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Protein concentrations in regions with fenestrated and continuous blood capillaries and in initial and collecting lymphatics
MICROVASCULAR
RESEARCH
12,245-251(1976)
Protein Concentrations in Regions with Fenestrated and Continuous Blood Capillaries and in Initial and Collecting Lymphatics J. R. CASLEY-SMITH Electron
Microscope
AND
M. A. SIMS
Unit, University of Adelaide, Box 4980, South Australia, 5001 Australia
Received
October
GPO,
Adelaide,
I, 1975
Radioactive iodinated serum albumin was injected into mice, who were killed 24 hr later. Autoradiographs of sections of the tips, bases, and serosa/muscle region of the jejuna and of the gastrocnemius muscle of the leg were examined electron microscopically. It was found, in confirmation of calculations, that in the arterial limbs of fenestrated capillaries the concentration of protein is very high in the pericapillary space, falls rapidly across the basement membrane, is low in the adjacent tissues, but rises to high values near the venous-limb capillaries, where it is also high in the basement membrane and pericapillary spaces. In continuous capillaries, the concentration is considerably lower in all these regions. The protein in the lymph in the initial lymphatics was considerably more concentrated than in the connective tissue fluid. It was shown that this lymph is once more diluted in the collecting lymphatics. These tend to confirm a hypothesis about the functioning of the initial lymphatics. INTRODUCTION
It has been suggested (Casley-Smith, 1970a, 1976a-c; Casley-Smith et al., 1976a) that in regions with fenestrated capillaries there is a considerable local circulation of fluid and proteins out of the fenestrae on the arterial limbs of the capillaries, through the tissues, and back into the blood via the venous-limb fenestrae. Direct and indirect evidence in favour of this has been reviewed (Casley-Smith, 1967a, b). It depends on the proteins being able to exert much of their normal colloidal osmotic pressures across pores many times larger than their diameters; this has been shown to occur in vitro (Casley-Smith and Bolton, 1973), as well as having strong indirect evidence in favour of it in vitro (Casley-Smith, 1976a, b). The suggestion also depends on there being a net uptake of proteins, against their concentration gradients, produced by the solvent drag of the inflowing fluid into the venous-limb fenestrae; in favour of this there is some in vitro evidence (Casley-Smith, 1976a), indirect in vivo evidence (Casley-Smith, 1976a, b; Casley-Smith et al., 1975a), and theoretical arguments (Casley-Smith, 1975, 1976~; Perl, 1975; cf. Michel, 1974). A mathematical model based on this hypothesis predicts the protein concentrations from the arterial-limb fenestrae to those on the venous limbs (Casley-Smith, 1976~). The concentration can be calculated to rise in the arterial-limb pericapillary space, fall sharply across its basement membrane, and rise slowly (and linearly) along the connective tissue channels; in the venous limb basement membranes and pericapillary spaces it should approximate plasma. The protein Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
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removed via the venous-limb fenestrae would be less concentrated than that in the connective tissue channels, so the lymphatics must remove the excess protein which would otherwise accumulate, just as these vessels do in regions with continuous capillaries. It was shown that this removal would be more efficient if the lymphatics concentrated the lymph. The concentration of lymph in the initial lymphatics has been suggested as the basic mechanism which causes the normal functioning of these vessels (Casley-Smith, 1970b, 1976a, b; Elhay and Casley-Smith, 1976). There is considerable evidence in favour of this. Direct estimation of protein concentrations in the initial lymphatics shows that the mean concentration is some three times higher than in the connective tissue, with the peak concentration (during tissue compression) being even higher and the low (during tissue relaxation) being lower (Arfors, 1976; Casley-Smith, 1970b, 1976a, b, d; Jonsson et al., 1970; Rusznyak et al., 1967; Witte, 1975). This hypothesis also depends on the inflow of proteins (caused by solvent drag) up a concentration gradient, whose effective colloidal osmotic pressure difference produces the fluid flow. It includes a corollary that the concentrated lymph ejected into the collecting lymphatics must be rapidly diluted again (Casley-Smith, 1970b, 1976a, b). While there is considerable evidence that this is possible and some evidence that it in fact occurs (reviewed in Casley-Smith, 1970b, 1976a, b), some recent experiments (Nicolaysen et al., 1975; Staub et al., 1975; Vriem et al., 1975) seem to show that this dilution does not occur, or is minimal. Arguments against this interpretation of these experiments are presented later. We thought that it would be worthwhile to test both parts of the hypothesis once more, using radio-iodinated albumin and electron-microscopical autoradiographs. The opportunity was also taken to test some of the conclusions from the model of protein passage through the tissues of regions with fenestrated capillaries; concentrations were also observed in a tissue with continuous capillaries. White mice were used as the experimental animal, and the jejunum and gastrocnemius muscle were studied. It has been shown (Casley-Smith, 1971) that in the mouse the villi tips usually contain venous-limb capillaries with many fenestrae, while the villi bases have arterial-limb capillaries with only a few fenestrae. The initial lymphatics could be found in this latter region, while the collecting lymphatics were studied in the serosa/muscle region of the outer part of the jejunal wall. MATERIALS
AND
METHODS
Five white mice were injected intraperitoneally with 0.5 ml of normal saline containing 1 mCi/kg of 1251-labelled bovine serum albumin (Amersham, U.K.). Twenty-four hours later they were anaesthetised with ether, and samples were taken from their jejuna and gastrocnemius muscles. The jejunum was ligated at two places 2-3 cm apart, and fixative was injected to just fill it, then the segment was placed in more fixative. This was 4 % glutaraldehyde in Millonig’s buffer (1961), to which 7 g % sucrose and 4 g% dextran (MW 40,000) had been added to preserve the crystaloidal and colloidal osmotic pressures in the tissues at the moment of fixation (Bohman, 1970). The tissues were cut into I-mm3 pieces, postfixed in 2 g % osmium tetroxide, dehydrated in ascending ethanols, and embedded in Araldite. Silver-gold sections were stained
