128

Lymphology 11 (1978) 128- 132 Anatomy of the Interstitial Tissue H. Haljamae, M.D., Ph. D.

Departments of Anesthesiology I and , Universi ty of Goteborg, Goteborg, Sweden - Summary Fibers Aspects on composition and function of the intersti­ Three types of fibers - collagenous, reticular tial tissue have been given. The of and elastic ones, are present in the interstitium. the interstitium have at physiological pH a net nega­ tive charge and are osmotically active. They restrict These fibers are synthetized by mesenchymal free diffussion through the interstitium. The ground cells, , osteogenic and chondrogenic substance phase can be further subdivided in to a cells, and possibly also smooth muscle cells colloid-rich subphase and a colloid-poor subphasc. (elastic fibers; 1). Molecular is secreted The latter seems to constitute the true tissue fluid phase of the interstitium. The functional importance from the cells into the interstitial ground sub­ of the interstitium on exchange processes between the stance, where it is polymerized to collagenous vascular and the cellular compartments is discussed. and reticular fibers (2). The reticular fibers Changes in aggregation and hydration of the ground form thin networks around cells and they are substance change the physico-chemical properties also closely related to basement membranes and the functional characteristics of the interstitium. and the of e.g. lymphoid organs. Colla­ genous fibers of various dimensions are demon­ The interstitial compartment surrounds most strable in the interstitial phase of most tissues cells of the organism. It consists of fibrillar while the networks of elastic fibers are not so structures embedded in a tissue fluid contain­ common. The functional importance of the ing amorphous ground substance. The fluid various fibers is mainly mechanical support. volume of this compartment is approximately The isoelectric points of the fibrillar constituents three times that of plasma and the exchange lie between neutrality and pH 5.0 (3). Therefore, rate of fluid between these two extracellular at physiological pH the number of charged compartments is rapid. All substances that sites is small and the influence of these fibrillar are exchanged between blood and tissue cells structures on the distribution of other ions have to pass through the interstitium. The in­ relatively limited. terstitial content of acid macromolecular com­ ponents may be assumed to affect the com­ Ground substance position of the filtrate reaching the The amorphous ground substance or gellike cells. An evaluation of the true environmen­ matrix of the interstitium is produced by the tal milieu of tissue cells therefore makes it same cell types as the fibrillar components. It necessary to take the physico-chemical proper­ contains several different glycosaminoglycans ties of these various interstitial components (mucopolysaccharides) the more common ones into consideration. Structural and functional of which are , chondroitin-4- aspects of the intersti tium will be dealt with sulphate, chondroitin-6-sulphate, dermatan in the following presentation. sulphate and keratan sulphate. The proportions The fundamental structure of the interstitial of the different glycosaminoglycans vary in the phase is schematically shown in Fig. I. The interstitium of various tissues. Hyaluronate is main components are fibers and the interstitial present in most places, while the chondroitin­ ground substance, which may be subdivided sulphates are mainly present in and into a colloid-rich and a water-rich phase. . The glycosaminoglycans are composed of repeat units of two diffe rent saccharides,

