728 Relationship of Aortic Wall and Baroreceptor Properties during Development in Normotensive and Spontaneously Hypertensive Rats MICHAEL C. ANDRESEN, JANE M. KRAUHS, AND ARTHUR M. BROWN SUMMARY We studied the relationship between aortic baroreceptor function and aortic wall prop- erties in normotensive (NTR) and spontaneously hypertensive (SHR) rats 10-20 weeks old. Barorecep- tor discharge, static pressure-volume (P-V), and pressure-radius relationships were measured in excised aortic segments. Histological studies of wall thickness and receptor numbers also were made. Circumferential wall stress and strain were calculated, as was the incremental elastic modulus (EINC). EINC in NTR's at 100 mm Hg was similar to values reported for in vivo human, dog, and rat aortas. At 10 weeks, SHR's had significantly elevated blood pressure, but SHR and NTR aortas had similar relationships among pressures, volumes, strains, and EINC'S. Differences arose subsequently and, at 20 weeks, NTR aortas had larger volumes, larger strains, and smaller EINC'S at equivalent pressures, whereas SHR aortas were unchanged. Thus the reduced distensibility of SHR relative to NTR aortas, rather than being due to retrogressive changes from normal, appeared to result from a failure to pass through a phase of increased distensibility. At 10 weeks, SHR baroreceptors showed resetting in both pressure-response and strain-response curves, and it was concluded that early hypertensive barore- ceptor resetting was due to primary changes in the receptors. At 20 weeks, the order of the strain- response curves for NTR and SHR baroreceptors was reversed due to a reduction in strain sensitivity of NTR baroreceptors. Resetting of NTR baroreceptors during development may have important implications as a mechanism of blood pressure control in development. BARORECEPTOR resetting is a crucial change in hypertension since it is impossible for the neural control system to operate effectively when the in- formation it receives concerning blood pressure is incorrect. Resetting has been attributed to reduced distensibility of vessel walls and/or destruction of baroreceptors, 1 " 3 but recently reported results ob- tained in spontaneously hypertensive rats (SHR's) suggested that these explanations might not apply, particularly in the early phases of hypertension. 4 " 6 The present experiments were designed to examine this question. We found that resetting in aortic baroreceptors of 10-week-old SHR's is likely to result from a defect in the receptors themselves. In addition, NTR baroreceptors showed interesting functional changes during development. Methods Sixty male NTR's (Wistar-Kyoto strain) and 60 male SHR's (Okamoto-Aoki strain 7 ) were studied at 2-week intervals between the ages of 10 and 20 weeks. Three additional older rats were included in From the Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston, Texas. Supported by National Institutes of Health Grants HL-16657, HL- 18798, HL-05550, and HL-05691. Address for reprints: Dr. Arthur M. Brown, Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston, Texas 77550. Received February 13, 1978; accepted for publication June 27, 1978. the histological studies to be described (Table 3). The tail pressure of each animal was measured by the occlusive cuff technique in conscious animals the week prior to use. The methods of exposing the aorta and aortic nerve have been reported. 4 6 The aorta was isolated from its root to beyond the left subclavian artery, and all branches were cut be- tween ligatures tied as closely to the aorta as pos- sible. The isolated aortic segment was excised, trimmed of fatty tissue, blotted dry, weighed, and then transferred to a perfusion chamber of the type described previously. 4 ' 6 Connections were made to the perfusion system, pressure wave form generator (shaker), and pressure transducer (strain gage) through metal cannulas and stiff polyethylene tub- ing; the total dead space volume was 1.54 ml. Mea- surements of pressure and nervous activity were made as previously described. 4 ' 6 In four of the ex- periments on wall properties, Ca 2+ was omitted from the perfusate and Ca 2+ activities were mea- sured directly using Ca 2+ selective electrodes kindly prepared for us by Dr. N. Hebert. The electrodes had selectivities over Mg 2+ , K + , and Na + of 3 x 10 4 , 1 X 10 5 , and 1 X 10 5 , respectively, and had slopes of 29 mV per 10-fold change of [Ca 2+ ] over the range of 1 X 10" 5 to 1 X 10~ 3 M and 20 mV per 10-fold change of [Ca 2+ ] over the range of 1 X 10~ 5 to 1 X 10~ 7 M. For statistical comparisons, Student's t-test was used. by guest on June 3, 2018 http://circres.ahajournals.org/ Downloaded from
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728
Relationship of Aortic Wall and BaroreceptorProperties during Development inNormotensive and Spontaneously
Hypertensive RatsMICHAEL C. ANDRESEN, JANE M. KRAUHS, AND ARTHUR M. BROWN
SUMMARY We studied the relationship between aortic baroreceptor function and aortic wall prop-erties in normotensive (NTR) and spontaneously hypertensive (SHR) rats 10-20 weeks old. Barorecep-tor discharge, static pressure-volume (P-V), and pressure-radius relationships were measured inexcised aortic segments. Histological studies of wall thickness and receptor numbers also were made.Circumferential wall stress and strain were calculated, as was the incremental elastic modulus (EINC).EINC in NTR's at 100 mm Hg was similar to values reported for in vivo human, dog, and rat aortas. At10 weeks, SHR's had significantly elevated blood pressure, but SHR and NTR aortas had similarrelationships among pressures, volumes, strains, and EINC'S. Differences arose subsequently and, at 20weeks, NTR aortas had larger volumes, larger strains, and smaller EINC'S at equivalent pressures,whereas SHR aortas were unchanged. Thus the reduced distensibility of SHR relative to NTR aortas,rather than being due to retrogressive changes from normal, appeared to result from a failure to passthrough a phase of increased distensibility. At 10 weeks, SHR baroreceptors showed resetting in bothpressure-response and strain-response curves, and it was concluded that early hypertensive barore-ceptor resetting was due to primary changes in the receptors. At 20 weeks, the order of the strain-response curves for NTR and SHR baroreceptors was reversed due to a reduction in strain sensitivityof NTR baroreceptors. Resetting of NTR baroreceptors during development may have importantimplications as a mechanism of blood pressure control in development.