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with lead citrate (pH 1l), coated with Ilford L4 emulsion (applied as a film formed across the open end of a tube-Sims, 1975) exposed for 60 days at 4”, and developed with Kodak Microdol-X developer for 5 min. The section thicknesses were measured (Casley-Smith and Cracker, 1975) to ensure that the sections were within 10% of the selested section thickness (80 nm) and to ensure that the few regions which had to be photographed without an adjacent blood capillary (lymphatics near the jejunal serosa) were of the correct thickness at that particular region. Photographs were taken at -5000x and enlarged to a constant 18,200(84) 25 times. (Here and elsewhere the standard error of a mean is given in parentheses after the mean and is followed by the number of observations in italics.) Four random blocks were examined from each region and random sections of each ‘were studied. (When more than one section of any block were used, they were at least 0.2 mm apart,) In the jejunal tissue, the regions were the villi tips, their bases, and the outer layers of the wall-the serosal/muscle region. Five blood capillaries per section were randomly selected and they and the surrounding tissue were photographed. Similarly, five random portions of lymphatics were studied in the bases of the villi and the serosal/muscle region of the jejuna. None were found in the tips, since the regions studied where distal to the lymphatics and the muscle samples of the leg were from deep within the muscle and so contained no lymphatics. Quantitative analysis of the data was performed according to the probability-circle method of Williams (1969), using circles with a 250-nm radius (Rogers, 1973). The area1 estimations were made using approximately twice the number of random circles and points as there were grains. Using the method of Williams, grains falling on the endothelium/pericapillary space and pericapillary space/basement membrane regions were allocated to determine the activity of the pericapillary space (i.e., the space between the endothelium and the basement membrane). Similarly, the pericapillary space/basement membrane and basement membrane/connective tissue grains were allocated to determine the activity of the basement membrane. In the connective tissue, grains were recorded separately over the collagen bundles, the “ground substance,” and the cells. It was found that all the cells-fibroblasts, epithelial cells, endothelium, and muscle cells-had approximately the same relative specific activities (SA’s), and these were grouped together. The background was relatively small (-5 % of the plasma values) and was subtracted from all the SA’s. In all regions the x2 test, as used by Williams, was highly significant (P < 0.001). It was necessary, however, to have a method for determining the standard errors (SE’s) of the SA’s of each region so that these could be compared with that of the plasma, and then these ratios could be compared from site to site. (The value of the plasma relative-specific-activity was thus used as a standard.) One way was to determine the ratios (g/a,) for each micrograph, wheregis the number of grains over region r, and a is the number of random probability circles. Then the mean ratio of these (g/a,) could be found, together with its SE, treating (g/a,) as an independent variable. This could even be carried further by using (g/a,)/(g/a),, where p refers to the plasma region. Unfortunately, such was the variation in the proportions of grains over the regions, and such was the variation in the proportions of hits with the random circles, that the SE’s found by these methods in a sample of the micrographs were quite large, especially those of the ratios of SAJSA,. It was found that an alternative method gave somewhat smaller SE’s. This used the fact, confirmed by plotting the’
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results, that the g’s and a’s are distributed in Poissonian distributions, as are their sums G, = 1’. g, and A, = xn a,, where n is the number of photographs per site. Hence, G, and A, are the best estimates of rz times the means & and ti,, and the SE’s of G, and A, are G,112and A:12, respectively. Using these, and the large number theory (Kendal and Stuart, 1936, p. 231), we could determine the SE’s of the ratios (G/A),. and also ofrhe ratio of this to (G/A),, i.e., (G/A),/(G/A),. Not only was it found that these SE’s were generally smaller than those found using the first method, but they were also obtained considerably more quickly. These ratios were then compared using t tests, with 198 degrees of freedom.
RESULTS The results are shown in Table 1 and illustrated in Figs. 1-5. It was confirmed that the muscle and serosa/muscle regions had continuous capillaries, while there were a few fenestrae at the bases of the villi and many at their tips. It can be seen that all the plasma specific activities (SA’s) are not significantly different, but it is better to compare the SA,/SA, values between regions and sites than just to use the SA, values, because the values for the various sites were obtained from different sections. It can be seen that the protein concentrations in the connective tissues, basement membranes, and periendothelial spaces are very significantly (P < 0.001) greater at the tips of the villi than in the serosa/muscle region and in the leg muscle. This also applies to the basement membrane and connective tissue concentrations of the bases of the villi, but here the periendothelial space concentration is considerably increased (P < 0.001) compared with the other two regions and with this region in the other two sites. It is of great interest that the concentration in the initial lymphatics in the bases of the villi is very significantly greater (P < 0.001) than that in the collecting lymphatics in the serosa/muscle region. This latter is not significantly different from that in the adjacent connective tissue. The concentration in the initial lymphatics is very significantly greater (P < 0.001) than the mean connective tissue concentration, as well as that in the actually adjacent connective tissue in the villi bases. It is also significantly (P < 0.01) greater than that in the villi tips. These findings indicate that the lymph in the initial lymphatics is more concentrated than the fluid in the connective tissue (aide infra) and imply that this is subsequently diluted by the time this fluid reaches the collecting lymphatics in the serosa/muscle region. It can be seen that in all the sites the concentration in the collagen bundles was not significantly different from that in the connective tissue in the regions where there was neither collagen nor cells.
