INTRAHEPATIC VASCULAR PATHWAYS. By W. H. H. ANDREWS

Transcription

1 INTRAHEPATIC VASCULAR PATHWAYS. By W. H. H. ANDREWS and I. DEL Rio LOZANO. From the Department of Pharmacology, St. Mary's Hospital Medical School, London, W. 2. (Received for publication 15th September 1962) Experiments were carried out on dogs, cats and rabbits. Under anaesthesia fine polythene cannulie were inserted so that injections of acetylcholine and catechol amines could be made into the portal venous, the systemic venous and the hepatic arterial systems. Carotid arterial pressure was recorded. Much larger amounts of acetylcholine were needed when injected into the portal vein than when injected into the hepatic artery to produce the same response in arterial pressure. With adrenaline and noradrenaline the difference between the two hepatic circulations was much less marked, except in some rabbits. In more than half the experiments in which it was carried out, cooling or section of the hepatic branch of the cceliac plexus altered the blood pressure response to drugs injected into the hepatic circulation, the arterial and portal systems being affected differently. Changes evoked by nerve cooling were reversible. Ganglionic blockade with hexamethonium bromide, and sympathetic blockade with choline 2:6 xylyl ether bromide or bretylium, also affected the intrahepatic circulation of blood, as did stimulation of the sympathetic nerve from the coeliac plexus. It was concluded that portal venous and hepatic arterial blood may, under some circumstances, flow through different pathways which are under autonomic control. SEVERAL authors, for example Kniseley, Block and Warner [1948], Wakim and Mann [1942], Seneviratne [1949] have investigated the circulation of normal livers with the technique of trans-illumination. In their papers they state that there was an intermittent flow through the sinusoids, and it can be deduced that various intrahepatic vascular pathways may exist, but they do not describe channelling through special vessels under the influence of nerves or injected substances. Daniel and Prichard [1951 a and b] describe a restricted (hilar) circulation in certain conditions, but consider that the blood passes through sinusoids in the deeper parts of the liver. Andrews, Hecker, Maegraith and Ritchie [1953] give physiological evidence for the shunting of arterial blood in perfused canine livers. Their conclusions have received some confirmation from the work of Szilagyi, Koscar and Kesztyus [1955] who left the liver in situ and measured the effect on general blood pressure of drugs injected into the liver circulation. In this study the work of Szilagyi et al. has been repeated with modifications, and also an attempt has been made to find whether the hepatic nerves affect intrahepatic vascular pathways. We present evidence that not only do various intrahepatic vascular pathways exist, but also that they are modified by the action of the hepatic sympathetic nerves. METHODS The method consisted basically of comparing the amounts of drug needed to give the same change in peripheral arterial pressure, after injecting a pharmacologically active substance into the hepatic artery and portal vein alternately. There are certain VOL. xlviii, NO

