We studied structural and functional changes of small muscular arteries from the mesenteric vascular bed of young spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) using a new morphometric protocol involving the use of confocal microscopy and a pressurized artery system. At 3 and 4 weeks of age, systolic pressure of SHR and WKY was similar; however, significant structural changes in the mesenteric vasculature were already present in SHR. Arteries fixed under pressure in vitro from SHR had a larger medial volume and increased number of smooth muscle cell layers but similar lumen size compared with arteries from WKY in maximally relaxed conditions. Functional studies showed that SHR arteries contracted more in response to stimulation by KCl and norepinephrine, resulting in a significantly smaller lumen size in these vessels than in those from WKY. SHR arteries precontracted with KCl were also able to maintain a smaller lumen diameter than WKY arteries when challenged with increasing pressure levels. No difference in the sensitivity of response of these arteries to norepinephrine stimulation was found. At 3 and 4 weeks of age, mesenteric arteries from some SHR and WKY were not responsive to periarterial nerve stimulation, and the number of responders was higher in the WKY than SHR. However, a greater degree of contraction was found in SHR arteries responding to field stimulation at 4 weeks than in WKY arteries. We conclude that there is a temporal difference in the rate of functional maturation of the innervation in SHR arteries compared with WKY arteries. Structural changes of the small muscular arteries, caused by an increase in the medial volume, and increased number of smooth muscle cell layers are primary changes that contribute to the development of hypertension in the SHR because these changes are present at the age when blood pressure is similar in SHR and WKY.
Structural and functional modifications of the resistance vasculature may be important causative factors in the development of essential hypertension. In SHR, a sustained elevation of BP compared with normotensive WKY controls was found at 11 and 17 weeks of age accompanied by an increase in total peripheral resistance, but there was no increase in cardiac output.1 Since BP is the product of total peripheral resistance and cardiac output, increased total peripheral resistance may be responsible for the sustained elevation of BP observed in SHR at these ages.
Thickening of resistance blood vessel walls has been associated with an increase in total peripheral resistance in several animal models of human essential hypertension, including Dahl salt-sensitive hypertensive rats, deoxycorticosterone-NaCl rats, and SHR,2 as well as in humans with essential hypertension.3 The finding of this type of structural change in the resistance vasculature is frequently concurrent with an elevation of BP. This was the case in the small muscular arteries of 12- and 28-week-old SHR, in which Lee et al4 found an increase in the medial and vessel wall cross-sectional areas, with a BP that was approximately 50 mm Hg higher than in WKY. The concurrence of BP elevation and medial thickening makes it impossible for one to assess whether the elevation of BP is the cause or the consequence of vessel wall hypertrophy.
It is possible that an increased intramural pressure in the hypertensive animal may cause an adaptive response in the blood vessels, resulting in wall thickening. Studies on the hypertrophic response of smooth muscle cells to increased intramural pressure in urinary bladder, small intestine, and ureter have found that hypertrophy of the smooth muscle layer was produced by increased intramural pressure.5 Similarly, thickening of the vessel wall due to smooth muscle hypertrophy was found in rat portal vein6 and aorta7 after coarctation. Some authors have suggested that increased intramural pressure resulting from elevated cardiac output due to volume expansion in young SHR may lead to the vessel wall hypertrophy found in these animals.8 The effect of wall hypertrophy increasing total peripheral resistance could then cause the sustained elevation of BP seen in SHR. If this were the case, BP in SHR would be expected to be elevated because of increased cardiac output before any thickening of the vessel wall had occurred.
At 4 weeks of age, SHR and WKY display little difference in BP.9 10 11 However, also at this age the BP begins to diverge in these two strains, so that a comparison of artery structure between SHR and WKY at this age should indicate whether some components of medial thickening in SHR are independent of pressure. Differences in the structure of small muscular arteries from 3- to 5-week-old SHR and WKY have been found. In maximally relaxed arteries fixed by perfusion at a perfusion pressure of approximately 20 mm Hg, significant increases in the cross-sectional area of the medial layer, the media-to-lumen ratio, and the number of smooth muscle layers in SHR over WKY were found9 ; however, the luminal areas of the arteries from SHR and WKY were the same.9 This seems to indicate that an increase in the volume of the small muscular artery wall has already occurred in SHR but does not yet encroach upon the lumen of the arteries. However, the lower than physiological level of perfusion pressure used during fixation in these experiments calls into question whether the lumen measurements recorded accurately reflect the lumen diameter of the arteries in the living animal.
