Mechanical and contractile properties of in situ localized mesenteric arteries in normotensive and spontaneously hypertensive rats

Mechanical and Contractile Properties of in situ Localized Mesenteric Arteries in Normotensive and Spontaneously Hypertensive Rats Hong Ying Qiu, Brttno Valtier, Institut National

Harry A. J. Struyker-Boudier,

and Bernard I. Levy

de la Sante’ et de la Recherche Me’dicale, H6pital Luriboisikre, Paris, France (H.Y.Q., B.V., B.I.L.) and Department of Pharmacology, University of Limburg Medical School, Maastricht, The Netherlands (H.S.-B.)

An in situ model was developed for studying mechanical properties of mesenteric arteries in rats. A branch of the mesenteric artery was exposed and dissected in normotensive (WKY) and spontaneously hypertensive rats (SHR). A catheter was introduced into the larger branch of the mesenteric artery and connected to a pressure chamber. The artery was submitted to transmural pressures ranging from 0 to 200 mmHg per steps of 25 mmHg and observed using a microscope-video-camera system. The diameter-pressure relations were established under basal conditions, under contraction (phenylephrine 10-6M), and after abolition of the smooth muscle tone by potassium cyanide (KCN, 0.1 mg/mL). The arterial segment was then fixed (glutaraldehyde 2.5%), and the wall cross-sectional areas were measured in transverse sections. Compliances, distensibility, wall tensions, and wall stresses were calculated from diameter, pressure, and media thickness values under three conditions. Active tension and active stress were defined as differences in wall stresses and wall tensions calculated under passive and active conditions. Comparision of WKY and SHR when arteries were studied at the respective operating pressure indicates (1) thicker and stiffer mesenteric arteries in SHRs than in WKY rats, (2) similar wall stresses in mesenteric arteries from WKY and SHRs despite larger wall tensions in the hypertensive group, and (3) larger contractility to phenylephrine in SHRs than in WKY mesenteric arteries. Keywords:

Hypertension;

Mesenteric

artery; Compliance;

Introduction The mechanisms underlying spontaneous hypertensive disease are complex. It is generally assumed that the resistance arteries that control most of the vasculature’s resistance to blood flow are of primary importance in the initiation and the maintenance of elevated blood pressure. Formerly, the resistance arteries were thought to consist of precapillary arterioles (diameter < 50 Fm). However, it is now clear that, in many organs, about one half of the precapillary resistances are located proximal to these arterioles, in small arteries with diameters ZG500 pm (Johnson and Hanson, 1962; Mulvany and Aalkjaer, 1990). Address reprint H6pital Lariboisitre, Received June

requests to Dr. Bernard Levy, INSERM 75010 Paris, France. 1994; revised and accepted November

Journal of Pharmacological and Toxicological Methods 0 1995 Else&r Science Inc. 655 Avenue of the Americas, New York, NY 10010

Unit

141,

1994.

33, 159-170

Distensibility;

Contraction.

The myograph is the most widely used method to assess the mechanical properties and pharmacological reactivity of small arteries. Briefly, this in vitro method uses segments of lOO-200~km arteries mounted as ring preparations on two fine wires, with the wires being clamped at each end to ensure that the response is isometric (Bevan and Osher, 1972; Mulvany and Halpem, 1976; Mulvany et al., 1978): A more recent approach entails in vitro cannulation of the vessels to allow control of the intraluminal pressure; the lumen diameter is then monitored (Duling et al., 1981; 0~01 and Halpem, 1985; Vanbavel et al., 1990) and pressurelumen relations determined. However, both in vitro methods have some disadvantages; especially, the longitudinal stress of the studied arteries is not kept at its in vivo value, and whatever the skillfulness of the experi-

(1995) SSDI

1056.8719/95/$9.50 1056-8719(94)00076-G

160

JPM Vol. 33, No. 3 June 1995:159-170

mentor, the excision procedure of the studied segment of artery cannot be atraumatic. We have developed an experimental model of in situ isolated rat mesenteric artery allowing us to determine the pressure-lumen relations in more physiological and potentially less traumatic conditions. The aim of this article is to describe this experimental model of in situ isolated mesenteric artery and to compare the static mechanical properties of its arterial wall under basal conditions as well under contracted and relaxed conditions in normotensive (WKY) and spontaneously hypertensive rats (SHR).

Methods Twenty-two lZweek-old SHRs were compared to 22 age-matched WKY animals. Body weights were (mean ? SD) 288 2 12 g and 320 + 15 g in SHRs and WKY rats, respectively. Experimental Model After anesthesia with intraperitoneal sodium pentobarbital (50 mg/kg), a catheter connected to a pressure transducer (Gould P23ID) was introduced into the right carotid artery in order to record the arterial blood pressure for each animal. A median laparotomy was then performed, and the last loop of small intestine was exposed on a laboratory-built plastic container (Figure 1). The preparation was immediately irrigated by a buffered Tyrode’s solution (pH = 7.4) maintained at 38°C. The preparation (intestine and mesenteric arteries) was just lying on the glass piece in the plastic container, allowing the preparation to be irrigated on both sides with the Tyrode’s solution. The mesenteric arteries and arcades were neither fixed nor stretched. The preparation was transilluminated by a light generator placed under the optic glass piece. A short segment of mesenteric artery (about 3 mm) of a second generation branch (diameter 400-500 pm) was then exposed and gently dissected under the binocular lens (Microcontrol, Evry, France). A video camera (Hitachi CCTV) was mounted on the binocular lens and allowed to record video images (video recorder S-VHS Blaupunkt 920PSW). The whole optical and recording system was previously calibrated using a glass frame. The final magnification of the system was x 100. The fat tissue was then removed and the mesenteric artery and vein were separated in order to obtain a clear image of the isolated segment of the observed artery with sharp and well-defined vessel edges on the video screen. Every arterial branch located downstream of the observed segment of artery, except one, was then ligated with silk thread 9/O. A removable microclamp was placed on the last branch. Finally, a polyethylene catheter (external diameter

