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The fundamental role of hyaluronidase in tissue
Medical
Hypotheses
THE FUNDAMENTAL
5: ll83-1200,
1979
ROLE OF HYALURONIDASE
IN TISSUE
An hypothesis on the protecting and restoring mechanisms of the "cellular unit" with their clinical consequences S.W.Bok. St-Lucas Ziekenhuis, Gassingel The Netherlands.
18,
9671
CX Winschoten,
ABSTRACT The interstitial ground substance plays an essential role in the physiological processes of the capillary and the cell. Together they form a fundamental unit of function in which the cell is the most important part. Therefore the fundamental unit is called: THE CELLULAR UNIT. It is proposed central role and that this enzyme is indispensible for normal healthy cells as well as for the reactive mechanism in critical situations. The reactive mechanisms can be divided into a local reactive mechanism and a general reactive mechanism. The local reactive mechanism has a function of RESTORATION. The general reactive mechanism is especially for PROTECTION AGAINST ISCHAEMIA and REDISTRIBUTION OF TOTAL BODY NUTRIENTS in order to stimulate the restoration. These two mechanisms may have important clinical consequences. Key words:
"cellular unit", hyaluronidase, protection, cell metabolism.
restoration,
INTRODUCTION From a clinical point of view the local reactive mechanism is well known. A short-term anoxia caused by tissue ischaemia is followed by a phase of hyperaemia; partly damaged organs can restore or regenerate and chronic irritation will lead to hyperaemia and cell-proliferation. This local reactive mechanism, characterised by an increased mitosis and hyperaemia, seems to be a mechanism of an increase in cellular metabolism. This directly implies that the energy requirement for the cells in this area will increase and also that the metabolic rate of a cell is not only dependent on the blood levels of special hormones. Recently, Erdmann et al (15, 26) were able to measure oxygen diffusion in the interstitial tissue. After short-term ischaemic anoxia, diffusion of oxygen in the interstitial ground substance is dramatically increased for a short period. Thus, the oxygen diffusion in the interstitial ground substance is variable and seems to be related to the cellular metabolism. What is the precise underlying mechanism of this local reaction? To explain this local reaction, we first have to know the relation between the nutrient supply to the cell and the cellular metabolism. Then perhaps we can explain this local reactive mechanism and describe also the efficiency of this 1183
mechanism. Perhaps it will then be possible also to explain the increased secretion of corticosteroids by the adrenal glands during stress. This can perhaps lead to a better knowledge of the functions of this hormone. Perhaps a knowledge of the precise underlying mechanism of these reactions will have important clinical consequences and lead to possible clinical application. NORMAL CELLULAR METABOLISM "The cellular unit". One of the well known standard works on physiology states that "the fundamental unit of function is the cell" (16). It is firmly established now that in any tissue the individual cell, collagen-, reticulum-, and elastin fibres, blood and lymph vessels are embedded in an amorphous gel or ground substance (10). This ground substance consists of mucopolysaccharide-protein complexes and the molecular weight of the mucopolysaccharides determines the viscosity of this complex (3). As we shall see later, it is better to talk about the "CELLULAR UNIT" as A FUNDAMENTAL UNIT OF FUNCTIONS, because every cell is embedded in this viscous gel and this gel plays a central role in the nutrient supply to the cell. This cellular unit then consists of the cell, the surrounding interstitial ground substance and the capillary endothelium with its pericapillary sheath (9) (figure 1). The movement of water within the interstitium. The functions of the cell can be devided into general functions, which are essential for continuation of cell life and the specific functions, which differ from organ to organ. All these functions have one thing in common and that is that they all require energy. The energy need for these functions is dependant on the metabolic rate of the cell. For an optimal nutrient supply to the cell the movement of water within the interstitium is essential. The classic work on this subject was published by Starling (32) and until now has remained almost unchanged. Interstitial movement of water is influenced by the following factors: The arterial and venous capillary blood-pressure, the size of the pores in the capillary endothelium, the permeability of the interstitium and the colloid osmotic blood-pressure. The permeability of the interstitium is dependent on the viscosity of the ground substance (3). In 1949 Opsahl (27) demonstrated that the viscosity of the ground substance is influenced by hormones particularly from the adrenal cortex. This conclusion was confirmed by others (6, 31). 1184
blood supply capillary endothelium peri capillary sheath cell groundsubstance.4
-
Figure
1.
THE CELLULAR
UNIT
This unit is of function.
proposed
1185
to
be the
THE CELLULAR UNIT
fundamental
unit
Hyaluronidase cell.
in relation to the ground substance and to the
Since the discovery of the enzyme-complex hyaluronidase by Duran-Reynals in 1928 much experimental work has been done. The name hyaluronidase was given by Meyer (25) after he found that this enzyme depolymerises hyaluronic acid. Shortly after hyaluronidase was found to be a depolymerising enzyme for the whole group of mucopolysaccharides that form the viscous gel around the cells, in other words the ground substance of the interstitium. The mucopolysaccharides are first depolymerised by this enzyme into oligosaccharides and then into monosaccharides, N-acetyl glucosamide and glucuroronic acid (22). These breakdown products diffuse away into the capillaries and can be detected in the urine (12, 30). The viscosity of the ground substance will decrease and become more water soluble and the permeability will increase as a result of this breakdown. Bensley (21) showed that the ground substance previously coloured with toluidine-blue, disappears after injection of hyaluronidase. This implies that the ground substance is not only depolymerised by this enzyme, but also dissolved. The elastin fibres in the interstitium are insensitive to this enzyme and this network can function as a frame (1). Recently Grossfeld (19) and Bollet (4) found that cells of many tissues produce this enzyme. Because all cells are embedded in this viscous gel of mucopolysaccharides, it seems highly probable that all cells produce this enzyme. THIS HYPOTHESIS IMPLIES THAT HYALURONIDASE SYNTHESIS IS A GENERAL FUNCTION OF ALL CELLS. For hyaluronidase to influence the surrounding ground substance it must move from the cell into the interstitium. Then there must be a continuous breakdown of the ground substance and consequently also a continuous replacement. The metabolic cycle has not yet been fully established, but after the isotope studies by Bostrom and studies by others much more is known about its probable form (5, 18). These experiments made clear, that replacement was the result of two different mechanisms. Firstly, the breakdown products, circulating in the blood, are partly used for replacement and secondly, the cells themselves produce these mucopolysaccharides. The breakdown is said to be due to hyaluronidase that is synthetized in the cells and then excreted into the interstitium. This implies that there is a balance between the breakdown and replacement of the ground substance in normal healthy cell units.
1186
Hyaluronidase
and the new formation of capillaries.
In 1947 Chambers and Zweifach (9) demonstrated that hyaluronidase has no direct effect on the capillary endothelium and its intercellular cement. Some years later the same authors (37) published microscopic observations on living tissue, infiltrated with hyaluronidase. Within a few minutes of injection the capillary walls showed an outward bulging followed by a rupture of the capillaries and sinusoids full of blood are formed. This is an indirect effect of hyaluronidase and is the consequence of the dissolution of the pericapillary sheath. The very thin layer of endothelial cells now cannot withstand the capillary blood-pressure and burst. In 1977 Kloner et al (21) published an experimental study on the treatment of myocardial infarction with high dosages of hyaluronidase, injected intravenously. The electronmicroscopical observation of myocardial tissue, 3 hours after treatment, showed a significant increase in the number of small vessels in the ischaemic areas by comparison with untreated animals. ft seems highly probable that the new formation of capillaries is the end result of sinusoid formation. Hwuronidase -
and the cellular metabolism.