2 128 Andrews and del Rio Lozano objections to this procedure which are considered in the discussion. Experiments were carried out on twenty-seven dogs, nine cats, ten rabbits, two rats and three guinea-pigs. Drugs chosen for aneesthesia were selected to give a constant depth in the different species used. In the case of pentobarbitone, injections were given at intervals as the drug is metabolized. The responses appeared to be independent of the aneesthetic agent used, which was: dogs, usually pentobarbitone, sometimes chloralose; cats, usually chloralose, occasionally pentobarbitone; rabbits, pentobarbitone together with chloralose; rats and guinea-pigs, urethane. Carotid arterial pressure was recorded. Through a mid-line abdominal incision the hepato-duodenal artery was exposed, cleaned and cannulated with a fine polythene tube. For small animals it was necessary to draw out the finest tubing, when heated, to obtain a cannula which would not damage the vessel wall. All other arterial branches were ligated so that substances injected into the hepato-duodenal artery would pass into the liver. In dogs a failure to demonstrate the difference between hepatic arterial and portal venous circulations was usually associated with a failure to ensure that the arterial injection passed in toto to the liver. For portal venous injections, a similar polythene cannula was introduced into a tributary of the superior mesenteric vein. In dogs and cats one of the leg veins was also cannulated. The pharmacologically active drugs injected into the arterial and venous systems were put in with sufficient force to cause turbulance and prevent stream-lining into the liver. Arterial injection was especially difficult in the rat and guinea-pig, because fluid could easily be forced into the aorta. In some experiments the hepatic branch of the coeliac ganglion was isolated, a procedure which is easy in the cat but difficult in most dogs, owing to the way in which the nerves sheath the hepatic artery. The hepatic nerves of rabbits are rather diffuse, and it is possible that not all the fibres were stimulated in our experiments. The nerve was placed on a silver-coated copper tube, bent in the shape of a double hook: the nerve was then cooled to approximately 10 C., by passing cold water through the tube. Care was taken not to cool the artery. Before the nerve was stimulated it was divided near the gaiiglion between ligatures. Stimulation was with a square wave, 4-5 volts, 035 msec. duration at a rate of 30/sec. RESULTS The method of using the animals' own blood pressure to assess the amount of pharmacologically-active drug reaching the general circulation was generally satisfactory. In some dogs, however, the arterial pressure showed spontaneous fluctuations and in a few others there appeared to be very active baroceptor reflexes, which made interpretation of results difficult. No attempt, therefore, was made to assess the results obtained in five of the twenty-seven dogs studied. There was some species variation, and also variation between members of the same species. Individual variation may be, in part, due to differences in operative technique, but it is thought that most of the variation noted was peculiar to the actual animal. For example, dogs resistant to the action of acetylcholine remained so throughout the experiment, and four rabbits consistently responded to smaller doses of adrenaline than did three others. Because of the variation, only a few firm conclusions are made, but tracings are shown which illustrate the fact that in many animals hepatic arterial blood appears to be exposed less to the influence of parenchymal cells than is portal venous blood, and that the difference between the two vessels is modified by nerve action.

3 Intrahepatic Vascular Pathways 129 Differences in the Effect of Drugs due to the Route of Administration In general, doses of drugs injected via different vessels were adjusted to give an equal response in systemic arterial pressure. For brevity the results are expressed as the ratio of the inverse quantity of drug required to produce a given effect when administered by two routes. Thus an hepatic arterial: portal venous ratio of 10: 1 signifies that arterial injection is ten times as effective as portal venous injection and that to produce a given response ten times the amount of drug is required when given by the portal venous route. In twenty out of twenty-seven experiments on dogs the hepatic FIG. 1.-Dog. Effect of drugs on arterial pressure when administered by different routes. Upper tracing. Response after injection of acetylcholine into the femoral vein (FV), hepatic artery (HA), and portal vein (PV). Lower tracing. Response after injection of carbachol. Figures denote the size of the dose in pug. Time in minutes. ACETYLCHOLINE. CARBACHOL, I arterial : portal venous ratio for injected acetylcholine was 3: 1 or greater, being about 10: 1 in seven dogs. Hepatic arterial injection of this drug was itself, only about one tenth as effective as injection into the femoral vein. A common result is shown in fig. 1, where the hepatic arterial: portal venous : femoral venous ratio was 10: 1: 100. The same figure shows that carbachol is almost equally effective in lowering arterial pressure when introduced into any of the three vessels. It is known that hepatic parenchyma contains choline esterases in large amounts, and it is thought that the difference in the effect of acetylcholine according to its route of administration is due to the degree of exposure of hepatic blood to these enzymes. Some dogs were refractory to the action of acetylcholine, and vast amounts were needed to affect the general circulation when injected into the portal vein. In one animal, 20 mg. lowered arterial pressure just appreciably, and in several animals the response to acetylcholine became less as the experiment proceeded, but not markedly so. In most experiments on dogs the character of the recorded tracing of the fall in blood pressure was the same for both hepatic arterial and portal venous administration, but this was not always pg.