0.6 mm, internal diameter 0.28 mm) was introduced and secured into the first generation branch of the mesenteric artery, at least 8 mm upstream of the observed segment. The catheter was filled with Tyrode’s solution with albumin (4%). The presence of protein in flushing and incubating solutions served to preserve the endothelium (Morisson et al., 1976) and maintained a physiological osmotic pressure across the vessel wall. The catheter was connected to a manometer with adjustable pressure levels. A three-way tap and a larger tube (diameter 2 mm), containing the same flushing solution, were connected between the manometer and the mesenteric catheter. After removing the clamp from the distal segment of the mesenteric artery, the observed mesenteric artery was filled with the flushing solution (Figure 2). To start the measurement, the segment of isolated artery was submitted to the atmospheric pressure for 5 min, and the external diameter of the artery was measured. The artery was then submitted to pressure steps of 25 mmHg from 0 to 200 mmHg during 5 min per step. The video image of the observed mesenteric artery was recorded during the last 30 set of each pressure step period.

Experimental Protocol Two sets of experiments were performed. In the first one (10 WKY rats and 10 SHRs), the mesenteric arterial diameter-pressure relation was first performed under basal conditions (superfusion with thermostated and buffered Tyrode’s solution). The transmural pressure was then returned to zero for 30 min, and the mesenteric artery was washed and flushed with another Tyrode’s albumin solution containing potassium cyanide (KCN 100 mg/L). The potassium cyanide solution was maintained in the mesenteric artery at a transmural pressure of 75 mmHG for 30 min, a period sufficient to poison the arterial smooth muscle and to totally abolish the smooth muscle tone (Dobrin and Rovick, 1969). In the second set of experiments (12 WKY rats and 12 SHRs), the preparation was superfused with Tyrode’s solution containing phenylephrine ( 1O-GM). After 10 min of incubation, the mesenteric arterial diameter was measured under active smooth muscle conditions for the same range of pressure (O-200 mmHg). During these measurements, the preparation was continuously superfused with the same phenylephrine solution (lo-CM). The transmural pressure was then returned to zero, phenylephrine was removed from the superfusion solution, and the studied mesenteric artery was incubated with KCN solution for 30 min. Using the same procedure, another series of diameter measurements was then performed under passive smooth muscle conditions. At the end of every experiment, the mesenteric artery was washed and filled with a solution containing glutar-

161

H. Y. QIU ET AL. PROPERTIES OF THE MESENTERIC ARTERY IN SHR

Monitor

Video

/

Video

recorder

‘Camera

v/

*-

____---

------

-

T.-L.. 4--

.

&-

__-______-

--------

-----

Optical

glass

Mesentery

Catheter

*--------

Illumination

system

Figure 1. Diagram of the experimental set-up. The mesentery, irrigated with thermostated Tyrode’s solution, was exteriorized and exposed on an optical glass for trans-illumination. The dissected segment of the mesenteric artery (dotted circle) was observed with a lens connected to a video camera. Images were displayed and recorded for further measurements.

aldehyde (2.5%) buffered with sodium cacodylate (0.1 M) and paraformaldehyde (2%). The vessel was maintained during 20 min at a distending pressure of 75 mmHg. The fixed studied mesenteric artery was then excised, embedded in specific medium, and transversal sections were performed using a cryostat (-20°C). Pictures of successive sections of each mesenteric artery were performed. The images of the arterial sections were magnified by projecting the slides onto a digitizer (Mac Tablet connected to a Macintosh SE) and outlining the luminal and external elastic lamella surfaces of the vessels. This procedure allowed us to calculate the vessel media cross sectional area.

*media thickness (h, km): Assuming a noncompressible arterial wall, h was calculated, for each level of distending pressure as: h = CSAl(n x 0,) The internal diameter (Di, pm) was De - 2.h, and the vessel volume (Vi, (IL) was calculated, for each transmural pressure: Vi = T x D,2/4 The compliance per unit length (C = p,L/mmHg mm) of the observed segment of mesenteric artery was defined, for each pressure step, as: C = AVIAP

Calculation of Mechanical Parameters Using the measured values of the in situ arterial external diameter (D,, pm) from 0 to 200 mrnHg and the media cross sectional area (CSA, km2) measured after the experiment, several parameters were calculated:

where AV is the volume change induced by a transmural pressure variation of AP (25 mmHg) in a segment of l-mm length of the artery. *The vessel distensibility (Dist, 10m3 mmHg-‘) was defined, for each step of transmural pressure (PZ-P2 =

162

JPM Vol. 33, No. 3 June 1995:159-170

variance with repeated measurements to provide evidence of differences related to strains and to treatment. Differences between groups were evaluated using the Newman-Keuls test (Zar, 1984).

