Acta physiol. scand. 1976. 98. 381-383 From the Department of Physiology a n d Biophysics, University of Lund, Sweden
Structural and Functional Changes in Rat Portal Veins after Experimental Portal Hypertension BY BORJEJOHANSSON
Blood vessels undergo structural adaptation in arterial hypertension and their smooth muscle develops functional changes of contractility and responsiveness to vasoactive agents (for ref. see e.g. Folkow et al. 1973, Bohr 1974). Some of these changes may be of a primary nature, contributing causally to the pressure rise while others may be secondary reactions of the vessels to the increased transmural stress. The purpose of the present study was to examine the response of a blood vessel to increased pressure in the absence of any kind of general hypertensive disease. Our laboratory has used for several years the rat portal vein as a model of myogenically active vascular smooth muscle. In view of our cumulated experience concerning electrophysiology, mechanics and pharmacology of this vessel a study of its response to a sustained pressure load seemed of interest. Partial obstruction of the two major branches of the portal vein in the hepatic hilus was produced in ether anesthetized Sprague-Dawley rats (230-300 g) by silk ligatures (4/0) tightened so that the portal vein was seen to bulge. (Complete ligction was tried but such animals did not survive.) Sham operations were performed by placing ligatures as loose loops around the portal branches. After a variable number of days the rats were again anesthetized, the abdomen opened and the portal pressure measured by puncturing the vein with a heparinized 16 gauge needle connected to a Statham P23Dc transducer. The rat was then killed and the portal vein dissected, cut open longitudinally, and mounted for recording of isometric contractions of the longitudinal smooth muscle. One preparation from a rat with experimental portal hypertension and one from a sham operated or unoperated control rat were studied simultaneously in the same bath. A set of 6 expts. on preparations from portal hypertensive rats operated 8-1 1 days earlier and from paired, sham operated controls was devoted to a study of the cumulative concentration-effect curve to noradrenaline (NA). Passive and active length-tension relations were determined in 7 pairs of preparations from control animals and from portal hypertensive rats operated 3-15 days earlier. After these muscles had accommodated in the bath at a preload of 5-6 mN for 1 h they were first shortened and then stretched in 0.2-0.5 mm steps by a micrometer screw. The passive force after stabilization at the respective lengths was read in the rest periods between spontaneous contractions whereas a submaximal active length-tension curve was obtained from the amplitude of the phasic contractions. In this way the approximate optimal length for active force development was found for each muscle and its maximal response to N A (10-6-10-5M) was then determined. The muscle was finally fixed at the optimal length in an isotonic formaldehyd solution and transverse histological sections were prepared. The cross sectional areas of these preparations and of their muscle layers were measured on microphotographs.
Fig. 1 summarizes our observations. Unoperated and sham-operated control animals showed portal pressures averaging 10.4 cm H,O compared to 20.8 cm H,O in rats with partial occlusions. These values, although influenced by the anesthesia and the abdominal surgery, should reflect the differences in pressure to which the portal veins had been exposed. Control preparations showed the usual pattern of spontaneous activity with 2 4 phasic
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BORJEJOHANSSON
Fig. 1. Diagram summarizing the differences found between portal veins from control rats and from rats with portal hypertension. Means? S.E. are indicated for portal pressure, frequency of spontaneous contractions, and maximal active force elicited by NA, whereas NA ED,, is shown only as mean values. Figures within parentheses below the bars represent the number of muscles in each group.
contractions per min while those from the portal hypertensive animals contracted at lower frequencies or were even devoid of spontaneous activity. The difference in frequency of spontaneous contractions was independent of the passive preload applied to the muscles. The relation between log[NA] and the contractile response (in per cent of maximum) of the hypertensive veins showed a parallel shift to the right of the control curves. As shown in Fig. 1 this subsensitivity corresponded to a mean change in ED,, from about 4.0 lo-' to about 6.9 x lo-' M. This change in NA sensitivity may be a non-specific phenomenon related to the lower spontaneous activity or it may be a specific effect of, for instance, changes in tissue uptake of NA. The optimal length for active force development in the length-tension study corresponded to a preload of about 5 mN in both control and hypertensive preparations. The greater cross-sectional area of the latter preparations (see below) implied, however, that their optimal length occurred at a passive force per cm2 which was only about a third of that for the control muscles. The maximal contractile force elicited by NA at the optimal lengths was 22.5 k2.9 mN in the 7 hypertensive veins compared to 11.2 i1.0 mN in the controls. Also the 6 hypertensive vessels of the dose-response study showed greater maximal contractions to NA than their sham-operated controls. Even if optimal length had not been determined individually in these muscles, all studied at a preload of 5 mN, the results indicate a difference in contractility similar to that found in the other set of 7 expts. In Fig. 1 the maximal contractile force is given for the entire material of 13 expts. showing mean values of 11.8 and 25.6 m N for control and hypertensive preparations, respectively. Fig. 2 shows hematoxyline-eosine stained sections of the portal vein from a partially ligated animal and from the sham-operated control rat both sacrificed on the 5th post-
PORTAL VEIN IN PORTAL HYPERTENSION
383
Fig. 2. Microphotographs of hematoxyline-eosine stained, transverse sections of portal vein from shamoperated (left) and portal hypertensive (right) rat.