BARORECEPTOR resetting is a crucial change inhypertension since it is impossible for the neuralcontrol system to operate effectively when the in-formation it receives concerning blood pressure isincorrect. Resetting has been attributed to reduceddistensibility of vessel walls and/or destruction ofbaroreceptors,1"3 but recently reported results ob-tained in spontaneously hypertensive rats (SHR's)suggested that these explanations might not apply,particularly in the early phases of hypertension.4"6
The present experiments were designed to examinethis question. We found that resetting in aorticbaroreceptors of 10-week-old SHR's is likely toresult from a defect in the receptors themselves. Inaddition, NTR baroreceptors showed interestingfunctional changes during development.
MethodsSixty male NTR's (Wistar-Kyoto strain) and 60
male SHR's (Okamoto-Aoki strain7) were studiedat 2-week intervals between the ages of 10 and 20weeks. Three additional older rats were included in
From the Department of Physiology and Biophysics, University ofTexas Medical Branch, Galveston, Texas.
Supported by National Institutes of Health Grants HL-16657, HL-18798, HL-05550, and HL-05691.
Address for reprints: Dr. Arthur M. Brown, Department of Physiologyand Biophysics, University of Texas Medical Branch, Galveston, Texas77550.
Received February 13, 1978; accepted for publication June 27, 1978.
the histological studies to be described (Table 3).The tail pressure of each animal was measured bythe occlusive cuff technique in conscious animalsthe week prior to use. The methods of exposing theaorta and aortic nerve have been reported.4 6 Theaorta was isolated from its root to beyond the leftsubclavian artery, and all branches were cut be-tween ligatures tied as closely to the aorta as pos-sible. The isolated aortic segment was excised,trimmed of fatty tissue, blotted dry, weighed, andthen transferred to a perfusion chamber of the typedescribed previously.4'6 Connections were made tothe perfusion system, pressure wave form generator(shaker), and pressure transducer (strain gage)through metal cannulas and stiff polyethylene tub-ing; the total dead space volume was 1.54 ml. Mea-surements of pressure and nervous activity weremade as previously described.4'6 In four of the ex-periments on wall properties, Ca2+ was omittedfrom the perfusate and Ca2+ activities were mea-sured directly using Ca2+ selective electrodes kindlyprepared for us by Dr. N. Hebert. The electrodeshad selectivities over Mg2+, K+, and Na+ of 3 x 104,1 X 105, and 1 X 105, respectively, and had slopes of29 mV per 10-fold change of [Ca2+] over the rangeof 1 X 10"5 to 1 X 10~3 M and 20 mV per 10-foldchange of [Ca2+] over the range of 1 X 10~5 to 1 X10~7 M. For statistical comparisons, Student's t-testwas used.
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AORTIC WALL AND BARORECEPTOR PROPERTIESMrcdresen 729
Measurements of the Wall Properties ofExcised Aortas
Dimensional measurements of excised aortaswere made with an ocular micrometer (OM) and/ora piezoelectric sonomicrometer (SM), which has aflat (±5%) frequency response to 40 Hz.6 With thefirst method, a dissecting microscope at 12x mag-nification was used; minor divisions of the OM wereequivalent to 83 jim. Before removal from the rat,the length, / , of the aortic segment was measuredwith an ocular micrometer at zero pressure. The invivo distance between the right and left carotidbranches, as well as the cannula-to-cannula dis-tance, were recorded. When the aortic segment wastransferred to the perfusion chamber, it was fixedso as to correspond as closely as possible to its invivo length by the metal cannulas. Diameter wasmeasured approximately midway between the leftcarotid and left subclavian arteries as shown by thearrows in Figure 1. When the OM was used, oneperson produced random step-wise changes in pres-sure from 0 to 200 mm Hg and a second person towhom the pressures were unknown read off themicrometer readings. The runs were repeated untiltwo to four readings at each pressure agreed within5%. When SM was used, the piezoelectric crystalswere fixed to measure a diameter normal to thatmeasured under OM (Fig. 1).
Pressure-volume measurements were made in thesteady state using injected volumes of Krebs-Hen-seleit solution. The volumes were injected in 10-/ilsteps using a 250-jul Hamilton microsyringe withdivisions of 5 jul. To assess the initial or zero pressurevolume, the syringe was withdrawn until the aorticsegment was completely collapsed. The withdrawnvolume was used as the initial volume at zero pres-
A A
3mmAN
FIGURE 1 Experimental arrangement of the excisedaortic segment. INN, innominate artery; DA, descendingaorta; AA, ascending aorta; LSC, left subclavian; LCC,left common carotid arteries; AN, aortic nerve; R, re-cording electrodes. Arrows indicate the region in whichocular micrometer measurements were made and theshaded square represents the anterior crystal of the pairof sonomicrometer crystals.
sure, and was usually aspirated with pressures of-15 to -25 mm Hg.
Calculations of Stress, Strain, andIncremental Elastic Modulus
The factors acting on aortic baroreceptors maybe better understood when they are expressed aswall stress and wall strain rather than distendingpressure. In length-fixed aortas, the longitudinalstrain is relatively constant8 so that the primarystrain acting on the aortic segments we used wasconsidered to be circumferential. Circumferentialstress a, was calculated as
a = P r/h (1)
where P was the distending pressure; r, the meanradius; and h, the calculated wall thickness. Thelatter was determined as re—n where rj the internalradius was derived from re the external radius as
r; = (re2 -Vw/m( (2)
Vw is the volume of the excised aorta and wasassumed to be a right cylinder for calculations of r;and was calculated from its weight, using a densityof 1.06.9 Direct measurements of wall thickness, h',were also made in the histological studies describedin the next section.
Circumferential wall strain, e, in response to anincrement in pressure was calculated as the ratio ofthe change in radius Ar, and the initial zero pressureradius, r0.
To compare further the mechanical propertiesbetween NTR and SHR aortas, as well as amongaortas of other species, the incremental elastic mod-ulus (EINC) was calculated according to Bergel'sformula10 as follows:
E,NC = (AP/Ar)2(l - (3)
where 6 the Poisson ratio was assumed to be 0.5.EINC was determined for stepwise AP's of 20 mm Hgusing calculated values of n at each of these pres-sures.