:I:t is well documented that depolymerisation of the ground substance by local injection of hyaluronidase results in a tissue-cell proliferation, an increased mitosis and therefore an increased cellular metabolism (2, 23, 36). However hyaluronidase has no direct effect on the capillary endothelium. The outward filtration of fluid will increase rapidly after hyaluronidase infiltration because the pericapillary sheath is depolymerised (9). The water solubility and the permeability of the ground substance will also increase rapidly after hyaluronidase rnjection. The overall result then will be an increased movement of water. This was also described by Zweifach and Chambers (37). With the increased movement of water in the ground substance the nutrient supply to the cell will also increase. We know now that this enzyme is probably produced by every cell, that it is excreted in the surrounding interstitium and that it regulates the nutrient supply to the cell. The observation of tissue infiltrated with hyaluronidase shows an increased metabolism. May we now make the following hypothesis that: THE CELL REGULATES ITS METABOLIC RATE BY THE REGULATION OF THE PRODUCTION OF HYALURONIDASE. If we can accept this hypothesis, the conclusion will be that
hyaluronidase plays a central role in cellular life. This was also suggested by Cameron (7). 1187
The hormonal influences on the metabolic rate of the cell can then be explained by influences on the activity of this enzyme and/or on its rate of production. THE LOCAL REACTIVE MECHANISM Introduction. We know that locally injected hyaluronidase leads to hyperaemia and cell proliferation and if we accept that hyaluronidase plays a central role in the cellular metabolism, can we explain the local reactive mechanism by changes of the hyaluronidase concentration in the interstitial ground substance? This implies that the local reactive mechanism, for example hyperaemia after short-term ischaemic anoxia, is caused by an increased interstitial movement of water due to a higher hyaluronidase concentration. In this context it is hard to believe that one of the general functions - the hyaluronidase synthesis - will increase when the nutrient supply to the cell is diminished. To explain this local reactive mechanism we must first determine the hyaluronidase concentration in the cellular unit. Hyaluronidase
concentration
in the cellular unit.
Hyaluronidase is synthesized in the cell and transported into the interstitium. Therefore it has to pass through the cellular membrane. The cellular membrane is a lipoid-protein layer. The mechanisms of transport through this layer can be a passive transport, an active transport or facilitated diffusion (16). Passive transport is only possible for lipoid soluble substances or water soluble molecules smaller than urea (16). Hyaluronidase is a highly water soluble enzyme and although the exact molecular weight is not known, it is much bigger than urea (8). That implies that it cannot pass through the cellular membrane by a passive transport mechanism. The possibilities remaining are facilitated diffusion or active transport. If it is transported by facilitated diffusion the intracellular hyaluronidase concentration has to be much higher than the extracellular concentration. If it is passed through the cellular membrane by an active transport mechanism the concentration is dependent upon the activity of this transport. The exact mechanism is not yet known. When hyaluronidase is transported through the cellular membrane the interstitial movements of water will transport this enzyme from the cell to the capillary, since we know that hyaluronidase does not diffuse into tissue when there is no movement of water (20). 1188
Because the direction of movement of water is from the arterial side to the venous side, the hyaluronidase concentration has to be higher on the venous side. That might explain the fact that the permeability of the venous capillary is higher than the permeability of the arterial capillary as described by Zijlstra (39) recently. Hyaluronidase passes through the capillary endothelium without difficulty and is then excreted in the urine (30, 31). Only if we can accept that the intracellular hyaluronidase concentration is higher than the extracellular concentration, can we explain the local reactive mechanism fully, as we will see later. Figure 2 shows the hypothetical hyaluronidase concentration in the cellular unit. General aspects of the local reactive mechanism. Working with the cellular unit model and the proposed hyaluronidase concentration, the reaction to traumatic cell death, short-term and long-term ischaemia as well as the invasion of micro-organisms can be explained. In all these different pathologic circumstances the efficiency of this reaction can also be explained. During the discussion on the hyaluronidase concentration in relation to the cellular unit the hypothesis was made that there is a higher intracellular concentration of this enzyme. Especially in traumatic cell death an explanation of the local reaction would be impossible, if the reserve is true. The reaction following ischaemia is much more complicated than after traumatic cell death. In this situation the reserve energy of the cell plays an essential role. This idea of a reserve of energy should be accepted because the cell can survive a certain period of total nutrient and oxygen deficiency. In long-term ischaemia the pH decrease caused by the accumulation of the metabolic endproducts plays an essential role since we know that hyaluronidase is only active within a pH range of 4-9 (8). Some years after the first publication about hyaluronidase in 1928, this enzyme was found in many different bacteria (27). Duran-Reynals (14) demonstrated the relationship between the amount of bacterial hyaluronidase synthesis and the invasivity of the bacteria. Traumatic cell death. Traumatic cell death implies that the cellular membrane has been destroyed. If there is a defect of the cellular membrane, then the interstitial hyaluronidase concentration will increase, because the intracellular concentration of hyaluronidase is higher than the extracellular concentration.
,189
ARTERIAL SIDE OF THE CAPILLARY
VENOUS SIDE OF THE CAPILLARY
-
hyaluronidase concentration
Figure
2:
The
probable
normal local
hyaluronidase
ccl lular reactive
concentration Working
unit. mechanism
1190
can
with
this
be explained.
in
the
model
the
This increased hyaluronidase concentration increases the interstitial movement of water and nutrient supply to the cells surrounding the dead ones with as consequence decreased mitosis and RESTORATION OF THE TOTAL NUMBER OF CELLS. Another advantage of the increased movement of water will be an INCREASED CLEARING OF NECROTIC MATERIALS. Invasion by micro-organisms. It is well understood that the invasivity of bacteria is related to the capacity of these micro-organisms to dissolve the ground substance by the production of hyaluronidase. When the tissue concentration of hyaluronidase rises, the reaction of these tissues will also lead to new capillary formation as previously described. The periferal resistance in this infected area will decrease with, as a consequence, hyperaemia. This local hyperaemia leads to optimalization of the defence mechanisms against these bacteria. The increase in locally circulating immune factors, as well as the restoration of the total number of cells, together with the increased clearance of necrotic materials all play important roles in this local reaction. To rationalize further, it can be seen that the magnitude of the fall in periferal resistance is related to the numbers and the type of hyaluronidase producing bacteria. The severity of the phenomenon of septic shock caused by some types of bacteria can now be explained by this mechanism. Conversely, when hyperaemia cannot occur because of vascular insufficiency (e.g. arteriosclerosis) or when the level of circulating immune factors is depressed then this local defence mechanism will fail. This would explain why bad11 vascularised tissues or malnourished people are more susceptible to infection. Short-term
ischaemia.
During the ischaemic period following arterial occlusion, the capillary blood pressure decreases to zero. This directly implies that there will be no further movement of water in the interstitium. As a consequence the nutrient supply to the cell will be cut off and the cell has to function on the reserve energy. The period that the cell can function on reserve energy is dependant upon the amount of reserve energy and the metabolic rate of the cell. During this time all the functions of the cell are stiil normal, including the hyaluronidase synthesis as well as its transport across the cell membrane. Because there is no movement of water in the interstitium to the capillary and then the pericellular hyaluronidase concentration will rise. The metabolic endproducts will accumulate and the pH will decrease. 1191
This implies that in the period of ischaemia there is no cellular reaction. THE CELL, THUS, IS UNABLE TO REACT DIRECTLY TO ISCHAEMIA. After restoration of the circulation the movement of water is restored and then the high pericellular hyaluronidase concentration can diffuse into the interstitium. This will lead to an increased movement of water which will mean that the cell firstly can RESTORE THE CELLULAR ENERGY RESERVE and secondly the metabolic endproducts can diffuse away more rapidly and THE pH WILL BE RESTORED. Long-term
ischaemia.