Studies on the functional responses of small muscular arteries from 4- to 6-week-old SHR and WKY have been performed but have focused on the response of perfusion pressure in isolated mesenteric vascular beds to various agonists.10 12 The exact anatomic site of response and relationship to arterial structure remain obscure. In 4-week-old animals, no difference in the response of perfusion pressure to norepinephrine was found between SHR and WKY.10 In 5- to 6-week-old SHR and WKY, perfusion pressures were found to increase to higher maximum levels in SHR in response to norepinephrine, KCl, serotonin, and vasopressin, with a reduced ED50 to norepinephrine in SHR in the presence of reuptake inhibition by cocaine.12
Our purpose in this study was to assess the relationship between the development of hypertension and changes in the structure and functions of small muscular arteries from young SHR. The advent of the confocal microscope makes it possible to produce multiple optical sections separated by a precisely known distance without the distortions generally associated with light microscopy. We have therefore developed a morphometric protocol using the new staining technique described in this article in order to measure directly the medial volume per unit length of artery fixed at a physiological pressure. Moreover, we also quantified the luminal area of vessel per unit length at midsection and number of smooth muscle layers. With this information, we assessed vessel hypertrophy and its relationship to lumen diameter and to BP development. We related these structural parameters to functional responses of the arteries on a pressure myograph apparatus to KCl, norepinephrine, and electrical field stimulations.
Young male SHR and WKY were used for structural and functional analyses. These rats were obtained from colonies maintained at the Animal Quarter of McMaster University. The SHR colony was started with rats from Ayerst Laboratory (Montreal, Quebec, Canada) in 1976, and the WKY colony was started with rats from Canadian Breeding Farms (Montreal) in 1983. Both colonies were derived from the strains from Charles River Laboratories (Wilmington, Mass), and we have maintained these colonies in our institute by continuous inbreeding. The care of these rats was in accordance with the guidelines of the Canadian Council on Animal Care. The rats were chosen in such a way as to result in equal mean ages between the groups. Origins of the rats were noted with regard to individual, litter, and strain so that results could be analyzed to see whether strain or litter of origin had any significant effect on the parameters measured.
The rats were weighed and their systolic BPs measured with a tail-cuff occlusion method (model PE300, Narco BioSystems) when rats were conscious. For each rat, BP was measured several times until a consistent result was obtained. Arteries were sampled from the thirdmost distal first-order branches of the superior mesenteric artery (Fig 1⇓). These arteries have been referred to previously as large mesenteric arteries.4 9 13
Preparation of the Vessels for Morphometry
In rats anesthetized with sodium pentobarbital (65 mg/kg IP), mesenteric vessels were cleared of blood by perfusion as follows. An infusion cannula was placed in the abdominal aorta, distal to the origin of the superior mesenteric artery. The aorta was clamped just below the diaphragm, proximal to the origin of the superior mesenteric artery. An exit for the perfusate was cut into the portal vein. This allowed the perfusate, oxygenated HBSS containing 1 μmol/L sodium nitroprusside, to clear the vasculature in the abdominal viscera. The arteries were perfused at a flow rate of 1 mL/min per 100 g body weight for 5 minutes, resulting in maximal relaxation of the arteries.
During sampling of the arteries, we tried to maintain the in vivo length of the arteries in the following way. Arteries to be sampled were tied with 10-0 suture ties at either end. A suture was placed along the artery between these two ties to mark the original length of the artery. The arteries were removed by cutting between the ties at either end (Fig 2⇓). They were placed in a Petri dish containing oxygenated HBSS and 1 μmol/L sodium nitroprusside. The surrounding fat tissue on the arteries was removed by fine dissection under a dissecting microscope. The arteries were then transferred to an organ bath containing oxygenated HBSS. The suture tied at one end of the artery was loosened so the artery could be slipped on a micropipette where it was resecured and pressurized. It was found that a pressure of approximately 70 mm Hg was required to expand the arteries to such an extent as to make the filament connecting the two ties regain its original length. These pressurized arteries were fixed and prepared for morphometric measurements with the use of confocal microscopy.