Histological StudiesPreparation of aortic specimens for histological
examination was done after measuring their physi-cal properties. A piece of aortic wall from the regioninnervated by the aortic nerve at the arrows shownin Figure 1 was excised and pinned without stretch-ing onto a small piece of Sylgard. Preparation of theaortic nerve for measurements of the ratios of my-elinated and unmyelinated axons was done in sep-arate experiments. The aortic nerve was cut closeto the arch, and this end was marked with thread.The nerve then was stretched slightly and pinnedto Sylgard. In both sets of experiments the speci-mens were placed in vials of fixative consisting of3% glutaraldehyde in 0.1 M piperazine-N,N'-bis(2-ethane-sulfonic acid) (PIPES) buffer, pH 7.6. After
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730 CIRCULATION RESEARCH VOL. 43, No. 5, NOVEMBER 1978
overnight fixation at 4°C, the specimens were rinsedin buffer, postfixed for lh in 1% OsO4 in 0.1 MPIPES, dehydrated in ethanol, and embedded inEpon. Specimens of nerve were removed from theSylgard just before the last change in 100% ethanol.Sections 1 /on thick were cut on a DuPont MT-2Bultramicrotome and stained with methylene blueand azure II. The sections of aorta were examinedand measured with an Olympus EH light micro-scope. For counting fibers in the aortic nerve, thin(80-nm) sections were cut from the end closest tothe arch and mounted on grids coated with collo-dion and carbon. All portions of the nerves werephotographed with a Phillips 300 electron micro-scope, and a large composite photograph of eachnerve cross-section was made. The fibers werecounted with a device we constructed which regis-tered a count when a probe was pushed through theprint to make electrical contact with a metal plate.
ResultsBlood Pressures in Young SHR's and NTR's
At ages between 10 and 20 weeks, mean valuesfor the tail systolic blood pressures were signifi-cantly greater in SHR's. Figure 2 shows that, inWistar-Kyoto controls, these pressures remained atabout 135 mm Hg throughout the study. However,in SHR's, these pressures were 166 mm Hg at 10weeks and rose to about 200 mm Hg at 20 weeks.The results are consistent with previous reportsthat blood pressure in SHR's is elevated soon afterbirth and continues to rise eventually reaching aplateau.7
Static Pressure-Volume (P-V) Characteristicsof NTR and SHR Aortas
Figure 3 compares the mean static P-V relation-ships for aortic arch segments from groups of NTR'sand SHR's at 10 and 20 weeks of age. Aortas from10-week-old rats of both groups had similar, almostlinear, P-V relationships between zero and 200 mmHg (Fig. 3A). By 14-16 weeks, however, significantdifferences began to emerge and, at 20 weeks, theNTR volumes exceeded those of SHR's at equiva-lent pressures >80 mm Hg, and these increases werestatistically significant (Fig. 3B). At 20 weeks, theP-V relationship of SHR's showed a small increasein slope and remained almost linear between zeroand 200 mm Hg. However, the relationship inNTR's had become increasingly sigmoidal with amaximum distensibility near 100 mm Hg. Similarlyshaped curves of relative radius vs. pressure werereported for NTR's by Berry and Greenwald.11 Thevolumes at any pressure were smaller than thosedescribed previously. That study used larger aorticsegments in which zero pressure volumes were notestimated.4 The present P-V curves were not alteredwhen zero Ca2+ solutions, in which measured Ca2+
activities were 10~6 M, were used. To summarize, at10 weeks, NTR and SHR aortas were equally dis-
cioo
2 0 0
180
160
140
120
100 1 1 1 1 1 i i
10 12 14 16 18 20
RAT AGE (weeks)
FIGURE 2 Relationship between tail systolic blood pres-sure and age. Data points are means for a given age;bars are SE; filled circles, SHR's; filled squares, NTR's.Lines were fit to the data by eye.
tensible but, at 20 weeks, NTR aortas had becomeclearly more distensible.
Stress-Strain Characteristics of NTR andSHR Aortas
The weights and lengths of excised aortic seg-ments from 10- and 20-week-old rats are summa-rized in Table 1. The values for re were equivalent,using either SM or OM, and are shown in Table 2.The average value for h' measured directly and theweights for similar lengths were greater in SHR's.Values for re were also generally larger in SHR's.Larger values of h were calculated in SHR's asshown in Table 2. The values for / , h, and Vw areless than those reported by Berry and Greenwald11
for thoracic and abdominal aortas from normoten-sive Wistar rats 12-20 weeks of age. Our animalsalso weighed less than theirs. The relative wallthickness, h/re, ranged from 0.04 to 0.10, and thesevalues are similar to those reported earlier for ratand dog aortas.10' n Aortic wall stress as a functionof distending pressure is shown in Figure 4, usingvalues for mean r and h from Table 2. When the
lOwl,
0 50 100 150 200 0 50 100 150 200
PRESSURE ( • • H g ! PRESSURE (mmHg)
FIGURE 3 Relationship between aortic pressure andvolume. Data points are means, n = 4. Vertical bars are± 1 SE. In this and subsequent figures, squares identifyNTR's; circles, SHR's. Unfilled symbols represent 10-week-old rats and filled symbols represent 20-week-oldrats. Asterisks designate statistically significant differ-ences between NTR and SHR groups (P < 0.05).
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AORTIC WALL AND BARORECEPTOR PROPERTIESMrcdresen 731
TABLE 1 Dimensions of the Aortic Arch in NTR's and SHR's
Wt of excised Length of excisedAge aortic segment aortic segment Measured wall No. of muscle(wk) Animal wt (g) (mg) (mm) thickness h' (/im) layers media
NTR (n = 8)SHR (n = 8)
20 346.4 ± 2.920 327.4 ± 5.5
9.68 ± 0.6313.97 ± 1.08*
8.07 ± 0.258.85 ± 0.44
162 ± 12.4204 ± 25.7
7.6 ± 0.57.1 ±0.6
Values are means ± SEM and were taken at zero pressure.' P < 0.02.
