J. R. Levick

Human lymphatic pumping measured in healthy and lymphoedematous arms by lymphatic congestion lymphoscintigraphy

Axillary surgery for breast cancer partially obstructs lymph outflow from the arm, chronically raising the lymphatic smooth muscle afterload. This may lead to pump failure, as in hypertensive cardiac failure, and could explain features of breast cancer treatment‐related lymphoedema (BCRL) such as its delayed onset. A new method was developed to measure human lymphatic contractility non‐invasively and test the hypothesis of contractile impairment. 99mTc‐human IgG (Tc‐HIG), injected into the hand dermis, drained into the arm lymphatic system which was imaged using a gamma‐camera. Lymph transit time from hand to axilla, ttransit, was 9.6 ± 7.2 min (mean ±s.d.) (velocity 8.9 cm min−1) in seven normal subjects. To assess lymphatic contractility, a sphygmomanometer cuff around the upper arm was inflated to 60 mmHg (Pcuff) before 99mTc‐HIG injection and maintained for >> ttransit. When Pcuff exceeded the maximum pressure generated by the lymphatic pump (Ppump), radiolabelled lymph was held up at the distal cuff border. Pcuff was then lowered in 10 mmHg steps until 99mTc‐HIG began to flow under the cuff to the axilla, indicating Ppump≥Pcuff. In 16 normal subjects Ppump was 39 ± 14 mmHg. Ppump was 38% lower in 16 women with BCRL, namely 24 ± 19 mmHg (P= 0.014, Students unpaired t test), and correlated negatively with the degree of swelling (12–56%). Blood radiolabel accumulation proved an unreliable measure of lymphatic pump function. Lymphatic congestion lymphoscintigraphy thus provided a quantitative measure of human lymphatic contractility without surgical cut‐down, and the results supported the hypothesis of lymphatic pump failure in BCRL.

Microvascular architecture and exchange in synovial joints.

The microcirculation of the synovial lining of joints displays many fascinating adaptations to function. One primary function is to supply nutrients to the avascular cartilage, whose chondrocytes are metabolically active but are relatively vast distances from the nearest capillary (> 1 cm in the center of a human knee). Exchange is facilitated by a high density of fenestrated capillaries situated very close to the synovial surface (an arrangement disrupted in rheumatoid synovium) with fenestrations preferentially oriented toward the joint cavity. Even so, diffusion alone is too slow to supply central chondrocytes with glucose. The problem is solved by the synovial microcirculation generating intra‐articular fluid (synovial fluid) that transports glucose by convection during joint movement. Synovial fluid is a plasma ultrafiltrate into which hyaluronan has been secreted, and it also serves to lubricate the joint.

