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Arteriolar network morphology in gracilis muscle of rats with salt-induced hypertension
MICROVASCULAR
RESEARCH
Atteriolar
4,
Network Morphology with Salt-Induced MATTHEW
Department
169- 178 (1990)
of Medicine,
in Gracilis Muscle of Rats Hypertension
A. BOEGEHOLD AND THEODORE A. KOTCHEN West
Virginia University School West Virginia 26506
Received
February
of Medicine,
Morgantown,
15, 1990
The purpose of this study was to determine if structural rarefaction of arterioles occurs in the gracilis muscle of Dahl salt-sensitive (DS) rats with salt-induced hypertension. Arteriolar network architecture was studied in cleared muscles removed from DS fed either a high (7% NaCI) or low-normal (0.45% NaCI) salt diet for 4 weeks. Muscles removed from Dahl salt-resistant (DR) rats on high and low-normal salt diets served as controls. The 7% NaCl diet had no effect on arterial pressure in DR, but caused marked hypertension in DS. The density of arcade arterioles was significantly lower in DS than in DR (0.77 vs 1.26 segments/mg tissue, respectively) and was unrelated to either dietary salt content or mean arterial pressure in both strains. The number of transverse arterioles/mm parent vessel was 19% lower in DS on 7% NaCl than in DS on 0.45% NaCl and DR on either diet. These data indicate that compared to normotensive DR, the DS rat with salt-induced hypertension exhibits a lower vascular density within both the arcading and the transverse portions of the gracilis muscle arteriolar network. The lower arcade vessel density reflects an inherent characteristic of the DS strain, whereas the lower transverse arteriole density reflects a true structural rarefaction associated with salt-induced hypertension. 01990 Academic
Press, Inc.
INTRODUCTION The Dahl salt-sensitive (DS) rat develops hypertension in response to high dietary salt intake, whereas its salt-resistant counterpart, the DR rat, does not (Dahl et al., 1962a,b). Similar to other forms of hypertension, the arterial pressure increase induced by high salt intake in DS is associated with an increase in total peripheral resistance (Ganguli et al., 1979; Pfeffer et al., 1984). Intravital microscopy studies in various models of hypertension suggest that this resistance increase may be due at least in part to either a reduction in arteriolar diameters or a decrease in the number of arterioles (rarefaction) (see Prewitt et al., 1987, for review). The extent to which either of these changes occurs is variable, depending on the vascular bed, the form of hypertension, and the stage at which hypertension is studied (Meininger et al., 1984; Prewitt et al., 1987). The rarefaction of arterioles in hypertension may occur through either a reversible closure of existing arterioles (functional rarefaction) or their permanent loss from the vascular bed (structural rarefaction). In the spontaneously hypertensive rat (SHR) and in rats with renovascular 169 @X26-2862/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in U.S.A.
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AND
KOTCHEN
hypertension, studies on cremaster muscle and gracilis muscle indicate that structural rarefaction of arterioles occurs after hypertension is well established (Lombard et al., 1989; Ono et al., 1989; Prewitt ef al., 1982, 1984). Structural rarefaction may therefore play an important role in maintaining elevated peripheral resistance in animals with chronic hypertension. However, there is also evidence to suggest that structural rarefaction does not occur in all muscle types. Engelson and co-workers (1986) have reported that arteriolar density is actually greater in the spinotrapezius muscle of mature SHR than in that of normotensive WistarKyoto controls. It is not known whether the structural rarefaction of arterioles varies with muscle type in other models of hypertension. In agreement with findings in the SHR spinotrapezius muscle, we have recently reported that the anatomic density of arterioles is not reduced in the spinotrapezius muscle of the DS rat with NaCl-induced hypertension (Boegehold and Kotchen, 1990). The current study was undertaken to determine whether this observation reflects a general characteristic of skeletal muscle in hypertensive DS or whether, as in the SHR, the development of structural rarefaction in this model is dependent on muscle type. In this study, the arteriolar network of the gracilis muscle was analyzed in DS and DR rats maintained on high (7% NaCl) and low-normal (0.45% NaCl) salt diets. This muscle was chosen for study because its vascular bed has been previously shown to undergo arteriolar rarefaction in two other models of hypertension: the SHR and rats with one-kidney, one-clip hypertension (Prewitt ef al., 1982, 1984). MATERIALS
AND METHODS
Weanling male DR and DS rats (Brookhaven type, 27-31 days old) were obtained from Harlan Sprague Dawley (Indianapolis, IN) and immediately placed on a natural grain diet containing 0.45% NaCl by weight (TD8831; Teklad, Madison, WI). After 1 week, one-half of the animals of each strain were placed on a 7% NaCl diet with the other half remaining on the 0.45% NaCl diet. The 7% NaCl diet was made by supplementing the Teklad diet with additional NaCl. Animals from all four experimental groups (DR, 0.45% NaCl; DR 7% NaCl, DS 0.45% NaCl, and DS 7% NaCl) were studied between 4 and 5 weeks after initiation of the 7% NaCl diet. All animals were anesthetized with sodium thiopental (100 mg/kg, ip) and placed on a heating mat to maintain a 37” rectal temperature. The trachea was intubated to ensure a patent airway and the right carotid artery was cannulated for direct measurement of systemic arterial pressure. The gracilis muscle vascular bed was then filled with a silicone rubber contrast medium (Microfil, Canton Bio-Medical, Boulder, CO) to allow identification of all arteriolar segments. An abdominal incision was made and the right iliac artery was tied off immediately distal to its bifurcation from the descending aorta. The descending aorta was then cannulated caudally with large bore polyethylene tubing, and the catheter tip advanced into the left iliac artery. Next, the animal’s blood was isovolumetrically replaced with a heparinized electrolyte solution containing 0.04 mg/ml papaverine HCl to maximally dilate the peripheral vasculature. The displaced blood was drained through the cannulated carotid artery. A 10% buffered formaldehyde solution (formalin) was then infused over a lo-min period to fix the vasculature in situ, followed by Microfil infusion. The
ARTERIOLAR
NETWORK
IN
DAHL
RATS
171
1 mm
FIG. 1. (Top) Line drawing from a cleared specimen illustrating the arcade arteriole network in gracilis muscle. A single arcade loop is identified by the segment a-b-c-d-a. (Bottom) Higher magnification view illustrating a transverse arteriole network branching from an arcade arteriole. *Origin of a capillary bundle.