It has been shown that a combination of the fluorescent DNA-specific dyes ethidium bromide, which is membrane impermeant, and Hoechst 33342, which is membrane permeant, is useful in elucidating artery structure and cell vitality in florescence microscopy.14 However, the limited range of laser excitation available (wavelengths of 488, 512, or 543 nm) in most commercial confocal microscopes, including our own in this facility, precludes the use of the Hoechst dye (maxex=346 nm). We have therefore carried out experiments with different concentrations of ethidium bromide (maxex=510 nm) to find a suitable condition in which to produce a membrane-permeant molecule for the elucidation of artery structure.
It was observed that trace amounts of sodium borhydride, when left in the organ bath after washing, were capable of reducing ethidium bromide. Reduction of the ethidium dye produced a membrane-permeant molecule that allowed the elucidation of artery structure so that the intimal, medial, and adventitial layers of the arteries could be distinguished (Fig 3A and 3B⇓⇓). This was the necessary prerequisite for medial volume quantification under the confocal microscope. The nuclei of smooth muscle cells in the artery wall were also clearly visible, which aided in the counting of the number of smooth muscle layers in either group (Fig 3C⇓).
The fixative generally used in our laboratory contains 2.5% glutaraldehyde,4 which produces a large amount of nonspecific fluorescence in tissue. We have found that a fixative containing 3.5% formaldehyde and 0.75% glutaraldehyde in 0.05 mol/L phosphate buffer allows preservation of artery structure with little nonspecific fluorescence. We therefore used the following method to prepare arteries for confocal microscopy. Arteries were fixed for 1 hour with a fixative containing 3.5% formaldehyde and 0.75% glutaraldehyde in 0.05 mol/L phosphate buffer at pH 7.4. The fixative was washed from the organ bath by repeated changes with HBSS (pH 7.4). Free aldehyde groups remaining after fixation were reduced by the addition of 1 mg/mL sodium borhydride to the bath for 5 minutes. The tissue was washed with HBSS with 0.1% Triton X-100 at a pH of 8.0. Reduced ethidium bromide (75 mg/mL) was then added to the bath. Ethidium bromide reduction was indicated by the loss of deep orange color from ethidium bromide. Arteries were stained for 20 minutes and removed from the organ bath for washing in HBSS at pH 7.4. After washing, arteries were mounted in 1:1 glycerol/HBSS (pH 7.4). Exposure to ultraviolet light for 10 minutes redeveloped the orange fluorescence characteristic of ethidium bromide in the nuclei of the tissue. At this stage, the arteries were ready for viewing under the confocal microscope.
Arteries in 1:1 glycerol/HBSS (pH 7.4) were placed on concave microscope slides for viewing. A Carl Zeiss LSM 10 system was used for confocal microscopy. The optical sections obtained from this system were approximately 0.2 μm thick. The system was equipped with an external argon laser with emission lines at 488 and 514 nm. The spectral line at 488 nm produced the best excitation of the ethidium dye with the least nonspecific fluorescence, resulting in an optimal signal-to-noise ratio. An 8–second per frame dwell time and 16-line averaging were used to improve image quality. Images were saved to electronic media and transferred to an Image 1 system for morphometric analysis. Micrographs were obtained from images with a 35-mm camera attached to a video frame printer.
The medial volume was calculated with a Cavalierian estimator of volume (see equation below). This estimator of volume does not rely on any assumptions about tissue or cell shape and is independent of tissue orientation.15 Multiple optical sections were taken longitudinally through the arteries. We used the ×20 objective with an electronic zoom factor of 20 times to produce images with a total magnification of ×400. Sectioning began at a random location more than 10 μm above the artery. Sections were separated by 10-μm intervals, and sectioning continued until the artery was completely traversed. This produced from 20 to 25 optical sections per artery. The sections covered approximately a 500-μm length of artery. Ethidium bromide staining allowed the medial layer to be clearly distinguished from the adventitial layer and endothelium plus internal elastic lamina of the arteries. The area of the medial layer in each optical section was determined by a computer-aided tracing method allowing the calculation of medial volume as in the following equation (Cavalierian estimator of volume): Volume=Σ(Area)×δT, where volume is the calculated volume of the medial layer, area is the area of the medial layer found on each optical section, and δT is the distance between each optical section.
