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(Hypertension. 1997;30:1455-1464.)
© 1997 American Heart Association, Inc.
Articles |
Cellular Aspects of Vascular Remodeling in Hypertension Revealed by Confocal Microscopy
From the Clinical Research Initiative in Heart Failure (S.M.A., S.M., J.C.M.), Institute of Biomedical and Life Sciences, and Department of Medicine and Therapeutics (A.F.D.), Western Infirmary, University of Glasgow, UK; Department of Physiology (C.G.), Faculty of Medicine, Universidad Aut-noma de Madrid, Spain; and Department of Biological Sciences (C.H.), Glasgow Caledonian University, UK.
Correspondence to Dr S.M. Arribas, Clinical Research Initiative in Heart Failure, Institute of Biomedical and Life Sciences, West Medical Building, University of Glasgow, Glasgow G12 8QQ, UK. E-mail S.Arribas{at}bio.gla.ac.uk
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Abstract |
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Abstract Cellular aspects of remodeling in intact arteries have not been fully investigated, mainly due to the lack of an appropriate methodology that allows for simple measurements. The aim of this study was to develop a method based on laser scanning confocal microscopy (LSCM), compare it with previous methodology, and apply it to the study of remodeling in hypertension. The morphology of mesenteric resistance arteries from stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar-Kyoto rats (WKY) was determined with wire myography on one segment with a standardized diameter setting (0.9d100) and with perfusion myography on a second segment from the same artery at the calculated equivalent pressure. The second segments were stained with the nuclear dye Hoechst 33342 (live tissue) or propidium iodide (fixed tissue) and measured with LSCM and MetaMorph software. Compared with wire myography, perfusion myography showed similar differences from those previously reported. Compared with LSCM, perfusion myography showed a similar lumen but significantly smaller wall thickness in both live and fixed tissue, probably due to measurement underestimation. In the study with LSCM, arteries from SHRSP compared with those from WKY showed (1) reduced lumen, (2) altered cell density that was significantly increased in the adventitia, decreased in the media, and unchanged in the intima, (3) significantly increased medial volume, (4) significantly smaller endothelial cell nuclei, and (5) adventitial-like cells in the media. We conclude that (1) LSCM is a reliable and straightforward method to study morphology in intact vessels, (2) it provides new information on the cellular changes in remodeling, (3) adventitia might play an active role in the process of remodeling in hypertension, and (4) endothelium "remodels" in hypertension.
Key Words: adventitia • endothelium • microscopy, confocal • remodeling • resistance arteries • rats, inbred SHR
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Introduction |
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The vascular wall is an active organ composed of endothelial cells, SMCs, and adventitial cells, which are coupled to each other. The wall is able to change its structure through a process known as remodeling. Remodeling is usually an adaptive process in response to long-term changes in hemodynamic conditions, as occurs during development,1 but it can also contribute to the pathophysiology of vascular diseases and circulatory disorders.2 Over the past decade, there has been extensive research concerning the structure of vessels and the process of remodeling in both physiological and pathological conditions and with special interest at the level of the resistance vasculature.1–5
Two methods are currently used to study gross morphology and the function of live small precapillary arteries with diameters of <500 µm: wire and perfusion myography. In the wire myograph, the vessel is mounted on two wires passed through the lumen, and measurements of wall thickness and lumen are taken with an eyepiece attached to a light microscope.6,7 In the perfusion system,8,9 similar measurements of vessel wall and internal diameter are obtained with a VDA from a pressurized artery.
Despite this extensive research and the increasing interest in the remodeling process in pathological situations, little is known at the cellular level about the morphology of intact arteries and the structural changes associated with them. Cellular changes in remodeling have been mainly studied in SMCs in culture. Cell culture studies, although valuable, lack critical interactions with other vascular cell types and natural matrices found in the vessel wall, so the results cannot be extrapolated to the changes in intact vessels. On the other hand, histological information on vessel cellular structure also has several limitations: the tissue must be sectioned and then reconstructed, so it is very labor intensive and has the disadvantage of being unable to provide a study of live tissue.