operative day. The marked dilatation of the hypertensive vessel is evident as well as its increased wall thickness involving both muscle layer and adventitial connective tissue. In the 6 pairs of muscles used for the dose-response study, the total cross-sectional area was calculated from length and wet weight of the preparations using a density of 1.05 g/cmR. This method also revealed a marked increase in wall area in the portal hypertensive group. The greater absolute values of maximal contractile force in the hypertensive veins (Fig. 1) is evidently related to their greater quantity of muscle. However, when active force was expressed per unit area of the vessel wall or of the muscle layer itself the hypertensive portal veins gave lower values than the controls (Fig. 1). This qualitative change in the direction of reduced contractility may indicate impairment of the contractile elements due to distension (cf. Gardner and Matthews 1969), immaturity of the proliferating smooth muscle cells, or reorientation of the cells in a more circular direction as suggested by some of the histological sections. The present study has revealed a rapid structural response of the portal vein to increased pressure load associated with changes in responsiveness and contractility of the smooth muscle which will be subjected to further analysis. The study was supported by the Swedish Medical Research Council (04X-00028). I am very grateful to Professor Dora Jacobsohn and Mrs Clary Persson for their kind help with the histological work.
References BOHR,D. F., Reactivity of vascular smooth muscle from normal and hypertensive rats: effect of several cations. Fed. Proc. 1974. 33. 127-132. FOLKOW,B., M. HALLBACK, Y. LUNDGREN, R. SIVERTSSON and L. WEISS,Importance of adaptive changes in vascular design for establishment of primary hypertension, studied in man and in spontaneously hypertensive rats. Circular. Res. 1973. 32. Suppl. I. 1-13. GARDNER, D. L. and N. A. MATTHEWS, Ultrastructure of the walls of small arteries in early experimental Pathol. . 1969. 97. 51-62. rat hypertension. .I
Structural and Functional Changes in Rat Portal Veins after Experimental Portal Hypertension BY BORJEJOHANSSON
Blood vessels undergo structural adaptation in arterial hypertension and their smooth muscle develops functional changes of contractility and responsiveness to vasoactive agents (for ref. see e.g. Folkow et al. 1973, Bohr 1974). Some of these changes may be of a primary nature, contributing causally to the pressure rise while others may be secondary reactions of the vessels to the increased transmural stress. The purpose of the present study was to examine the response of a blood vessel to increased pressure in the absence of any kind of general hypertensive disease. Our laboratory has used for several years the rat portal vein as a model of myogenically active vascular smooth muscle. In view of our cumulated experience concerning electrophysiology, mechanics and pharmacology of this vessel a study of its response to a sustained pressure load seemed of interest. Partial obstruction of the two major branches of the portal vein in the hepatic hilus was produced in ether anesthetized Sprague-Dawley rats (230-300 g) by silk ligatures (4/0) tightened so that the portal vein was seen to bulge. (Complete ligction was tried but such animals did not survive.) Sham operations were performed by placing ligatures as loose loops around the portal branches. After a variable number of days the rats were again anesthetized, the abdomen opened and the portal pressure measured by puncturing the vein with a heparinized 16 gauge needle connected to a Statham P23Dc transducer. The rat was then killed and the portal vein dissected, cut open longitudinally, and mounted for recording of isometric contractions of the longitudinal smooth muscle. One preparation from a rat with experimental portal hypertension and one from a sham operated or unoperated control rat were studied simultaneously in the same bath. A set of 6 expts. on preparations from portal hypertensive rats operated 8-1 1 days earlier and from paired, sham operated controls was devoted to a study of the cumulative concentration-effect curve to noradrenaline (NA). Passive and active length-tension relations were determined in 7 pairs of preparations from control animals and from portal hypertensive rats operated 3-15 days earlier. After these muscles had accommodated in the bath at a preload of 5-6 mN for 1 h they were first shortened and then stretched in 0.2-0.5 mm steps by a micrometer screw. The passive force after stabilization at the respective lengths was read in the rest periods between spontaneous contractions whereas a submaximal active length-tension curve was obtained from the amplitude of the phasic contractions. In this way the approximate optimal length for active force development was found for each muscle and its maximal response to N A (10-6-10-5M) was then determined. The muscle was finally fixed at the optimal length in an isotonic formaldehyd solution and transverse histological sections were prepared. The cross sectional areas of these preparations and of their muscle layers were measured on microphotographs.