formalin and Microfil infusions were made at a pressure equivalent to the animal’s mean arterial pressure. The left anterior gracilis muscle was then exposed through a skin incision and formalin was super-fused over its surface to complete muscle fixation. The muscle was removed, weighed, and secured at its in situ length in a petri dish by ligatures embedded in a perimeter of modeling clay. After dehydration in progressively increasing concentrations of ethyl alcohol, the muscle was cleared with methyl salicylate. Arteriolur network analysis. The cleared muscle specimen was photographed with a 35mm camera through an Olympus BHMJ videomicroscope at 40x magnification, and the individual prints were assembled into a montage of the vascular bed. The exact magnification of the photomontage was established by photography of a stage micrometer under the same conditions as the muscle specimen. Vessels were classified according to location within the arteriolar network. The gracilis muscle receives its blood supply from both the saphenous artery and the muscular branch of the femoral artery (Greene, 1955). Distributing arterioles enter the muscle from both arteries and, together with their immediate branches, anastomose to form an arcading network which extends throughout the muscle (Fig. 1, top). Arising directly from these arcade arterioles are the transverse arterioles, which do not anastomose with collateral vessels and whose branches perfuse discrete regions of tissue. In distinction to the arcade network, a transverse arteriole and all of its distal branches are collectively referred to as a transverse network (Fig. 1, bottom). Vessels within transverse networks were assigned branch orders according to the method of Strahler (1957). By this method, a terminal arteriole giving direct rise to capillaries is classified as a firstorder arteriole. Two first-order arterioles arise from a second-order arteriole, and this pattern continues proximally with two like-order vessels arising from a
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parent vessel of the next highest order. When two vessels of different branch order meet, the parent segment is assigned the higher order. The entire arcade arteriole network was traced onto an acetate sheet overlying the montage. Muscle specimens with incomplete network filling were excluded from analysis. The identification of all arcade segments in the montage was verified through repeated comparison with the cleared specimen under the microscope at 40x and 100x magnification. A rectangular region in the center of the specimen was then outlined on the acetate overlay, and the node-to-node length of each individual arcade segment within the region was measured. For the purposes of this analysis, a node is defined as any branch point formed by the junction of three arcade segments. The number of arcade “loops” lying within the region was also determined. An arcade loop is defined as a series of anastomosing segments joined end-to-end in such a manner as to circumscribe a discrete region of tissue (see Fig. 1, top). Ten arcade segments were then randomly selected and examined in the cleared specimen at higher (200 x) magnification to count the transverse arterioles branching from each segment. The lumenal diameter of arcade and transverse arterioles was also measured directly from the video monitor of the microscope, at a final screen magnification of 760 x . Diameter measurements were only made in vessels with clearly visible walls. Randomly selected transverse networks were then mapped in their entirety, and the total number of vessels of each branch order was determined for each network. Because the Strahler method of assigning branch orders requires the identification of all terminal vessels, only those transverse networks whose distal branches were filled down to the capillaries were included in this analysis. Otherwise, no special selection criteria were used. For each muscle, the total arcade vessel length, number of individual arcade segments, and number of arcade loops within the counting region were normalized to tissue mass and expressed as per milligram wet tissue weight. The average number of transverse arterioles/mm of arcade segment length was calculated by dividing the total number of transverse arterioles counted by the cumulative length of their parent arcade segments. Statistics. All data are expressed as means 2 SEM. For each variable, simultaneous multiple comparison of group means was made using analysis of variance in a 2 x 2 factorial design to evaluate treatment effects (strain, diet) and potential strain-diet interaction. Post hoc analysis of any differences was made using the Newman-Keuls multiple range procedure. In all tests, significance was assessed at the 95% confidence level (P < 0.05). RESULTS Arteriolar networks were analyzed in a total of 12 DR (6 on 0.45% NaCl, 6 on 7% NaCl) and 12 DS (6 on 0.45% NaCl, 6 on 7% NaCl). Within each strain, the mean body weight of animals on the 7% NaCl diet was significantly less than that of animals on the 0.45% NaCl diet (Table 1). The 7% NaCl diet had no effect on mean arterial pressure (MAP) in DR but produced marked hypertension (MAP = 163 & 8 mm Hg) in DS. Mean arterial pressure in DS on 0.45% NaCl (134 -t- 3 mm Hg) tended to be higher than that of DR on either 0.45 or 7% NaCl (118 2 9 and 119 2 14 mm Hg, respectively), but the differences were
ARTERIOLAR
CHARACTERISTICS
Experimental group
n
DR, 0.45% NaCl DR, 7% NaCl DS, 0.45% NaCl DS, 7% NaCl
6 6 6 6
Note. DR, Dahl salt-resistant * Significantly different from
NETWORK
IN DAHL
TABLE 1 OF EXPERIMENTAL ANIMALS
70 69 72 69
2 -c ? -+
(lid 2 1 1 2
304 273 325 268
k 2 k _’
AT THE TIME OF STUDY Mean arterial pressure (mm Hg)
Body weight
Age (days)
173
RATS
7 6* 20 6*
rats. DS, Dahl salt-sensitive same strain on 0.45% NaCl
118 119 134 163
k t -e k
rats. Values (P < 0.05).
9 14 3 8* are means
Gracilis muscle wet weight (ms) 148 130 163 122
k t + ”
12 10 11 6*
2 SEM.
not significant. Also shown in Table 1 is the average gracilis muscle wet weight in each group. Consistent with the lower body weights of DR and DS maintained on the high salt diet, the gracilis muscle tended to be smaller in those animals as well. This difference was significant in DS. Averaged across all experimental groups, the lumenal diameter for passive, fixed vessels was 57 + 4 pm for arcade arterioles and 23 * 2 ,um for transverse arterioles. No significant differences in either arcade or transverse diameters were found among individual groups. The morphometric characteristics of the arcade network in each group are illustrated in Fig. 2. Within each strain, the high salt diet had no significant effect on arcade network density; animals on 7% NaCl did not differ from those on 0.45% NaCl in terms of total arcade length/mg tissue (top), number of arcade segments/mg tissue (middle), or number of arcade loops/mg tissue (bottom). However, there was a significant straindependent difference in each of these variables, with the values for DS significantly less than those for DR, regardless of diet. Total arcade length/mg tissue (average for both diets) was 22% lower in DS than in DR, with the average number of arcade segments and arcade loops/mg tissue 38 and 36% lower in DS, respectively. The average number of transverse networks branching from each millimeter of arcade segment is illustrated in Fig. 3. The level of dietary salt intake had no effect on this value in DR. DS maintained on 0.45% NaCl had essentially the same number of transverse arterioles/mm arcade as DR, but in contrast to DR, high salt intake in DS was associated with a reduction in this number. DS on 7% NaCl were found to have 19% fewer transverse arterioles/mm arcade than DS on 0.45% NaCl. This difference was statistically significant. In each experimental group, fifteen transverse networks were randomly selected and all of the vessels within those networks were mapped in their entirety. Transverse network size was variable in each group, with a total of 7 to 28 capillary bundles supplied by the distal branches of a single transverse arteriole. Despite this size range, each network examined was found to contain either three or four branching generations (as determined by the Strahler method) from its point of origin at the arcade arteriole to the origin of the capillaries. The average number of vessels of each branch order per transverse network is shown for each group in Table
174
BOEGEHOLD
AND
0 DR, 0.45% NaCl EZi DR, 7% NaCl El DS, 0.45% NaCl I DS, 7% NaCl
1.4
kg CE s; 4 .z g!”