DISCUSSION There is one important potential source of error. This is how much connective tissue space is occupied by the ground substance, which is invisible in the electron microscope, but which denies space to the proteins. Garlick and Renkin (1970) found that the overall exclusion of macromolecules in the dog paw would correspond to a mean hyaluronic acid concentration of 0.2 g/l00 ml, i.e., it excludes ~10% of albumin (Laurent, 1970). This is, however, only a mean figure. According to Laurent (1970, 1972), there is likely
PROTEIN CONCENTRATIONS IN CAPILLARIES AND LYMPHATICS
249
FIG. 1. A venous-limb capillary near the tip of a villus. The autoradiographic grains are shown in cikles, inside of which there is a 0.5 probability that the radiation originated. Many fenestrae are visible. 15,000x.
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FIG. 2. An arterial-limb capillary near the base of a villus. This has no fenestrae and rather fewer grains in the connective tissue, but it is impossible to ascertain this unless many micrographs are examined. 15,000~.
PROTEIN
CONCENTRATIONS
IN CAPILLARIES
AND
LYMPHATICS
FIG. 3. A capillary in the leg. Grains are more prevalent over the lumen and the connective tissue than over the muscle. 15,000x.
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FIG. 4. An initial lymphatic near the base of a villus. There are many grains over the lumen, which also has a high electron-opacity density due to the protein it contains. 15,000x.
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FIG. 5. A portion of a collecting lymphatic in the serosa/muscle region of the jejunum. There are fewer grains over its lumen than in Fig. 4; the electron-optical density of its contents is less also, but this must be measured on the plate, not on the print. 15,000x.
to be quite a large difference in concentration of proteins between the gel and sol phases of the ground substance. He presents data showing that the gel is much tighter near the collagen fibres. The results of Hauck (1972), Hauck and Schroer (1969), Merker and Gunther (1972), Witte and Hagel (1971), and Casley-Smith et al. (1975a) indicate that fluid and protein move through the tissue in many quite large channels (of some 60-nm radius). If this is so, it would seem that the ground substance, while perhaps being occasionally present in the channels, is basically around them, forming their walls and permeating the rest of the connective tissue, especially near the collagen fibres. Thus, the rest of the tissue would be available to water and small molecules (except where there were cells or collagen fibres), but not so readily available to proteins, nor for most fluid flow (because of Poiseulle’s Law), nor for fluid diffusion (because of the varying
280
443 456
241 233
36
561 139 120
309 83 71
A
0.13(0.023)
0.54(O.W) 0.51(0.029)
0.55(0.039) 0.60(0.083) 0.59(0.089)
SA
tips
0.24(0.045)
0.98(0.11) 0.93(0.10)
1.1 (0.17) 1.1 (0.17)
SA,/SA,
24 690
47 72
214 112 57
G
188 767
276 329
357 152 163
A
SA
bases
0.13(0.028) 0.9qO.047)
0.17(0.027) 0.22(0.028)
0.60(0.052) 0.74(0.092) 0.35(0.054)
Villi
0.22(0.050) 1.5 (0.14)
0.28(O.OSl) 0.37(0.056)
1.2 (0.19) 0.58(0.10)
SAJSA,
1
75 273
91 50
383 42 46
G
377 618
261 130
581 161 139
A
0.20(0.025) 0.44(0.037)
0.35(0.042) 0.38(0.059)
0.66(0.043) 0.26(0.045) 0.33(0.056)
SA
Serosa/muscle
0.30(0.043) 0.67(0.065)
0.5310.072) 0.57(0.089)
0.39(0.073) 0.50(0.091)
SAJSA,,
132
46 62
251 25 28
G
878
312 248
502 126 131
A
O.lS(O.014)
0.22(0.035) O.ZS(O.035)
O.SO(O.039) 0.2O(O.C43) 0.21(0.045)
SA
Leg muscle
’ The labels for the columns are: G, total grains counted; A, total area of the region as datermined by randomly superimposed probability circles; SA, relative specific activity; relative specific activity of a region divided by that of the plasma. b The mean SA in the connective tissue of the villi is SAJSA,, which is 0.63(0.061). c It can be seen that the SAJSA, of the initial lymphatics (in the villi bases) is 2.2(0.30) that of the collecting lymphatics (in the serosa/muscle region). Also, that of the initial divided by that of the mean of the connective tissue is 2.4(0.32).