Results observed

l-r*--------Catheter

segment

(0:0.6

mm)

4l=i!l Pressure chamber

r;FrlLigatures m Fatty tissue

P

Figure 2. Schematic view of the mesenteric arterial tree. A polyethylene catheter was introduced into the upper part of the MO branch and connected to a pressure chamber system. A short segment of Ml was dissected for optical observation. All lateral and terminal branches of the mesenteric artery were sutured except one on which a removable clamp was placed.

25 mmHg), as the compliance (0 calculated between PI and P2 divided by the initial internal volume calculated at the pressure PI. Dist = CIVi

*According to Laplace’s law, the wall tension (T, N/m), the tangential wall force per unit length was given by T = P x Dj2

*The tangential wall stress (s, kPa) was given by: s = T/h

According to Mulvany under isometric conditions (Mulvany and Halpem, 1977) and Cox (1979) from in vitro pressure-diameter relationship, the active wall stress was calculated as the difference in wall stress calculated, at a given pressure, under active (incubation with phenylephrine) and passive (KCN poisoning) conditions. Similarly, active wall tension was calculated from active and passive wall tension values. Statistical

Analysis

Results are expressed as mean +SEM. mental

design

allowed

us to use a two-way

The experianalysis of

The operating arterial pressure was recorded after surgical preparation of the isolated segment of the mesenteric artery about 1 hr after induction of anesthesia. The systolic, diastolic, and mean arterial pressure values were 128 2 11, 95 t 9, and 104 + 11 mmHg in WKY rats, and 182 2 16, 145 k 14, and 154 k 15 mmHg in SHRs. We defined the mean operating pressure as the mean arterial pressure of the animals, roughly 100 mmHg in WKY and 150 mmHg in SHR. The mesenteric artery external diameters measured at operating pressures were significantly larger in SHRs than in WKY rats (p < .Ol). The medial hypertrophy was evidenced by an increased cross-sectional area in SHRs compared to WKY rats: 19033 k 342 pm2 and 14563 +- 208 p,m2, respectively (p < .OOl). Figure 3 shows the diameter-pressure relation in both strains and under the three experimental conditions: basal, active, and passive smooth muscle conditions. At low pressures, the vessels exhibited large changes in diameters with each step change in pressure, whereas at high pressures, diameter changes per pressure increase were minor. This pattern was more pronounced under basal conditions and particularly after poisoning with KCN, whereas a much more gradual curve was observed after treatment with phenylephrine. There were significant differences between the diameter-pressure curves from WKY rats and SHRs under basal conditions (p < .Ol) and under passive smooth muscle conditions (JJ < .OOl) but not under active muscle conditions. The contraction by the active muscle can be estimated at each pressure by subtracting the diameter after treatment with phenylephrine from the diameter after poisoning with KCN. Maximum active muscular contraction occured at 50 mmHg in WKY’s mesenteric arteries and between 50 and 100 mmHg in SHRs. The active muscle contraction was significantly higher in SHRs than in WKY rats (p < .OOl). Figure 4 summarizes the magnitude of active muscular contraction for all the vessels studied. The maximum contraction represents 13% of the diameter of the KCNpoisoned vessel at 50 mmHg in WKY rats and 16% of the diameter of the KCN-poisoned vessel at 75 mmHg in SHRs. Figure 5 and Figure 6 show the compliance and the distensibility of the mesenteric artery under the three experimental conditions. Under basal conditions and under active and passive muscle conditions, the compliance and distensibility curves were not significantly different in both strains over the whole range of pres-

H. Y. QIU ET AL. PROPERTIES OF THE

163 MESENTERIC

ARTERY

IN SHR

JI

600 -

pco.01

0

50

150

100

Pressure

200

(mmHg)

600 -1 Ns 500 -

Active 0

P:kksure

100 (mmHg)

600 -

T I T

150

200

T 1 1

T 1 T

p~o.001

Passive I 50

I 100

Pressure

I 150

I 200

(mmHg)

Figure 3. Mean values (+-SEM) of external diameters of the mesenteric arteries from nonnotensive WKY rats (open symbols) and SHRs (closed symbols) for pressure values from 0 to 200 mmHg per step of 25 mmHg. Upper graph: control conditions; middle graph: active conditions (phenylephrine 10e6M); lower graph: passive conditions (poisoning of the smooth muscle cells by potassium cyanide). The white and black arrows indicate the levels of the operating pressures in WKY and SHR groups, respectively. The p values given in every experimental condition indicate the differences between curves from WKY and SHRs for the whole range of pressure (NS = nonsignificant).

sure. The maximal values of all curves were observed at the low pressure range (between 25 and 75 mmHg). At higher, more physiological pressures, mesenteric compliance and distensibility were lower (< 0.001

~L/mmHg per mm artery and < 0.005 mmHg-‘, respectively). Figure 7 presents the tangential wall tension calculated in both strains under the different experimental

164

JPM Vol. 33, No. 3 June 1995:159-170

Basal

2 : sz! 'L 2

40

20

y

500

0 C

0 I

pco.001

50

100

Pressure

150

(mmHg)

0 0

50

100

150

200

PRESSURE (mm Hg) Figure 4. Mean values (+-SEM) of active muscle contraction plotted as a function of transmural pressure. These were calculated by subtracting diameters after KCN from diameters after phenylephrine.