Revision of the Starling principle: new views of tissue fluid balance

Tissue fluid balance, plasma volume regulation and clinical oedema formation are governed by the Starling principle of microvascular fluid exchange. This states that transendothelial filtration is driven by capillary pressure (Pc) and interstitial protein osmotic pressure (πi), while a counteracting absorptive force is exerted by plasma protein osmotic pressure (πp) and interstitial pressure (Pi). Since Pc falls along a capillary, the plausible concept of filtration from arterial capillaries and sustained reabsorption into venous capillaries has become embedded in the literature. Most of us learned this as first year undergraduates and took it to be a well proven, somewhat fossilized truth. In recent years, however, this ‘accepted’ view has undergone substantial experimental and theoretical re-evaluation; see the Classical Perspective by Michel (2004) in this issue of The Journal of Physiology. In particular, a landmark study of the Pc–filtration relation by Michel & Phillips (1987) demonstrated that although absorption occurs transiently at Pc < πp, absorption cannot be sustained, probably because πi increases with time. A further problem was the ‘low lymph flow paradox’, namely that net capillary filtration rate calculated from tissue-averaged Starling forces (including πi) is much greater than the tissue lymph production. These observations, along with new structure–function findings, led to the proposal of novel endothelial filtration models by Michel and by Weinbaum (Michel, 2004 and below). Now Adamson et al. (2004), also in this issue of The Journal of Physiology, report a direct investigation of the little-studied effect of πi on fluid exchange, with results that conflict dramatically with classical Starling predictions but support the Michel–Weinbaum model. In an elegant, rigorous study, Adamson et al. (2004) measured trans-endothelial fluid flux in cannulated postcapillary venules (a non-fenestrated ‘model’ for capillaries) in the rat mesentery and changed πi by albumin superfusion. The key finding was that raising πi increased the filtration rate by only a small fraction of that predicted by the Starling principle. The fraction was 25% under their specific experimental conditions but depends on the filtration rate (see below). Continuous endothelium thus displays osmotic asymmetry, unlike the symmetrical Starling principle. Indeed, in an analogous study in frogs Professor Roy Currys group found that altering πi had virtually no effect at all on filtration rate (Hu et al. 2000). Adamson et al. (2004) combined their experiments in vivo with confocal imaging of interstitial albumin distribution, reconstruction of the endothelial intercellular pathway geometry by serial electron micrography and a sophisticated mathematical model of the exchange pathway, to explain their results quantitatively. The semipermeable membrane (selective pores) across which protein osmotic pressure is exerted is the luminal glycocalyx of endothelium (Fig. 1). The outside of this membrane is not in direct contact with interstitial fluid (πi) but is connected to it by a long, narrow but open paracellular cleft. The subglycocalyx fluid is of lower protein concentration than the interstitial fluid because it is dominated by a continuously formed ultrafiltrate (osmotic pressure πg). Protein concentration is higher in the bulk interstitial fluid (30–60% of plasma concentration) because plasma proteins cross the endothelial barrier by a separate pathway, the large pore system. To have any effect on the glycocalyx, interstitial protein has to diffuse against the current of fluid sweeping out through the intercellular cleft. Figure 1 Changing nature of Starling principle for fluid exchange across non-fenestrated endothelium Can the concept of osmotic asymmetry be generalized to other tissues? In the structurally different fenestrated capillary, the exit from the ultrafilter (glycocalyx overlying fenestral membrane apertures) is much less enclosed. Here an analogous but less extreme osmotic asymmetry has been observed; changes in πi have 50% of the effect of changes in πp. This is again due to an abluminal protein gradient, in this case around the filtering fenestrations (Levick, 1994). However, in the only other study of continuous (non-fenestrated) endothelium and πi (Smaje et al. 1970) the results seem at first to conflict with the asymmetry model. Smaje et al. varied πi around rat cremaster and rabbit omental capillaries perfused with blood at normal pressures and found that filtration rate increased linearly with πi, in approximately the amount expected from the capillary filtration coefficient. Adamson et al. (2004) argue that this may be because native Pc and filtration velocity are low, allowing interstitial protein to diffuse up the cleft into the subglycocalyx space. (Control filtration rate was deliberately set high in the Adamson et al. study to test the concept of a ‘protected’ subglycocalyx space.) The authors estimate that 70–90% of the bulk πi may be effective in the subglycocalyx space (πg) under conditions of low Pc at heart level. The new paradigm has important implications for fluid balance and oedema formation. First, it renders untenable the popular argument that sustained venular absorption accounts largely for tissue fluid balance. During fluid absorption the subglycocalyx πg will increase quickly, due to reverse ultrafiltration, and thus prevent sustained absorption – as Michel & Phillips (1987) proved experimentally. To explain tissue fluid balance we must now focus increasingly on lymphatic function. Second, the findings may help to resolve the low lymph flow paradox (see earlier). Use of bulk πi overestimates the net filtration force and hence the lymph production, because the effective abluminal osmotic pressure πg is smaller than πi. The size of the difference is itself a function of filtration rate. Third, reduction of bulk πi is traditionally considered a major part of the ‘safety margin’ against oedema formation. This is evidently untrue at high filtration rates. Physiological filtration rates in many tissues, however, are probably slower than in the Adamson et al. experiment, so it remains possible that increases in filtration from low initial rates are buffered by reduced πg. Indeed the authors argue that the effectiveness of this buffering process is enhanced. This could be tested by repeating their study over a range of πi and Pc values. The paper by Adamson et al. (2004) is an important step forward but it also raises a new, medically important puzzle; how is the life-supporting reabsorption of interstitial fluid sustained in clinical shock? In human hypovolaemic shock ∼500 ml of interstitial fluid can be absorbed over ∼30 min, topping up the depleted circulation, as Starling himself noted. Hyperglycaemic hyperosmolarity may influence the fluid shift in the whole animal. However, even in an isolated perfused cat or dog hind-limb, the absorption process can continue for 15 min. The steady-state model of Adamson et al. (2004) does not address the time course of reabsorption; but since the intercellular cleft volume is extremely small, a very short time constant might be implied, possibly seconds. The time course of absorption remains inadequately understood and constitutes a medically important challenge for the future.