tissue CdlS Lymph’
Plasma Periendothelial space Basement membrane Non-collagenous connective tissuP Collagenous connective
G
Villi
Jejunum
TABLE
lymphat&
SAJSA,,
0.30(0.036)
O/%4(0.078) 0.50(0.080)
0.40(0.091) 0.42(0.010)
sA,ISA,
h
Fs
z
E
*
PROTEIN
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dimensions of the interstices, blind pockets, etc.). Unfortunately, the inaccuracies of the autoradiographic method are such that this division, if present, could not be revealed. In any event, it is evident that this potential source of error is unlikely to be important. The results confirm the calculations made about the banking-up at the arterial-limb basement membrane and the considerable sieving it causes (Casley-Smith, 1976~). In the venous-limb fenestrated capillaries the concentration of protein is much higher in the connective tissue and basement membrane: in the periendothelial space it is about equal to that in the plasma, which is higher than in the continuous capillaries. Again this confirms the calculations (Casley-Smith, 1976~). These suggested that the arterial-limb basement membrane would be a considerable barrier to protein (although probably not to smaller molecules-Casley-Smith et al., 1975b), but that at the venous limbs, where the inflow is likely to be very slow, the membranes would probably not cause much banking-up. This appears to be the case, but the limits of accuracy of our present measurements are such that asmall amount of banking-up could not be detected. Similarly, it does not appear that the fenestrae on either limb cause any significant obstruction to the protein (as was again suggested by the calculations), but once more we are hampered by the limits of accuracy. The calculations also predicted the great differences we find here between the connective tissue protein concentrations near the arterial and venous limbs. Certainly, the present results confirm some of the main features of the calculations and do not disagree with any part of them. It can also be seen, in the two sites where there are continuous capillaries (the leg and the serosa/muscle), that here the basement membrane does not seem to offer much impedance to the passage of protein, compared with the endothelium itself. In the passage of protein via either the endothelial vesicles or the junctions, there would be considerable molecular sieving; these paths, rather than the basement membrane, evidently limit the passage of protein (reviewed by Casley-Smith, 1976a; Casley-Smith et al., 1975b). This does not imply that here the basement membrane is not a barrier, but just that the endothelium is a considerably greater one. The present results strongly indicate that the initial lymphatics concentrate the fluid they receive from the tissues. The value in the initial lymphatics is only a mean and hence is probably considerably lower than the peak concentration, since the periods of compression are only brief and those of relaxation are long, during which the concentration probably falls exponentially (Casley-Smith, 1976b, d; Elhay and Casley-Smith, 1976). Hence, this aspect of the initial lymphatic hypothesis is again confirmed. Our results also indicate that there is a dilution between these initial lymphatics and the collecting lymphatics. Here the results are at variance with those of Staub’s group (Nicolaysen et al., 1975; Staub et al., 1975; Vreim et al., 1975) who did not find this in the mouse or sheep lung in some experiments, although they did find it in others. In these latter they used chemical fixation, while in the former they used freezing. However, there are two differences between their experiments and the present ones. Although we used chemical fixation, we occluded all the collecting lymphatics so that their contents could not escape, which was considered to have happened in the lung. Second, they only examined the “adjacent” collecting lymphatics (Casley-Smith, 1976b, d). All the lung lymphatics are fairly uniformly compressed during expiration, so one would not expect great variations in concentration along their lengths. In the lung, the true
256
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(“remote”) collecting vessels, in the sense of having substantially different internal hydrostatic pressures from the initial lymphatics (Casley-Smith, 1976b, d), are not in the lung itself. This similarity in the mean concentrations in the initial lymphatics and the adjacent collectors has also been seen elsewhere (Casley-Smith, 1976d), where it was also shown that there was a considerable redilution in the remote collectors. In the jejunum the vessels in the serosa/muscle region are remote collectors since the villous muscles compress only the lacteals. Hence, redilution can occur. ACKNOWLEDGMENTS
We are most grateful for the skilful technical assistance of Mr. K. W. J. Cracker, the support of the Australian Research Grants Committee, and the statistical assistance of Mr. P. I. Leppard. REFERENCES
K-E. (1976). In “Proc. IV Int. Cong. Lymphology, Tucson” (M. Witte and C. Witte, eds.). University of Arizona Press, in press. BOHMAN, S. 0. (1970). Effects on tissue fine structure of variations in colloidal osmotic pressure of glutaraldehyde fixatives. J. Ultrastrue. Res. 30,195-208. CASLEY-SMITH, J. R. (1970a). The functioning of endothelial fenestrae on the arterial and venous limbs of capillaries, as indicated by the differing directions of passage of protein. Experientia 26, 852-854. CASLEY-SMITH, J. R. (1970b). Osmotic pressure: A probably important force for theentrance ofmaterial into lymphatics. In “Proc. III Int. Cong. Lymphology, Brussels” (J. Gruwez, ed.). Int. Sot. Lymphology, 148 (abstract). CASLEY~MITH, J. R. (1971). Endothelial fenestrae in intestinal villi: Differences between the arterial and venous ends of the capillaries. Microvasc. Res. 3,49-68. CASLEY-SMITH, J. R. (1975). A theoretical support for the transport of macromolecules by osmotic flow across a leaky membrane against a concentration gradient. Microvasc. Res. 9,43-48. CASLEY-SMITH, J. R. (1976a). Lymphatics and lymph. In “Microcirculation” (G. Kaley and B. Altura, eds.). University Park Press, Baltimore, Md., in press. CASLEY-SMITH, J. R. (1976b). The functioning and interrelationships of blood capillaries and lymphatics. Experientia 32, l-12. CASLEY-SMITH, J. R. (1976~). Calculations relating to the passage of fluid and protein out of arteriallimb fenestrae, through the basement membranes and tissue channels, and into venous-limb fenestrae and lymphatics. Microvasc. Res., 12, 13-34. CASLEY-SMITH, J. R. (1976d). The concentrating of proteins in the initial lymphatics and their rediluting in the collecting lymphatics. Folia Angiologica., submitted for publication. CASLEY-SMITH, J. R., AND BOLTON, T. (1973). The presence of large effective colloidal osmotic pressures across large pores. Microvasc. Res. $213-216. CASLEY-SMITH, J. R., AND CROCKER, K. W. J. (1975). Estimation of section thickness, etc., by quantitative electron microscopy. J. Microscopy 103, 351-368. CASLEY-SMITH, J. R., O’DONOGHLJE, P. J., AND CROCKER, K. W. J. (1975a). The quantitative relationships between fenestrae on jejunal capillaries and connective tissue channels: Proof of “tunnelcapillaries.” Microvasc. Res. 9,78-100. CASLEY-SMITH, J. R., GREEN, H. S., HARRIS, J., AND WADEY, P. (1975b). The quantitative morphology of skeletal muscle capillaries in relation to permeability. Microvasc. Res. 10,43-64. ELHAY, S., AND CASLEY-SMITH, J. R. (1976). A mathematical model of the initial lymphatic. Microvase. ARFORS,
Res., 12,121-140. GARLICK, D. G., AND RENKIN, E. M. (1970). Transport of large molecules from plasma to interstitial fluid and lymph in dog. Amer. J. Physiol. 219,1595-1600. HAUCK, G. (1972). Pathways between capillaries and lymphatics. Pfliigers Arch., Suppl. 336, S55S57. HAUCK, G., AND SCHR~ER, H. (1969). Vitalmikroskopische Untersuchungen zur Lokalisation der Eiweisspermeabilitlt an der Endstrombahn von Warmbltltern. Pjiigers Arch. 312,32-40.