Fig. 1 summarizes our observations. Unoperated and sham-operated control animals showed portal pressures averaging 10.4 cm H,O compared to 20.8 cm H,O in rats with partial occlusions. These values, although influenced by the anesthesia and the abdominal surgery, should reflect the differences in pressure to which the portal veins had been exposed. Control preparations showed the usual pattern of spontaneous activity with 2 4 phasic
381
382
BORJEJOHANSSON
Fig. 1. Diagram summarizing the differences found between portal veins from control rats and from rats with portal hypertension. Means? S.E. are indicated for portal pressure, frequency of spontaneous contractions, and maximal active force elicited by NA, whereas NA ED,, is shown only as mean values. Figures within parentheses below the bars represent the number of muscles in each group.
contractions per min while those from the portal hypertensive animals contracted at lower frequencies or were even devoid of spontaneous activity. The difference in frequency of spontaneous contractions was independent of the passive preload applied to the muscles. The relation between log[NA] and the contractile response (in per cent of maximum) of the hypertensive veins showed a parallel shift to the right of the control curves. As shown in Fig. 1 this subsensitivity corresponded to a mean change in ED,, from about 4.0 lo-' to about 6.9 x lo-' M. This change in NA sensitivity may be a non-specific phenomenon related to the lower spontaneous activity or it may be a specific effect of, for instance, changes in tissue uptake of NA. The optimal length for active force development in the length-tension study corresponded to a preload of about 5 mN in both control and hypertensive preparations. The greater cross-sectional area of the latter preparations (see below) implied, however, that their optimal length occurred at a passive force per cm2 which was only about a third of that for the control muscles. The maximal contractile force elicited by NA at the optimal lengths was 22.5 k2.9 mN in the 7 hypertensive veins compared to 11.2 i1.0 mN in the controls. Also the 6 hypertensive vessels of the dose-response study showed greater maximal contractions to NA than their sham-operated controls. Even if optimal length had not been determined individually in these muscles, all studied at a preload of 5 mN, the results indicate a difference in contractility similar to that found in the other set of 7 expts. In Fig. 1 the maximal contractile force is given for the entire material of 13 expts. showing mean values of 11.8 and 25.6 m N for control and hypertensive preparations, respectively. Fig. 2 shows hematoxyline-eosine stained sections of the portal vein from a partially ligated animal and from the sham-operated control rat both sacrificed on the 5th post-
PORTAL VEIN IN PORTAL HYPERTENSION
383
Fig. 2. Microphotographs of hematoxyline-eosine stained, transverse sections of portal vein from shamoperated (left) and portal hypertensive (right) rat.
operative day. The marked dilatation of the hypertensive vessel is evident as well as its increased wall thickness involving both muscle layer and adventitial connective tissue. In the 6 pairs of muscles used for the dose-response study, the total cross-sectional area was calculated from length and wet weight of the preparations using a density of 1.05 g/cmR. This method also revealed a marked increase in wall area in the portal hypertensive group. The greater absolute values of maximal contractile force in the hypertensive veins (Fig. 1) is evidently related to their greater quantity of muscle. However, when active force was expressed per unit area of the vessel wall or of the muscle layer itself the hypertensive portal veins gave lower values than the controls (Fig. 1). This qualitative change in the direction of reduced contractility may indicate impairment of the contractile elements due to distension (cf. Gardner and Matthews 1969), immaturity of the proliferating smooth muscle cells, or reorientation of the cells in a more circular direction as suggested by some of the histological sections. The present study has revealed a rapid structural response of the portal vein to increased pressure load associated with changes in responsiveness and contractility of the smooth muscle which will be subjected to further analysis. The study was supported by the Swedish Medical Research Council (04X-00028). I am very grateful to Professor Dora Jacobsohn and Mrs Clary Persson for their kind help with the histological work.
References BOHR,D. F., Reactivity of vascular smooth muscle from normal and hypertensive rats: effect of several cations. Fed. Proc. 1974. 33. 127-132. FOLKOW,B., M. HALLBACK, Y. LUNDGREN, R. SIVERTSSON and L. WEISS,Importance of adaptive changes in vascular design for establishment of primary hypertension, studied in man and in spontaneously hypertensive rats. Circular. Res. 1973. 32. Suppl. I. 1-13. GARDNER, D. L. and N. A. MATTHEWS, Ultrastructure of the walls of small arteries in early experimental Pathol. . 1969. 97. 51-62. rat hypertension. .I