0024-7766/78 1600-0128 S 04.00 © 1978 Geo rg Thieme Publishers

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COLLOID-RICH THE INTER STIT IAL PH ASE ! ...... • . . . . CAPILLARY Fig. 1 Schematic presentation of the . . anatomy of the interstitium. usually hexosamine and hexuronic acid. The disaccharide units contain charged anionic groups such as carboxylate and sulphate groups. The density varies from one (hyalurona­ te) to four (heparin) of these anionic groups per disaccharide unit. The mucopolysacchari­ des have low isoelectric points (between pH 2.0 and 3.0) and at physiological pH there is consequently a high density of negative colloidal charge wi thin the interstitial ground substance. It has been suggested, however, that the extent of aggregation of the polymers varies within the interstitium as schematically shown in Fig. 1. On the basis of this concept a colloid­ rich water-poor phase coexists with a water- Fig. 2 Density pf charged coll~id:U m_acromo ~ ecu l es rich colloid-poor phase (4). This hypothesis in the water-rich and the collo1d-nch mterstitial phases. predicts the existence of areas with highly ag­ gregated ground substance and a high negative Important functional effects of th~ coll~id~ charge density alternating with areas which phase are restriction of free diffuswn, bmdmg have a low content of disaggregated ground and immobilization of cations and anions, and su bstance and a low negative charge density involvement in ion-exchange reactions. It is (Figs. 1 and 2). In the highly aggregated areas obvious that the extent of these interstitial the charge density and the large domains of effects is dependent upon the degree of colloi­ the macromolecules will result in steric exclu­ dal aggregation and tissue hydration (Fig. 3). sion of other large molecules (5). Therefore, Oedema causes an increased hydration and it is considered that e.g. plasma proteins pas­ depolymerization of the ground substance. The sing the capillary walls will be mainly restrict­ result will be a decrease in the colloidal-depen- ' ed to a random network of interstitial channels dent restriction of free diffusion as well as in corresponding to the colloid-poor water-rich the binding capacity of cations (9). Tissue areas (6, 7, 8). The relative mobilities and the dehydration, on the other hand, increase~ the distribution of water as well as of small diffusilr colloidal density and thereby restricts free ble cations and anions in the interstitium will diffusion and increases the binding capacity of also be affected by the high negative charge cations ( 10) . density of the g1ycosaminog1ycan aggregates (3, 4). Permission granted for single print for individual use. Reproduction not permitted without permission of Journal LYMPHOLOGY. 130 H. Haljamae, M.D. Ph. D.

CO NTROL OED EM I\ DEHYDRATI O N ----=----/ 4JI) . - ~ - - -· ~ ~ '------. COL lOIDAl nf N~I lY • • IIH I Dtl , USION

OSMOf iC flit CiS • ElECTROlYTES Fig. 3 Changes in inters tilial functio­ · ANION S • nal characteristics in commection with tissue oedema or dehydration.

Interstitial tissue fluid Table I The distribution of electrolytes ( K, Na and The existance of an repre­ Cl) and proteins between plasma (P) and interstitial tissue Ou id (TF ). (From ref. s. 11 , 12, 13 and unpub­ senting a mere dialysate of blood was questio­ lished data) ned already in 1960 by Gersh and Catchpole p ( 4). It seems reasonable to believe that the TF TF- P TF:P free fluid phase i.e. the colloid-poor water­ K mmol/1 4.67 3.78 +0.89 1.25 rich phase is the true interstitial fluid. This Na mmol/1 15 3 142 +11 L.08 Cl mmol/1 91.2 99.5 - 8.2 0.92 fluid phase will receive products from the vas­ Total protein 0.32 cular as well as from the intracellular compart­ Albumin 0.36 ments and its composition will be modified by Globulin 0.24 the equilibrium with the colloid-rich phase. It may be assumed that cellular membranes are tl13.11 1.0 aJld that of ens smaller than 1.0 mainly in contact with this movable fluid (Table 1). This higher content of K and Na in phase and tllis phase, therefo re, represents the tissue fluid is probably due to binding effects true environmental milieu of tissue cells. on cati ons caused by tile colloidal anionic Techniques for direct sampliJlg of nanoliter polyelectrolytes of the interstitium. The low quantities of this interstitial tissue fluid phase Cl con tent is consequently due to the presence have been developed ( 11 ) and the protein ( 12) of t11esc colloidal anionic polyelectrolytes. as well as the electrolyte (I 1, 13) composition The total protein content as well as the albumin: of the flu id has been studied using microme­ globulin ratio of tile sampled fluid are also in tllods (14, 15). agreement wi th hypothetical interstitial fluid The content of this movable concentrations ( 12). tissue fluid phase is shown to be low in com­ Experimental data obtained from direct analy­ parison to that o bserved in fluid obtained from ses of sampled interstitial tissue flu id, therefore, in1planted capsules. The content in such implan­ are in agreement with the hypothesis that the ted capsules may be assumed to represent total coll oid-poor water-rich phase is the true environ­ interstitial content (13). The low content of mental mil ieu of the cells. The nu trients to and glycosaminoglycans in the fluid obtained witl1 metabolites from cells pass mainly through tllis the micropuncture techniques, therefore, phase , but the interplay with the colloid-rich favours the concept that it is a true representa­ phase prevents major fluctuations in the cellular tive for the colloid-poor water-rich interstitial milieu. Extensive loads of e.g. metabolites fluid phase. during tissue hypoxia are buffe red in the in­ The electrolyte content of tl1e interstitial flui d terstitium due to ionic binding to and ion-ex­ is also markedly different from a hypotlletical change reactions with the colloid-rich phase. ultrafiltrate of plasma. The tissue fluid : plasma This interplay between the two interstitial ratios of K+ and Na+ are consi derably large r phases may be considered as an important de-