NTR's were between 10 and 20 weeks of age, themean wall stress increased significantly (P < 0.05)at pressures above 140 mm Hg. This resulted froma larger increase in radius compared to wall thick-ness during this period (Table 2). In SHR's between10 and 20 weeks of age, however, the mean wallstress dropped significantly (P < 0.05) above 140mm Hg due to large increases in wall thickness {P< 0.01), whereas radius remained relatively con-stant. At both ages, the NTR's had significantly (P< 0.05) greater wall stresses at pressures above 140mm Hg.
Circumferential wall strain as a function of dis-tending pressure is plotted in Figure 5. The pres-sure-strain relationships did not differ at 10 weeks,but by 20 weeks NTR's required much lower pres-sures to attain equivalent wall strains. The pres-sure-strain curves were similar among SHR's 10and 20 weeks of age and NTR's 10 weeks of age,whereas NTR's 20 weeks of age showed signifi-cantly greater distensibility. The increase in disten-sibility was detected as early as 14 weeks, but we
have not followed its total time course. Similarincreases in distensibility were reported for NTRaortas by Berry et al.,12 although these occurred atabout 10-14 weeks in their study and were followedby reductions in distensibility with further aging.
The importance of using derived values of wallthickness, h, was assessed by comparing the valuesfor wall stress and EINC using h or a single value forwall thickness, h', which was measured directly.The use of a constant value h' in determining wallstress and EINC did not change the shapes of theirrelationships to pressure and strain, respectively(see for example, Figure 6A), nor the conclusionsdrawn from them.
Incremental Elastic Modulus of NTR andSHR Aortas
EINC at 100 mm Hg was similar for aortas takenfrom both NTR's and SHR's (3.3-4.4 X 106
dynes/cm2) and was similar to values reported foraortas of dogs, rats, and humans at the same dis-tending pressure.111314 Values for EINC in SHR's
TABLE 2 Means of Calculated Values of Internal Radius, Wall Thickness and EINC atDifferent Pressures in the Aortic Arch ofNTR's and SHR's
732 CIRCULATION RESEARCH VOL. 43, No. 5, NOVEMBER 1978
8.0
f6.0
4 .0
2.0
10 wksa NrRO S H R
20wks• NTR• SHR
D
8
40 80
PRESSURE
120
mm Hg '.
160 200
FIGURE 4 Relationship between pressure and circum-ferential wall stress calculated as P • r/h where P ispressure, r is the mean radius of the vessel wall, and his the vessel wall thickness. Significant differences (P <0.05) exist between SHR's and NTR's of the same agefor pressures above 140 mm Hg. The differences between10-week-old and 20-week-old rats are also significantabove this pressure. Error bars are omitted for clarity.See text for details.
and NTR's 10 and 20 weeks of age over pressuresranging from 0 to 200 mm Hg are shown in Table 2.
A more complete comparison was obtained byplotting EINC as a function of strain. EINC was rela-tively flat initially and began to increase at strainsof about 0.3. The curves were similar for NTR's andSHR's 10 weeks in age (Fig. 6A) and, as alreadynoted, there was very little difference when a singlevalue of h' was used rather than the calculated hvalues using constant wall volume assumptions. At20 weeks, EiNC for NTR's began to increase at muchhigher wall strains than those required in 20-week-old SHR's and 10-week-old NTR's and SHR's (Fig.6B). As we shall see, the thresholds for dischargeoccurred at strains greater than about 0.3 so thatthe receptors are activated when EINC is undergoingits greatest change.
Relationship between Aortic WallCharacteristics and Aortic BaroreceptorDischarge in NTR's and SHR's
Resetting and reduced sensitivity as a function ofdistending pressure have been demonstrated re-peatedly in SHR baroreceptors.3' "•15 The pertinentrelationships, however, are those among dischargeand wall stress and/or wall strain. We have evalu-ated these relationships as shown in Figure 7. First,steady state discharge was plotted as a function ofstatic distending pressure (Fig. 9A). The curveswere predicted from an equation which fits theactual values for threshold and maximum asymp-totic discharge of 11 NTR baroreceptors and 9 SHRbaroreceptors to distending pressure (Equation 1and Table 1 of Reference 4). Resetting and reducedsensitivity are evident in the SHR baroreceptorcurve. Similar results have been reported in subse-quent experiments5 and were observed presently,although in less detail. These earlier receptor stud-ies used rats 16-20 weeks of age, and the dischargecan be compared to the vessel wall measurementsmade presently in rats 20 weeks of age. The steadystate discharge as a function of wall stress or wallstrain is shown in parts B and C, respectively, ofFigure 7. The wall stresses of NTR and SHR aorticsegments were quite different in 20-week-old rats,and the plot of steady state discharge (Fss) vs. wallstress in Figure 7B shows that the differences be-tween NTR and SHR baroreceptors were not somarked as in the Fss-pressure curves. Moreover,curves of discharge vs. wall strain were now re-versed, the SHR curve being to the left of the NTRcurve (Fig. 7C). The variability in the computedcircumferential wall strains for 20-week-old animalswas so large that the differences were not statisti-cally significant. Note that the relationship betweenFss and wall strain was more linear than the rela-tionship between Fss and either pressure or wallstress.
The results in 16- to 20-week-old SHR's indicatedthat alterations in aortic wall characteristics wereresponsible for resetting, and it appeared that SHR
0.6
2 04
0.2
0.6
0.4
0.2
" 0 50 100 150 200 0 50 100 150 200
PRESSURE (mmHg) PRESSURE (mmHg)
FIGURE 5 Relationship between pressure and circumferential wall strain Ar/ro defined in text.