Hyaluronan Secretion into the Synovial Cavity of Rabbit Knees and Comparison with Albumin Turnover

1 Hyaluronan is not only a lubricant but also enhances the synovial linings resistance to fluid outflow. This finding led to the proposal that hyaluronan (> 2 × 106Da, ∼210 nm radius) may escape across the synovial lining less freely than smaller solutes (e.g. albumin, 6.7 × 104 Da, 3.6 nm radius) or water. Here multiple washouts were used to measure intra‐articular hyaluronan mass and secretion rate in rabbit knees, leading to an estimate of hyaluronan turnover time. Plasma albumin permeation into the joint cavity was also measured to enable comparison of turnover times between molecules of very disparate size. 2 Endogenous hyaluronan mass in the joint cavity, analysed by high performance liquid chromatography of joint washes, was 182 ± 9.9 μg (mean ± S.E.M; n= 21). Since hyaluronan concentration in synovial fluid averages 3.62 ± 0.19 μg μl−1, the endogenous synovial fluid volume was calculated to be 50 μl (mass/concentration), about double the aspiratable volume. 3 The hyaluronan secretion rate over 4h was 4.80 ± 0.77μg h−1 (n= 5). The rate was significantly higher in contralateral joints expanded by 2 ml Ringer solution (5.80 ± 0.84 μg h −1, n= 5, P= 0.01, Students paired t test), indicating a stretch/hydration sensitive secretory mechanism. The newly secreted chains ((2.05–2.48) × 108 Da) were not significantly different in length from the endogenous chains (2.95 × 108 Da). 4 Hyaluronan turnover time, calculated as mass/secretion rate, was 31.4–37.9 h. This is more than an order of magnitude longer than turnover time for intra‐articular albumin. The latter, determined from the intra‐articular albumin mass and plasma‐to‐cavity permeation rate was 1.8 h (95% confidence intervals 1.2–3.5 h, n= 9). The big difference in turnover times support the view that, relative to albumin and water, hyaluronan is partially sieved out and retained in the joint cavity by the synovial lining. The lining cell layer is discontinuous, so it appears that interstitial matrix itself acts as a leaky size‐selective molecular filter.

Starling pressures in the human arm and their alteration in postmastectomy oedema.