At the beginning of a period of long-term ischaemia there exists of course the same situation as in short-term ischaemia. After a certain period of anoxia the cellular energy reserve is consumed and the cellular functions slow down. The accumulation of the metabolic end products increases. The pH decreases so much that hyaluronidase becomes inactive. After restoration of the circulation this inactive hyaluronidase will not give rise to any reaction by contrast with the situation during short-term ischaemia. Because the cellular functions are already decreased there will be a decrease in the activity of the mechanisms of the sodium/potassium pump and the cell will become oedematous. This is unfavourable for function as well as affecting the availability of the nutrient supply. Restoration is impossible, irreversible changes occur and the cell will die. In this situation there will be no reaction to cellular death, in contrast to traumatic cell death, because hyaluronidase is inactive. The final conclusion
about the local reactive mechanism.
The local reactive mechanism, characterized by hyperaemia and cell proliferation can be explained by the hyaluronidase interactions. The effects of this local reaction are as follows: 1. RESTORATION OF THE TOTAL NUMBER OF CELLS (=regeneration) 2. INCREASED CLEARING OF NECROTIC MATERIALS 3. RESTORATION OF THE CELLULAR ENERGY RESERVE 4. RESTORATION OF THE pH. All these actions can be collectively described as tissue restoration. THUS, HYALURONIDASE IS RESPONSIBLE FOR THE RESTORATION. A new aspect in this approach is that the hyperaemia is not only caused by dilatation of the vessels but also by THE FORMATION OF NEW CAPILLARIES. Perhaps the collateral vessel growth, frequently seen in poorly vascularized extremities, is based on the same reaction. also essential in this hypothesis is that THE CELL ITSELF HAS NO DIRECT DEFENCE AGAINST ISCHAEMIA (aSHOCK). 1192
CORTICOSTEROIDS
AND THE GENERAL REACTIVE MECHANISM
Introduction. We know that the body reacts in stressful circumstances with a very complex general reaction. Only a small part of this general reaction is the increase in the blood levels of corticosteroids. The essential effect of the increased circulating corticosteroids in stress is thought to lead to protection. We now know much more about the interactions that these hormones have on cellular metabolism and the probable actions on capillary membranes. However the exact mechanism of cellular protection is unknown. We will discuss the effects of corticosteroids on the cellular unit as a whole and not the effects on the individual cell or capillary as have frequently been described in the past. The effects of corticosteroids
on the cellular unit.
In 1949 Opsahl (27) demonstrated that hyaluronidase in combination with corticosteroids decreased the spreading effect of this hormone. Seifter (31) and many others (6) found the same antagonistic effects in their experiments. The indirect effect of corticosteroids on the mucopolysaccharides in the cellular unit then will be polymerisation. The viscosity of the ground substance will increase and the movement of interstitial water will decrease. If the cellular metabolism is dependent on the nutrient supply, as previously proposed, then corticosteroids automatically will lead to a decreased cellular metabolism. This is probably the case as many investigators have found, that corticosteroids lead to a decreased oxygen consumption, a decreased protein synthesis, and a decreased mitosis (17, 33, 34, 35). We know that many patients who have been taking corticosteroids over a long period have disturbed wound healing. This could be explained because hyaluronodase and corticosteroids are antagonists. That implies that corticosteroids decrease the restoration effect of hyaluronidase. Corticosteroids decrease the invasion of micro-organisms by polymerisation of the ground substance and by inactivation of bacterial hyaluronidase. It seems to be a favourable effect, but the defence mechanism against micro-organisms will also decrease. Corticosteroids are said to have a stabilising effect on capillary membranes (29, 35). This stabilisation seems only to be due to a polymerisation of the pericapillary sheath. The same contradictory effect of corticosteroid therapy in the treatment of bacterial invasion is also in evidence j_n the treatment of oedema with this hormone. 1193
The development of oedema will decrease when the capillary permeability is decreased, but the restoring mechanism is also influenced negatively. This would suggest that there may be a better treatment for oedema which will also stimulate restoration at the same time. The capillary permeability should be so exceptionally good, that polypeptides, protein and corpuscles can diffuse away as easily as possible. This situation can be created by using hyaluronidase instead of corticosteroids. Cellular protection
by corticosteroids.
Many theories have been suggested regarding the protective mechanism of the corticosteroids. In this context I will discuss one of these theories published by Doumont and Cannisre (13). They stated that the decreased cellular metabolism, caused by the increased levels of corticosteroids, gives the cell the opportunity to live longer on reserve energy. This seems to be a very attractive approach, but in this approach to the cellular metabolism it seems illogical to suggest that the cellular metabolism decreases when there is still energy in reserve. Therefore it would appear that this theory is not acceptable, working with this hypothesis. It is known that corticosteroids polymerise the ground substance and that the viscosity will rise as a result. One of the most energy consuming processes of the cell is the sodium/potassium pump (11, 38). This active transport is necessary for the exceptional difference in the intra- and extracellular sodium and potassium concentrations. However these molecules can diffuse through the cellular membrane very easily. When the ground substance is polymerised, this passive sodium/ potassium exchange will decrease and energy is saved from this active transport. This energy saving effect together with the energy reserve leads to protection against ischaemia. When this energy reserve is consumed the cellular metabolism will adapt. Thus, this protecting effect against anoxia is maximal when the energy reserve is maximal. CORTICOSTEROIDS LEAD TO MAXIMAL PROTECTION AGAINST ISCHAEMIA WHEN GIVEN JUST BEFORE THE ONSET OF THE ISCHAEMIC PROCESS. Therefore the blood levels of corticosteroids increase in shock as well as a complex mechanism of redistribution of blood in order to perfuse the most important organs as long as possible. The final conclusion
about corticosteroids.
The major beneficial effect of corticosteroids increase on the cellular unit in stressful circumstances is PROTECTION AGAINST ISCHAEMIA (=SHOCK) FOR THE NORMAL CELL. The period of protection is dependent upon the period of cellular adaption from a high to a low metabolic rate. 1194
THE RELATION BETWEEN THE LOCAL AND GENERAL REACTIVE MECHANISM In critical situations the body seems to have two apparent opposing reactive mechanisms, a local reactive mechanism and a general reactive mechanism. Ir; the local reactive mechanism hyaluronidase plays a central role. This mechanism is characterized by an increased cellular metabolism, AN ENERGY CONSUMING PROCESS. The function of this mechanism is restoration. In the general reactive mechanism corticosteroids, antagonists of hyaluronidase, play a central role at the cellular level. This mechanism is characterized by a decreased cellular metabolism, AN ENERGY SAVING PROCESS. The period of adaptation to this decreased metabolic rate yives the healthy cells PROTECTION against "nutrient cut off". Because the general reactive mechanism is energy saving, there will be extra energy available for the restoration. These two mechanisms together then can influence THE REDISTRIBUTION OF THE TOTAL BODY NUTRIENTS. Healthy cells use less glucose and oxygen and the damaged cells can be restored. THIS IS THUS A FIRST CLASS "FIGHT OF FLIGHT" REACTION THE BODY COULD HAVE. THE CLINICAL CONSEQUENCES If this hypothesis is true the clinical consequences will be substantial. We can give PROTECTION TO CELL JUST BEFORE A CRITICAL SITUATION OR STIMULATE THE REDISTRIBUTION OF NUTRIENTS using corticosteroids and we can STIMULATE THE RESTORATION with hyaluronidase. Perhaps we will be able now to give a better explanation for the clinical course of various illnesses. For example, a very attractive explanation for pulmonary distress, multiple organ failure and irreversible shock could be related to long-term ischaemia of the cellular unit. That would directly imply that this illness could be prevented and/or treated with hyaluronidase. Because this is a general theory a further discussion of all the other factors will not be made. In this context only some general remarks about the activation of one of these mechanisms is made. The proposed treatment with hyaluronidase requires further discussion. THE GOALS OF HYALURONIDASE TREATMENT ARE STIMULATION OF THE RESTORATION, STIMULATION OF THE VASCULARISATION OR A COMBINATION OF BOTH. In the normal body reactive mechanisms, hyaluronidase and corticosteroids together lead to maximal restoration, because of redistribution of nutrients.