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AORTIC WALL AND BARORECEPTOR PROPERTIES/Andresen 733
25
CM
E
c
"D•O
O
uz
20
15
10
5
• A
10 wksD NTR
- oSHR
-
n n o _Q IT-CS " — ^ 3
1 1 t_
O
/
/ °
1 1 I
. 6
STRAIN r̂o
25 I- B
15
o— 10
? 5
20wks• NTR• SHR
2 4
STRAIN £-r
. 6
FIGURE 6 Relationship between aortic incrementalelastic modulus, in 10-week-old SHR's and NTR's (A)and 20-week-old rats (B). Emc and aortic wall straincalculated as described in text. Values for EINC (mean± SE) are given in Table 2. Curves drawn through pointsby eye. At 10 weeks (A), the curves for NTR's and SHR'sare identical. The dashed lines are calculations using asingle value for wall thickness, h', which was determineddirectly. See text for details.
baroreceptors at 20 weeks were if anything moresensitive to strain than NTR baroreceptors. Sincedifferences in the pressure-volume and stress-strainrelationships of NTR and SHR aortic segmentswere not present at 10 weeks of age, we examinedFas in these younger rats for the possibility of reset-ting. Eleven baroreceptors from 6 NTR's and 17baroreceptors from 8 SHR's were examined. AsFigure 8 shows, thresholds for pressure and strainwere higher in 10-week-old SHR's. The mean pres-sures at threshold discharge were 137 ± 27 and 103
6 0
< 40
20
20wks
100 120 140 160 180
PRESSURE (mmHg)
200
B 60
1 40
a.~ 20
20wk>NTR
2 4 6 8
STRESS r (X106dyn»/cm2)
6 0
< 40
20
20wks
SHR
.40 .50 .60
STRAIN
.70
FIGURE 7 Relationships between aortic baroreceptordischarge and in A, aortic pressure, in B, aortic wallstress, and in C, aortic wall strain in 16- to 20-week-oldrats. Relationship in A was computed from previousexperiments on rats.
Fas =(P- P,s)/[a, + ao(P - P,.)] + Fo
where F,s is static discharge rate, P», is threshold pres-sure for discharge, P is suprathreshold pressure, Fo isminimum discharge, l/ao is asymptotic discharge, anda\ is the shape factor* Discharge data in the presentexperiments were similar but less complete, and theearlier discharge data were used for the plots B and C.The points in B and C relating discharge to stress andstrain used measured values of stress and strain corre-sponding to appropriate pressures in A. Bars are ± SE,and the lines in C are least squares best fits, NTR r2 =0.993, SHR r2 = 0.972. Note the reversal of SHR andNTR curves in C.
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FIGURE 8 Histogram displaying the distribution of baroreceptor thresholds for pressure and strain in 10-week-oldNTR's and SHR's.
± 18 mm Hg for SHR's and NTR's, respectively,and these differences were highly significant (P <0.01). The means of the strain thresholds were 0.301± 0.021 and 0.370 ± 0.025 for NTR's and SHR's,and these differences were also highly significant.Almost half of the SHR baroreceptors recordedfrom had thresholds above 140 mm Hg, which wasthe highest threshold pressure recorded in the 10-week-old NTR's that were examined (Fig. 8). Thusresetting had clearly occurred at a time when aorticwall characteristics were unchanged. The bimodalthreshold distributions for NTR's probably re-flected the presence of receptors with myelinatedaxons (lower thresholds) and unmyelinated axons(higher thresholds), and the two modes had valuessimilar to mean values previously reported for re-ceptors having myelinated and unmyelinated axons,respectively.4'16 The bimodal distributions forSHR's probably had a similar explanation, judgingfrom the recent report of Jones and Thoren.17
The respective threshold pressures for 10-week-old NTR's and SHR's were almost identical torespective values reported earlier for rats that were16-20 weeks old. Maximum asymptotic dischargeswere recorded only infrequently in the present ex-periments but corresponded to earlier values also.4
Therefore it was of interest to examine the relation-ship between static discharge and either pressure,stress, or strain, as was done for the 16- to 20-week-old rats in Figure 7. The procedure was identicaland the results are shown in Figure 9. The signifi-cant difference between NTR and SHR barorecep-
tors is evident by comparing parts C of these twofigures. At 10 weeks, the curve for SHR barorecep-tor discharge vs. strain is displaced to the right ofthe curve for NTR baroreceptors, significantly so,at threshold. The scatter at suprathreshold strainsis so large that the differences are not significant.By contrast, between 16 and 20 weeks, the SHRcurve was displaced to the left of the NTR curve.Note, however, that the SHR curve had changedvery little between 10 and 20 weeks, whereas theNTR strain curve had shifted considerably to theright indicating, surprisingly, that resetting of NTRbaroreceptors had occurred during this period.
Histological StudiesThe values for wall thickness were larger for SHR
aortas (Tables 1 and 2). The number of laminas inthe media were not different between the twogroups, and their thickness appeared similar. Theadventitia averaged about 0.75 times the thicknessof the media, and wall thickness was calculated as1.75 times the media thickness. Our histologicalstudies (Table 1) showed that h' measured at zeromm Hg distending pressure was larger for SHRaortas at zero mm Hg at 20 weeks, but the differencewas not significant statistically. Values for h com-pared favorably with the measured value, h' (Tables1 and 2). There were no obvious differences in theappearance of the receptors in NTR and SHRaortas, but a quantitative comparison was notmade. There was no evidence of receptor degener-ation or increased Schwann cell activity in the SHR
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AORTIC WALL AND BARORECEPTOR PROPERTIES/Andresen 735
e
a
60
40
20
lOwks
100 120 140 160 180
PRESSURE (tnmHg)
200
60
40
20
lOwks
SHR
2 4 6 8
STRESS £-r (X 10" dyne/cm*)
60
4 0
20
lOwks
.25 35
STRAIN -
.4 5 .55
FIGURE 9 Relationship for 10-week-old rats amongaortic baroreceptor discharge and aortic pressure (A),aortic wall stress (B), and aortic wall strain (C). Thecurves were computed as for Figure 7 and further de-scribed in the text. The lines in C are least square fits,NTR's, r2 = 0.968, SHR's, r2 = 0.998.
aortas. The lower threshold receptors have myeli-nated axons,16 and if resetting were due to selectivedestruction of low threshold fibers and degenerationextended backward to the axons, it might be antic-ipated that differences in the ratios of unmyelin-ated-to-myelinated axons between SHR and NTRaortic nerves might be present. However, no suchdifferences were found (P < 0.95), and the average
ratios were 9.4 ± 3.3 in five NTR's and 9.8 ± 3.7 infive SHR's (Table 3). In addition, there were nomorphological differences between SHR and NTRreceptor endings in the vessel wall.