1. Surgery and radiotherapy to axillary lymph nodes during breast cancer treatment is often followed, commonly years later, by chronic postmastectomy oedema (PMO). PMO is considered a ‘high protein’ oedema due to reduced axillary lymph drainage. Since oedema formation also depends on fluid input (capillary filtration), we studied the Starling pressures in the affected and contralateral arm. Colloid osmotic pressure was measured in patient serum (pi p) and interstitial fluid (pi i). Subcutis fluid was collected from PMO arms by both wick and aspiration methods, and from the control arm by the wick method only. Interstitial hydraulic pressure (P(i)) was measured by the wick‐in‐needle method. 2. Oedema pi i was 19.2 +/‐ 4.1 cmH2O (n = 13, wick) to 16.3 +/‐ 4.4 cmH2O (n = 41, aspirate; difference not significant; mean +/‐ S.D. throughout). This was significantly lower than pi i in the control arm (21.4 +/‐ 3.8 cmH2O, n = 14, P < 0.01, analysis of variance). Also, there was a negative correlation between oedema pi i and the percentage increase in arm volume (correlation coefficient r = ‐0.35, P < 0.05) in contrast to conventional expectation. 3. Oedema P(i) (1.9 +/‐ 2.0 cmH2O, n = 28) exceeded the subatmospheric control P(i) (‐2.8 +/‐ 3.0 cmH2O; P < 0.01). Venous and arterial pressures were normal but pi p was subnormal (31.1 +/‐ 2.7 cmH2O, n = 47). 4. Net pressure opposing capillary blood pressure, P(o), was calculated as P(i) + sigma (pi p‐pi i) for a reflection coefficient, sigma, of 0.90‐0.99.(ABSTRACT TRUNCATED AT 250 WORDS)

Differences in lymph drainage between swollen and non-swollen regions in arms with breast-cancer-related lymphoedema

Recent research indicates that the pathophysiology of breast-cancer-related lymphoedema (BCRL) is more complex than simple axillary lymphatic obstruction as a result of the cancer treatment. Uneven distribution of swelling (involvement of the mid-arm region is common, but the hand is often spared) is puzzling. Our aim was to test the hypothesis that local differences in lymphatic drainage contribute to the regionality of the oedema. Using lymphoscintigraphy, we measured the removal rate constant, k (representing local lymph flow per unit distribution volume, VD), for 99mTc-labelled human immunoglobulin G in the oedematous proximal forearm, and in the hand (finger web) in women in whom the hand was unaffected. Tracer was injected subcutaneously, and the depot plus the rest of the arm was monitored with a gamma-radiation camera for up to 6 h. VD was assessed from image width. Contralateral arms served as controls. k was 25% lower in oedematous forearm tissue than in the control arm (BCRL, -0.070+/-0.026% x min(-1); control, -0.093+/-0.028% x min(-1); mean+/-S.D.; P=0.012) and VD was greater. In the non-oedematous hand of the BCRL arm, k was 18% higher than in the control hand (BCRL, -0.110+/-0.027% x min(-1); control, -0.095+/-0.028% x min(-1); P=0.057) and 59% higher than forearm k on the BCRL side (P=0.0014). VD did not differ between the hands. Images of the BCRL arm following hand injection showed diffuse activity in the superficial tissues, sometimes extending almost to the shoulder. A possible interpretation is that the hand is spared in some patients because local lymph flow is increased and diverted along collateral dermal routes. The results support the hypothesis that regional differences in surviving lymphatic function contribute to the distribution of swelling.

Characterization of the effect of high molecular weight hyaluronan on trans‐synovial flow in rabbit knees