1195
If hyaluronidase is injected intravenously in order to stimulate the restoration, this redistribution effect is lost and the result can be the opposite. The best stimulation of the restoration will be a simulation of the normal reactive mechanisms: local "infiltration" of hyaluronidase together with intravenous injection of corticosteroids. When nutrient supply to the cell is decreased because of the development of oedema a better local condition for restoration will be required when this oedema at first is "dissolved" by local or intravenous injection of hyaluronidase. In the STIMULATION OF THE RESTORATION the increased cellular metabolism plays a central role. An increased cellular metabolism implies an increased utilisation of glucose, oxygen consumption and protein synthesis. Because the cellular metabolism is dependant upon the nutrient supply the best stimulation for restoration will theoretically be: HYALURONIDASE
+ EXTRA GLUCOSE + EXTRA OXYGEN
(Infusion of glucose 30-50%) (Hyperbaric 02, mechanical ventilation with positive end expiratory pressure (PEEP)) + EXTRA AMINOACIDS.
With regard to THE STIMULATION OF THE VASCULARISATION the situation is different. Nothing needs to be restored. We only need to use one of the capacities of hyaluronidase: new capillary formation. The problem here is whether this new capillary formation will be definitive and not just temporary. From a clinical point of view we know that the growth of collateral vessels in badly vascularised extremities can be stimulated by "walktraining". On the other hand we know that unused muscles will become weak and badly vascularised. If the mechanism for this adaptation is also based on the influence of hyaluronidase, then the optimal treatment in order to stimulate the vascularisation will be: HYALURONIDASE + ACTIVATION OF THAT SPECIAL ORGAN. Probably in clinical practise the combination of both would be most frequently carried out. CONCLUSION Although much research into hyaburonidase has been done since the first publication by Duran-Reynals in 1928, this enzyme is rarely used in patient management. If this hypothesis about the cellular metabolism as well as the protecting and restoring and mechanisms can be proven then the clinical consequences would be a change in our approach to many problems in medicine. Hyaluronidase may well prove its value in the future. 1196
REFERENCES 1. Banga I, Bolo J. Connective Tissue - a symposium. p 254 (RE Tunbridge ed) Blackwell, Oxford, 1957. 2. Bensley SH. Histological studies of the reactions of cells and intercellular substances of loose connective tissue to the spreading factor of testicular extracts. Ann N Y Acad Sci. 52: 983, 1950. 3. Blumberg BS, Ogstron AG. Chemistry and Biology of Mucopolysaccharides. p 22 (GEW Wolstenholme ed) Churchill, London, 1958. 4. Bollet AJ, Bonner WM, Nance JL. The presence of hyaluronidase in various mammilian tissue. J Biol Chem 238: 3522, 1963. 5. Bostrom H, Garde11 S. Uptake of sulphates in mucopolysaccharides esterified with sulphuric acid in the skin of adult rats after intrapentoneal injection of 535 labelled sodium sulphate. Acta Chem Stand. 7: 216. 1953. 6. Campbell A, Cameron E, Mollison A. Seromucoid, the ground substance and hypothyroidism. J Clin Path. 17: 655, 1964. 7. Cameron E. Hyaluronidase and Cancer. p 32 (E Cameron ed) Pergamon Press, Oxford-London-New York-Toronto-ParijsFrankfurt, 1966. 8. Christie R et al. International commission for the standarisation of Pharmaceutical Enzymes. J Mond Pharm. 8: 5, 1965. 9.
Chambers R, Zweifach BW. Intercellular cement and capillary permeability. Phys Rev. 27: 436, 1947.
10. Deutsche HF. Some properties of a human serum hyaluronic acid. J Biol Chem 224: 767, 1957. 11. De Robertis EDP et al. Cellbiology. p 361 (De Robertis Nawinski Saez ed) Saunders WB Company, Philadelphia and London, 1965. 12. Di Ferrante N. Urinary excretion of mucopolysaccharides by patients with rheumatoid arthritis. J Clin Invest. 36: 1516, 1957. 13. Doumont JM, De Canniere P. Les cercles vivieux du choc, Their treatment with high dosages of hydrocortisone hemisuccinate combined with Isoprenaline. Brux Med. 52: 621, 1972.
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14. Duran-Reynals F. Tissue permeability and the spreading factors in infection A contribution to the host: parasite problem. Bact Rev. 6: 197, 1942. 15. Erdmann W et al. Decrease of oxygen diffusion velocity after ischaemic anoxia, a cause of prolonged cell anoxia. Exe Med. 452: 1978. 16. Friedmann JJ. Physiology. Brown and Company, 1963.
p 3 (E Selkurt ed) Little
17. Friedman M, Strangl LB. Effect of longterm corticosteroids and corticotrophin on the growth of children. Lancet 2: 568, 1966. 18. Gersh I, Catchpole HR. The organisation of ground substance and basement membrane and its significante in tissue injury, disease and growth. Amer J Anat. 85: 457, 1949. 19. Grossfeld H. Production of hyaluronic acid by fibroblasts growing from explants of Walker Tumour 256: production of hyaluronidase by tumor cells. J Nat Cancer Inst. 27: 543, 1961. 20. Hechter 0. Mechanisms of spreading N Y Acad Sci. 52: 1028, 1950.
factor action. Ann
21. Kloner RA et al. Effect of Hyaluronidase During the Early Phase of Acute Myocardial Ischemia An Ultrastructure and Morphometric Analysis. Amer J Card. 40: 43, 1977. 22. Linker A, Meyer K, Weissmann B. Enzymatic formation of monosaccharides from hyaluronidase. J Biol Chem. 213: 237, 1955. 23. MC Clean D. A factor in culture filtrates of certain pathogenic bacterias which increases the permeability of tissue. J Path and Bact. 42: 477, 1936. 24. MC Clean D. Studies on diffusing factors: methods of assay of hyaluronidase and their correlation with skin diffusion activity. Biochem J. 37: 169, 1943. 25. Meyer K. The biological significans of hyaluronic and hyaluronidase. Physiol Rev. 27: 335, 1947.
acid
26. Morawetz R. Strong E, Clark DK, Erdmann W. Oxygen Transport to Tissue III. p 629 (JA Silver et al ed) Plenum Press, New York-London, 1978.
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27. Opsahl JC. The influences of hormones from the adrenal cortex on the dermal spread of indian ink with and without hyaluronidase: prelimirary report. Yale J Biol Med. 21: 255, 1949. 28. Rogers HJ. The complexity of hyaluronidase produced by microorganisms. Biochem J. 42: 633, 1948. 29. Rovit RL, Hagan R. Steroids and cerebral oedema. The effects of glucocorticosteroids on abnormal capillary permeability following cerebral injury in cats. J Neuropath Exp Neurol. 27: 277, 1968. 30. Schiller S, Dewey KF. Isolation of chondroitinsulfuric acid from normal human plasma. Fed. Proc. 15: 348, 1956. 31. Seifter J. Baeder DH, Dervinis A. Alterations in permeability of some membranes by hyaluronidase and inhibition of this effect by steroids. Ann N Y Acad Sci 52: 1141, 1950. 32. Starling EH. On the absorption of fluids from the connective tissue space. J Physiol London 19: 312, 1896. 33. Stoppani AOM, De Brignone CH, De Brignone JA. Structural requirements for the action of steroids as inhibitor of electron transfer. Arch Biochem Biophys. 127: 463, 1968. 34. Sweikert HU et al. Biochemie, Physiologie und klinische Abwendung der Cortisols und Corticoide. Sweiz Apoth Zeit. 109: 111, 1971. 35. Wied D de. Algemene Farmacotherapie. p 754 (W Lammers ed) Stafleu's Wetenschappelijke Uitg. Leiden, 1968. 36. Williams RG. The effects of continuous local injection of hyaluronidase on skin and subcutaneous tissue in rats. Anat Rec. 122: 349, 1955. 37. Zweifach BW, Chambers R. The actions of hyaluronidase extracts on the capillary wall. Ann N Y Acad Sci. 52: 1047, 1950. 38. Zijlstra WG et al. Fysiologie van het milieu interieur. p 379 (WG Zijlstra et al edf Van Gorkum and Comp. BV, Assen, 1973. 39.