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J., ARFORS, K-E., AND HINT, H. C. (1971). Studies on relationships between the blood and lymph systems within the microcirculation. In “VIth Europ. Conf. Microcirculation, Aalborg,” pp. 214-218. Karger, Basel. KENDALL, M. G., AND STUART, A. (1966). “The Advanced Theory of Statistics,” Vol. 1, 2nd ed., p. 231. Griffin, London. LAURENT, T. C. (1970). The structure and function of the intercellular polysaccharides in connective tissue. In “Capillary Permeability” (C. Crone and N. A. Lassen, eds.), pp. 261-277. Academic Press, New York and London. LAURENT, T. C. (1972). Physico-chemical properties of interstitial tissue. Pfigers Arch., Suppl. 366, S21LS33. MERKER, H. J., AND GUNTHER, T. (1972). Ultrastructure of interstitial tissue. Pfltigers Arch., Suppl. 336,S33-S35. MICHEL, C. (1974). The transport of solute by osmotic flow across a leaky membrane and a note on the reflection coefficient. Microvasc. Res. 8, 122-125. NICOLAYSEN, G., NICOLAYSEN, A., AND STAUB, N. C. (1975). A quantitative radioautographic comparison of albumin concentration in different sized lymph vessels in normal mouse lungs. Microvasc. Res. 10,138-152. PERL, W. (1975). Convection and permeation of albumin between plasma and interstitium. Microvasc. Res. 10,83-94. ROGERS, A. W. (1973). “Techniques of Autoradiography,” 2nd ed. Elsevier, New York. RUSZNYAK, I., F~LDI, M., AND SZAB~, G. (1967). “Lymphatics and Lymph Circulation,” 2nd ed. Pergamon, London. SIMS, M. A. (1975). An electron microscopic autoradiographic investigation of the accumulation of Zince5 by a species of Eutreptia. Experientia 31,426-428. STAUB, N. C., NICOLAYSEN, G., AND NICOLAYSEN, A. (1975). Constant albumin concentration along lung lymphatics in normal mice. In “Proc. V. Int. Cong. Lymphology, Buenos Aires and Rio de Janeiro” (C. A. Grandval, ed.), pp. 49-50 (abstract.) VREIM, C. E., DEMLING, R. H., AND STAUB, N. C. (1975). Protein composition of lung fluids during edema. In “Proc. V. Int. Cong. Lymphology, Buenos Aires and Rio de Janeiro” (C. A. Grandval, ed.), pp. 47-48 (abstract). WILLIAMS, M. A. (1969). The assessment of electron microscopic autoradiographs. In “Advances in Optical and Electron Microscopy” (R. Barer and V. E. Cosslett, eds.), Vol. 3, pp. 219-272. Academic Press, New York and London. WITTE, S. (1975). Protein exchange between blood and lymph. In “VIII Scientific Workshop of the Society for Manual Lymph Drainage, Schluchsee.” WITTE, S., AND HAGEL, F. (1971). Quantitative ultraviolet vitalmicroscopy. In “VI Europ. Conf. Microcirculation”, pp. 86-89. Karger, Basel.
JONSWN,
RESEARCH
12,245-251(1976)
Protein Concentrations in Regions with Fenestrated and Continuous Blood Capillaries and in Initial and Collecting Lymphatics J. R. CASLEY-SMITH Electron
Microscope
AND
M. A. SIMS
Unit, University of Adelaide, Box 4980, South Australia, 5001 Australia
Received
October
GPO,
Adelaide,
I, 1975
Radioactive iodinated serum albumin was injected into mice, who were killed 24 hr later. Autoradiographs of sections of the tips, bases, and serosa/muscle region of the jejuna and of the gastrocnemius muscle of the leg were examined electron microscopically. It was found, in confirmation of calculations, that in the arterial limbs of fenestrated capillaries the concentration of protein is very high in the pericapillary space, falls rapidly across the basement membrane, is low in the adjacent tissues, but rises to high values near the venous-limb capillaries, where it is also high in the basement membrane and pericapillary spaces. In continuous capillaries, the concentration is considerably lower in all these regions. The protein in the lymph in the initial lymphatics was considerably more concentrated than in the connective tissue fluid. It was shown that this lymph is once more diluted in the collecting lymphatics. These tend to confirm a hypothesis about the functioning of the initial lymphatics. INTRODUCTION
It has been suggested (Casley-Smith, 1970a, 1976a-c; Casley-Smith et al., 1976a) that in regions with fenestrated capillaries there is a considerable local circulation of fluid and proteins out of the fenestrae on the arterial limbs of the capillaries, through the tissues, and back into the blood via the venous-limb fenestrae. Direct and indirect evidence in favour of this has been reviewed (Casley-Smith, 1967a, b). It depends on the proteins being able to exert much of their normal colloidal osmotic pressures across pores many times larger than their diameters; this has been shown to occur in vitro (Casley-Smith and Bolton, 1973), as well as having strong indirect evidence in favour of it in vitro (Casley-Smith, 1976a, b). The suggestion also depends on there being a net uptake of proteins, against their concentration gradients, produced by the solvent drag of the inflowing fluid into the venous-limb fenestrae; in favour of this there is some in vitro evidence (Casley-Smith, 1976a), indirect in vivo evidence (Casley-Smith, 1976a, b; Casley-Smith et al., 1975a), and theoretical arguments (Casley-Smith, 1975, 1976~; Perl, 1975; cf. Michel, 1974). A mathematical model based on this hypothesis predicts the protein concentrations from the arterial-limb fenestrae to those on the venous limbs (Casley-Smith, 1976~). The concentration can be calculated to rise in the arterial-limb pericapillary space, fall sharply across its basement membrane, and rise slowly (and linearly) along the connective tissue channels; in the venous limb basement membranes and pericapillary spaces it should approximate plasma. The protein Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
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AND
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removed via the venous-limb fenestrae would be less concentrated than that in the connective tissue channels, so the lymphatics must remove the excess protein which would otherwise accumulate, just as these vessels do in regions with continuous capillaries. It was shown that this removal would be more efficient if the lymphatics concentrated the lymph. The concentration of lymph in the initial lymphatics has been suggested as the basic mechanism which causes the normal functioning of these vessels (Casley-Smith, 1970b, 1976a, b; Elhay and Casley-Smith, 1976). There is considerable evidence in favour of this. Direct estimation of protein concentrations in the initial lymphatics shows that the mean concentration is some three times higher than in the connective tissue, with the peak concentration (during tissue compression) being even higher and the low (during tissue relaxation) being lower (Arfors, 1976; Casley-Smith, 1970b, 1976a, b, d; Jonsson et al., 1970; Rusznyak et al., 1967; Witte, 1975). This hypothesis also depends on the inflow of proteins (caused by solvent drag) up a concentration gradient, whose effective colloidal osmotic pressure difference produces the fluid