0.6
am -5
0.4
E
+rl 5%
KOTCHEN
1.2 1.0 0.6
0.2 0.0 1.6
$
2
1.4
.g
1.2
iiF
1.o
%E
0.0
2iQ ai9 2;E=
0.6 0.2 0.4
P
0.0
FIG. 2. Structural characteristics of the arcade network in each experimental group. Values are means ? SEM and are normalized to tissue mass. (Top) Total arcade segment length/mg; (middle) number of individual arcade segments/mg; (bottom) number of arcade loops/mg. Values grouped under bars are not significantly different. *Significantly different from DR on same diet (P < 0.05).
2. The number of vessels of each branch order was similar in all groups, with no significant strain- or diet-dependent differences observed. DISCUSSION The current observations demonstrate a marked difference between the DS and DR rat strains in the architecture of the arcade arteriole network in gracilis muscle. In addition, our results suggest that the development of salt-induced hypertension in the DS strain is accompanied by a structural rarefaction of transverse arterioles in this vascular bed. Compared to normotensive DR, the arcading portion of the network in hypertensive DS on 7% NaCl was found to be less dense as judged by a lesser
ARTERIOLAR
Y 52 z$ a ZiE CQJ Q) au) ul
3.5 3.0
2.0
22 @a b;y-
1.5
E GE a, 5 g
IN
DAHL
175
RATS
0 ESS El m
DR, DR, DS, DS,
O&i”h NaCl 7% NaCl 0.45% NaCl 7% NaCl
2.5
g;
%
NETWORK
1.0 0.5
z 0.0
3. Number of transverse networks/mm arcade segment length. Values are means ? SEM. Values grouped under bar are not significantly different. *Significantly different from DS on 0.45% NaCl (P < 0.05). Frc.
total vascular length and fewer individual segments and vascular loops/mg of tissue (Fig. 2). In contrast, arcade network values for DS on 7% NaCl were virtually identical to those for DS on 0.45% NaCl, despite a significant difference between these groups in mean arterial pressure (Table 1). This suggests that the observed difference in arcade network density between hypertensive DS and normotensive DR reflects an inherent difference in vascular architecture between these strains that is independent of either the level of dietary salt intake or its effect on arterial pressure. In addition, on an individual animal basis, we found no statistical correlation between mean arterial pressure and the total arcade length (P = 0.80), number of arcade segments (P = 0.55), or number of vascular loops (P = 0.55) per milligram tissue, further demonstrating the lack of relationship between arcade network density and mean arterial pressure. These findings suggest that although DS and DR are descended from the same parent strain (Dahl et al., 1962a,b), the genetic isolation of these two lines brought about by years of selective breeding has given rise to some degree of genetic drift, producing differences in arcade network architecture that are apparently unrelated to the phenomenon of salt-induced hypertension. In contrast to the arcade arterioles, the reduced number of transverse arterioles/mm arcade segment in DS on 7% NaCl was not seen in DS on 0.45% NaCl (Fig. 3), suggesting that this is a true rarefaction of vessels related to the saltinduced increase in arterial pressure. The average number of distal branches arising from existing transverse arterioles was found to be equivalent in all experimental groups (Table 2), which indicates that a reduction in the number of branches within a transverse network does not occur independent of rarefaction of the parent vessel. Therefore, the vascular unit which becomes rarefied at this stage of salt-induced hypertension appears to be the entire transverse network. Data from longitudinal studies in SHR and rats with renovascular hypertension suggest that structural rarefaction of arterioles in these forms of hypertension is due to a loss of preexisting vessels (Ono et al., 1989; Prewitt et
176
BOEGEHOLD AND KOTCHEN
NUMBER
Experimental group DR, DR, DS, DS,
0.45% NaCl 7% NaCl 0.45% NaCl 7% NaCl
OF VESSELS
OF EACH
TABLE 2 BRANCH ORDERPERTRANSVERSENETWORK Number of vessels
1st Order 12.3 14.5 14.5 14.1
-c ? f 2
1.5 1.4 2.2 1.3
2nd Order
3rd Order
4th Order
4.6 5.2 5.4 5.3
1.7 1.5 1.6 1.5
0.6 0.3 0.4 0.5
2 ‘_ 2 +
0.5 0.5 0.8 0.6
k -c k 2
0.2 0.2 0.3 0.2
5 “_ 2 k
0.1 0.1 0.2 0.1
Note. DR, Dahl salt-resistant rats. DS, Dahl salt-sensitive rats. Values are means k SEM. Branch orders are assigned according to Strahler (1957): 1st order, most distal vessels, and 4th order, most proximal vessels.