conditions. These curves were very similar in both strains: There were no differences for the whole range of pressure, but, when calculated at the operating pressure levels, the wall tension was markedly increased in SHRs whatever the contractile state of the arterial smooth muscle (p < .Ol). The wall stress-pressure curves (Figure 8) were very similar in active and passive conditions in WKY rats and SHRs. Under basal conditions, the wall stresses were significantly larger in WKY than in SHR (p < .OS). When calculated at the operating pressure in each strain (arrows in Figure S), the wall stress values were almost identical in both strains. Figure 9A reports the active wall tension, representing the magnitude of muscular contraction in both strains. Active wall tension is markedly larger in SHRs than in WKY rats (p < .Ol). It means that the maximally contracted mesenteric arterial wall from SHRs was able to exert about three-times-larger forces than that from WKY rats. In the same way, the active wall stress (wall tension normalized by wall thickness) was significantly larger (about twice) in SHRs than in WKY rats @ < .Ol, Figure 9B). This results suggest that the same volume of smooth muscle cells from SHRs mesenteric artery was able to exert about two-times-larger forces than that from WKY rats. Table one shows the mesenteric arterial diameters and the mechanical parameters measured and calculated at the mean operating pressure under basal, active, and passive conditions.

Discussion The principal method for in vitro small artery measurement comprises a scaled-down organ bath called a

=: z

2500 -

E

2000 -

2 .

1500-

E

1000 -

2 c, F

500 -

Active

T V

0

0

I 0

50

I

I

I

100

rs'o

200

Pressure =: t

2500

T

(mmHg)

T

Passive

50

100

500 Ns

0 0

0

Pressure

150

200

(mmHg)

Figure 5. Mean values (5 SEM) of compliance of the mesenteric arteries from normotensive WKY rats (open symbols) and SHRs (closed symbols) for pressure values from 0 to 200 mmHg per step of 25 mmHg. Upper graph: control conditions; middle graph: active conditions (phenylephrine 10e6M); lower graph: passive conditions (poisoning of the smooth muscle cells by potassium cyanide). The white and black arrows indicate the levels of the operating pressures in WKY and SHR groups respectively. The p values given in every experimental condition indicate the differences between curves from WKY and SHRs for the whole range of pressure (NS = nonsignificant).

myograph, in which the vessel segment is held on two wires passed through the lumen (Bevan and Osher, 1972; Mulvany and Halpem, 1977). Most often, the

H. Y. QIU ET AL. PROPERTIES OF THE

165 MESENTERIC

ARTERY

IN SHR

Basal

Ns 8

Basal

d

100

50

Pressure

150

200

0

50

100

Pressure

(mmHg)

150

200

(mmHg)

8-

Active d

50

100

Pressure 30 -

150

0

200

Passive

5;

2;o

Pressure

(mmHg)

(mmHg)

8-

820 -

IO -

0

Pressure

(mmHg)

50

100

Pressure

150

200

(mmHg)

Figure 6. Mean values (+SEM) of distensibility of the mesenteric arteries from normotensive WKY rats (open symbols) and SHRs (closed symbols) for pressure values from 0 to 200 mmHg per step of 25 mmHg. Upper graph: control conditions; middle graph: active conditions (phenylephrine 10m6M); lower graph: passive conditions (poisoning of the smooth muscle cells by potassium cyanide). The white and black arrows indicate the levels of the operating pressures in WKY and SHR groups respectively. The p values given in every experimental condition indicate the differences between curves from WKY and SHRs for the whole range of pressure (NS = nonsignificant).

Figure 7. Mean values (?SEM) of tangential wall tension of the mesenteric arteries from normotensive WKY rats (open symbols) and SHRs (closed symbols) for pressure values from 0 to 200 mmHg per step of 25 mmHg. Upper graph: control conditions; middle graph: active conditions (phenylephrine 10-6M); lower graph: passive conditions (poisoning of the smooth muscle cells by potassium cyanide). The white and black arrows indicate the levels of the operating pressures in WKY and SHR groups respectively. The p values given in every experimental condition indicate the differences between curves from WKY and SHRs for the whole range of pressure (NS = nonsignificant).

activation level of the smooth muscle cells is evaluated by measurement of the force exerted on the isometrically maintained wires. This very useful technique carries certain limitations, as recently pointed out by Halpern

and Kelly (199 1). For example, the inevitable and unequal damage done to the endothelial cells by the passage of the wires must be considered. Also, it has been shown that vessel wall distension can affect the sensitivity of resis-

JPM Vol. 33, No. 3 June 1995:159-170

098

p
0

50

100

Pressure

150

0

200

50

(mmHg)

100

Pressure

“‘1

150

200

(mmHg)

B

400-

Active 0

50

100

Pressure

150

p
(mmHg) 50

800

Pressure

100

150

(mmHg)

Figure 9. A. Active tangential wall tension (?SEM) developed at pressures varying from 0 to 200 mmHg by the mesenteric artery from WKY rats (open symbols) and SHRs (closed symbols). B. Active tangential wall stress (?SEM) developed at pressures varying from 0 to 200 mmHg by the mesenteric artery from WKY rats (open symbols) and SHRs (closed symbols).