DiscussionThe major findings of the present experiments
are that hypertensive baroreceptor resetting in itsearliest stages occurs without any reduction in ves-sel wall distensibility and that baroreceptor func-tion is altered considerably in normotensive ratsbetween 10 and 20 weeks of age. These two resultsare discussed separately in the subsequent sections.
Hypertensive Baroreceptor ResettingFour possible mechanisms for hypertensive bar-
oreceptor resetting exist: (1) decreased vessel walldistensibility leading to reduced strain or deforma-tion of the receptors, (2) selective destruction of lowthreshold receptors, (3) uncoupling of the receptorsfrom the vessel wall, or (4) resetting of the receptorsthemselves. The present experiments seem to ex-clude the first two possibilities because, in 10-week-old SHR's, resetting is present although distensibil-ity is unchanged and, in SHR's 20 weeks of age,receptor destruction is absent. It appears from thethreshold histograms (Fig. 8) that a relatively largerproportion of receptors having unmyelinated axonswas sampled in the SHR's, so true threshold differ-ences may be even greater, since it has recentlybeen reported that hypertensive resetting does notshift threshold pressure as much for receptors withunmyelinated axons.17 Since these results lead toconclusions regarding resetting contrary to thosegenerally proposed,1'2'18 it is important to assess theadequacy of the methods we have used to measurewall stress, wall strain, and receptor destruction.We already have noted that the pressure-responsecharacteristics of in vitro and in vivo aortic baro-receptors are similar4 and that EINC'S of in vitro andin vivo aortae are similar.14
The determination of static wall characteristicsrelies on our measurements of pressure, aortic di-mensions, and aortic wall thicknesses. The pressuremeasurements were made with standard straingauge manometers, and there is no possibility thatsystematic errors between SHR's and NTR's weremade. The dimensional measurements were madein excised aortas using optical and sonar methods,and the cross-sectional dimension of the aortic archbetween the left carotid and left subclavian arterieswas similar with either method. Loading of thevessel wall by the piezoelectric crystals therefore isprobably insignificant. In addition, calculations ofwall thickness h which depend on re measurementswere in reasonable agreement with histologicalmeasurements, h'.
The pressure-volume curves were not changedwhen Ca2+ activity in the perfusate was about 10~6
M. If Ca2+ activity outside the aortic smooth muscle
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736 CIRCULATION RESEARCH VOL. 43, No. 5, NOVEMBER 1978
TABLE 3 Ratio of Unmyelinated to Myelinated Axons in Rat Aortic Nerve
Rat
12345
Average
No. of mye-linated fi-
bers
8655632068
58.4
NTR's
No. of un-myelinated
fibers
70571619263
1379
611
UM
7.613.03.13.2
20.3
9.4 ± 3.3
Age(wk)
2416161620
No. ofmyelinated
fibers
61354273
226
87.4
SHR's
No. of un-myelinated
fibers
191289313
17721308
775
UM
3.18.37.6
24.35.8
9.8 ± 3.7
Age(wk)
2416161634
cells is also about 10 6 M, this suggests that Ca2+-dependent smooth muscle tone has not affectedvessel wall properties measured presently and sup-ports the conclusion that the curves reflect passivewall properties.
There are differences between the measured val-ues for re used to calculate the strain data in Figure5 and the radius which might be expected based onthe volume data in Figure 3. Thus in 10-week-oldNTR's at 200 mm Hg, r2Oo = 1.6 ro and the predictedV200 assuming a right cylinder should be about (1.6)2
X Vo or 3.0 Vo. It is about 4.5 in Figure 3, however.The discrepancy is probably due to the assumptionabout the geometrical shape in distended aorticarch segments. The true volume is better describedby a truncated oblate spheroid than a right cylinder.However, we did not feel that this discrepancywarranted using the more complicated volume for-mula.
The shapes of our strain-pressure curves and thevalues of our EINC'S are consistent with those re-ported earlier for NTR's of the Wistar strain byBerry and Greenwald," given the differences inmethods and animals. Thus they measured n usinghypertonic radio-opaque dyes (95% barium sul-phate), after flushing the entire aorta at high pres-sure (300 mm Hg), and examined the entire thoracicand abdominal aortas. Their Wistar control animalswere larger than our Wistar-Kyoto controls, andtheir values for relative wall thickness, segmentlength, and aortic weight were also greater. Theymeasured strain differently and, using their re-ported values for related radius, we calculated strainaccording to our method and found their animalshad larger values than ours. They also producedhypertension by nephrectomy plus implanted de-oxycorticosterone acetate so that their hypertensivedata were not comparable to ours. However, ourresults with NTR's are in general agreement so thatour data on SHR's are also likely to be reliable andlead us to conclude that, at 10 weeks, the aortic wallproperties of SHR's and NTR's are similar but, at20 weeks NTR aortas have become more distensi-ble, whereas SHR aortas have not changed. In anearlier publication, Berry et al.12 also found thataortic distensibility initially increased in young an-imals and subsequently decreased. We have not
made systematic measurements in older NTR's, butthe few we have made are consistent with a reduc-tion in distensibility in animals older than 25-30weeks. Biochemical differences such as those re-ported by others19'20 were not examined.
The observation that SHR receptors appearednormal in the light and electron microscopes is notinvalidated by the fact that the observation is qual-itative. The fact that the ratio of unmyelinated-to-myelinated axons in the aortic nerve is similar inNTR's and SHR's suggests that low threshold re-ceptors have not been selectively destroyed, assum-ing that degeneration might extend backward suf-ficiently for it to be measured with this method.Even if this were not the case, there was no evidenceof damage closer to or around the parts of thereceptors within the vessel wall. The increasedSchwann cell activity which often accompanies de-generation21'22 was also not observed. Abnormali-ties of receptor discharge patterns might also havebeen expected but, again, this was not observed.