al., 1982, 1984). The structural rarefaction of transverse arterioles in the present study could reflect either a loss of vessels or a suppression of normal new vessel growth in developing DS on the high salt diet. Because younger animals were not also examined in the present study, we cannot distinguish between these two possibilities. The number of transverse arterioles/mm arcade segment was 19% lower in the hypertensive group than in the other groups (Fig. 3). Assuming that the rarefied networks were similar in size to those present, our data suggest that when normalized to arcade length, there is a 19% reduction of all distal arterioles in hypertensive DS. Our data also indicate that in addition to the rarefaction associated with hypertension, there is an inherent difference between DS and DR in distal arteriolar density. The spacing of transverse arterioles along arcade segments is identical in DR and DS on 0.45% NaCl (Fig. 3), but the inherently lower arcade network density in DS results in a calculated transverse network density that is 28% lower than that in DR (2.35 2 0.26 vs 3.25 t 0.29 networks/mg tissue, P < 0.05). In DS on 7% NaCl, with 19% fewer vessels per millimeter arcade, transverse network density is further reduced to 2.06 IC_0.12 per milligram tissue, or 37% below that of DR on 0.45% NaCl. The extent of structural rarefaction found in the present study is somewhat less than that reported in the gracilis muscle of SHR (56%) and rats with one-kidney, one-clip hypertension (50%) (Prewitt et al., 1982, 1984). However, the data from SHR were obtained in mature animals (16-18 weeks) and the one-kidney, one-clip data were obtained 8-10 weeks after renal artery clipping. In contrast, the DS studied here were observed after only 4 weeks on the high salt diet. It is possible that the lesser extent of structural rarefaction found in the current study simply reflects a shorter duration of hypertension in these animals. To our knowledge, there is little information available on the relative contribution of different microvascular segments to total gracilis muscle vascular resistance. Consequently, the importance of transverse network rarefaction in increasing whole-organ resistance in hypertensive DS is open to speculation. Greene and co-workers (1989) have recently developed a mathematical model of the arteriolar network to estimate the hemodynamic effect of arteriolar constriction and rarefaction in hypertensive animals. The design of their network closely resembles that of the gracilis muscle, and the relationship between rarefaction and network resistance derived from that model predicts that a 19%
ARTERIOLAR NETWORK IN DAHL RATS
177
rarefaction of distal arterioles would only increase total network resistance in DS by approximately 10%. In comparing hypertensive DS to DR, the estimated 37% reduction in distal arteriolar density would increase network resistance by approximately 20%, with the lower arcade network density in DS also contributing to elevated network resistance. Our finding of a reduced arteriolar density in the gracilis muscle of DS rats with salt-induced hypertension is at variance with our recent report that arteriolar density is normal in the spinotrapezius muscle of these animals (Boegehold and Kotchen, 1990). In that study, arcade and transverse network density in hypertensive DS on 7% NaCl was found to be similar to that of DS on 0.45% NaCl and DR on either diet. The number of distal branches per transverse network was also equivalent in all groups. The experimental groups in the recent study were identical to those studied here, in terms of both age and length of time on diet. In the current study, the reduction in arteriolar density in hypertensive DS appears to be both of genetic origin (at the arcade level, Fig. 2) and a response to the salt-induced rise in arterial pressure (at the transverse level, Fig. 3). The occurrence of hypertension-dependent structural rarefaction in the gracilis muscle but not in the spinotrapezius muscle is consistent with earlier findings in the SHR. The 56% rarefaction of small arterioles in SHR gracilis muscle reported by Prewitt and co-workers (1982) contrasts sharply with a report that rarefaction does not occur in the spinotrapezius muscle of SHR at the same age (Engelson et al., 1986). In fact, Engelson and co-workers found that both arcade network density and the number of transverse arterioles were actually greater in SHR than in age-matched normotensive WKY. These observations were made in animals of up to 20 weeks old, which suggests that hypertension-dependent rarefaction may never occur in the rat spinotrapezius muscle. With respect to the arcade network, it is unclear why an inherent difference in vascular density between the DS and DR strains would occur in the gracilis muscle but not in the spinotrapezius muscle. This heterogeneity in strain-dependent differences, coupled with the suggestion that structural rarefaction may not occur in all muscle types, highlights the importance of studying different vascular beds within any one model of hypertension. Conclusions on hypertension-dependent changes based on observations from a single vascular bed, even in the case of skeletal muscle, could be misleading. In summary, the anatomic density of gracilis muscle arcade arterioles was found to be inherently lower in the DS rat strain than in the DR rat strain. This difference appears to be unrelated to the salt sensitivity of blood pressure in the DS rat. In contrast, the anatomic density of more distal transverse arterioles in the gracilis muscle was significantly lower in hypertensive DS than in normotensive DS, suggesting that these vessels undergo a structural rarefaction associated with the development of salt-induced hypertension. The structural rarefaction observed in this muscle was not found in an earler study on the spinotrapezius muscle, suggesting that this is not a characteristic common to all vascular beds in the DS model of hypertension. ACKNOWLEDGMENT This investigation was supported by a Grant-in-Aid Virginia affiliate.
from the American Heart Association, West
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REFERENCES M. A., AND KOTCHEN, T. A. (1990). Effect of dietary salt on the skeletal muscle microvasculature in Dahl rats. Hypertension 15, 420-426. DAHL, L. K., HEINE, M., AND TASSINARI, L. (1962a). Effects of chronic excess salt ingestion: Evidence that genetic factors play an important role in susceptibility to experimental hypertension. J. Exp. Med. 115, 1173-1190. DAHL, L. K., HEINE, M., AND TASSINARI, L. (1962b). Role of genetic factors in susceptibility to experimental hypertension due to chronic excess salt ingestion. Nature(London)194, 480-482. ENGELSON, E. T., SCHMID-SHONBEIN, G. W., AND ZWEIFACH, B. W. (1986). The microvasculature in skeletal muscle II: Arteriolar network anatomy in normotensive and spontaneously hypertensive rats. Microvasc. Res. 31, 356-374. GANGULI, M., TOBIAN, L., AND IWAI, J. (1979). Cardiac output and peripheral resistance in strains of rats sensitive and resistant to NaCl hypertension. Hypertension 1, 3-7. GREENE, A. S., TONELLATO, P. J., LUI, J., LOMBARD, J. H., AND COWLEY, A. W., JR. (1989). Microvascular rarefaction and tissue vascular resistance in hypertension. Amer. J. Physiol. 256, H126-H131. GREENE, E. C. (1955). “The Anatomy of the Rat.” Hafner, New York. LOMBARD, J. H., HINOIOSA-LABORDE, C., AND COWLEY, A. W., JR. (1989). Hemodynamics and microcirculatory alterations in reduced renal mass hypertension. Hypertension 13, 128-138. MEININGER, G. A., HARRIS, P. D., AND JOSHUA, I. G. (1984). Distributions of microvascular pressure in skeletal muscle of one-kidney, one-clip, two-kidney, one-clip, and deoxycorticosterone-salt hypertensive rats. Hypertension 6, 27-34. ONO, Z., PREWITT, R. L., AND STACY, D. L. (1989). Arteriolar changes in developing and chronic stages of two-kidney, one-clip hypertension. Hypertension 14, 36-43. PFEFFER,M. A., PFEFFER,J., MIRSKY, J., AND IWAI, J. (1984). Cardiac hypertrophy and performance of Dahl hypertensive rats on graded salt diets. Hypertension 6, 475-481. PREWITT, R. L., CHEN, I. I. H., AND DOWELL, R. (1982). Development of microvascular rarefaction in the spontaneously hypertensive rat. Amer. J. Physiol. 243, H243-H251. PREWITT, R. L., CHEN, I. I. H., AND DOWELL, R. F. (1984). Microvascular alterations in the onekidney, one-clip renal hypertensive rat. Amer. J. Physiol. 246, H728-H732. PREWITT, R. L., HASHIMOTO, H., AND STACY, D. L. (1987). Microvascular alterations in hypertension. In “Microvascular Perfusion and Transport in Health and Disease” (P. McDonagh, Ed.), pp. 31-59. Karger, Basel. SCHMID-SCHOENBEIN, G. W., ZWEIFACH, B. W., AND KOVALCHECK, S. (1977). The application of stereological principles to morphometry of the microcirculation in different tissues. Microvasc.
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16. STACY, D. L., AND PREWITT, R. L. (1989). Attenuated microvascular alterations in coarctation hypertension. Amer. J. Physiol. 256, H213-H221. 17. STRAHLER,A. N. (1957). Quantitative analysis of watershed geomorphology. Trans. Amer. Geophys.