4 130 Andrews and del Rio Lozano so, and on four occasions arterial injection produced a more prolonged reaction which could be due to arterial blood passing through more than one pathway and discharging acetylcholine into the general circulation at intervals (fig. 2). In the dog only a slight difference was noted between portal venous and hepatic arterial circulations when adrenaline and noradrenaline were injected, possibly owing to a much slower rate of destruction or removal by the hepatic parenchyma. Frequently the ratio was only 2: 1 or even less, the hepatic >>; xl d 3 8 o 8 38 E V NM C- l N cm O ('4 CM IT BEFORE COOLING. DURING COOLING. AFTER COOLING. FIG. 2.-Dog. Effect of acetylcholine on blood pressure when injected into the hepatic circulation, and of cooling the hepatic nerve. In the control period the arterial pressure was lowered by approximately the same amount by injections of acetylcholine 4 mg. into the portal vein, 20,ug. into the femoral vein and 200 jpg. into the hepatic artery. While the nerve was cooled to about 100 C. the response to arterial injections was increased relative to the response to femoral venous injections, and the effect of portal venous injections was diminished. After the nerve was allowed to warm the original relationship returned. Abbreviations are as for fig. 1. The scale on the left indicates the arterial pressure in mm. Hg. arterial injection being the more effective in producing a rise in arterial pressure. Results obtained on cats were more variable and less conclusive than those obtained on dogs. In some cats hepatic arterial and portal venous injections of acetylcholine were equally effective in lowering the general arterial pressure: occasionally the difference was marked (fig. 3) but in general arterial injection was two to five times as effective as portal venous injection. Hepatic arterial injection of acetylcholine frequently produced a temporary rise in blood pressure before the fall took place, but a rise seldom took place after portal venous injection. It was thought possible that the rise of blood pressure was due to the liberation of sympathin by injected acetylcholine, and that the arterial circulation is more intimately connected with the sympathetic nerves than is the portal venous. Accordingly sufficient atropine was injected to block the muscarinic effect of injected acetylcholine whilst leaving the nicotinic effect unchanged. It is seen in fig. 4 that this interpretation is probably correct, and it is also shown, by the

5 I u in CONTROL. d a: O <0 d > 0 I CY,,, ia -W ~ u 60 so 40 NERVE COOLED. AFTER C6. FIG. 3.-Cat. Effect of acetylcholine on arterial pressure when injected into the hepatic circulation, and of cooling the hepatic nerve. Top tracing. Control period. Lower left tracing. During cooling of the hepatic nerve. Lower right tracing. After administration of hexamethonium bromide, 10 mg./kg. Abbreviations as for fig. 1. Cooling of the hepatic nerve appeared to alter the response to portal venous injections of acetylcholine. 0r a. 0. ā> o-5. ob, u X -r a. X 0 0. < O o0 O o O -'0 FIG. 4.-Cat. Effect of atropine on the response of the arterial pressure to injections of acetylcholine. It is shown that after atropine (1 mg./kg. body weight) the response to injected acetylcholine becomes pressor, and that injections into the hepatic artery have a greater effect than injections into the portal vein or into the general circulation.

6 132 Andrews and del Rio Lozano response to acetylcholine injected into the femoral vein, that the sympathin is liberated in the liver and not at nerve endings in other parts of the body. Ten rabbits were studied, and there appeared to be a considerable difference between the hepatic arterial and portal venous circulations. In one rabbit, for example, 50,ug. of acetylcholine was injected into the portal vein without effect on the peripheral blood pressure, but the same dose put into the hepatic artery produced irreversible cardiac arrest. In one respect five of the eight rabbits in which catechol amines were used differed considerably from dogs and cats, namely in the amount of adrenaline which the liver appeared to be able to remove from the blood. This point, together with the fact that there is a definite difference in the response according to the vessel of injection, is shown in fig. 5. A similar though less well marked FIG. 5.-Rabbit. Effect of adrenaline on arterial 120 pressure when injected into the liver circulation. Upper tracing. Injections into the 1O- and the portal vein. Arterial hepatic injection artery appeared 80o- to be about sixteen times as effective as portal venous. Lower tracing. After administration of choline 2: 6 xylyl ether bromide which prevents liberation of sympathin by nerve endings. The sensitivity i n- to arterial injections is slightly increased, but to -- portal venous injections is greatly increased difference was noted in guinea-pigs between responses to portal and hepatic injections, but it was not possible to exclude artifacts, so the experiments were discontinued: no difference in the two circulations was seen in the two rats studied. Effect of Nerve Block by Cooling or Section The results of mechanical block of the nerve were variable. In dogs (8 experiments), the difference between the effects of hepatic arterial and portal venous injections usually became greater (fig. 2). In cats, cooling or section of the hepatic nerves was without effect in two experiments (fig. 6), but not in another three (fig. 3). Changes evoked by cooling the nerves were reversible. Effect of Drug-induced Nerve Block Hexamethonium bromide was injected into eight dogs, and in four the difference in the amounts required to produce the same response in blood pressure with portal venous and hepatic arterial injections either disappeared or became very much less (fig. 7), but only a slight effect was noted in the other