1 The effect of a rooster comb hyaluronan (3.6–4.0 g l−1) of similar chain length to rabbit synovial fluid hyaluronan, on the trans‐synovial escape of fluid from the joint cavity in the steady state (Q̇s) was studied in 29 rabbit knees at controlled intra‐articular pressures (Pj). 2 Rooster hyaluronan caused the pressure‐flow relation to flatten out as pressure was raised. At 10–20 cmH2O the slope of the quasi‐plateau, 0.05 ± 0.01 μl min−1 cmH2O−1 (mean ±s.e.m.), was 1/39th that for Ringer solution (1.94 ± 0.01 μl min−1 cmH2O−1). 3 Bovine synovial fluid had a similar effect to hyaluronan in Ringer solution. 4 The quasi‐plateau was caused by increasing opposition to outflow; the pressure required to drive unit outflow increased 4.4‐fold between 5 and 20 cmH2O. The increased opposition to outflow at 20 cmH2O was equivalent to an effective osmotic pressure of 13–17 cmH2O at the interface. Since the infusates osmotic pressure was only 0.9 cmH2O, this implied concentration polarization to 15–18 g l−1 hyaluronan at the interface. 5 Mechanical perforation of the lining, or enzymatic degradation of the interstitial matrix by chymopapain, abolished the quasi‐plateau. Hydrational expansion of the matrix by /2‐fold did not. The increased opposition to outflow was reversible by washing out the hyaluronan, or by reducing Pj. It was unaffected by interruption of tissue blood flow or synoviocyte oxidative metabolism. These properties are compatible with a concentration polarization mechanism, i.e. flow‐induced concentration of hyaluronan at the synovial interface due to molecular reflection. 6 A concentration polarization theory was developed for a partially reflected solute. Numerical solutions supported the feasibility of this osmotic explanation of the quasi‐plateau. Additional mechanisms may also be involved. 7 It is concluded that native‐size hyaluronan helps to retain synovial fluid in the joint cavity when pressure is raised and acts, at least in part, by exerting osmotic pressure at the interface between synovial matrix and a concentration polarization layer.

Glycosaminoglycan concentration in synovium and other tissues of rabbit knee in relation to synovial hydraulic resistance.

1. The hydraulic resistance of the synovial lining of a joint, a key coupling coefficient in synovial fluid turnover, is thought to depend on the concentration of biopolymers (glycosaminoglycans (GAGs) and collagen) in the synovial intercellular spaces, because these polymers create hydraulic drag. The primary aim of this study was to obtain microscopically separated, milligram samples of the very thin synovium from eight rabbit knees, and to analyse these quantitatively for GAGs (chondroitin sulphate, heparan sulphate and hyaluronan) and collagen to allow comparison with published hydraulic resistance data. Synovial fluid and femoral cartilage were also studied. 2. Synovium comprised 73 +/‐ 3% water by weight (mean +/‐ S.E.M.). Of the 270 mg solid per gram of wet tissue, protein formed 136 mg (by automated amino acid analysis), and of this 94 mg was collagen by hydroxyproline analysis. From the collagen mass and fibril volume fraction (0.153 of tissue by morphometry), fibrillar specific volume was calculated to be 1.43 ml per gram of molecular collagen, and fibril water content 47% by volume. 3. The concentration of chondroitin 4‐sulphate (C4S) plus chondroitin 6‐sulphate (C6S), measured by capillary zone electrophoresis was 0.55 mg per gram of synovium‐‐much greater than in synovial fluid (0.04 mg g‐1) and much less than in cartilage (27.8 mg g‐1). The C4S/C6S ratio in synovium (7.3) differed from that in cartilage (0.7), indicating that different proteoglycans predominated in synovium. The heparan sulphate concentration, assayed by radioactive Ruthenium Red binding, was 0.92 mg per gram of synovium (synovial fluid, 0.08 mg g‐1; cartilage, 0.72 mg g‐1). 4. In contrast to sulphated GAGs, the hyaluronan concentration was highest in synovial fluid (3.53 mg g‐1; biotinylated G1 domain binding assay). The concentration in synovial interstitium was only 0.56 mg g‐1 (corrected for interstitial volume fraction, 0.66), even though there is open contact between synovial interstitium and synovial fluid. This may be due to exclusion or washout. 5. Total GAG mass was approximately 4 mg per gram of synovial interstitium. A model of trans‐synovial flow indicated that a uniform GAG concentration of 4 mg g‐1 is less than 1/3rd of that required to explain the experimental estimate of synovial hydraulic resistance. Errors in the resistance estimate do not appear to be large enough to resolve the problem. It is possible, therefore, that additional polymeric material in the interstitium, such as glycoproteins and proteoglycan core protein, may contribute to the hydraulic resistance.