If our methods for evaluating wall strain, wallstructure, and receptor destruction are acceptable,then the results indicate that hypertensive baro-receptor resetting in 10-week-old rats must be dueeither to receptor uncoupling (mechanism 3) or toa primary receptor change (mechanism 4). We al-ready have shown that the dynamic properties ofbaroreceptors from 20-week-old SHR's and NTR'sdo not differ6 and, since the dynamics reflect cou-pling properties, mechanism 3 seems unlikely. Weare left, therefore, with mechanism 4, a primarychange in the receptors. One possibility might bethat the membrane sodium-to-potassium conduct-ance ratio in hypertensive baroreceptors is lowerthan normal, and we have recently shown that sucha difference could in principle account for resettingand reduced sensitivity.5 Another possibility mightbe differences in activity of the electrogenic Na+
pump known to operate in baroreceptors.23 Thelikelihood that baroreceptor membranes of SHR'sare different may be supported by the observationsthat membrane permeabilities and transport of Na+
and K+ are altered in vascular smooth muscle24'25
and red blood cells of SHR's.26'27 The observationson RBC's have been recently extended to humanswith hypertension.28
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AORTIC WALL AND BARORECEPTOR PROPERTIESMndreserc 737
Primary changes in SHR baroreceptors might beproduced by genetic or pressure-induced differ-ences, and a common feature might be that SHRbaroreceptors are genetically susceptible to in-creases in pressure. This idea is not dissimilar fromthe theory regarding essential hypertension pro-posed by Folkow et al.29 This theory proposes thatSHR arterioles are genetically susceptible to func-tional and, subsequently, structural alterations pro-duced by increases in pressure. If threshold andsensitivity of SHR baroreceptors were also geneti-cally susceptible to alterations in blood pressure,then it is possible that fluctuations in pressureoccurring on a chance basis could lead to resetting.It is also of interest that the increase in distensibilityof NTR aortas that occurs with age does not occurin SHR aortas, possibly for similar reasons. Therelative contributions of genetic and pressure fac-tors to resetting are currently under examination.
Baroreceptor Function during Developmentin NTR's and SHR's
The pressure thresholds for discharge in the pres-ent experiments were almost identical to valuesobtained in three separate sets of experiments de-scribed already." 5 16 The justification for using ear-lier values for maximum asymptotic discharge isthat similar values were obtained in the presentexperiments although in fewer instances. The re-producibility of the results probably reflects boththe uniformity of experimental animal models,SHR's and NTR's, and the standard conditions ofour in vitro methods. It is a considerable advantage,since a large data base can be accumulated.
As we have noted already, at 10 weeks of age, thewall characteristics of NTR and SHR aortas areequivalent. Between 10 and 20 weeks, NTR aortasbecome more distensible but SHR aortas do not. Itappears as if the hypertensive disorder, rather thanincreasing aortic wall stiffness, interferes with thenormal development of distensibility. Whatever thereason, at 20 weeks, SHR aortas are less distensibleand EINC is greater. The threshold strains are ac-tually reversed as are the suprathreshold discharge-strain curves (Fig. 7). This raises the possibility thatbaroreceptors in stiff vessels may be set to dischargeat smaller strains; that is, the receptors themselvesare actually more sensitive under these circum-stances.
However, the most surprising finding in the pres-ent experiments was that NTR baroreceptors be-came less sensitive to strain between 10 and 20weeks of age. NTR aortas have a phase of increaseddistensibility during the same period as previouslyreported.12 The rats undergo puberty at about 12weeks of age,12 so that it seems reasonable to referto these changes as developmental. This resettingof NTR baroreceptors should be considered in thelight of the following facts. As NTR's develop be-tween 10 and 20 weeks, their aortas become larger
(Table 2) and more distensible, producing a largeincrease in wall strain. Without resetting, NTRbaroreceptor discharge would be increased at theoperating pressure and this would tend to reduceblood pressure reflexly. However, NTR barorecep-tors become less sensitive to strain, which serves tomaintain their pressure-response curves constantduring this period. This adaptive response is prob-ably of great importance because it indicates that,during normal development, some process acts tomaintain a constant relationship between barore-ceptor activity and blood pressure despite changingconditions in the vessel wall.
AcknowledgmentsThe authors appreciate the help of Dr. W.R. Saum in the
early experiments.
References1. Kezdi P: Resetting of the carotid sinus in experimental renal
hypertension. In Baroreceptors and Hypertension, edited byP Kezdi. Oxford, Pergamon Press, 1967, pp 301-306
2. Angell-James JE: Characteristics of single aortic and rightsubclavian baroreceptor fiber activity in rabbits with chronicrenal hypertension. Circ Res 32: 149-161, 1973
3. Nosaka S, Wang SC: Carotid sinus baroreceptor functions inthe spontaneously hypertensive rat. Am J Physiol 222:1079-1084, 1972
4. Brown AM, Saum WR, Tuley FH: A comparison of aorticbaroreceptor discharge in normotensive and spontaneouslyhypertensive rats. Circ Res 39: 488-496, 1976
5. Saum WR, Ayachi S, Brown AM: Actions of sodium andpotassium on baroreceptors of normotensive and sponta-neously hypertensive rats. Circ Res 41: 768-774, 1977
6. Brown AM, Saum WR, Yasui S: Baroreceptor dynamics andtheir relationship to afferent fiber type and hypertension.Circ Res 42: 694-702, 1978
7. Okamoto K, Yamori Y, Ooshima A, Park C, Haebara H,Matsumoto M, Tanaka T, Okuda T, Hazama F, KyogokuM: Establishment of the inbred strain of the spontaneouslyhypertensive rat and genetic factors involved in hyperten-sion. In Spontaneous Hypertension, chap I, edited by KOkamoto. New York, Springer-Verlag, 1972, pp 1-8
8. Patel DJ, Fry DL: The elastic symmetry of arterial segmentsin dogs. Circ Res 24: 1-8, 1969
9. McDonald DA: Blood Flow in Arteries, ed 2, chap 10. Lon-don, Arnold, 1974, p 279