7 Intrahepatic Vascular Pathways 133 FIG. 6.-Dog. Effect of acetylcholine on arterial pressure before and after administration of hexamethonium bromide. Left tracing. Control period. Right tracing. After administration of hexamethonium bromide, 10 mg./kg. body weight. Neither the rate of fall of arterial pressure nor the rate of recovery due to injection of acetylcholine into the femoral vein is much affected by ganglion blockade, and the prolonged effect after injection into the liver circulation appears to be due to delay in passage of acetylcholine through the liver I CJ 0 U- c L. CW C U 1% i19, I I 19 L 4, C 0 u -S a z 0 0 U) * I 0. U - un >. 0 0 ul - I0 O i < I EIo 0 0-0xu a.1: n - FIG. 7.-Cat. Effect of acetylcholine on arterial pressure after cooling, section and stimulation of the hepatic nerve. In the control periods acetylcholine, 50 jug. injected into the portal vein and 10,Mg. injected into the hepatic artery produced the same effect on general arterial pressure. Cooling or section of the nerves did not affect the ratio of the amount of drug given to produce the same effect but during stimulation the ratio altered considerably.

8 134 Andrews and del Rio Lozano four. In two cats hexamethonium bromide was entirely without effect on the ratio of effectiveness of portal venous and hepatic arterial injection, but not in another three cats (fig. 3). In this figure it is shown that hexamethonium bromide abolishes the rise in blood pressure which often occurred in cats after administration of acetylcholine, especially when injected into the hepatic artery. Choline 2: 6 xylyl ether bromide was administered to three rabbits and four cats; also in two dogs bretylium tosylate was given. Analysis of so few experiments is not possible, but in all cases the ratio of effectiveness between portal venous and hepatic arterial injection was affected, and it was concluded that sympathetic blockade probably can effect the routes by which blood traverses the liver. Effect of Nerve Stimulation Nerve stimulation was carried out in three dogs, three cats and three rabbits. In all experiments excepting one (on a cat), there was considerable difference in the ratio of effectiveness of injections into the hepatic artery and portal vein during the actual stimulation, when compared with the resting state. In five animals, arterial injection became less effective when compared with portal venous, but in three it became more effective. Daniel and Prichard [1951], found that hepatic nerve stimulation changed angiographic patterns in some livers only; it was considered that the electrical stimulation of the total sympathetic supply to the liver was not specific enough to control certain vessels only, and further experiments were not carried out. DIscussIoN In perfused canine livers, injection of acetylcholine produces contraction of the hepatic vein. The contraction increases liver volume and decreases the blood flow; an indication of the amount of acetylcholine passing through the liver can therefore be obtained by suitable measurements, and a clear difference between portal venous and hepatic arterial circulations when this drug was injected, could be demonstrated [Andrews et al., 1953]. It was hoped that similar, clear-cut results could be obtained by means of the technique used in this study, but the results have been less definite, possibly because of the more complex systems involved. Szilagyi et al. [1955] do not describe their technique in detail, and it is possible that the long operative time which we found necessary to isolate the hepatic arterial circulation from the portal venous, produced various changes in the activity of the vasomotor centre and rendered our preparations less stable than theirs. The use of pharmacologically active drugs to measure differences between two circulations has limitations, but if the object is to demonstrate that a difference exists, and to obtain some indication of factors which accentuate or reduce the difference, the method would appear to be acceptable. The difference of the effect of a drug on the arterial pressure according to its route of administration observed in this study, cannot be due to local constriction