Union
38, 913-920.
RESEARCH
Atteriolar
4,
Network Morphology with Salt-Induced MATTHEW
Department
169- 178 (1990)
of Medicine,
in Gracilis Muscle of Rats Hypertension
A. BOEGEHOLD AND THEODORE A. KOTCHEN West
Virginia University School West Virginia 26506
Received
February
of Medicine,
Morgantown,
15, 1990
The purpose of this study was to determine if structural rarefaction of arterioles occurs in the gracilis muscle of Dahl salt-sensitive (DS) rats with salt-induced hypertension. Arteriolar network architecture was studied in cleared muscles removed from DS fed either a high (7% NaCI) or low-normal (0.45% NaCI) salt diet for 4 weeks. Muscles removed from Dahl salt-resistant (DR) rats on high and low-normal salt diets served as controls. The 7% NaCl diet had no effect on arterial pressure in DR, but caused marked hypertension in DS. The density of arcade arterioles was significantly lower in DS than in DR (0.77 vs 1.26 segments/mg tissue, respectively) and was unrelated to either dietary salt content or mean arterial pressure in both strains. The number of transverse arterioles/mm parent vessel was 19% lower in DS on 7% NaCl than in DS on 0.45% NaCl and DR on either diet. These data indicate that compared to normotensive DR, the DS rat with salt-induced hypertension exhibits a lower vascular density within both the arcading and the transverse portions of the gracilis muscle arteriolar network. The lower arcade vessel density reflects an inherent characteristic of the DS strain, whereas the lower transverse arteriole density reflects a true structural rarefaction associated with salt-induced hypertension. 01990 Academic
Press, Inc.
INTRODUCTION The Dahl salt-sensitive (DS) rat develops hypertension in response to high dietary salt intake, whereas its salt-resistant counterpart, the DR rat, does not (Dahl et al., 1962a,b). Similar to other forms of hypertension, the arterial pressure increase induced by high salt intake in DS is associated with an increase in total peripheral resistance (Ganguli et al., 1979; Pfeffer et al., 1984). Intravital microscopy studies in various models of hypertension suggest that this resistance increase may be due at least in part to either a reduction in arteriolar diameters or a decrease in the number of arterioles (rarefaction) (see Prewitt et al., 1987, for review). The extent to which either of these changes occurs is variable, depending on the vascular bed, the form of hypertension, and the stage at which hypertension is studied (Meininger et al., 1984; Prewitt et al., 1987). The rarefaction of arterioles in hypertension may occur through either a reversible closure of existing arterioles (functional rarefaction) or their permanent loss from the vascular bed (structural rarefaction). In the spontaneously hypertensive rat (SHR) and in rats with renovascular 169 @X26-2862/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in U.S.A.
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AND
KOTCHEN
hypertension, studies on cremaster muscle and gracilis muscle indicate that structural rarefaction of arterioles occurs after hypertension is well established (Lombard et al., 1989; Ono et al., 1989; Prewitt ef al., 1982, 1984). Structural rarefaction may therefore play an important role in maintaining elevated peripheral resistance in animals with chronic hypertension. However, there is also evidence to suggest that structural rarefaction does not occur in all muscle types. Engelson and co-workers (1986) have reported that arteriolar density is actually greater in the spinotrapezius muscle of mature SHR than in that of normotensive WistarKyoto controls. It is not known whether the structural rarefaction of arterioles varies with muscle type in other models of hypertension. In agreement with findings in the SHR spinotrapezius muscle, we have recently reported that the anatomic density of arterioles is not reduced in the spinotrapezius muscle of the DS rat with NaCl-induced hypertension (Boegehold and Kotchen, 1990). The current study was undertaken to determine whether this observation reflects a general characteristic of skeletal muscle in hypertensive DS or whether, as in the SHR, the development of structural rarefaction in this model is dependent on muscle type. In this study, the arteriolar network of the gracilis muscle was analyzed in DS and DR rats maintained on high (7% NaCl) and low-normal (0.45% NaCl) salt diets. This muscle was chosen for study because its vascular bed has been previously shown to undergo arteriolar rarefaction in two other models of hypertension: the SHR and rats with one-kidney, one-clip hypertension (Prewitt ef al., 1982, 1984). MATERIALS
AND METHODS
Weanling male DR and DS rats (Brookhaven type, 27-31 days old) were obtained from Harlan Sprague Dawley (Indianapolis, IN) and immediately placed on a natural grain diet containing 0.45% NaCl by weight (TD8831; Teklad, Madison, WI). After 1 week, one-half of the animals of each strain were placed on a 7% NaCl diet with the other half remaining on the 0.45% NaCl diet. The 7% NaCl diet was made by supplementing the Teklad diet with additional NaCl. Animals from all four experimental groups (DR, 0.45% NaCl; DR 7% NaCl, DS 0.45% NaCl, and DS 7% NaCl) were studied between 4 and 5 weeks after initiation of the 7% NaCl diet. All animals were anesthetized with sodium thiopental (100 mg/kg, ip) and placed on a heating mat to maintain a 37” rectal temperature. The trachea was intubated to ensure a patent airway and the right carotid artery was cannulated for direct measurement of systemic arterial pressure. The gracilis muscle vascular bed was then filled with a silicone rubber contrast medium (Microfil, Canton Bio-Medical, Boulder, CO) to allow identification of all arteriolar segments. An abdominal incision was made and the right iliac artery was tied off immediately distal to its bifurcation from the descending aorta. The descending aorta was then cannulated caudally with large bore polyethylene tubing, and the catheter tip advanced into the left iliac artery. Next, the animal’s blood was isovolumetrically replaced with a heparinized electrolyte solution containing 0.04 mg/ml papaverine HCl to maximally dilate the peripheral vasculature. The displaced blood was drained through the cannulated carotid artery. A 10% buffered formaldehyde solution (formalin) was then infused over a lo-min period to fix the vasculature in situ, followed by Microfil infusion. The
ARTERIOLAR
NETWORK
IN
DAHL
RATS
171
1 mm
FIG. 1. (Top) Line drawing from a cleared specimen illustrating the arcade arteriole network in gracilis muscle. A single arcade loop is identified by the segment a-b-c-d-a. (Bottom) Higher magnification view illustrating a transverse arteriole network branching from an arcade arteriole. *Origin of a capillary bundle.