600

400

200

0

0

50

100

Pressure

150

200

(mmHg)

Figure 8. Mean values (+-SEM) of tangential wall stress of the mesenteric arteries from normotensive WKY rats (open symbols) and SHRs (closed symbols) for pressure values from 0 to 200 mmHg per step of 25 mmHg. Upper graph: control conditions; middle graph: active conditions (phenylephrine IOm6M); lower graph: passive conditions (poisoning of the smooth muscle cells by potassium cyanide). The white and black arrows indicate the levels of the operating pressures in WKY and SHR groups respectively. The p values given in every experimental condition indicate the differences between curves from WKY and SHRs for the whole range of pressure (NS = nonsignificant).

tance arteries to pharmacological agonists (Nilsson and Sjijblom, 1985). The most commonly used method for setting vessel stretch is to calculate the forces corresponding to an effective pressure according to Laplace’s

law. This is only a poor approximation because of the noncircular shape of the stretched ring and its relatively short length. Furthermore, the absence of arterial longitudinal stresses in these experimental conditions introduces other differences between in vivo and in vitro conditions. For these reasons, Halpern and Kelly proposed cannulated vessel methods that offer a more physiological and versatile approach. The use of cannulated in vitro and our in situ methods provide the following advantages and possibilities: (1) diameter is allowed to change, (2) shape is cylindrical, (3) a transmural pressure exists across the wall, and thus the filtration rate through the vessel wall can be measured in nearly physiological conditions, (4) physiological axial length and geometry of the vascular tree are conserved, (5) pharmacological agents can be perfused or superfused. Basically, our in situ method even offers more advantages than the in vitro cannulated arteries with further interesting characteristics: (1) The innervation of

H. Y. QIU ET PROPERTIES

AL. OF THE

167 MESENTERIC

Table 1. Mechanical SHR = 1.50 mmHg)

ARTERY

Characteristics

IN SHR

of the Mesenteric Artery (mean + SEM) at the Operating WKY Basal

External diameter (pm) Media thickness (km) Compliance (10-6 pUnmHgmm Distensibility (10-3 mmHg- 1) Wall tension (N/m) Wall stress @Pa) Active wall tension (N/m) Active wall stress @Pa)

490 8.9 art)

k 13 2 0.2

897 -f 195 4.75 2 1.03 3.36 2 0.08 260 f 18

SHR (P =

(P = 100 mmHg) Phenylephrine

KCN

466 2 21 10 ? 0.5

503 2 21 8.5 2 0.14

531 5 168 3.59 t 1.33 2.97 + 0.14 303 2 22 0.26 2 0.06

383 2 87 2.16 t- 0.45 3.26 t 0.14

54 ?

13

the mesenteric arterial tree is conserved, and (2) the normal length and geometry of the arteries are conserved for the whole experimental procedure. Actually, for the in vitro arterial cannulation, the vessel must be excised (collapsed and retracted), cannulated, and then positioned at its original axial length and pressure. As previously reported for larger arteries (Baldwin et al., 1982; Tedgui and Lever, 1984), the maintenance of axial and radial stresses during the surgical preparation are of major importance to preserve the endothelial functional and histological integrity. When compared to Mulvany’s results obtained in smaller mesenteric arteries (195 + 10 Frn and 206 ? 12 pm in SHRs and WKY rats, respectively, i.e., twice smaller as in the present study), our results evidenced much smaller active wall tensions (roughly 7 times smaller in SHR and 11 times smaller in WKY rats). In view of the finding that the active wall tension is proportional to the difference in radius under active and passive conditions, it is surprising that, in our study, larger arteries evidenced smaller active tensions. In a preliminary series of experiments, we tested the dose-response effect of phenylephrine; 10-6M corresponded to 82 + 8% in SHR and 81 ? 7% in WKY of the phenylephrine-induced maximal contraction. Discrepancies between our results and that obtained by myograph method could be related to differences in experimental models and/or to differences in the size of the studied arteries. However, Lash et al. (1991), who studied even smaller (90 pm) intestinal arteries localized in situ and in vivo, reported active tension of 0.27 N/m in normotensive animals, that is, very close to those obtained in the present study. Therefore, the differences in experimental techniques used to record the radius-pressure relationship seem to be of major importance. One of the major methodological concerns of the present work is to compare the arterial wall mechanical behavior of hypertensive and normotensive rats at their respective operating pressure and not at the same level of pressure. Although the comparison of the mechanical properties of two different materials must be performed at the same stress or pressure, we believe that the addition of comparison of their mechanical properties at

Pressure (WKY

357 + 28

Basal

5 2 -t -c

mmHg)