Zijlstra WG. Transcapillair 52: 2041, 1978.
Transport. Ned. Tijds v Gen.
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ACKNOWLEDGEMENTS It is a great pleasure to thank Professor Peter Safar M.D., Head and Chairman of the Resuscitation Research Center, Pittsburgh University, Pittsburgh, USA, and Professor Wilhelm Erdmann M.D., Department of Anaesthesiology, University of Groningen, the Netherlands for all there courteous stimulation to publish this hypothesis as well as Peter H. Robinson, F.R.C.S., Department of Plastic Surgery, University of Groningen, the Netherlands, for the excellent editory remarks and suggestions. I would also like to thank Mrs. A. van der Velde, Secretary of the Department of General Surgery, University of Groningen, the Netherlands, who was responsible for the time consuming task involved in preparing and typing this manuscript. My thanks to D.Buiter, medical artist of the Department of Neurosurgery, University of Groningen, the Netherlands, who kindly supplied the excellent line drawings.
1200
Hypotheses
THE FUNDAMENTAL
5: ll83-1200,
1979
ROLE OF HYALURONIDASE
IN TISSUE
An hypothesis on the protecting and restoring mechanisms of the "cellular unit" with their clinical consequences S.W.Bok. St-Lucas Ziekenhuis, Gassingel The Netherlands.
18,
9671
CX Winschoten,
ABSTRACT The interstitial ground substance plays an essential role in the physiological processes of the capillary and the cell. Together they form a fundamental unit of function in which the cell is the most important part. Therefore the fundamental unit is called: THE CELLULAR UNIT. It is proposed central role and that this enzyme is indispensible for normal healthy cells as well as for the reactive mechanism in critical situations. The reactive mechanisms can be divided into a local reactive mechanism and a general reactive mechanism. The local reactive mechanism has a function of RESTORATION. The general reactive mechanism is especially for PROTECTION AGAINST ISCHAEMIA and REDISTRIBUTION OF TOTAL BODY NUTRIENTS in order to stimulate the restoration. These two mechanisms may have important clinical consequences. Key words:
"cellular unit", hyaluronidase, protection, cell metabolism.
restoration,
INTRODUCTION From a clinical point of view the local reactive mechanism is well known. A short-term anoxia caused by tissue ischaemia is followed by a phase of hyperaemia; partly damaged organs can restore or regenerate and chronic irritation will lead to hyperaemia and cell-proliferation. This local reactive mechanism, characterised by an increased mitosis and hyperaemia, seems to be a mechanism of an increase in cellular metabolism. This directly implies that the energy requirement for the cells in this area will increase and also that the metabolic rate of a cell is not only dependent on the blood levels of special hormones. Recently, Erdmann et al (15, 26) were able to measure oxygen diffusion in the interstitial tissue. After short-term ischaemic anoxia, diffusion of oxygen in the interstitial ground substance is dramatically increased for a short period. Thus, the oxygen diffusion in the interstitial ground substance is variable and seems to be related to the cellular metabolism. What is the precise underlying mechanism of this local reaction? To explain this local reaction, we first have to know the relation between the nutrient supply to the cell and the cellular metabolism. Then perhaps we can explain this local reactive mechanism and describe also the efficiency of this 1183
mechanism. Perhaps it will then be possible also to explain the increased secretion of corticosteroids by the adrenal glands during stress. This can perhaps lead to a better knowledge of the functions of this hormone. Perhaps a knowledge of the precise underlying mechanism of these reactions will have important clinical consequences and lead to possible clinical application. NORMAL CELLULAR METABOLISM "The cellular unit". One of the well known standard works on physiology states that "the fundamental unit of function is the cell" (16). It is firmly established now that in any tissue the individual cell, collagen-, reticulum-, and elastin fibres, blood and lymph vessels are embedded in an amorphous gel or ground substance (10). This ground substance consists of mucopolysaccharide-protein complexes and the molecular weight of the mucopolysaccharides determines the viscosity of this complex (3). As we shall see later, it is better to talk about the "CELLULAR UNIT" as A FUNDAMENTAL UNIT OF FUNCTIONS, because every cell is embedded in this viscous gel and this gel plays a central role in the nutrient supply to the cell. This cellular unit then consists of the cell, the surrounding interstitial ground substance and the capillary endothelium with its pericapillary sheath (9) (figure 1). The movement of water within the interstitium. The functions of the cell can be devided into general functions, which are essential for continuation of cell life and the specific functions, which differ from organ to organ. All these functions have one thing in common and that is that they all require energy. The energy need for these functions is dependant on the metabolic rate of the cell. For an optimal nutrient supply to the cell the movement of water within the interstitium is essential. The classic work on this subject was published by Starling (32) and until now has remained almost unchanged. Interstitial movement of water is influenced by the following factors: The arterial and venous capillary blood-pressure, the size of the pores in the capillary endothelium, the permeability of the interstitium and the colloid osmotic blood-pressure. The permeability of the interstitium is dependent on the viscosity of the ground substance (3). In 1949 Opsahl (27) demonstrated that the viscosity of the ground substance is influenced by hormones particularly from the adrenal cortex. This conclusion was confirmed by others (6, 31). 1184
blood supply capillary endothelium peri capillary sheath cell groundsubstance.4
-
Figure
1.
THE CELLULAR
UNIT
This unit is of function.
proposed
1185
to
be the
THE CELLULAR UNIT
fundamental
unit
Hyaluronidase cell.
in relation to the ground substance and to the
Since the discovery of the enzyme-complex hyaluronidase by Duran-Reynals in 1928 much experimental work has been done. The name hyaluronidase was given by Meyer (25) after he found that this enzyme depolymerises hyaluronic acid. Shortly after hyaluronidase was found to be a depolymerising enzyme for the whole group of mucopolysaccharides that form the viscous gel around the cells, in other words the ground substance of the interstitium. The mucopolysaccharides are first depolymerised by this enzyme into oligosaccharides and then into monosaccharides, N-acetyl glucosamide and glucuroronic acid (22). These breakdown products diffuse away into the capillaries and can be detected in the urine (12, 30). The viscosity of the ground substance will decrease and become more water soluble and the permeability will increase as a result of this breakdown. Bensley (21) showed that the ground substance previously coloured with toluidine-blue, disappears after injection of hyaluronidase. This implies that the ground substance is not only depolymerised by this enzyme, but also dissolved. The elastin fibres in the interstitium are insensitive to this enzyme and this network can function as a frame (1). Recently Grossfeld (19) and Bollet (4) found that cells of many tissues produce this enzyme. Because all cells are embedded in this viscous gel of mucopolysaccharides, it seems highly probable that all cells produce this enzyme. THIS HYPOTHESIS IMPLIES THAT HYALURONIDASE SYNTHESIS IS A GENERAL FUNCTION OF ALL CELLS. For hyaluronidase to influence the surrounding ground substance it must move from the cell into the interstitium. Then there must be a continuous breakdown of the ground substance and consequently also a continuous replacement. The metabolic cycle has not yet been fully established, but after the isotope studies by Bostrom and studies by others much more is known about its probable form (5, 18). These experiments made clear, that replacement was the result of two different mechanisms. Firstly, the breakdown products, circulating in the blood, are partly used for replacement and secondly, the cells themselves produce these mucopolysaccharides. The breakdown is said to be due to hyaluronidase that is synthetized in the cells and then excreted into the interstitium. This implies that there is a balance between the breakdown and replacement of the ground substance in normal healthy cell units.