The optical section at the center of the stack was used for determination of the midsection lumen area per length of artery. An electronic zoom factor of 40 times was used with the ×20 objective (×800) so that the number of smooth muscle layers present in the media of the artery could be counted.
We used a pressurized myograph apparatus similar to that described by Halpern et al16 to study the functional response of the mesenteric arteries from 3-, 4-, and 6-week-old SHR and WKY. The apparatus consisted of an arterial myograph system in a heated organ bath in which temperature was maintained at 37°C. One end of the arteries was tied off with a 10-0 suture. The other end was secured to the tip of a micropipette, which was connected to a pressure reservoir, so that arteries could be inflated with physiological solution at different pressures. A microscope with a video camera attached was mounted on the bath, allowing the recording of external diameter and lumen diameter of arteries. Arteries were taken directly from living rats anesthetized with ketamine (75 mg/kg IP) and urethane (0.25 g/kg SC) and were mounted on the micropipette in the bath. Arteries in the bath were maintained in a Krebs' solution consisting of (mmol/L) NaCl 116, NaHCO3 9.3, d-glucose 11, KCl 4.6, MgSO4 1.2, NaH2PO4 1.2, and CaCl2 2.5, pH 7.4, and aerated with 95% O2/5% CO2. Periarterial nerve stimulation was provided by a stimulator (model S4, Grass Medical Instruments) with an output of monophasic pulses at 150 V of 0.85 millisecond duration and a train of 10 seconds. Arteries from 3-, 4-, and 6-week-old rats were stimulated at 20 Hz over a range of pressure from 30 to 250 mm Hg for assessment of the contractile response of the arteries to nerve stimulation. Tetrodotoxin was added to the bath for determination of the component of contraction due to periarterial nerve stimulation and that due to direct muscle stimulation. Dose-response curves to norepinephrine were constructed over the concentration range of 2×10−7 to 1×10−3 mol/L with β-blockade (1.5×10−6 mol/L propranolol). Dose response to KCl was determined over a concentration range of 2×10−3 to 2.4×10−1 mol/L. Yielding pressure in arteries maximally contracted by KCl was determined over a pressure range of 50 to 250 mm Hg in both SHR and WKY.
Values given are mean±SE. Data analysis was performed with a computer-based statistical package (SAS Institute Inc). ANOVA was used to determine whether differences existed for BP, medial volume, lumen area, and number of smooth muscle cell layers. Change in lumen size in response to the contractile agonists norepinephrine and KCl, contraction produced by electrical stimulation, and sensitivity to norepinephrine stimulation were compared by Bonferroni's corrected Student's unpaired t tests at each point. Significance was recognized at the 95% level. Pearson's correlation was performed between medial volume and number of smooth muscle cell layers. Power analysis of F tests were performed to determine the probability that the parameters being measured were equal.
BP at 4 Weeks
BP was similar in SHR and WKY at 4 weeks of age (127±3 [n=42] and 119±3 [n=38] mm Hg, respectively) (Fig 4⇓). Mixed model ANOVA with fixed effects of strain type and random effects of litter type nested inside of strain type showed that the 8–mm Hg difference in systolic BP found between the strains was significant (P=.02). Power analysis of the F test revealed that there was an 80% chance that BP differed by less than 20 mm Hg between the strains. The ANOVA also showed that litters within the strains differed significantly in BP (P=.007). The model describing the variation in BP as the result of differences in strains and litters accounted for 69% of the variation in BP measured. Of that variation, 6% was found between the strains and 63% between the litters within the strains; 31% of the variation in BP remained unexplained by the model. BP of the smaller subsets of SHR and WKY used for morphometry and functional studies showed means similar to those displayed in the general population (115±5 [n=10] and 110±4 [n=10] mm Hg, respectively) (Fig 4⇓).