flow. It includes a corollary that the concentrated lymph ejected into the collecting lymphatics must be rapidly diluted again (Casley-Smith, 1970b, 1976a, b). While there is considerable evidence that this is possible and some evidence that it in fact occurs (reviewed in Casley-Smith, 1970b, 1976a, b), some recent experiments (Nicolaysen et al., 1975; Staub et al., 1975; Vriem et al., 1975) seem to show that this dilution does not occur, or is minimal. Arguments against this interpretation of these experiments are presented later. We thought that it would be worthwhile to test both parts of the hypothesis once more, using radio-iodinated albumin and electron-microscopical autoradiographs. The opportunity was also taken to test some of the conclusions from the model of protein passage through the tissues of regions with fenestrated capillaries; concentrations were also observed in a tissue with continuous capillaries. White mice were used as the experimental animal, and the jejunum and gastrocnemius muscle were studied. It has been shown (Casley-Smith, 1971) that in the mouse the villi tips usually contain venous-limb capillaries with many fenestrae, while the villi bases have arterial-limb capillaries with only a few fenestrae. The initial lymphatics could be found in this latter region, while the collecting lymphatics were studied in the serosa/muscle region of the outer part of the jejunal wall. MATERIALS
AND
METHODS
Five white mice were injected intraperitoneally with 0.5 ml of normal saline containing 1 mCi/kg of 1251-labelled bovine serum albumin (Amersham, U.K.). Twenty-four hours later they were anaesthetised with ether, and samples were taken from their jejuna and gastrocnemius muscles. The jejunum was ligated at two places 2-3 cm apart, and fixative was injected to just fill it, then the segment was placed in more fixative. This was 4 % glutaraldehyde in Millonig’s buffer (1961), to which 7 g % sucrose and 4 g% dextran (MW 40,000) had been added to preserve the crystaloidal and colloidal osmotic pressures in the tissues at the moment of fixation (Bohman, 1970). The tissues were cut into I-mm3 pieces, postfixed in 2 g % osmium tetroxide, dehydrated in ascending ethanols, and embedded in Araldite. Silver-gold sections were stained
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with lead citrate (pH 1l), coated with Ilford L4 emulsion (applied as a film formed across the open end of a tube-Sims, 1975) exposed for 60 days at 4”, and developed with Kodak Microdol-X developer for 5 min. The section thicknesses were measured (Casley-Smith and Cracker, 1975) to ensure that the sections were within 10% of the selested section thickness (80 nm) and to ensure that the few regions which had to be photographed without an adjacent blood capillary (lymphatics near the jejunal serosa) were of the correct thickness at that particular region. Photographs were taken at -5000x and enlarged to a constant 18,200(84) 25 times. (Here and elsewhere the standard error of a mean is given in parentheses after the mean and is followed by the number of observations in italics.) Four random blocks were examined from each region and random sections of each ‘were studied. (When more than one section of any block were used, they were at least 0.2 mm apart,) In the jejunal tissue, the regions were the villi tips, their bases, and the outer layers of the wall-the serosal/muscle region. Five blood capillaries per section were randomly selected and they and the surrounding tissue were photographed. Similarly, five random portions of lymphatics were studied in the bases of the villi and the serosal/muscle region of the jejuna. None were found in the tips, since the regions studied where distal to the lymphatics and the muscle samples of the leg were from deep within the muscle and so contained no lymphatics. Quantitative analysis of the data was performed according to the probability-circle method of Williams (1969), using circles with a 250-nm radius (Rogers, 1973). The area1 estimations were made using approximately twice the number of random circles and points as there were grains. Using the method of Williams, grains falling on the endothelium/pericapillary space and pericapillary space/basement membrane regions were allocated to determine the activity of the pericapillary space (i.e., the space between the endothelium and the basement membrane). Similarly, the pericapillary space/basement membrane and basement membrane/connective tissue grains were allocated to determine the activity of the basement membrane. In the connective tissue, grains were recorded separately over the collagen bundles, the “ground substance,” and the cells. It was found that all the cells-fibroblasts, epithelial cells, endothelium, and muscle cells-had approximately the same relative specific activities (SA’s), and these were grouped together. The background was relatively small (-5 % of the plasma values) and was subtracted from all the SA’s. In all regions the x2 test, as used by Williams, was highly significant (P < 0.001). It was necessary, however, to have a method for determining the standard errors (SE’s) of the SA’s of each region so that these could be compared with that of the plasma, and then these ratios could be compared from site to site. (The value of the plasma relative-specific-activity was thus used as a standard.) One way was to determine the ratios (g/a,) for each micrograph, wheregis the number of grains over region r, and a is the number of random probability circles. Then the mean ratio of these (g/a,) could be found, together with its SE, treating (g/a,) as an independent variable. This could even be carried further by using (g/a,)/(g/a),, where p refers to the plasma region. Unfortunately, such was the variation in the proportions of grains over the regions, and such was the variation in the proportions of hits with the random circles, that the SE’s found by these methods in a sample of the micrographs were quite large, especially those of the ratios of SAJSA,. It was found that an alternative method gave somewhat smaller SE’s. This used the fact, confirmed by plotting the’
248
CASLEY-SMITH
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results, that the g’s and a’s are distributed in Poissonian distributions, as are their sums G, = 1’. g, and A, = xn a,, where n is the number of photographs per site. Hence, G, and A, are the best estimates of rz times the means & and ti,, and the SE’s of G, and A, are G,112and A:12, respectively. Using these, and the large number theory (Kendal and Stuart, 1936, p. 231), we could determine the SE’s of the ratios (G/A),. and also ofrhe ratio of this to (G/A),, i.e., (G/A),/(G/A),. Not only was it found that these SE’s were generally smaller than those found using the first method, but they were also obtained considerably more quickly. These ratios were then compared using t tests, with 198 degrees of freedom.