Confocal microscopy produces "optical sections" through semitransparent tissue without the need for cutting thin slices. It eliminates the blur and flare of out-of-focus planes in an object, has improved axial resolution that enables accurate three-dimensional reconstruction, and has rapid speed of image acquisition.10–12 With LSCM, we recently developed methods that allow the study of the structure-function relationship in resistance arteries, as close as possible to the cellular level.13–15
The aim of the present work was to use LSCM to study in intact resistance arteries the cellular changes associated with remodeling in hypertension. We first assessed the ability of the method to obtain simple dimensional measurements compared with the two methods presently available: wire and perfusion myography. We then examined the cellular arrangement of the coats within the vascular wall by LSCM and found several features, not described before, in the three layers of mesenteric resistance arteries from SHRSP, including important changes in the adventitia and endothelium, layers usually neglected in structural studies in hypertension.
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Methods |
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Animals
Rats were obtained from the Glasgow inbred colonies that have been maintained in the Department of Medicine and Therapeutics, University of Glasgow, since 1991.16 Thirteen WKY and eight SHRSP male or female 8- to 10-month-old rats were used. Systolic blood pressure was measured by the tail-cuff method (custom made; Department of Clinical Physics and Bio-Engineering, Western Infirmary, University of Glasgow). The rats were killed through inhalation of 3% to 4% halothane in oxygen for each experiment, according to institutional guidelines. The mesenteric bed was carefully removed and placed on a petri dish with PSS at 4°C for dissection of the third order branch of the superior mesenteric artery. Each artery was divided in two segments—one to be used on the wire and the other to be used on the perfusion myograph and subsequently on LSCM.
Drugs and Solutions
All experiments were performed with PSS of the following
composition (in mmol/L): NaCl 118.4, KCl 4.7, CaCl2
2.5, KH2PO4 1.2, MgSO4 1.2,
NaHCO3 25, and glucose 11.1. The solution was bubbled with
95% O2/5% CO2 to pH 7.4 at 37°C. Propidium
iodide and Hoechst 33342 trihydrochloride were purchased from Sigma
Chemical Co, and halothane was purchased from ICI Pharmaceuticals.
Wire Myography
A 2-mm-long segment was mounted on a wire myograph
(Myo-interface, model 410A; Aarhus). Briefly, two 40-µm tungsten
wires were threaded through the lumen of the segment. One wire was
attached to the stationary support driven by a micrometer,
and the other was attached to an isometric force transducer and this in
turn to a chart recorder (Linseis L6512B; Belmont Instruments) to
measure force development. After a 30-minute equilibration period at
37°C, the morphological measurements were determined on the vessels
held under just enough tension to hold the walls separated. In each
segment, the following dimensions were measured with a light microscope
(40x water-immersion objective, with a filar eyepiece, Zeiss; Welwyn):
wall thickness (average of six measurements), distance between the
inner edges of the wires (average of three measurements), and length
(average of two measurements). The resting tension–internal
circumference relation was determined for each segment according to
Mulvany and Halpern.7 From this relation, we calculated the
theoretical internal circumference of the artery if relaxed and under a
transmural pressure of 100 mm Hg (L100). The corresponding
internal diameter (d100) was calculated as L100/
. Each artery was
then set to 0.9d100 calculated with a modified computer
program. At this internal diameter wall thickness, wall-to-lumen ratio
and equivalent pressure were calculated. CSA, equivalent to wall volume
per unit length, was also calculated with the computer, assuming
constant wall volume.17
Perfusion Myography
Mesenteric resistance arteries were prepared for morphological
measurements with the use of pressure-perfusion myography (Living
Systems).8 In brief, the arterial segment was
secured between two glass cannulae using single strands of surgical
nylon suture. One cannula was closed with a syringe and the other was
connected to a system containing PSS, which in turn was linked to a
pressure-servo unit. This system allowed the pressure (or flow) in the
system to be precisely controlled and altered at will. The myograph was
a 10-mL vessel chamber with an input-and-output channel to allow
perfusion of PSS. One of the cannulae was attached to a
micrometer, permitting adjustment to allow changes in
longitudinal dimension of the mounted vessel without stretch or
buckling. The artery was imaged using a video camera and
analyzed with a VDA (Living Systems), which was linked to a
chart recorder (Linseis L6512B; Belmont Instruments). Each segment
was then pressurized at the equivalent pressure calculated from wire
myograph experiments, as described before. After a 30-minute
equilibration period at 37°C in oxygenated PSS, wall
thickness and internal lumen diameter were measured at six equidistant
points along the vessel segment with a VDA. At the end of the
experiment, the arteries were fixed under pressure with 10%
formal-saline solution for 30 minutes at room temperature and stored
for further LSCM analysis. Fig 1A
shows a photograph of the arterial wall from a perfusion
myograph–mounted segment measured with VDA.