Volume of Medial Layer and Luminal Area
Two optical sections taken longitudinally at the midpoint of the artery, one from SHR and one from WKY (Fig 5⇓), were displayed to illustrate the thickness of the medial layer in both groups. The mean value for medial volume in the SHR group was 16 790 μm3/μm length of artery (n=10) and for the WKY group was 11 250 μm3/μm length (n=10, Fig 6⇓). ANOVA showed a significant difference between SHR and WKY with regard to medial volume (P=.0002). The difference between the means represents a 49% greater medial volume in the SHR than WKY.
Lumen area was not significantly different between SHR and WKY (95 060 and 95 460 μm2, respectively, for a 500-μm length of artery) (P=.84, ANOVA) (Fig 6⇑). Power analysis revealed that there was an 87% chance that the lumen areas differed between the strains by less than 5%.
Number of Smooth Muscle Layers and Relationship to Medial Volume
The number of smooth muscle cell layers present in the media differed significantly between the strains (P=.0009, ANOVA), with 4.1 in SHR and 2.7 in WKY (n=8 in each group, Fig 6⇑). Pearson's correlation performed between the values obtained for medial volume and the number of smooth muscle cell layers in the media showed a highly significant result (P=.0001). This analysis showed that the increase in smooth muscle cell layers accounted for 81% of the increase in medial volume.
Functional Analysis of Hypertrophied Vessels
Unstimulated SHR and WKY arteries had similar lumen diameters. Dose-response curves to KCl showed that the area under the curve (AUC) was significantly greater for WKY than SHR arteries (n=6 each, P<.05), indicating that over the entire range of KCl concentrations, SHR arteries had a smaller lumen diameter than WKY arteries. Moreover, the maximum contractile response for the SHR arteries was significantly greater than that derived from the WKY arteries (P<.01) (Fig 7A⇓). The pressure at which arteries maximally contracted with KCl could no longer hold their contraction was lower in WKY than SHR. In the WKY arteries, lumen diameter increased linearly in response to pressure, whereas SHR arteries were able to maintain their maximally contracted lumen diameter until a pressure of approximately 150 mm Hg was exceeded. When curves were fitted between the lumen diameter response and increasing pressure, it was found that the inflection point of the fit (ie, yielding point) was 154 mm Hg for the WKY and 184 mm Hg for the SHR arteries (n=6 each, P<.05) (Fig 7B⇓). Norepinephrine dose-response curves when normalized as a percentage of maximum contraction due to KCl showed no significant difference in the reactivity of the arteries to norepinephrine stimulation (Fig 8A⇓). However, when the absolute diameter of the lumen was used to express the response to a dose of norepinephrine, the fitted curves again showed a significantly greater AUC for WKY than SHR arteries, indicating that over the entire range of norepinephrine concentrations, SHR arteries had smaller lumen diameters (n=6 each, P<.01, Fig 8B⇓). These results suggest that the increased muscle mass on the SHR arteries had a functional manifestation as an increased contractile response to KCl and norepinephrine.
Nerve-Stimulated Contractile Response
Mesenteric arteries responsive to electrical field stimulation were present in 0 of 10 SHR and 3 of 10 WKY at 3 weeks of age. At 4 weeks, arteries from 4 of 15 SHR and 8 of 15 WKY responded to electrical field stimulation. These results were analyzed by a log linear analysis with three factors: strain (SHR versus WKY), age (3 versus 4 weeks), and responsiveness (responders versus nonresponders). The two-factor interaction between strain and responsiveness was significant (χ2=5.20, P<.05), indicating that there was a different response in the two strains. The two-factor interaction between age and responsiveness was also significant (χ2=4.19, P<.05), indicating an overall greater responsiveness at 4 weeks than 3 weeks. Of the nonresponding arteries, all were found to contract to stimulation with norepinephrine (10−3 mol/L) after unsuccessful field stimulation. Finally at 6 weeks, all the arteries from six each of SHR and WKY responded to electrical field stimulation.
When SHR arteries that were responsive to electrical field stimulation were compared with those from WKY over a pressure range of 30 to 250 mm Hg, SHR arteries were found to contract more than WKY arteries. This was true for arteries from both 4-week-old (P=.08, Fig 9A⇓) and 6-week-old (P<.001, Fig 9B⇓) groups. The AUC for SHR arteries was significantly greater than for WKY arteries, indicating that over the entire pressure range, SHR arteries contracted to produce smaller lumen diameters. These responses were blocked by tetrodotoxin, indicating that these responses were due to nerve stimulation.