1186
Hyaluronidase
and the new formation of capillaries.
In 1947 Chambers and Zweifach (9) demonstrated that hyaluronidase has no direct effect on the capillary endothelium and its intercellular cement. Some years later the same authors (37) published microscopic observations on living tissue, infiltrated with hyaluronidase. Within a few minutes of injection the capillary walls showed an outward bulging followed by a rupture of the capillaries and sinusoids full of blood are formed. This is an indirect effect of hyaluronidase and is the consequence of the dissolution of the pericapillary sheath. The very thin layer of endothelial cells now cannot withstand the capillary blood-pressure and burst. In 1977 Kloner et al (21) published an experimental study on the treatment of myocardial infarction with high dosages of hyaluronidase, injected intravenously. The electronmicroscopical observation of myocardial tissue, 3 hours after treatment, showed a significant increase in the number of small vessels in the ischaemic areas by comparison with untreated animals. ft seems highly probable that the new formation of capillaries is the end result of sinusoid formation. Hwuronidase -
and the cellular metabolism.
:I:t is well documented that depolymerisation of the ground substance by local injection of hyaluronidase results in a tissue-cell proliferation, an increased mitosis and therefore an increased cellular metabolism (2, 23, 36). However hyaluronidase has no direct effect on the capillary endothelium. The outward filtration of fluid will increase rapidly after hyaluronidase infiltration because the pericapillary sheath is depolymerised (9). The water solubility and the permeability of the ground substance will also increase rapidly after hyaluronidase rnjection. The overall result then will be an increased movement of water. This was also described by Zweifach and Chambers (37). With the increased movement of water in the ground substance the nutrient supply to the cell will also increase. We know now that this enzyme is probably produced by every cell, that it is excreted in the surrounding interstitium and that it regulates the nutrient supply to the cell. The observation of tissue infiltrated with hyaluronidase shows an increased metabolism. May we now make the following hypothesis that: THE CELL REGULATES ITS METABOLIC RATE BY THE REGULATION OF THE PRODUCTION OF HYALURONIDASE. If we can accept this hypothesis, the conclusion will be that
hyaluronidase plays a central role in cellular life. This was also suggested by Cameron (7). 1187
The hormonal influences on the metabolic rate of the cell can then be explained by influences on the activity of this enzyme and/or on its rate of production. THE LOCAL REACTIVE MECHANISM Introduction. We know that locally injected hyaluronidase leads to hyperaemia and cell proliferation and if we accept that hyaluronidase plays a central role in the cellular metabolism, can we explain the local reactive mechanism by changes of the hyaluronidase concentration in the interstitial ground substance? This implies that the local reactive mechanism, for example hyperaemia after short-term ischaemic anoxia, is caused by an increased interstitial movement of water due to a higher hyaluronidase concentration. In this context it is hard to believe that one of the general functions - the hyaluronidase synthesis - will increase when the nutrient supply to the cell is diminished. To explain this local reactive mechanism we must first determine the hyaluronidase concentration in the cellular unit. Hyaluronidase
concentration
in the cellular unit.
Hyaluronidase is synthesized in the cell and transported into the interstitium. Therefore it has to pass through the cellular membrane. The cellular membrane is a lipoid-protein layer. The mechanisms of transport through this layer can be a passive transport, an active transport or facilitated diffusion (16). Passive transport is only possible for lipoid soluble substances or water soluble molecules smaller than urea (16). Hyaluronidase is a highly water soluble enzyme and although the exact molecular weight is not known, it is much bigger than urea (8). That implies that it cannot pass through the cellular membrane by a passive transport mechanism. The possibilities remaining are facilitated diffusion or active transport. If it is transported by facilitated diffusion the intracellular hyaluronidase concentration has to be much higher than the extracellular concentration. If it is passed through the cellular membrane by an active transport mechanism the concentration is dependent upon the activity of this transport. The exact mechanism is not yet known. When hyaluronidase is transported through the cellular membrane the interstitial movements of water will transport this enzyme from the cell to the capillary, since we know that hyaluronidase does not diffuse into tissue when there is no movement of water (20). 1188
Because the direction of movement of water is from the arterial side to the venous side, the hyaluronidase concentration has to be higher on the venous side. That might explain the fact that the permeability of the venous capillary is higher than the permeability of the arterial capillary as described by Zijlstra (39) recently. Hyaluronidase passes through the capillary endothelium without difficulty and is then excreted in the urine (30, 31). Only if we can accept that the intracellular hyaluronidase concentration is higher than the extracellular concentration, can we explain the local reactive mechanism fully, as we will see later. Figure 2 shows the hypothetical hyaluronidase concentration in the cellular unit. General aspects of the local reactive mechanism. Working with the cellular unit model and the proposed hyaluronidase concentration, the reaction to traumatic cell death, short-term and long-term ischaemia as well as the invasion of micro-organisms can be explained. In all these different pathologic circumstances the efficiency of this reaction can also be explained. During the discussion on the hyaluronidase concentration in relation to the cellular unit the hypothesis was made that there is a higher intracellular concentration of this enzyme. Especially in traumatic cell death an explanation of the local reaction would be impossible, if the reserve is true. The reaction following ischaemia is much more complicated than after traumatic cell death. In this situation the reserve energy of the cell plays an essential role. This idea of a reserve of energy should be accepted because the cell can survive a certain period of total nutrient and oxygen deficiency. In long-term ischaemia the pH decrease caused by the accumulation of the metabolic endproducts plays an essential role since we know that hyaluronidase is only active within a pH range of 4-9 (8). Some years after the first publication about hyaluronidase in 1928, this enzyme was found in many different bacteria (27). Duran-Reynals (14) demonstrated the relationship between the amount of bacterial hyaluronidase synthesis and the invasivity of the bacteria. Traumatic cell death. Traumatic cell death implies that the cellular membrane has been destroyed. If there is a defect of the cellular membrane, then the interstitial hyaluronidase concentration will increase, because the intracellular concentration of hyaluronidase is higher than the extracellular concentration.
,189
ARTERIAL SIDE OF THE CAPILLARY
VENOUS SIDE OF THE CAPILLARY
-
hyaluronidase concentration
Figure
2:
The
probable
normal local
hyaluronidase
ccl lular reactive
concentration Working
unit. mechanism
1190
can
with
this
be explained.
in
the
model
the
This increased hyaluronidase concentration increases the interstitial movement of water and nutrient supply to the cells surrounding the dead ones with as consequence decreased mitosis and RESTORATION OF THE TOTAL NUMBER OF CELLS. Another advantage of the increased movement of water will be an INCREASED CLEARING OF NECROTIC MATERIALS. Invasion by micro-organisms. It is well understood that the invasivity of bacteria is related to the capacity of these micro-organisms to dissolve the ground substance by the production of hyaluronidase. When the tissue concentration of hyaluronidase rises, the reaction of these tissues will also lead to new capillary formation as previously described. The periferal resistance in this infected area will decrease with, as a consequence, hyperaemia. This local hyperaemia leads to optimalization of the defence mechanisms against these bacteria. The increase in locally circulating immune factors, as well as the restoration of the total number of cells, together with the increased clearance of necrotic materials all play important roles in this local reaction. To rationalize further, it can be seen that the magnitude of the fall in periferal resistance is related to the numbers and the type of hyaluronidase producing bacteria. The severity of the phenomenon of septic shock caused by some types of bacteria can now be explained by this mechanism. Conversely, when hyperaemia cannot occur because of vascular insufficiency (e.g. arteriosclerosis) or when the level of circulating immune factors is depressed then this local defence mechanism will fail. This would explain why bad11 vascularised tissues or malnourished people are more susceptible to infection. Short-term
ischaemia.