Phenylephrine

571 2 11 10.7 2 0.2 219 0.85 5.41 311

150

= 100 mmHg,

83 0.4 0.11 17

526 + 11.9 2 529 2 2.61 -+ 4.52 431 0.55 102

k 2 -c 2

23 1 130 0.76 0.56 68 14 28

KCN 581 10.5 149 0.63

2 2 k k

11 0.2 66 0.29

5.63 2 0.13 548 -c 38

their operating pressure gives more realistic further informations. This could be one of the more important reasons explaining the discrepancy between the results obtained in small resistance arteries by Mulvany (1989), evidencing no differences in passive compliance measured at the same level of effective pressure (100 mmHg) in SHRs and WKY rats and those reported by Pate1 et al. (1988) showing that small arteries were stiffer in hypertensive patients than in normotensive subjects when measured at their respective operating pressures. The results of the present experiments show a number of differences in mechanical and geometrical properties of in situ mesenteric small arteries from WKY rats and SHRs under control conditions and under conditions of both passive and active smooth muscle. As reported by Dobrin and Rovick (1969), we used potassium cyanide instead of vasodilators generally used in physiological studied to obtain smooth muscle passive condition. The use of this method arises two questions: (1) Was the relaxation maximum after KCN poisoning? (2) Was the arterial wall stiffer after KCN poisoning due to rigor mortis? To address these questions we compared, in pilot experiments, the diameters of mesenteric submitted first to several combinations of vasodilatory and finally to KCN. We never obtained larger diameters with vasodilators than those observed after KCN poisoning. Furthermore, the diameters measured during superfusion with sodium nitroprusside were not stable: In particular, myogenic contractions occurred when pressure steps were applied. Finally, in every experiment, we verified that the arterial diameters measured, for null transmural pressure under passive conditions (KCN poisoning), were not different before and after the procedure of inflation from 0 to 200 mmHg. This last observation suggests that no significant rigor mortis occurred during the 45 min period of measurements (5 n-tin x 9 pressure steps). Abolition of vascular smooth muscle tone by KCN induced significant but slight increase in mesenteric arterial diameter both in WKY (2.6%) and in SHR (1.7%). In microcirculatory studies, markedly larger vasomotor tone is currently observed under control conditions. However, in microcirculatory studies much

168

smaller mesenteric arteries are observed. Le Noble et al. (1990) reported that for arterioles ranging from 20 to 50 p.rn the maximum arteriolar dilation induced by adenosine ranged from 20% to 40% in WKY and in SHR. For larger arterioles (80 pm), the maximum dilation was lower than 15% in both strains. In agreement, the present study evidenced lower maximum dilation in larger arteries (300-500 pm). Values for basal and passive mechanical properties demonstrate that the distensibility, compliance, and wall tension curves for WKY rats and SHRs did not differ when studied over the entire pressure range of O-200 mmHg. When measured at operating pressure, SHR mesenteric small arteries have reduced compliance and distensibility, whereas wall tension was increased. Previous in vitro measurements of WKY and SHR mesenteric resistance artery mechanical characteristics showed similar passive mechanical properties for transmural pressures up to 100 mmHg (Mulvany, 1989). Our study extends these observations over the entire physiological pressure range up to 200 mmHg. Furthermore, our data show that the increase in SHR passive wall tension at operating pressure levels was primary due to the increase in blood pressure. The lack of difference in passive wall stress at operating pressure (Figure 8) is, in contrast, due to the increased vessel wall thickness in SHRs. This set of data suggests that increase in vessel wall mass may be an important adaptive mechanism to maintain a near-normal wall stress in arteries from hypertensive animals (Leung et al., 1977). With respect to compliance and distensibility, we observed no major differences at low pressure levels, where compliance and distensibility were highest in both WKY and SHR. In the present study, the mesenteric compliance measured in WKY rats at operating pressure (roughly 0.001 pL/mmHg per mm artery) was approximately 10 times smaller than that of the WKY rat carotid artery reported, by our group, in several previous studies (Levy et al., 1990; 1991). It is widely admitted that the reservoir (or windkessel) function of the arterial system is mainly located in the aorta and the major large arteries (Nichols and O’Rourke, 1990). However, if the total length of the large branches of the mesenteric artery (more than 10 cm in the rat mesentery) is considered, the role of the mesenteric arterial network in the windkessel function of the arterial system could be of major importance. At operating pressure levels, compliance and distensibility were significantly smaller in SHRs when compared to WKY rats. Similar results were previously reported on compliance of isolated segments of mesenteric (Mulvany, 1989), carotid (Cox, 1979), and cerebral arteries (Brayden et al., 1983). However, the data are at variance with those of Baumbach et al. (1988) who reported increased distensibility of cerebral arterioles in vivo in 6-8-month-old stroke-prone SHRs. In their

JPM Vol. 33, No. 3 June 1995:159-170

paper, they already discussed several possible reasons for the discrepancies, including the methodology of measurements or the unique sensitivity of cerebral arterioles to hypertension. As an additional explanation, we propose that 6-8-month-old stroke-prone SHR may represent a special subset of this strain that, due to high vascular distensibility, survives the early fulminating hypertension. Active mechanical measurements demonstrated an increase in both active wall tension and stress in SHRs over almost the entire pressure range (Figure 9). These data confirm previous in vitro observations on active mechanical properties of isolated mesenteric (Mulvany and Halpem, 1977), carotid (Cox, 1979) and cerebral arteries (Brayden et al., 1983) from SHRs and WKY rats. Larger active wall tension in SHRs’ mesenteric artery wall can be attributed to larger smooth muscle mass in arteries from SHRs than from WKY rats and/or to larger forces exerted by smooth muscle cells from SHRs than from WKY. The increased active wall tension in SHRs compared to WKY rats (about three times larger) indicates that mesenteric artery reactivity is larger in hypertensive rats. The increased media crosssectional area in SHR evidenced a larger muscle mass in SHRs than in WKY rats by a factor of 1.3. Normalization of active wall stress relative to media thickness allowed us to evidence significantly larger active wall stress in SHRs than in WKY rats. These results reflect that the increased force exerted by the contracted arterial wall was due to more effective contraction of each smooth muscle cell in SHRs than in WKY rats and not only to the increased smooth muscle mass in SHRs. Several factors could be involved in the increase in active wall tension and stress. The two most likely factors are the increased smooth muscle content of the SHR vessel wall and the increased sensitivity of SHR vascular smooth muscle cells to pressor substances, such as phenylephrine (Mulvany and Aalkjaer, 1990). Further research, using pharmacological tools, is in progress in our model to better define factors that contribute to active wall tension and stress in SHR mesenteric small arteries. An unexpected result from our study comes from the geometrical measurements. The increase we observed in SHR mesenteric small artery CSA is in line with previous observations (Lee et al., 1983; Brayden et al., 1983), whereas the small but significant diameter increase in SHR over the entire pressure range was somewhat surprising. Diameter measurements on isolated vascular segments show a decreased internal diameter in SHR aorta (Amer and Uvelius, 1982), carotid artery (Cox, 1979), basilar artery (Winquist and Bohr, 1983) small mesenteric (Mulvany et al., 1978), femoral (Mulvany, 1982), renal (Mulvany, 1986), and cerebral arteries (Brayden et al., 1983). No change or even a slight increase in SHR internal diameters were found in the