The major findings of this study were as follows. SHR arteries displayed significant structural alterations compared with WKY control arteries at a time when only a small difference in BP existed. These alterations included an increase in the volume of the media layer and increase in the number of smooth muscle cell layers but no change in lumen diameter in the relaxed state. Moreover, these structural alterations had a functional manifestation. SHR arteries underwent increased amounts of contraction that resulted in a significantly smaller lumen size in response to stimulation by KCl or norepinephrine. SHR arteries precontracted with KCl were also able to maintain a smaller lumen size than WKY arteries at comparable intramural pressures. These changes were due to the presence of an increased medial volume and an increase in the number of smooth muscle cell layers. However, the number of arteries exhibiting a tetrodotoxin-sensitive contractile response to electrical field stimulation was less in SHR than WKY at 3 and 4 weeks of age. This difference may provide the explanation for the similar BP in these rats at these ages despite the presence of significant vascular structural changes in the mesenteric arteries, as discussed below.
Structural and Functional Observations
The finding that hypertrophy of the medial layer had already occurred before any large difference in BP existed between SHR and WKY indicates that the thickening of the medial layer in SHR is a primary change in the development of hypertension,17 in agreement with the results of previous studies.9 12 18 19 20 21 Therefore, it appears that some unknown process occurring before the elevation of BP in SHR is causing medial thickening. Studies on cultured smooth muscle cells from SHR have shown a greater growth response than cells derived from WKY.22 23 24 The increased vascular smooth muscle mass in mature SHR may then produce the higher BP observed in these animals by increasing total peripheral resistance. In young SHR, however, the BP was similar to that of age-matched WKY despite the presence of a hypertrophied medial wall and an increase in the media-to-lumen ratio in the mesenteric arteries and arterioles.9 12 20 21 It was suggested that the BP increase in SHR may lag behind the thickening of the muscle layer because of the late maturation of neuromuscular coupling in young SHR, preventing the hypertrophied muscle layer from fully expressing itself functionally.9 Results from the present study have now provided evidence to this effect. In young SHR, despite the presence of a high innervation density in the mesenteric arteries compared with WKY,9 a tetrodotoxin-sensitive response to electrical field stimulation was absent at 3 weeks and less frequently found at 4 weeks of age in the SHR than WKY arteries. Such a temporal difference in the rate of functional maturation of innervation in the mesenteric arteries from SHR compared with those from WKY may explain why some young SHR did not express their genetic predisposition to increased total peripheral resistance by exhibiting a higher BP than WKY. These results are also consistent with a previous study in which functional maturation of the nerve terminal–smooth muscle complex (levator palpebrae in the eyelid) was retarded in SHR relative to WKY during postnatal weeks 2 and 3.25
At 4 weeks of age when some arteries from both SHR and WKY displayed a tetrodotoxin-sensitive contractile response, a comparison of the percent contraction of the responding arteries could be made. It was found that SHR arteries that responded to electrical stimulation contracted to a greater degree than WKY arteries. This indicates that when functional innervation is present in SHR, the genetic predisposition to higher total peripheral resistance is expressed as a greater contraction of small resistance vessels. At 6 weeks when all the arteries in both SHR and WKY responded to field stimulation, the same pattern of a greater degree of contraction in SHR arteries than WKY arteries was again present, therefore showing the important role of vascular structural changes in the control of total peripheral resistance.