formalin and Microfil infusions were made at a pressure equivalent to the animal’s mean arterial pressure. The left anterior gracilis muscle was then exposed through a skin incision and formalin was super-fused over its surface to complete muscle fixation. The muscle was removed, weighed, and secured at its in situ length in a petri dish by ligatures embedded in a perimeter of modeling clay. After dehydration in progressively increasing concentrations of ethyl alcohol, the muscle was cleared with methyl salicylate. Arteriolur network analysis. The cleared muscle specimen was photographed with a 35mm camera through an Olympus BHMJ videomicroscope at 40x magnification, and the individual prints were assembled into a montage of the vascular bed. The exact magnification of the photomontage was established by photography of a stage micrometer under the same conditions as the muscle specimen. Vessels were classified according to location within the arteriolar network. The gracilis muscle receives its blood supply from both the saphenous artery and the muscular branch of the femoral artery (Greene, 1955). Distributing arterioles enter the muscle from both arteries and, together with their immediate branches, anastomose to form an arcading network which extends throughout the muscle (Fig. 1, top). Arising directly from these arcade arterioles are the transverse arterioles, which do not anastomose with collateral vessels and whose branches perfuse discrete regions of tissue. In distinction to the arcade network, a transverse arteriole and all of its distal branches are collectively referred to as a transverse network (Fig. 1, bottom). Vessels within transverse networks were assigned branch orders according to the method of Strahler (1957). By this method, a terminal arteriole giving direct rise to capillaries is classified as a firstorder arteriole. Two first-order arterioles arise from a second-order arteriole, and this pattern continues proximally with two like-order vessels arising from a
172
BOEGEHOLD
AND
KOTCHEN
parent vessel of the next highest order. When two vessels of different branch order meet, the parent segment is assigned the higher order. The entire arcade arteriole network was traced onto an acetate sheet overlying the montage. Muscle specimens with incomplete network filling were excluded from analysis. The identification of all arcade segments in the montage was verified through repeated comparison with the cleared specimen under the microscope at 40x and 100x magnification. A rectangular region in the center of the specimen was then outlined on the acetate overlay, and the node-to-node length of each individual arcade segment within the region was measured. For the purposes of this analysis, a node is defined as any branch point formed by the junction of three arcade segments. The number of arcade “loops” lying within the region was also determined. An arcade loop is defined as a series of anastomosing segments joined end-to-end in such a manner as to circumscribe a discrete region of tissue (see Fig. 1, top). Ten arcade segments were then randomly selected and examined in the cleared specimen at higher (200 x) magnification to count the transverse arterioles branching from each segment. The lumenal diameter of arcade and transverse arterioles was also measured directly from the video monitor of the microscope, at a final screen magnification of 760 x . Diameter measurements were only made in vessels with clearly visible walls. Randomly selected transverse networks were then mapped in their entirety, and the total number of vessels of each branch order was determined for each network. Because the Strahler method of assigning branch orders requires the identification of all terminal vessels, only those transverse networks whose distal branches were filled down to the capillaries were included in this analysis. Otherwise, no special selection criteria were used. For each muscle, the total arcade vessel length, number of individual arcade segments, and number of arcade loops within the counting region were normalized to tissue mass and expressed as per milligram wet tissue weight. The average number of transverse arterioles/mm of arcade segment length was calculated by dividing the total number of transverse arterioles counted by the cumulative length of their parent arcade segments. Statistics. All data are expressed as means 2 SEM. For each variable, simultaneous multiple comparison of group means was made using analysis of variance in a 2 x 2 factorial design to evaluate treatment effects (strain, diet) and potential strain-diet interaction. Post hoc analysis of any differences was made using the Newman-Keuls multiple range procedure. In all tests, significance was assessed at the 95% confidence level (P < 0.05). RESULTS Arteriolar networks were analyzed in a total of 12 DR (6 on 0.45% NaCl, 6 on 7% NaCl) and 12 DS (6 on 0.45% NaCl, 6 on 7% NaCl). Within each strain, the mean body weight of animals on the 7% NaCl diet was significantly less than that of animals on the 0.45% NaCl diet (Table 1). The 7% NaCl diet had no effect on mean arterial pressure (MAP) in DR but produced marked hypertension (MAP = 163 & 8 mm Hg) in DS. Mean arterial pressure in DS on 0.45% NaCl (134 -t- 3 mm Hg) tended to be higher than that of DR on either 0.45 or 7% NaCl (118 2 9 and 119 2 14 mm Hg, respectively), but the differences were
ARTERIOLAR
CHARACTERISTICS
Experimental group
n
DR, 0.45% NaCl DR, 7% NaCl DS, 0.45% NaCl DS, 7% NaCl
6 6 6 6
Note. DR, Dahl salt-resistant * Significantly different from
NETWORK
IN DAHL
TABLE 1 OF EXPERIMENTAL ANIMALS
70 69 72 69
2 -c ? -+
(lid 2 1 1 2
304 273 325 268
k 2 k _’
AT THE TIME OF STUDY Mean arterial pressure (mm Hg)
Body weight
Age (days)
173
RATS
7 6* 20 6*
rats. DS, Dahl salt-sensitive same strain on 0.45% NaCl
118 119 134 163
k t -e k
rats. Values (P < 0.05).
9 14 3 8* are means
Gracilis muscle wet weight (ms) 148 130 163 122
k t + ”
12 10 11 6*
2 SEM.
not significant. Also shown in Table 1 is the average gracilis muscle wet weight in each group. Consistent with the lower body weights of DR and DS maintained on the high salt diet, the gracilis muscle tended to be smaller in those animals as well. This difference was significant in DS. Averaged across all experimental groups, the lumenal diameter for passive, fixed vessels was 57 + 4 pm for arcade arterioles and 23 * 2 ,um for transverse arterioles. No significant differences in either arcade or transverse diameters were found among individual groups. The morphometric characteristics of the arcade network in each group are illustrated in Fig. 2. Within each strain, the high salt diet had no significant effect on arcade network density; animals on 7% NaCl did not differ from those on 0.45% NaCl in terms of total arcade length/mg tissue (top), number of arcade segments/mg tissue (middle), or number of arcade loops/mg tissue (bottom). However, there was a significant straindependent difference in each of these variables, with the values for DS significantly less than those for DR, regardless of diet. Total arcade length/mg tissue (average for both diets) was 22% lower in DS than in DR, with the average number of arcade segments and arcade loops/mg tissue 38 and 36% lower in DS, respectively. The average number of transverse networks branching from each millimeter of arcade segment is illustrated in Fig. 3. The level of dietary salt intake had no effect on this value in DR. DS maintained on 0.45% NaCl had essentially the same number of transverse arterioles/mm arcade as DR, but in contrast to DR, high salt intake in DS was associated with a reduction in this number. DS on 7% NaCl were found to have 19% fewer transverse arterioles/mm arcade than DS on 0.45% NaCl. This difference was statistically significant. In each experimental group, fifteen transverse networks were randomly selected and all of the vessels within those networks were mapped in their entirety. Transverse network size was variable in each group, with a total of 7 to 28 capillary bundles supplied by the distal branches of a single transverse arteriole. Despite this size range, each network examined was found to contain either three or four branching generations (as determined by the Strahler method) from its point of origin at the arcade arteriole to the origin of the capillaries. The average number of vessels of each branch order per transverse network is shown for each group in Table
174
BOEGEHOLD
AND
0 DR, 0.45% NaCl EZi DR, 7% NaCl El DS, 0.45% NaCl I DS, 7% NaCl
1.4
kg CE s; 4 .z g!”