H. Y. QIU ET AL. PROPERTJES OF THE

169 MESENTERIC

ARTERY

IN SHR

tail artery (Cox, 1981) and a number of arterioles smaller than 100 pm (Mulvany and Aalkjaer, 1990). Histological measurements have given variable results. In the mesenteric vascular bed fixed under relaxed conditions with a perfusion pressure of about 20 mmHg, Lee et al. (1983) found no difference in the internal diameter of SHR and WKY vessels, although the media was thicker. We have considered several explanations for these different results. First, we measured external diameter as opposed to internal diameters in most other studies. Because all thickness represents only 2-5% of the external diameter in the studied arteries (300-500 pm), it is highly unlikely that this difference in methodology explains the varying results. A second possible explanation is the unique location of our vascular segment between the usually studied resistance segment (< 200 pm) and large arteries (> 0.8 mm). In view of the continuous nature of the vascular system and the gradual course of physical variables over its structure, it is not clear why and how this segment of the circulation should behave differently. Nevertheless, the vessels examined in the present study were 2-bifurcation proximal to those investigated by Mulvany and colleagues. Thus, it is possible that this difference in location could partially explain the different results. This leaves a third, more methodological explanation. Whereas most previous diameter measurements were performed on isolated vessels (i.e., calculated from the length of the stretched arterial ring), we observed diameters directly, on vessels that remained in situ. The removal of vessels from the vascular tree and subsequent perfusion at nonphysiological pressures or stretching them to a certain tension may create artifacts if SHR and WKY vessels react differently to these manipulations. Using an original pulsed ultrasound echo-tracking system, several works reported significantly larger arterial diameter in hypertensive than in normotensive subjects when recorded at the same level of arterial pressure (Hayoz et al., 1992; Laurent et al., 1994). The balance between the relaxing and constricting factors released by the endothelial cells is widely affected in the SHR (Ltischer et al., 1988); therefore, damage of endothelium, inevitable during the dissection of the vessel segment, could have different effects on the tone of arterial smooth muscle of isolated vessels in WKY and SHR. Finally, the discrepancies may be due to a definition of the type of vessel used. In our study, we always used a second-generation artery counted from the feeding artery (first generation) in each preparation. Although these vessels represent anatomically reproducible segments, it is not certain whether they are identical in SHR and WKY from a developmental point of view. In conclusion, the present method for studying the mechanical and contractile properties of the in situ localized mesenteric artery (300-500 Frn) offers a new possibility to explore the behavior of small arteries in

the intact rat. This in situ method provides results markedly different of those obtained from in vivo myographmounted arteries. Comparative studies of normotensive WKY rats and SHRs indicate (1) stiffer arteries in SHRs than in WKY rats when mechanical properties were compared at their respective physiological operating pressure values, (2) an increase in tangential forces measured at physiological operating pressure values in arteries from the hypertensive strain, and (3) similar wall stress (forces normalized by the media thickness) in both normotensive and hypertensive rats despite large differences in operating arterial pressure. The contractility of the mesenteric arteries to phenylephrine was larger in SHR than in WKY. This increased contractility in SHRs was due to increased smooth muscle mass in the wall of arteries and to enhanced contractility of each smooth muscle cell to phenylephrine.

References Amer A, Uvelius B (1982) Force-velocity characteristics and active tension in relation to content and orientation of smooth muscle celles in aortas from nonnotensive and spontaneously hypertensive rats. Circ Res 50:812-821. Baldwin AL, Winlove CP, Caro CG (1982) Structural response of the rabbit thoracic aorta and inferiorvena cava to in situ collapse. Artery 10:420-439. Baumbach CL, Dobrin PB, Hart MN, Heistad DD (1988) Mechanics of cerebral arterioles in hypertensive rats. Circ Res 62:56-64. Bevan JA, Osher JV (1972) A direct method for recording tension changes in the wall of small blood vessels in vitro. Agents Action 2:257-260. Brayden JE, Halpem W, Brann LR (1983) Biochemical properties of resistance arteries from normotensive rats. Hypertension 5: 17-25. Cox RH (1979) spontaneously Cox RH (1981) spontaneously

and mechanical and hypertensive

Comparison of arterial mechanics in normotensive and hypertensive rats. Am J Physiol237:159-167. Basis for the altered arterial wall mechanics in the hypertensive rat. Hypertension 3:485493.