During the ischaemic period following arterial occlusion, the capillary blood pressure decreases to zero. This directly implies that there will be no further movement of water in the interstitium. As a consequence the nutrient supply to the cell will be cut off and the cell has to function on the reserve energy. The period that the cell can function on reserve energy is dependant upon the amount of reserve energy and the metabolic rate of the cell. During this time all the functions of the cell are stiil normal, including the hyaluronidase synthesis as well as its transport across the cell membrane. Because there is no movement of water in the interstitium to the capillary and then the pericellular hyaluronidase concentration will rise. The metabolic endproducts will accumulate and the pH will decrease. 1191
This implies that in the period of ischaemia there is no cellular reaction. THE CELL, THUS, IS UNABLE TO REACT DIRECTLY TO ISCHAEMIA. After restoration of the circulation the movement of water is restored and then the high pericellular hyaluronidase concentration can diffuse into the interstitium. This will lead to an increased movement of water which will mean that the cell firstly can RESTORE THE CELLULAR ENERGY RESERVE and secondly the metabolic endproducts can diffuse away more rapidly and THE pH WILL BE RESTORED. Long-term
ischaemia.
At the beginning of a period of long-term ischaemia there exists of course the same situation as in short-term ischaemia. After a certain period of anoxia the cellular energy reserve is consumed and the cellular functions slow down. The accumulation of the metabolic end products increases. The pH decreases so much that hyaluronidase becomes inactive. After restoration of the circulation this inactive hyaluronidase will not give rise to any reaction by contrast with the situation during short-term ischaemia. Because the cellular functions are already decreased there will be a decrease in the activity of the mechanisms of the sodium/potassium pump and the cell will become oedematous. This is unfavourable for function as well as affecting the availability of the nutrient supply. Restoration is impossible, irreversible changes occur and the cell will die. In this situation there will be no reaction to cellular death, in contrast to traumatic cell death, because hyaluronidase is inactive. The final conclusion
about the local reactive mechanism.
The local reactive mechanism, characterized by hyperaemia and cell proliferation can be explained by the hyaluronidase interactions. The effects of this local reaction are as follows: 1. RESTORATION OF THE TOTAL NUMBER OF CELLS (=regeneration) 2. INCREASED CLEARING OF NECROTIC MATERIALS 3. RESTORATION OF THE CELLULAR ENERGY RESERVE 4. RESTORATION OF THE pH. All these actions can be collectively described as tissue restoration. THUS, HYALURONIDASE IS RESPONSIBLE FOR THE RESTORATION. A new aspect in this approach is that the hyperaemia is not only caused by dilatation of the vessels but also by THE FORMATION OF NEW CAPILLARIES. Perhaps the collateral vessel growth, frequently seen in poorly vascularized extremities, is based on the same reaction. also essential in this hypothesis is that THE CELL ITSELF HAS NO DIRECT DEFENCE AGAINST ISCHAEMIA (aSHOCK). 1192
CORTICOSTEROIDS
AND THE GENERAL REACTIVE MECHANISM
Introduction. We know that the body reacts in stressful circumstances with a very complex general reaction. Only a small part of this general reaction is the increase in the blood levels of corticosteroids. The essential effect of the increased circulating corticosteroids in stress is thought to lead to protection. We now know much more about the interactions that these hormones have on cellular metabolism and the probable actions on capillary membranes. However the exact mechanism of cellular protection is unknown. We will discuss the effects of corticosteroids on the cellular unit as a whole and not the effects on the individual cell or capillary as have frequently been described in the past. The effects of corticosteroids
on the cellular unit.
In 1949 Opsahl (27) demonstrated that hyaluronidase in combination with corticosteroids decreased the spreading effect of this hormone. Seifter (31) and many others (6) found the same antagonistic effects in their experiments. The indirect effect of corticosteroids on the mucopolysaccharides in the cellular unit then will be polymerisation. The viscosity of the ground substance will increase and the movement of interstitial water will decrease. If the cellular metabolism is dependent on the nutrient supply, as previously proposed, then corticosteroids automatically will lead to a decreased cellular metabolism. This is probably the case as many investigators have found, that corticosteroids lead to a decreased oxygen consumption, a decreased protein synthesis, and a decreased mitosis (17, 33, 34, 35). We know that many patients who have been taking corticosteroids over a long period have disturbed wound healing. This could be explained because hyaluronodase and corticosteroids are antagonists. That implies that corticosteroids decrease the restoration effect of hyaluronidase. Corticosteroids decrease the invasion of micro-organisms by polymerisation of the ground substance and by inactivation of bacterial hyaluronidase. It seems to be a favourable effect, but the defence mechanism against micro-organisms will also decrease. Corticosteroids are said to have a stabilising effect on capillary membranes (29, 35). This stabilisation seems only to be due to a polymerisation of the pericapillary sheath. The same contradictory effect of corticosteroid therapy in the treatment of bacterial invasion is also in evidence j_n the treatment of oedema with this hormone. 1193
The development of oedema will decrease when the capillary permeability is decreased, but the restoring mechanism is also influenced negatively. This would suggest that there may be a better treatment for oedema which will also stimulate restoration at the same time. The capillary permeability should be so exceptionally good, that polypeptides, protein and corpuscles can diffuse away as easily as possible. This situation can be created by using hyaluronidase instead of corticosteroids. Cellular protection
by corticosteroids.
Many theories have been suggested regarding the protective mechanism of the corticosteroids. In this context I will discuss one of these theories published by Doumont and Cannisre (13). They stated that the decreased cellular metabolism, caused by the increased levels of corticosteroids, gives the cell the opportunity to live longer on reserve energy. This seems to be a very attractive approach, but in this approach to the cellular metabolism it seems illogical to suggest that the cellular metabolism decreases when there is still energy in reserve. Therefore it would appear that this theory is not acceptable, working with this hypothesis. It is known that corticosteroids polymerise the ground substance and that the viscosity will rise as a result. One of the most energy consuming processes of the cell is the sodium/potassium pump (11, 38). This active transport is necessary for the exceptional difference in the intra- and extracellular sodium and potassium concentrations. However these molecules can diffuse through the cellular membrane very easily. When the ground substance is polymerised, this passive sodium/ potassium exchange will decrease and energy is saved from this active transport. This energy saving effect together with the energy reserve leads to protection against ischaemia. When this energy reserve is consumed the cellular metabolism will adapt. Thus, this protecting effect against anoxia is maximal when the energy reserve is maximal. CORTICOSTEROIDS LEAD TO MAXIMAL PROTECTION AGAINST ISCHAEMIA WHEN GIVEN JUST BEFORE THE ONSET OF THE ISCHAEMIC PROCESS. Therefore the blood levels of corticosteroids increase in shock as well as a complex mechanism of redistribution of blood in order to perfuse the most important organs as long as possible. The final conclusion
about corticosteroids.