Enhanced cutaneous lymphatic network in the forearms of women with postmastectomy oedema.

Postmastectomy oedema (PMO) of the arm is a common aftermath of axillary lymphatic damage during treatment for breast cancer. The aim of the present study was to quantify the forearm dermal lymphatic capillaries in order to determine whether they exhibit adaptive responses to PMO. Both forearms were examined by fluorescence microlymphography in 16 patients with oedema following treatment for breast cancer (mean swelling 25 ± 4%) and 19 patients treated for breast cancer but without oedema. Delineated lymphatic networks were analysed stereologically. The main findings were: (1) lymphatic density at any specified distance from the injection site was greater in the swollen arm than the control arm (p < 0.01, t test); (2) taking into account the increased skin area, the total length of lymphatic capillaries in a 1-cm annulus of skin was 676 ± 56 cm (swollen), compared with 385 ± 30 cm (control) (p < 0.001, t test); (3) fluorescent marker was transported over a greater distance before draining deep in the swollen arm (2.74 ± 0.33 cm) than in the control arm (1.59 ± 0.24 cm) (p = 0.02); (4) there was no evidence of lymphatic dilatation in the swollen arm, and (5) in breast cancer patients without swelling, the arm on the side of radiotherapy/surgery (otherwise referred to as the unswollen arm) showed none of the above changes, indicating that the changes are linked to the oedema rather than being universal responses to breast cancer or its treatment. It is concluded that microlymphatic changes occur in the swollen arm, namely a local superficial rerouting of lymph drainage and either lymphangiogenesis and/or increased recruitment of dormant lymphatic vessels. Since blood capillary angiogenesis occurs in the swollen arms, and lymphangiogenesis occurs in experimental lymphoedema, there is a precedent for proposing lymphangiogenesis in PMO. An increased number of functional vessels would help to maintain the ratio of local tissue drainage capacity to filtration capacity.

Role of hyaluronan chain length in buffering interstitial flow across synovium in rabbits

1 Synovial fluid drains out of joints through an interstitial pathway. Hyaluronan, the major polysaccharide of synovial fluid, attenuates this fluid drainage; it creates a graded opposition to outflow that increases with pressure (outflow ‘buffering’). This has been attributed to size‐related molecular reflection at the interstitium‐fluid interface. Chain length is reduced in inflammatory arthritis. We therefore investigated the dependence of outflow buffering on hyaluronan chain length. 2 Hyaluronan molecules of mean molecular mass ≈2200, 530, 300 and 90 kDa and concentration 3.6 mg ml−1 were infused into the knees of anaesthetized rabbits, with Ringer solution as control in the contralateral joint. Trans‐synovial drainage rate was recorded at known joint pressures. Pressure was raised in steps every 30–60 min (range 2–24 cmH2O). 3 With hyaluronan‐90 and hyaluronan‐300 the fluid drainage rate was reduced relative to Ringer solution (P < 0.001, ANOVA) but increased steeply with pressure. The opposition to outflow, defined as the pressure required to drive unit outflow, did not increase with pressure, i.e. there was no outflow buffering. 4 With hyaluronan‐530 and hyaluronan‐2000 the fluid drainage rate became relatively insensitive to pressure, causing a near plateau of flow. Opposition to outflow increased markedly with pressure, by up to 3.3 times over the explored pressures. 5 Hyaluronan concentration in the joint cavity increased over the drainage period, indicating partial reflection of hyaluronan by synovial interstitium. Reflected fractions were 0.12, 0.33, 0.25 and 0.79 for hyaluronan‐90, ‐300, ‐530 and ‐2200, respectively. 6 Thus the flow‐buffering effect of hyaluronan depended on chain length, and shortening the chains reduced the degree of molecular reflection. The latter should reduce the concentration polarization at the tissue interface, and hence the local osmotic pressure opposing fluid drainage. In rheumatoid arthritis the reduced chain length will facilitate the escape of hyaluronan and fluid.