Permission granted for single print for individual use. Reproduction not permitted without permission of Journal LYMPHOLOGY. Anatomy of the interstitial Tissue 131 fence mechanism which prevents extensive 5 Laurent, T.C.: The interaction between polysac changes in the milieu outside the cell membra­ charides and other macromolecules. IX. The ex· ne and U1ereby also major cellular functi onal elusion of molecules from hyaluronic acid gels and solutions. 13iochem. J. 93 ( 1964) 106-1 12 disturbances in emergency situ lations. 6 Sobel, H., K. Fa rd. 1: Robuck: Transvascular pas· sage of albumin-I 13t in of mice. Proc. Soc. Disturbances of interstitial homeostasis Exp. Bioi. Med. 123 (1966) 519- 521 7 Casley-Smith, J.R.: Calculations relating to the The extent of aggregati on and disaggregation p a~sage of fluid and protein ou t of arterial-limb of the interstitial ground substance will affect fenestrae, tluough basement membranes and con· the interstitial homeostasis. Hormones, e.g. - nective tissue channels, and in to venous-limb fenestrae and lymphatics. Microvasc. Res. 12 estrogen, androgen and cortisone, have been ( 1976) 13-34 shown to affect the amount as well as the 8 Wiederhielm, C.A., J.R. Fox, D.R. Lee: Ground slate of aggregation of ground substance. Disag­ substance mucopolysaccharides and plasma proteins: gregation of the colloides lowers the density of their role in cap illary water balance. Am. J. Physiol. 230 (1976) 1121 - 1125 the colloidal charge and increases Ule osmo tic 9 Friedman, M.H., K. Green: Io n binding and Donnan effect. Thereby tissue water content increases equilibria in rabbit corneal stroma. Am. J. Physiol. and an edema situation is initiated ( 4, 16). In­ 221 (1971) 356-362 flammatory reactions increase the vascular I 0 Haljamiie, H.: " Hidden" cellular electrolyte respon· pem1eability and also cause a disaggregation of scs to hemorrhagic shock and their significance. Rev. Surg. 27 0970) 315- 324 the interstitial ground substance. Hyaluronida­ 11 Haljamiie, H.: Sampling of nanoliter volumes of se and collagenase induced decomposition of mammalian subcutaneous tissue fluid and ultra· interstitial components will d ran1atically in­ micro flame photome tric analyses of the K and Na crease the spread of substances through the in­ concentrations. Acta Physiol. Scand. 78 ( 1970) 1- 10 terstitium. Such changes favour activlation and 12 Haljamiie, H. , H. Freden: Comparative analysis of movement of ft)(.ed tissue as well the protein con tent of local subcutaneous tissue as the migration of inflammatory cells such as fluid and plasma. Microvasc. Res. 2 (1970) 163- 171 monocytes, leukocytes and lymphocytes into 13 Haljamiie, H., A. Linde, B. Amundson: Compara· the interstitial tissue. tive analyses of capsular fluid and interstitial flu id. Am. J. Physiol. 227 (1974) 1199- 1205 14 Haljamiie, H., S. Larsson: An uluamicro flame photometer for K and Na analyses of single cells and nanoliter quantities of biologic:tl fluids. Chern. References l ns uum. 1 (1968) 131- 144 15 Haljamiie, H., D.C. Jllood: An:tlysis of picomole I Ross, R., P. Bornstein: Elastic fibers in the body. quantities ofCa, Mg, K, Na, and Cl in biological Sci. Am. 224 (1971) 44-52 samples by uluamicro flame specuophotomeuy. 2 Miller, E. G. , V. G. Matukas: Biosynthesis of coUa­ Anal. Biochem. 42 (1971) 155-170 gen. Fed. Proc. 33 (1974) 11 97- 1204 16 Secher-Hansen, E.: Studies on subcutaneous ab· 3 Catchpole, H.R., N.R. Joseph, M.B. Engel: Thcr· sorption in mice. V. Absorption of water injected modynamic relations of polyelectrolytes and in to the skin of normal and oes tradiol-treated inorganic ions of . Fed. Proc. 25 animals. Acta pharmacol. e t toxicol. 26 (1968) (1966) 11 24 - 11 26 229-239 4 Gersh,!., H.R. Catchpole: The nature of ground 17 Day, T.D.: The permeabil ity of interstitial connecti· substance of connective tissue. Perspect. Bioi. ve tissue and the nature o f the interfibrillary sub· Med. 3 ( 1960) 282-319 stance. J. Physiol. ( Lond.) 117 ( 1952) 1-8