RESULTS The results are shown in Table 1 and illustrated in Figs. 1-5. It was confirmed that the muscle and serosa/muscle regions had continuous capillaries, while there were a few fenestrae at the bases of the villi and many at their tips. It can be seen that all the plasma specific activities (SA’s) are not significantly different, but it is better to compare the SA,/SA, values between regions and sites than just to use the SA, values, because the values for the various sites were obtained from different sections. It can be seen that the protein concentrations in the connective tissues, basement membranes, and periendothelial spaces are very significantly (P < 0.001) greater at the tips of the villi than in the serosa/muscle region and in the leg muscle. This also applies to the basement membrane and connective tissue concentrations of the bases of the villi, but here the periendothelial space concentration is considerably increased (P < 0.001) compared with the other two regions and with this region in the other two sites. It is of great interest that the concentration in the initial lymphatics in the bases of the villi is very significantly greater (P < 0.001) than that in the collecting lymphatics in the serosa/muscle region. This latter is not significantly different from that in the adjacent connective tissue. The concentration in the initial lymphatics is very significantly greater (P < 0.001) than the mean connective tissue concentration, as well as that in the actually adjacent connective tissue in the villi bases. It is also significantly (P < 0.01) greater than that in the villi tips. These findings indicate that the lymph in the initial lymphatics is more concentrated than the fluid in the connective tissue (aide infra) and imply that this is subsequently diluted by the time this fluid reaches the collecting lymphatics in the serosa/muscle region. It can be seen that in all the sites the concentration in the collagen bundles was not significantly different from that in the connective tissue in the regions where there was neither collagen nor cells.
DISCUSSION There is one important potential source of error. This is how much connective tissue space is occupied by the ground substance, which is invisible in the electron microscope, but which denies space to the proteins. Garlick and Renkin (1970) found that the overall exclusion of macromolecules in the dog paw would correspond to a mean hyaluronic acid concentration of 0.2 g/l00 ml, i.e., it excludes ~10% of albumin (Laurent, 1970). This is, however, only a mean figure. According to Laurent (1970, 1972), there is likely
PROTEIN CONCENTRATIONS IN CAPILLARIES AND LYMPHATICS
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FIG. 1. A venous-limb capillary near the tip of a villus. The autoradiographic grains are shown in cikles, inside of which there is a 0.5 probability that the radiation originated. Many fenestrae are visible. 15,000x.
250
CASLEY-SMITH
AND
SIMS
FIG. 2. An arterial-limb capillary near the base of a villus. This has no fenestrae and rather fewer grains in the connective tissue, but it is impossible to ascertain this unless many micrographs are examined. 15,000~.
PROTEIN
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IN CAPILLARIES
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FIG. 3. A capillary in the leg. Grains are more prevalent over the lumen and the connective tissue than over the muscle. 15,000x.
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CASLEY-SMITH
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SIMS
FIG. 4. An initial lymphatic near the base of a villus. There are many grains over the lumen, which also has a high electron-opacity density due to the protein it contains. 15,000x.
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FIG. 5. A portion of a collecting lymphatic in the serosa/muscle region of the jejunum. There are fewer grains over its lumen than in Fig. 4; the electron-optical density of its contents is less also, but this must be measured on the plate, not on the print. 15,000x.