Previous studies with McGregor perfusion of the isolated mesenteric vascular bed in 4-week-old SHR and WKY10 and isolated perfusion of the entire mesenteric vascular bed with the intestinal tract intact12 found that frequency-response curves to periarterial nerve stimulation, measured as changes in perfusion pressure, produced a greater response in SHR than WKY despite the lack of a significant difference in systolic BP measured at this age in these rats. This is in agreement with our comparison of responding vessels at 4 weeks of age when SHR vessels contract to a greater degree than WKY vessels. However, it is not known whether all the vascular beds examined from SHR at 4 weeks of age were responsive to electrical stimulation. At 5 weeks of age and with the wire myograph technique, electrical stimulation of small mesenteric arteries from SHR and WKY elicited similar pressure responses in these arteries.20 Differences in the study methods may account for the discrepancy between the results obtained in this study and those obtained with the isolated perfusion method in contrast to the wire myograph method. There are marked differences in the vasoconstrictor responses of the arteries between wire-mounted and pressurized preparations.26 The other possibility is that the arteries we used in the present study were larger than those used by Stephens et al.20 Nevertheless, by 12 weeks of age, SHR mesenteric arteries did show a greater response to electrical stimulation than WKY arteries.20
Lumen diameter in the maximally relaxed pressurized arteries from SHR and WKY was the same, consistent with the results obtained from morphometric measurement of mesenteric arteries fixed in situ9 and those calculated from the wire myographic measurements.20 21 This indicates that the hypertrophied media of the SHR arteries had not encroached on the lumen to reduce diameter. Large changes in medial thickness may be necessary to reduce lumen diameter because of the elasticity of the artery wall. Small amounts of wall thickening may not decrease lumen diameter in vivo because the resulting decrease in flow would increase pressure, expanding the nonrigid vessel and restoring lumen diameter. Effective reduction in lumen diameter may only occur with a change in vessel wall compliance in the relaxed state or an increase in the ability of the muscle mass to generate active tension in the contracted state. Immaturity of innervation in the arteries from young SHR may prevent the full expression of this active reduction in lumen diameter in the animal so that BP was similar between WKY and SHR at 3 and 4 weeks of age.
However, the fact that the mesenteric arteries from these young SHR were able to produce smaller lumen diameters than WKY arteries in response to stimulation by norepinephrine and KCl indicates the functional significance of a larger medial mass in these arteries. Moreover, a comparison of the arteries responding to field stimulation at 4 weeks of age also indicated a greater percentage of contraction in SHR than WKY vessels. Therefore, with the complete functional maturation of the small muscular arteries in older SHR, it is inevitable that hypertension would develop due to an increase in total peripheral resistance because of the ability of the hypertrophied medial wall to generate more active tension. Previous studies with the wire myograph method involving small mesenteric arteries20 21 and isolated perfusion of the mesenteric vascular bed12 from young SHR (4 or 5 weeks old) found an enhanced contractile response to norepinephrine compared with arteries or vascular bed from WKY.
Consideration of the Methods
Confocal microscopy represents an important advance in light microscopy because it effectively removes the need for sectioning and eliminates the inherent distortion associated with embedding and sectioning for the viewing of biological materials. The fact that the confocal microscope is able to produce very thin optical sections of the same order of magnitude as thin sections used for electron microscopy (0.2 μm) makes it ideal for stereological techniques based on the use of two or more parallel sections because the distance between optical sections is precisely known.15 This enabled us to use a Cavalierian estimator of volume,15 involving the summation of 20 to 25 parallel sections over a 200- to 250-μm depth of tissue. Such a procedure would be very difficult to perform with ultramicrotomy.
Fixation of arteries by perfusion involves considerable difficulty in matching the hemodynamic parameters present in the living organism during fixation. Fixation with a low-viscosity fluid at a physiological flow rate results in low perfusion pressures, as were measured in the aorta.9 Increasing the flow rate by many folds (5 to 10 times) greater than physiological tissue flow rates produced only small increases in pressure (<twofold) in the large vessels, but these high flow rates may damage the walls of the small vessels, especially the endothelial cells. Moreover, the use of large molecular weight fractions, such as dextran, to increase fixative viscosity without affecting tonicity remains problematic because of the non-Newtonian properties of blood, in which viscosity is a function of tube diameter as in all suspensions and decreases in smaller arteries.27 This being the case, titration of viscosity to physiological pressures in larger vessels for a fixative solution with Newtonian properties, such as a fixative containing dextran, would produce a pressure too high in smaller vessels, thereby causing some indeterminate damage to the vessels. These problems in establishing ideal perfusion fixation conditions that reflect the hemodynamic parameters of flow and pressure in the living animal bring into question the data collected for luminal sizes of perfusion-fixed arteries. To address these problems, we removed from the body the small muscular arteries used in the present experiments and fixed them in vitro at a physiological pressure without flow.