H.Haljamiie, Department of Histology,Fack. S-.JOO 33 G6teborg 33 Sweden

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Discussion

Lasson: How would you explain your findings of very small. Therfore, in the capsule we do not probably higher K+ and Na+ in tissue fluid than in plasma? have a suborganization into a colloid-rich and a col­ loid-poor phase. The other thing we found was that Ha/jamiie: It is usually considered that there is a if we injected labelled substances in to the blood, the distribution of electrolytes between the vascular com­ exchange rate to the capsular fluid was small compa­ partment and the interstitial compartment according red to "true tissue fluid". Therefore we do not think to a Donnan equilibrium. The effects of the intersti­ that the capsular fluid is representative for tissue tial ground substance are, however, not taken intp fluid. It is a big lake, but what happens on the shores consideration. Therefore a lower content of potassium of t11at lake does only to a minor extent affect its and sodium is assumed to exist in the interstitial fluid composition. The sampling procedure of "true in­ than in blood plasma. In the interstitium, however, terstitial fluid" I described in 1970 in a paper in you have a higher content of colloidal mucopolysac· Acta Physiologica Scandinavica. The sampling is per­ charidcs than in the plasma. The charged macromolc· formed under liquid parafin. Subcutanous tissue fluid cules of the interstitium will affect the distribution of is obtained from connective tissue by the use of thin all small cations and anions. Especially in situations . With this technique it is possible to get with changing tissue hydration mucopolysaccharides nanoliter quantities of the fluid. A fresh surface area, seem to participate in ion-exchange reactions of ma­ i.e. untouched, is always used to prevent capillary jor importance for the control of the milieu at the leakeage. The electrolyte and protein content of re­ level. peated samples from the same animal is very constant indicating a good reproducibility. A uk/and: If you could compare local capillary plasma ionic contration wi th that of interstitial fluid your Question: Is there a difference in the composition values might be closer than when you compare plasma of and of 'the tissue fluid collected with your from large veins. method?

Haljamiie: What we arc using is the regional venous Haljamiie: I think that fluid within the lymphatic blood. This is the closest what we can get with-out vessels docs not reflect the changes in the interstitial disturbing t11c homeostasis across the capillary wall. fl uid. In the lymphatics we do not have the equili­ We arc also trying to study the local in tcrstitial chan­ brium between the two colloidal phases, which we ge by the use of other techniques. As I mentioned, have in the interstitium i.e. between the colloid-poor we are using local tissue pH registrations and com­ and colloid-rich phase. Therefore, lymph does not paring to simult:meous regional blood changes. We reflect changes in the immediate cellular milieu to are also trying to compare interstitial electrolyte such an extent as does local interstitial fluid. changes with changes of cellular transmembrane po­ tentials. Olszewski: During micropipetting you damage cells. I would also like to come back to the q uestion about Is not the high potassium centcnt the result of capsular fluid. We have tried to analyze capsular release of this ion from damaged cells? fluid and we used titanium capulcs which we implan­ ted subcutanously in rabbits. We compared the elec­ Haljamiie: If the difference in potassium distribution trolyte and the glycosaminoglycan composition of was only due to cellular damage during sampling t11e capsular fluid with that what we call " true inter­ then at the same time, t11e sodium content would stitial fluid". We found profound differences in com­ have been affected. Due to entry into the cells sodium position and I t11ink that t11e results point to the fact, would decrease. What we have found was a high con­ that in the capsule there is a large pool of fluid, tent of potassium as wciJ as of sodium and a low and t11e number of capillaries per volume of fluid is content of chloride in interstitial fluid.

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