to be quite a large difference in concentration of proteins between the gel and sol phases of the ground substance. He presents data showing that the gel is much tighter near the collagen fibres. The results of Hauck (1972), Hauck and Schroer (1969), Merker and Gunther (1972), Witte and Hagel (1971), and Casley-Smith et al. (1975a) indicate that fluid and protein move through the tissue in many quite large channels (of some 60-nm radius). If this is so, it would seem that the ground substance, while perhaps being occasionally present in the channels, is basically around them, forming their walls and permeating the rest of the connective tissue, especially near the collagen fibres. Thus, the rest of the tissue would be available to water and small molecules (except where there were cells or collagen fibres), but not so readily available to proteins, nor for most fluid flow (because of Poiseulle’s Law), nor for fluid diffusion (because of the varying
280
443 456
241 233
36
561 139 120
309 83 71
A
0.13(0.023)
0.54(O.W) 0.51(0.029)
0.55(0.039) 0.60(0.083) 0.59(0.089)
SA
tips
0.24(0.045)
0.98(0.11) 0.93(0.10)
1.1 (0.17) 1.1 (0.17)
SA,/SA,
24 690
47 72
214 112 57
G
188 767
276 329
357 152 163
A
SA
bases
0.13(0.028) 0.9qO.047)
0.17(0.027) 0.22(0.028)
0.60(0.052) 0.74(0.092) 0.35(0.054)
Villi
0.22(0.050) 1.5 (0.14)
0.28(O.OSl) 0.37(0.056)
1.2 (0.19) 0.58(0.10)
SAJSA,
1
75 273
91 50
383 42 46
G
377 618
261 130
581 161 139
A
0.20(0.025) 0.44(0.037)
0.35(0.042) 0.38(0.059)
0.66(0.043) 0.26(0.045) 0.33(0.056)
SA
Serosa/muscle
0.30(0.043) 0.67(0.065)
0.5310.072) 0.57(0.089)
0.39(0.073) 0.50(0.091)
SAJSA,,
132
46 62
251 25 28
G
878
312 248
502 126 131
A
O.lS(O.014)
0.22(0.035) O.ZS(O.035)
O.SO(O.039) 0.2O(O.C43) 0.21(0.045)
SA
Leg muscle
’ The labels for the columns are: G, total grains counted; A, total area of the region as datermined by randomly superimposed probability circles; SA, relative specific activity; relative specific activity of a region divided by that of the plasma. b The mean SA in the connective tissue of the villi is SAJSA,, which is 0.63(0.061). c It can be seen that the SAJSA, of the initial lymphatics (in the villi bases) is 2.2(0.30) that of the collecting lymphatics (in the serosa/muscle region). Also, that of the initial divided by that of the mean of the connective tissue is 2.4(0.32).
tissue CdlS Lymph’
Plasma Periendothelial space Basement membrane Non-collagenous connective tissuP Collagenous connective
G
Villi
Jejunum
TABLE
lymphat&
SAJSA,,
0.30(0.036)
O/%4(0.078) 0.50(0.080)
0.40(0.091) 0.42(0.010)
sA,ISA,
h
Fs
z
E
*
PROTEIN
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dimensions of the interstices, blind pockets, etc.). Unfortunately, the inaccuracies of the autoradiographic method are such that this division, if present, could not be revealed. In any event, it is evident that this potential source of error is unlikely to be important. The results confirm the calculations made about the banking-up at the arterial-limb basement membrane and the considerable sieving it causes (Casley-Smith, 1976~). In the venous-limb fenestrated capillaries the concentration of protein is much higher in the connective tissue and basement membrane: in the periendothelial space it is about equal to that in the plasma, which is higher than in the continuous capillaries. Again this confirms the calculations (Casley-Smith, 1976~). These suggested that the arterial-limb basement membrane would be a considerable barrier to protein (although probably not to smaller molecules-Casley-Smith et al., 1975b), but that at the venous limbs, where the inflow is likely to be very slow, the membranes would probably not cause much banking-up. This appears to be the case, but the limits of accuracy of our present measurements are such that asmall amount of banking-up could not be detected. Similarly, it does not appear that the fenestrae on either limb cause any significant obstruction to the protein (as was again suggested by the calculations), but once more we are hampered by the limits of accuracy. The calculations also predicted the great differences we find here between the connective tissue protein concentrations near the arterial and venous limbs. Certainly, the present results confirm some of the main features of the calculations and do not disagree with any part of them. It can also be seen, in the two sites where there are continuous capillaries (the leg and the serosa/muscle), that here the basement membrane does not seem to offer much impedance to the passage of protein, compared with the endothelium itself. In the passage of protein via either the endothelial vesicles or the junctions, there would be considerable molecular sieving; these paths, rather than the basement membrane, evidently limit the passage of protein (reviewed by Casley-Smith, 1976a; Casley-Smith et al., 1975b). This does not imply that here the basement membrane is not a barrier, but just that the endothelium is a considerably greater one. The present results strongly indicate that the initial lymphatics concentrate the fluid they receive from the tissues. The value in the initial lymphatics is only a mean and hence is probably considerably lower than the peak concentration, since the periods of compression are only brief and those of relaxation are long, during which the concentration probably falls exponentially (Casley-Smith, 1976b, d; Elhay and Casley-Smith, 1976). Hence, this aspect of the initial lymphatic hypothesis is again confirmed. Our results also indicate that there is a dilution between these initial lymphatics and the collecting lymphatics. Here the results are at variance with those of Staub’s group (Nicolaysen et al., 1975; Staub et al., 1975; Vreim et al., 1975) who did not find this in the mouse or sheep lung in some experiments, although they did find it in others. In these latter they used chemical fixation, while in the former they used freezing. However, there are two differences between their experiments and the present ones. Although we used chemical fixation, we occluded all the collecting lymphatics so that their contents could not escape, which was considered to have happened in the lung. Second, they only examined the “adjacent” collecting lymphatics (Casley-Smith, 1976b, d). All the lung lymphatics are fairly uniformly compressed during expiration, so one would not expect great variations in concentration along their lengths. In the lung, the true
256
CASLEY-SMITH
AND SIMS
(“remote”) collecting vessels, in the sense of having substantially different internal hydrostatic pressures from the initial lymphatics (Casley-Smith, 1976b, d), are not in the lung itself. This similarity in the mean concentrations in the initial lymphatics and the adjacent collectors has also been seen elsewhere (Casley-Smith, 1976d), where it was also shown that there was a considerable redilution in the remote collectors. In the jejunum the vessels in the serosa/muscle region are remote collectors since the villous muscles compress only the lacteals. Hence, redilution can occur. ACKNOWLEDGMENTS
We are most grateful for the skilful technical assistance of Mr. K. W. J. Cracker, the support of the Australian Research Grants Committee, and the statistical assistance of Mr. P. I. Leppard. REFERENCES
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JONSWN,