphatic and vascular responses during longer (15-20

min) periods of stimulation (see Fig. 3) show that the lymphatic response was shortlived while the vascular response was well maintained. The fact that the duration ofthe two responses differed would tend to suggest that the increased lymph flow wasnot dependent upon an effect on blood vessels, thus supporting the notion that it wasdue to direct lymphatic stimulation. It is not clear why the lymph flow increase wasshort lived, but is seems likely that this was related to the quantity of lymphcontained within the system. Following the initial increase, afferent flow was alwaysdepressed after about 10 min of stimulation, which again would be consistent withthem having expelled most of their contents at a time when lymph formation wasreduced by vasoconstriction. It is interesting to note in this context that the responseof the efferent vessels tended to be both greater and of longer duration than that ofthe afferent (compare Figs 1 and 6). This might reflect the fact that the afferents werecannulated much closer to the main site of lymph formation (i.e. the foot) than werethe efferents. Thus, in the case of the latter, the quantity of preformed lymphavailable for clearance from the system would have been greater.The physiological importance of lymphatic innervation is still a matter for

speculation. It may be that neurohormonal mechanisms serve to increase return oflymph in times of stress. For example, emotional stimulation (McHale & Thornbury,1986) and haemorrhage (Hayashi, Johnston, Nelson, Hamilton & McHale, 1986)increase pumping in double-cannulated mesenteric lymphatics in the sheep. It ispossible that this could increase protein return and assist in restoring the plasmavolume during haemorrhagic shock. However, much future work is required beforethe exact nature and significance of these effects can be determined.

The authors wish to acknowledge support from the Wellcome Trust, the Nuffield Foundationand the Department of Health and Social Services, Northern Ireland. We are indebted to MrGeorge Creighton and his staff at the Queen's University Medical Research Unit for surgical andtechnical assistance.

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