9 Intrahepatic Vascular Pathways or dilatation of the hepatic vessels, for the change in arterial pressure did not begin until about 20 sec. after the drug had been administered. Moreover, in several cats it was shown that the fall of arterial pressure produced by injection of acetylcholine into the liver circulation was preceded by a rise, which was due to stimulation of the sympathetic system of the liver. Acetylcholine is destroyed in the blood, but the time taken for an injection to reach the liver from the tip of the cannula was less than 5 sec., and it was shown experimentally that the proportion of injected acetylcholine destroyed during this time was negligible. When acetylcholine is used as a test drug, there appears to be a marked difference between the hepatic arterial and portal venous circulations in most of the animals studied, and the difference cannot be ascribed to technical imperfections. When adrenaline or noradrenaline was used as a test drug, the difference between the two circulations was very much less in both dogs and cats. Experiments on perfused canine livers, [Andrews et al., 1955], showed that catechol amines probably close the vessels by which arterial blood may by-pass the parenchyma. The results on dogs and cats were therefore not unexpected, but rabbits differed considerably. Not only were differences seen both with acetylcholine and adrenaline in the two hepatic circulations, but also some rabbits were able to tolerate injections of much larger amounts of adrenaline into the portal circulation than were the other species studied. It has also been shown in this study that the hepatic nerve can influence intrahepatic pathways of blood. Many authors have observed the effect of nerve stimulation on total blood-flow through the liver, but the only published indication that nerves may alter radically the path taken by portal blood in the liver, concerns the development of hilar flow which was first noted by Daniel and Prichard [1951 a and b]. In their experiments, stimulation of the hepatic nerve gave rise to a restricted circulation on some animals, but not others. Brauer, Holloway, Krebs, Leong and Carroll [1959], found a restricted hilar circulation exists with regard to the portal vein in about 40 per cent of rats under pentobarbitone anesthesia during rewarming after hypothermia. These authors also found about the same percentage of rats showed a diminished hepatic uptake of colloidal chromium phosphate during the same period. The irregularity with which changes are noted, is an agreement with our results in which it appears that the hepatic circulation is under constant nervous control in some animals, but not in others. On the basis of our experiments, one may, however, deduce with a reasonable degree of certainty, that not only can the sympathetic nerve alter the flow of blood through the liver, but also that it may do so in a fairly complex manner, a prediction made largely on theoretical grounds by Brauer [1958]. Burton-Opitz [1912], suggested that stimulation of the sympathetic nerves to the liver had no direct effect on the portal vein, but acted indirectly by affecting the arterial flow; later [1914], he found evidence that there was, in fact, a direct effect. Daniel and Prichard [1951 b], used serial angiograms, and confirmed that the portal vein was directly affected by nerves. It was 135

10 136 Andrews and del Rio Lozano found in this study and in related (unpublished) experiments, that, after atropine, hepatic arterial injections of acetylcholine produced a greater rise in general arterial pressure than did portal venous injections. The effect is almost certainly due to liberation of sympathin within the liver. Neither translobular nerve fibres nor a sympathetic supply to the hepatic vein has been described, and it appears that the sympathetic nerves are more closely related to the hepatic artery than to the portal vein. But, as can be seen from fig. 3, the amount of sympathin liberated by injected acetylcholine can be affected by nerve action, presumably by diversion through various vascular pathways. McIndoe [1928], described by-passes from the portal to the hepatic vein in cirrhotic livers. He considered that the by-passes could act as a series of minute intrahepatic Eck fistulae, a conception which has been endorsed by modern authors [e.g. Conn, 1961; Potvin, Rappaport and Scott, 1961], who have shown that ammonium salts are removed more readily from the hepatic arterial than from the portal venous circulation in normal dogs. Most, possibly all of the difference, appears to be due to the better oxygenation of arterial blood, but the experiments demonstrate conclusively that there is not full mixing of arterial and portal blood at the periphery of the hepatic lobule. Recently a translobular vein has been observed in apparently normal laboratory animals [del Rio Lozano and Andrews, to be published]. Theoretically, the translobular vein could carry the flow of blood which occurs in a hilar flow, and also could act as an internal Eck fistula. It is likely that in the near future much of the research on the hepatic circulation will be concentrated on intrahepatic vascular pathways in both health and disease, and it would appear to be of importance to recognize that the pathways may be modified by the nerve action. ACKNOWLEDGMENT The work was supported by a Grant from the Medical Research Council.

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