0.6
am -5
0.4
E
+rl 5%
KOTCHEN
1.2 1.0 0.6
0.2 0.0 1.6
$
2
1.4
.g
1.2
iiF
1.o
%E
0.0
2iQ ai9 2;E=
0.6 0.2 0.4
P
0.0
FIG. 2. Structural characteristics of the arcade network in each experimental group. Values are means ? SEM and are normalized to tissue mass. (Top) Total arcade segment length/mg; (middle) number of individual arcade segments/mg; (bottom) number of arcade loops/mg. Values grouped under bars are not significantly different. *Significantly different from DR on same diet (P < 0.05).
2. The number of vessels of each branch order was similar in all groups, with no significant strain- or diet-dependent differences observed. DISCUSSION The current observations demonstrate a marked difference between the DS and DR rat strains in the architecture of the arcade arteriole network in gracilis muscle. In addition, our results suggest that the development of salt-induced hypertension in the DS strain is accompanied by a structural rarefaction of transverse arterioles in this vascular bed. Compared to normotensive DR, the arcading portion of the network in hypertensive DS on 7% NaCl was found to be less dense as judged by a lesser
ARTERIOLAR
Y 52 z$ a ZiE CQJ Q) au) ul
3.5 3.0
2.0
22 @a b;y-
1.5
E GE a, 5 g
IN
DAHL
175
RATS
0 ESS El m
DR, DR, DS, DS,
O&i”h NaCl 7% NaCl 0.45% NaCl 7% NaCl
2.5
g;
%
NETWORK
1.0 0.5
z 0.0
3. Number of transverse networks/mm arcade segment length. Values are means ? SEM. Values grouped under bar are not significantly different. *Significantly different from DS on 0.45% NaCl (P < 0.05). Frc.
total vascular length and fewer individual segments and vascular loops/mg of tissue (Fig. 2). In contrast, arcade network values for DS on 7% NaCl were virtually identical to those for DS on 0.45% NaCl, despite a significant difference between these groups in mean arterial pressure (Table 1). This suggests that the observed difference in arcade network density between hypertensive DS and normotensive DR reflects an inherent difference in vascular architecture between these strains that is independent of either the level of dietary salt intake or its effect on arterial pressure. In addition, on an individual animal basis, we found no statistical correlation between mean arterial pressure and the total arcade length (P = 0.80), number of arcade segments (P = 0.55), or number of vascular loops (P = 0.55) per milligram tissue, further demonstrating the lack of relationship between arcade network density and mean arterial pressure. These findings suggest that although DS and DR are descended from the same parent strain (Dahl et al., 1962a,b), the genetic isolation of these two lines brought about by years of selective breeding has given rise to some degree of genetic drift, producing differences in arcade network architecture that are apparently unrelated to the phenomenon of salt-induced hypertension. In contrast to the arcade arterioles, the reduced number of transverse arterioles/mm arcade segment in DS on 7% NaCl was not seen in DS on 0.45% NaCl (Fig. 3), suggesting that this is a true rarefaction of vessels related to the saltinduced increase in arterial pressure. The average number of distal branches arising from existing transverse arterioles was found to be equivalent in all experimental groups (Table 2), which indicates that a reduction in the number of branches within a transverse network does not occur independent of rarefaction of the parent vessel. Therefore, the vascular unit which becomes rarefied at this stage of salt-induced hypertension appears to be the entire transverse network. Data from longitudinal studies in SHR and rats with renovascular hypertension suggest that structural rarefaction of arterioles in these forms of hypertension is due to a loss of preexisting vessels (Ono et al., 1989; Prewitt et
176
BOEGEHOLD AND KOTCHEN
NUMBER
Experimental group DR, DR, DS, DS,
0.45% NaCl 7% NaCl 0.45% NaCl 7% NaCl
OF VESSELS
OF EACH
TABLE 2 BRANCH ORDERPERTRANSVERSENETWORK Number of vessels
1st Order 12.3 14.5 14.5 14.1
-c ? f 2
1.5 1.4 2.2 1.3
2nd Order
3rd Order
4th Order
4.6 5.2 5.4 5.3
1.7 1.5 1.6 1.5
0.6 0.3 0.4 0.5
2 ‘_ 2 +
0.5 0.5 0.8 0.6
k -c k 2
0.2 0.2 0.3 0.2
5 “_ 2 k
0.1 0.1 0.2 0.1
Note. DR, Dahl salt-resistant rats. DS, Dahl salt-sensitive rats. Values are means k SEM. Branch orders are assigned according to Strahler (1957): 1st order, most distal vessels, and 4th order, most proximal vessels.