10. Bergel DH: The static elastic properties of the arterial wall.J Physiol (Lond) 156: 445-457, 1961
11. Berry CL, Greenwald SE: Effects of hypertension on thestatic mechanical properties and chemical composition ofthe rat aorta. Cardiovasc Res 10: 437-451, 1976
12. Berry CL, Greenwald SE, Rivett JF: Static mechanical prop-erties of the developing and mature rat aorta. CardiovascRes 9: 669-678, 1975
13. Patel DJ, Austen WG, Greenfield JC, Tindall GT: Imped-ence of certain large blood vessels in man. Ann NY Acad Sci115: 1129-1139, 1964
14. Patel DJ, Vaishnav RN: The rheology of large blood vessels.In Cardiovascular Fluid Dynamics, vol 2, chap 11, edited byDH Bergel. New York, Academic Press, 1972, pp 1-64
15. Sapru HN, Wang SC: Modification of aortic baroreceptorresetting in the spontaneously hypertensive rat. Am J Phys-iol 230: 664-674, 1976
16. Thoren P, Saum WR, Brown AM: Characteristics of rataortic baroreceptors with nonmedullated afferent nerve fi-bers. Circ Res 40: 231-237, 1977
17. Jones JV, Thoren P: Characteristics of non-medullated af-
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ferents arising from the aortic arch in rabbits with renovas-cular hypertension. J Physiol (Lond) 272: 81P-82P, 1977
18. McCubbin JW, Green JH, Page IH: Baroreceptor functionin chronic renal hypertension. Circ Res 4: 205-210, 1956
19. Wolinsky H: Response of the rat aortic media to hyperten-sion. Circ Res 26: 507-522, 1970
20. Fischer GM: Effects of spontaneous hypertension and ageon arterial connective tissue in the rat. Exp Gerontol 11:209-215, 1976
21. Raisman G, Matthews MR: Degeneration and regenerationof synapses. In The Structure and Function of NervousTissue, vol 5, edited by GH Bourne. New York, AcademicPress, 1972, pp 61-104
22. Janesco G, Kiraly E, Janesco-Gabor A: Pharmacologicallyinduced selective degeneration of chemosensitive primarysensory neurones. Nature 270: 741-743, 1978
23. Saum WR, Brown AM, Tuley FH: An electrogenic sodiumpump and baroreceptor function in normotensive and spon-taneously hypertensive rats. Circ Res 39: 497-505, 1976
24. Hermsmeyer K: Electrogenesis of increased norepinephrine
sensitivity of arterial vascular muscle in hypertension. CircRes 38: 362-367, 1976
25. Jones AW: Altered ion transport in vascular smooth musclefrom spontaneously hypertensive rats: Influences of aldos-terone, norepinephrine, and angiotensin. Circ Res 33:563-572, 1973
26. Friedman SM, Wahashima M, Mclndoe RA, Friedman CL:Increased erythrocyte permeability to Li and Na in sponta-neously hypertensive rats. Experientia 32: 476-478, 1976
27. Postnov YV, Orlov S, Gulak P, Shevchenko A: Alteredpermeabilityof the erythrocyte membrane for sodium andpotassium ions in spontaneously hypertensive rats. PfluegersArch 365: 257-263, 1976
28. Postnov YV, Orlov S, Shevchenko A, Adler A: Alteredsodium permeability, calcium binding and Na-K-ATPaseactivity in red blood cell membranes in essential hyperten-sion. Pfluegers Arch 371: 263-269, 1977
29. Folkow B, Hallback M, Lundgren Y, Weiss L: Structurallybased increase of flow resistance in spontaneously hyperten-sive rats. Acta Physiol Scand 79: 373-378, 1970
The Distribution of Blood RheologicalParameters in the Microvasculature
of Cat Mesentery
HERBERT H. LIPOWSKY, STEVEN KOVALCHECK, AND BENJAMIN W. ZWEIFACH
SUMMARY In vivo studies of the rheological behavior of blood in the microcirculation were conductedby direct in situ measurements in cat mesentery. Upstream to downstream pressure drops weremeasured in unbranched arterioles, capillaries, and venules, with diameters from 7 to 58 fim. Simulta-neous measurements of red cell velocity and vessel geometry facilitated computation of bulk velocity,pressure gradient, apparent viscosity, wall shear stress, and resistance. Arteriovenous distributions ofthese parameters revealed the following. Maximum pressure gradient (0.015 cm H2O/;im) occurs in thetrue capillaries (7 /im in diameter); intravascular wall shear stress averaged 47.1 dynes/cm2 in arteriolesand 29.0 dynes/cm2 in venules. Extreme values as great as 200 dynes/cm2 were observed in a fewshunting arterioles. Apparent viscosity averaged 3.59 cP in arterioles, 5.15 cP in venules, and 4.22 cPoverall. Intravascular resistance per unit length of microvessel varied with luminal diameter as apower law function with exponents of —4.04 for arterioles, -3.94 for venules, and —3.99 for all vesselscombined. This apparent maintenance of Poiseuille's law is attributed to the opposing processes ofhematocrit reduction and decreasing shear rate as blood is dispersed in successive arteriolar segments,and the converse action of these processes in the venous confluences which lessen the extent of networkvariations in apparent viscosity. Reductions in bulk velocity from the normal flow state to below 0.5mm/sec resulted in increases in apparent viscosity by a factor of 2 to 10, which are attributed primarilyto obstruction of the lumen by leukocyte-endothelium adhesion.
CONTROL of the peripheral circulation, irrespec-tive of whether it is neurogenic, metabolic, or my-ogenic in origin, is manifest principally by a changein the vascular resistance to blood flow. Althoughit is understood clearly that hemodynamic resist-
From AMES-Bioengineering, University of California, San Diego, LaJolla, California.
Supported by Grant HL-10881 from the U.S. Public Health Service.Dr. Lipowsky's present address is: Department of Physiology, Colum-
bia University, New York, New York.Address for reprints: Dr. H. H. Lipowsky, Department of Physiology,
Columbia University, New York, New York 10032.Received October 31, 1977; accepted for publication June 13, 1978.
ance in the microcirculation is determined by bothvascular (topographical and geometric) and intra-vascular (rheological) factors, the interrelationshipbetween the two has not been well defined fornormal and pathological flow states. In large part,this situation can be attributed to the lack of asuitable constitutive relationship for describing therheological behavior of blood in the microcircula-tion.
In an attempt to elucidate the interaction be-tween microvascular topography and hemorheol-ogy, Landis1 used the classical studies of Poiseuille2
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