Dobrin PB, Rovick AA (1969) Influence contractile mechanics and elasticity 217:1644-1651.

of vascular smooth of arteries. Am

muscle on J Physiol

Duling BR, Gore RW, Dacey RG, Damon DN (1981) Methods for isolation, cannulation, and in vitro study of single microvessels. Am J Physiol241:H108-H116. Halpem W, Kelly M (1991) In vitro methodology for resistance arteries. Blood Vessels 28:245-25 1. Hayoz D, Rutschmann B, Perret F, et al. (1992) Conduit artery compliance and distensibility are not necessarily reduced in hypertension. Hypertension 20: l-6. Johnson PC, Hanson venous resistance

KM (1962) Effect of arterial of intestine. / Appl Physiol

pressure on arterial 17:503-508.

Lash JM, Bohlen G, Waite L (1991) Mechanical characteristics tension generation in rat intestinal arterioles. Am 260:H1551-H1574. Laurent S, Caviezel and distending 23:878-883.

and

and active J F’hysiol

B, Beck L, et al. (1994) Carotid artery distensibility pressure in hypertensive humans. Hypertension

Lee RMKW, Garfield RE, Forrest JB, Daniel EE (1983) Morphometric study of structural changes in the mesenteric blood vessels of spontaneously hypertensive rats. Blood Vessels 20x57-71,

JPM Vol. 33, No. 3 June 1995:159-170

170

Le Noble JLML, Tangelder GJ, Slaaf DW, Essen HV, Reneman RS, Struyker-Boudier HAJ (1990) A functional morphometric study of the cremaster muscle microcirculation in young spontaneously hypertensive rats. J Hypertens 8:741-748.

Mulvany MJ, Halpem W (1976) Mechanical properties smooth muscle cells in situ. Nature 260:617-619.

of

vascular

Levy

Mulvany MJ, Halpem W (1977) Contractile properties of small arterial resistance vessels in spontaneously hypertensive and normotensive rats. Circ Res 41: 19-26. Mulvany MJ, Hansen PK, Aalkjaer C (1978) Direct evidence that the greater contratility of resistance vessels in spontaneously hypertensive rats is associated with a narrowed lumen, a thickened media, and an increased number of smooth muscle cell layers. Circ Res 43:845-864.

Liischer TF, Diederich D, Weber E, Vanhoutte PM, Buhler FR (1988) Endothelium dependent responses in carotid and renal arteries of normotensive and hypertensive rats. Hypertension 11573-578.

Nichols WM, O’Rourke MF (1990) McDonald’s Blood Flow in Arteries. 3rd ed. London: E. Arnold Ed. Nilsson H, SjGblom N (1985) Distension-dependent changes in noradrenaline sensitivity in small arteries from the rat. Acta Physiol Stand 125:429435. 0~01 G, Halpem W (1985) Myogenic properties of cerebral blood vessels from normotensive and hypertensive rats. Am J Physiol 249:H914H921.

Morisson AD, Berwick L, Orci L, Winegrad AI (1976) Morphology and metabolism of an aortic intima-media preparation in which an intact endothelium is preserved. J Clin Invest 57:650-660.

Pate1 DJ, Vaishnav RN, Coleman BR, Teamey (1988) A theoritical method for estimating in man. Circ Res 63572-576.

Mulvany MJ (1982) Are isolated femoral resistance vessels or tail arteries good models for the hindquarter vasculature of spontaneously hypertensive rats. Acta Physiol Stand 116:275-283.

Tedgui A, Lever rabbit thoracic

Leung DYM, Glagow S, Mathews MB (1977) Elastin and collagen accumulation in rabbit ascending aorta and pulmonary trunk during postnatal growth. Correlation of cellular synthetic response with medial tension. Circ Res 41:316-323. BI, Benessiano J, Poitevin P, Safar ME (1990) Endothelium dependent mechanical properties of the carotid artery in WKY and SHR: Role of angiotensin converting enzyme inhibition. Circ Res 66:321-328. Levy BI, Poitevin P, Safar ME (1991) Effects of alpha-l blockade on arterial compliance in normotensive and hypertensive rats. Hypertension 17:53&540.

Mulvany MJ (1986) Role of vascular strecture in blood pressure development of the spontaneously hypertensive rat. J Hypertens 4(suppl 3):61-63. Mulvany MJ (1989) Compliance of isolated resistance vessels from spontaneously hypertensive rats. In Vascular Dynamics Physiological Perspectives. NATO AS1 Series. Ed., N Westerhof and DR Gross. New York: Plenum Press, pp. 125-134. Mulvany MJ, Aalkjaer C (1990) Physiol Rev 70:922-961.

Structure

and function

of small arteries.

RJ, Cothran LN, Curry CL small vessel distensibility

MJ (1984) Filtration through damaged aorta. Am J Physiol247:H784-H791.

and undamaged

Vanbavel E, Mooij T, Giezemann MJMM, Spaan JAE (1990) Oxygen and adenosine sensitivity of isolated cannulated porcine and coronary resistance arteries. Ear J Pharmacol 183: 1080-108 1. Winquist RJ, Bohr DF (1983) Structural and functional changes in cerebral arteries from spontaneously hypertensive rats. Hypertension 5 1292-297. Zar JH (1984) Biostatistical Analysis. Englewood Cliffs, NJ: Prentice Hall.