al., 1982, 1984). The structural rarefaction of transverse arterioles in the present study could reflect either a loss of vessels or a suppression of normal new vessel growth in developing DS on the high salt diet. Because younger animals were not also examined in the present study, we cannot distinguish between these two possibilities. The number of transverse arterioles/mm arcade segment was 19% lower in the hypertensive group than in the other groups (Fig. 3). Assuming that the rarefied networks were similar in size to those present, our data suggest that when normalized to arcade length, there is a 19% reduction of all distal arterioles in hypertensive DS. Our data also indicate that in addition to the rarefaction associated with hypertension, there is an inherent difference between DS and DR in distal arteriolar density. The spacing of transverse arterioles along arcade segments is identical in DR and DS on 0.45% NaCl (Fig. 3), but the inherently lower arcade network density in DS results in a calculated transverse network density that is 28% lower than that in DR (2.35 2 0.26 vs 3.25 t 0.29 networks/mg tissue, P < 0.05). In DS on 7% NaCl, with 19% fewer vessels per millimeter arcade, transverse network density is further reduced to 2.06 IC_0.12 per milligram tissue, or 37% below that of DR on 0.45% NaCl. The extent of structural rarefaction found in the present study is somewhat less than that reported in the gracilis muscle of SHR (56%) and rats with one-kidney, one-clip hypertension (50%) (Prewitt et al., 1982, 1984). However, the data from SHR were obtained in mature animals (16-18 weeks) and the one-kidney, one-clip data were obtained 8-10 weeks after renal artery clipping. In contrast, the DS studied here were observed after only 4 weeks on the high salt diet. It is possible that the lesser extent of structural rarefaction found in the current study simply reflects a shorter duration of hypertension in these animals. To our knowledge, there is little information available on the relative contribution of different microvascular segments to total gracilis muscle vascular resistance. Consequently, the importance of transverse network rarefaction in increasing whole-organ resistance in hypertensive DS is open to speculation. Greene and co-workers (1989) have recently developed a mathematical model of the arteriolar network to estimate the hemodynamic effect of arteriolar constriction and rarefaction in hypertensive animals. The design of their network closely resembles that of the gracilis muscle, and the relationship between rarefaction and network resistance derived from that model predicts that a 19%
ARTERIOLAR NETWORK IN DAHL RATS
177
rarefaction of distal arterioles would only increase total network resistance in DS by approximately 10%. In comparing hypertensive DS to DR, the estimated 37% reduction in distal arteriolar density would increase network resistance by approximately 20%, with the lower arcade network density in DS also contributing to elevated network resistance. Our finding of a reduced arteriolar density in the gracilis muscle of DS rats with salt-induced hypertension is at variance with our recent report that arteriolar density is normal in the spinotrapezius muscle of these animals (Boegehold and Kotchen, 1990). In that study, arcade and transverse network density in hypertensive DS on 7% NaCl was found to be similar to that of DS on 0.45% NaCl and DR on either diet. The number of distal branches per transverse network was also equivalent in all groups. The experimental groups in the recent study were identical to those studied here, in terms of both age and length of time on diet. In the current study, the reduction in arteriolar density in hypertensive DS appears to be both of genetic origin (at the arcade level, Fig. 2) and a response to the salt-induced rise in arterial pressure (at the transverse level, Fig. 3). The occurrence of hypertension-dependent structural rarefaction in the gracilis muscle but not in the spinotrapezius muscle is consistent with earlier findings in the SHR. The 56% rarefaction of small arterioles in SHR gracilis muscle reported by Prewitt and co-workers (1982) contrasts sharply with a report that rarefaction does not occur in the spinotrapezius muscle of SHR at the same age (Engelson et al., 1986). In fact, Engelson and co-workers found that both arcade network density and the number of transverse arterioles were actually greater in SHR than in age-matched normotensive WKY. These observations were made in animals of up to 20 weeks old, which suggests that hypertension-dependent rarefaction may never occur in the rat spinotrapezius muscle. With respect to the arcade network, it is unclear why an inherent difference in vascular density between the DS and DR strains would occur in the gracilis muscle but not in the spinotrapezius muscle. This heterogeneity in strain-dependent differences, coupled with the suggestion that structural rarefaction may not occur in all muscle types, highlights the importance of studying different vascular beds within any one model of hypertension. Conclusions on hypertension-dependent changes based on observations from a single vascular bed, even in the case of skeletal muscle, could be misleading. In summary, the anatomic density of gracilis muscle arcade arterioles was found to be inherently lower in the DS rat strain than in the DR rat strain. This difference appears to be unrelated to the salt sensitivity of blood pressure in the DS rat. In contrast, the anatomic density of more distal transverse arterioles in the gracilis muscle was significantly lower in hypertensive DS than in normotensive DS, suggesting that these vessels undergo a structural rarefaction associated with the development of salt-induced hypertension. The structural rarefaction observed in this muscle was not found in an earler study on the spinotrapezius muscle, suggesting that this is not a characteristic common to all vascular beds in the DS model of hypertension. ACKNOWLEDGMENT This investigation was supported by a Grant-in-Aid Virginia affiliate.
from the American Heart Association, West
178
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AND
KOTCHEN
REFERENCES M. A., AND KOTCHEN, T. A. (1990). Effect of dietary salt on the skeletal muscle microvasculature in Dahl rats. Hypertension 15, 420-426. DAHL, L. K., HEINE, M., AND TASSINARI, L. (1962a). Effects of chronic excess salt ingestion: Evidence that genetic factors play an important role in susceptibility to experimental hypertension. J. Exp. Med. 115, 1173-1190. DAHL, L. K., HEINE, M., AND TASSINARI, L. (1962b). Role of genetic factors in susceptibility to experimental hypertension due to chronic excess salt ingestion. Nature(London)194, 480-482. ENGELSON, E. T., SCHMID-SHONBEIN, G. W., AND ZWEIFACH, B. W. (1986). The microvasculature in skeletal muscle II: Arteriolar network anatomy in normotensive and spontaneously hypertensive rats. Microvasc. Res. 31, 356-374. GANGULI, M., TOBIAN, L., AND IWAI, J. (1979). Cardiac output and peripheral resistance in strains of rats sensitive and resistant to NaCl hypertension. Hypertension 1, 3-7. GREENE, A. S., TONELLATO, P. J., LUI, J., LOMBARD, J. H., AND COWLEY, A. W., JR. (1989). Microvascular rarefaction and tissue vascular resistance in hypertension. Amer. J. Physiol. 256, H126-H131. GREENE, E. C. (1955). “The Anatomy of the Rat.” Hafner, New York. LOMBARD, J. H., HINOIOSA-LABORDE, C., AND COWLEY, A. W., JR. (1989). Hemodynamics and microcirculatory alterations in reduced renal mass hypertension. Hypertension 13, 128-138. MEININGER, G. A., HARRIS, P. D., AND JOSHUA, I. G. (1984). Distributions of microvascular pressure in skeletal muscle of one-kidney, one-clip, two-kidney, one-clip, and deoxycorticosterone-salt hypertensive rats. Hypertension 6, 27-34. ONO, Z., PREWITT, R. L., AND STACY, D. L. (1989). Arteriolar changes in developing and chronic stages of two-kidney, one-clip hypertension. Hypertension 14, 36-43. PFEFFER,M. A., PFEFFER,J., MIRSKY, J., AND IWAI, J. (1984). Cardiac hypertrophy and performance of Dahl hypertensive rats on graded salt diets. Hypertension 6, 475-481. PREWITT, R. L., CHEN, I. I. H., AND DOWELL, R. (1982). Development of microvascular rarefaction in the spontaneously hypertensive rat. Amer. J. Physiol. 243, H243-H251. PREWITT, R. L., CHEN, I. I. H., AND DOWELL, R. F. (1984). Microvascular alterations in the onekidney, one-clip renal hypertensive rat. Amer. J. Physiol. 246, H728-H732. PREWITT, R. L., HASHIMOTO, H., AND STACY, D. L. (1987). Microvascular alterations in hypertension. In “Microvascular Perfusion and Transport in Health and Disease” (P. McDonagh, Ed.), pp. 31-59. Karger, Basel. SCHMID-SCHOENBEIN, G. W., ZWEIFACH, B. W., AND KOVALCHECK, S. (1977). The application of stereological principles to morphometry of the microcirculation in different tissues. Microvasc.
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16. STACY, D. L., AND PREWITT, R. L. (1989). Attenuated microvascular alterations in coarctation hypertension. Amer. J. Physiol. 256, H213-H221. 17. STRAHLER,A. N. (1957). Quantitative analysis of watershed geomorphology. Trans. Amer. Geophys.
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