In vitro fixation also involves potential pitfalls because arteries are elastic tissues and are under considerable longitudinal tension in vivo. As such, once they are removed from their tethering connective tissue, they shorten. This shortening was shown to be up to 40% in larger elastic vessels such as the iliac arteries and increased as vessel size decreased.28 We therefore used the pressurized myograph method to analyze the functional responses and also to fix the arteries under pressure for structural analysis. We took care to restore the in vivo length of the arteries during fixation because medial volume and lumen area were being quantified over certain known lengths of arteries for comparison between groups. In this way, more accurate results than those previously produced were obtainable.
In summary, using a new morphometric protocol involving confocal microscopy, we have shown that structural changes of small muscular arteries have already occurred in young SHR when BP is similar to that in WKY. There is also a temporal delay in the functional maturation of innervation in SHR arteries compared with WKY arteries. We therefore conclude that structural changes of the small muscular arteries, which are due to an increase in medial volume, and increased number of smooth muscle cell layers are primary changes that contribute to the development of hypertension in the SHR because these changes are present at the age when BP is similar in SHR and WKY.
Selected Abbreviations and Acronyms
|HBSS||=||Hanks' basic salt solution|
|SHR||=||spontaneously hypertensive rat(s)|
This work was supported by the Heart and Stroke Foundation of Ontario. We thank Dr Geoff R. Norman, Department of Clinical Epidemiology and Biostatistics, McMaster University, for his advice on statistical analyses.
Reprint requests to Dr R.M.K.W. Lee, Department of Anaesthesia (HSC-4V34), McMaster University, Hamilton, Ontario, Canada L8N 3Z5.
- Received June 18, 1996.
- Revision received July 29, 1996.
- Revision received September 25, 1996.
Smith TL, Hutchins PM. Central hemodynamics in the developmental stage of spontaneous hypertension in the unanesthetized rat. Hypertension. 1979;1:508-517.
Aalkjaer C, Heagerty AM, Petersen KK, Swales JD, Mulvany MJ. Evidence for increased media thickness, increased neuronal amine uptake, and depressed excitation-contraction coupling in isolated resistance vessels from essential hypertensives. Circ Res. 1987;61:181-186.
Rioux F, Berkowitz BA. Role of the thyroid gland in the development and maintenance of spontaneous hypertension in rats. Circ Res. 1977;40:306-312.
Kong JQ, Taylor DA, Fleming WW. Mesenteric vascular responses of young spontaneously hypertensive rats. J Pharmacol Exp Ther. 1991;258:13-17.
Halpern W, Osol G, Coy GS. Mechanical behaviour of pressurized in vitro prearteriolar vessels determined with a video system. Ann Biomed Eng. 1994;12:463-469.
Korner PI, Swales JD. Debate on the role of resistance arteries in hypertension. In: Mulvany MJ, Aalkjaer C, Heagerty AM, Nyborg NCB, Strandgaard S, eds. Resistance Arteries, Structure and Function. Amsterdam, Netherlands: Elsevier Science Publishers; 1991:39-43.
Miller BG, Conners BA, Bohlen HG, Evan AP. Cell and wall morphology of intestinal arterioles from 4-6 and 17-19 week old Wistar-Kyoto and spontaneously hypertensive rats. Hypertension. 1987;9:59-68.
Mulvany MJ, Aalkjaer C, Christensen J. Changes in noradrenaline sensitivity and morphology of arterial resistance vessels during development of high blood pressure in spontaneously hypertensive rats. Hypertension. 1980;2:664-671.
Stephens N, Bund SJ, Jagger C, Heagerty AM. Arterial neuroeffector responses in early and mature spontaneously hypertensive rats. Hypertension. 1991;18:674-682.
Rizzoni D, Castellano M, Porteri E, Bettoni G, Muiesan ML, Agabiti-Rosei E. Vascular structural and functional alterations before and after the development of hypertension in SHR. Am J Hypertens. 1994;7:193-200.
McDonald DA. Blood Flow in Arteries. 2nd ed. London, UK: Edward Arnold; 1974:64-66.
McDonald DA. Blood Flow in Arteries. 2nd ed. London, UK: Edward Arnold; 1974:256-259.