The major beneficial effect of corticosteroids increase on the cellular unit in stressful circumstances is PROTECTION AGAINST ISCHAEMIA (=SHOCK) FOR THE NORMAL CELL. The period of protection is dependent upon the period of cellular adaption from a high to a low metabolic rate. 1194
THE RELATION BETWEEN THE LOCAL AND GENERAL REACTIVE MECHANISM In critical situations the body seems to have two apparent opposing reactive mechanisms, a local reactive mechanism and a general reactive mechanism. Ir; the local reactive mechanism hyaluronidase plays a central role. This mechanism is characterized by an increased cellular metabolism, AN ENERGY CONSUMING PROCESS. The function of this mechanism is restoration. In the general reactive mechanism corticosteroids, antagonists of hyaluronidase, play a central role at the cellular level. This mechanism is characterized by a decreased cellular metabolism, AN ENERGY SAVING PROCESS. The period of adaptation to this decreased metabolic rate yives the healthy cells PROTECTION against "nutrient cut off". Because the general reactive mechanism is energy saving, there will be extra energy available for the restoration. These two mechanisms together then can influence THE REDISTRIBUTION OF THE TOTAL BODY NUTRIENTS. Healthy cells use less glucose and oxygen and the damaged cells can be restored. THIS IS THUS A FIRST CLASS "FIGHT OF FLIGHT" REACTION THE BODY COULD HAVE. THE CLINICAL CONSEQUENCES If this hypothesis is true the clinical consequences will be substantial. We can give PROTECTION TO CELL JUST BEFORE A CRITICAL SITUATION OR STIMULATE THE REDISTRIBUTION OF NUTRIENTS using corticosteroids and we can STIMULATE THE RESTORATION with hyaluronidase. Perhaps we will be able now to give a better explanation for the clinical course of various illnesses. For example, a very attractive explanation for pulmonary distress, multiple organ failure and irreversible shock could be related to long-term ischaemia of the cellular unit. That would directly imply that this illness could be prevented and/or treated with hyaluronidase. Because this is a general theory a further discussion of all the other factors will not be made. In this context only some general remarks about the activation of one of these mechanisms is made. The proposed treatment with hyaluronidase requires further discussion. THE GOALS OF HYALURONIDASE TREATMENT ARE STIMULATION OF THE RESTORATION, STIMULATION OF THE VASCULARISATION OR A COMBINATION OF BOTH. In the normal body reactive mechanisms, hyaluronidase and corticosteroids together lead to maximal restoration, because of redistribution of nutrients.
1195
If hyaluronidase is injected intravenously in order to stimulate the restoration, this redistribution effect is lost and the result can be the opposite. The best stimulation of the restoration will be a simulation of the normal reactive mechanisms: local "infiltration" of hyaluronidase together with intravenous injection of corticosteroids. When nutrient supply to the cell is decreased because of the development of oedema a better local condition for restoration will be required when this oedema at first is "dissolved" by local or intravenous injection of hyaluronidase. In the STIMULATION OF THE RESTORATION the increased cellular metabolism plays a central role. An increased cellular metabolism implies an increased utilisation of glucose, oxygen consumption and protein synthesis. Because the cellular metabolism is dependant upon the nutrient supply the best stimulation for restoration will theoretically be: HYALURONIDASE
+ EXTRA GLUCOSE + EXTRA OXYGEN
(Infusion of glucose 30-50%) (Hyperbaric 02, mechanical ventilation with positive end expiratory pressure (PEEP)) + EXTRA AMINOACIDS.
With regard to THE STIMULATION OF THE VASCULARISATION the situation is different. Nothing needs to be restored. We only need to use one of the capacities of hyaluronidase: new capillary formation. The problem here is whether this new capillary formation will be definitive and not just temporary. From a clinical point of view we know that the growth of collateral vessels in badly vascularised extremities can be stimulated by "walktraining". On the other hand we know that unused muscles will become weak and badly vascularised. If the mechanism for this adaptation is also based on the influence of hyaluronidase, then the optimal treatment in order to stimulate the vascularisation will be: HYALURONIDASE + ACTIVATION OF THAT SPECIAL ORGAN. Probably in clinical practise the combination of both would be most frequently carried out. CONCLUSION Although much research into hyaburonidase has been done since the first publication by Duran-Reynals in 1928, this enzyme is rarely used in patient management. If this hypothesis about the cellular metabolism as well as the protecting and restoring and mechanisms can be proven then the clinical consequences would be a change in our approach to many problems in medicine. Hyaluronidase may well prove its value in the future. 1196
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factor action. Ann
21. Kloner RA et al. Effect of Hyaluronidase During the Early Phase of Acute Myocardial Ischemia An Ultrastructure and Morphometric Analysis. Amer J Card. 40: 43, 1977. 22. Linker A, Meyer K, Weissmann B. Enzymatic formation of monosaccharides from hyaluronidase. J Biol Chem. 213: 237, 1955. 23. MC Clean D. A factor in culture filtrates of certain pathogenic bacterias which increases the permeability of tissue. J Path and Bact. 42: 477, 1936. 24. MC Clean D. Studies on diffusing factors: methods of assay of hyaluronidase and their correlation with skin diffusion activity. Biochem J. 37: 169, 1943. 25. Meyer K. The biological significans of hyaluronic and hyaluronidase. Physiol Rev. 27: 335, 1947.
acid
26. Morawetz R. Strong E, Clark DK, Erdmann W. Oxygen Transport to Tissue III. p 629 (JA Silver et al ed) Plenum Press, New York-London, 1978.
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27. Opsahl JC. The influences of hormones from the adrenal cortex on the dermal spread of indian ink with and without hyaluronidase: prelimirary report. Yale J Biol Med. 21: 255, 1949. 28. Rogers HJ. The complexity of hyaluronidase produced by microorganisms. Biochem J. 42: 633, 1948. 29. Rovit RL, Hagan R. Steroids and cerebral oedema. The effects of glucocorticosteroids on abnormal capillary permeability following cerebral injury in cats. J Neuropath Exp Neurol. 27: 277, 1968. 30. Schiller S, Dewey KF. Isolation of chondroitinsulfuric acid from normal human plasma. Fed. Proc. 15: 348, 1956. 31. Seifter J. Baeder DH, Dervinis A. Alterations in permeability of some membranes by hyaluronidase and inhibition of this effect by steroids. Ann N Y Acad Sci 52: 1141, 1950. 32. Starling EH. On the absorption of fluids from the connective tissue space. J Physiol London 19: 312, 1896. 33. Stoppani AOM, De Brignone CH, De Brignone JA. Structural requirements for the action of steroids as inhibitor of electron transfer. Arch Biochem Biophys. 127: 463, 1968. 34. Sweikert HU et al. Biochemie, Physiologie und klinische Abwendung der Cortisols und Corticoide. Sweiz Apoth Zeit. 109: 111, 1971. 35. Wied D de. Algemene Farmacotherapie. p 754 (W Lammers ed) Stafleu's Wetenschappelijke Uitg. Leiden, 1968. 36. Williams RG. The effects of continuous local injection of hyaluronidase on skin and subcutaneous tissue in rats. Anat Rec. 122: 349, 1955. 37. Zweifach BW, Chambers R. The actions of hyaluronidase extracts on the capillary wall. Ann N Y Acad Sci. 52: 1047, 1950. 38. Zijlstra WG et al. Fysiologie van het milieu interieur. p 379 (WG Zijlstra et al edf Van Gorkum and Comp. BV, Assen, 1973. 39.
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ACKNOWLEDGEMENTS It is a great pleasure to thank Professor Peter Safar M.D., Head and Chairman of the Resuscitation Research Center, Pittsburgh University, Pittsburgh, USA, and Professor Wilhelm Erdmann M.D., Department of Anaesthesiology, University of Groningen, the Netherlands for all there courteous stimulation to publish this hypothesis as well as Peter H. Robinson, F.R.C.S., Department of Plastic Surgery, University of Groningen, the Netherlands, for the excellent editory remarks and suggestions. I would also like to thank Mrs. A. van der Velde, Secretary of the Department of General Surgery, University of Groningen, the Netherlands, who was responsible for the time consuming task involved in preparing and typing this manuscript. My thanks to D.Buiter, medical artist of the Department of Neurosurgery, University of Groningen, the Netherlands, who kindly supplied the excellent line drawings.
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