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Comparison of Live and Fixed Tissue
Five arteries from WKY were used to compare perfusion myography
and LSCM measurements under live and fixed conditions. The arteries
were first set at 50 mm Hg (mean equivalent pressure
calculated from wire myograph previous experiments at
0.9d100). After a 30-minute equilibration period in PSS at
37°C, measurements were taken with the perfusion myograph VDA. The
PSS was then substituted for one containing 0.01 mg/mL concentration of
the vital dye Hoechst 3334218 for 30 minutes. To enhance
the stain of the endothelium, the PSS with the dye was
also perfused intraluminally for 2 minutes at a constant flow rate of
0.1 mL/min. The chamber was then placed on the stage of the LSCM, and
images of the wall and lumen were acquired as described below. Finally,
the vessel was fixed under pressure as previously described, and it was
measured again with VDA and LSCM, after several washouts with PSS.
LSCM
The fixed segments were incubated for 30 minutes in 10%
formal-saline solution containing 10 µmol/L of the nuclear dye
propidium iodide19 and washed overnight. The vessel was
placed on a slide and covered with a coverslip attached to the slide by
a thick layer of vacuum grease. This formed a small chamber for the
artery, avoiding contact of the coverslip with the segment and
therefore minimizing possible changes in wall shape. The vessel was
then placed under an upright LSCM (Odyssey LSCM; Noran Instruments)
with a Nikon microscope. The lumen was visualized with a 10x air
objective (Nikon, NA 0.5), and the wall was visualized with a 40x oil
immersion objective (Nikon, NA 1.3), using the argon ion 488-nm line
with a 515-nm LP barrier filter of the LSCM (pinhole aperture, 15
µm).
Live tissue was stained with Hoechst 33342 as previously described, and the artery was visualized while mounted and pressurized on the myograph. The lumen was visualized with a 10x water objective (Nikon, NA 0.3), and the wall was visualized with a 40x water objective (Zeiss, Welwyn, NA 0.75), using the 364-nm line (no barrier filter) of the LSCM (pinhole aperture, 15 µm).
Lumen images were taken in the X-Y plane by placing the
focus on the middle of the vessel (Fig 1
, schematic diagram). This
allowed visualization of the lumen and the cell nuclei in profile (Fig 1C). Six images of different regions were captured along the artery,
and they were stored and coded for further analysis. This
protocol was identical for fixed or live tissue.
Wall thickness measurements were taken in the X-Y plane
and/or the Z axis. For X-Y measurements, the
plane of focus was placed on the middle of the artery, and 12 images of
the wall were captured along the segment. This protocol was identical
for fixed or live tissue. The wall images clearly showed adventitial,
medial, and intimal layers (Fig 1B). However, the limits of the intima
and adventitia were not always clear, and only the media was measured.
A minimum of two measurements from each image of both lumen and media
were taken with MetaMorph software (Universal Imaging Corporation). To
determine the degree of consistency of X-Y
measurements, they were obtained by three different observers and
compared statistically with ANOVA test for interobserver
reliability.
The different layers of small vessels stained with nuclear dyes can be clearly distinguished with confocal microscopy according to the shape and orientation of the cell nuclei.14,15 Nuclear dyes also provide the advantage of showing the cells as individual single objects, allowing for direct simple measurements with image analysis software, including the number of cells in each layer and the endothelial cell nuclei area and shape factor (a value from 0 to 1 that represents how closely the object resembles a circle).
Such a quantitative analysis can be done on the assumption that
the dyes stain all nuclei of vascular cells. To prove this assumption,
the pattern of nuclei stained with Hoechst 33342 was compared with that
obtained with hematoxylin and eosin, a conventional
histological protocol used for morphometric studies.
For this purpose, an artery was stained first with Hoechst 33342 as
described before, fixed under pressure, embedded in liver, and
sectioned on a cryostat set at 10 µm. A section was then mounted
on a slide and visualized with LSCM (40x water objective, 364 nm, no
barrier filter). Ten optical slices (1 µm thick) were captured
in Z axis from several regions of the section, and a
three-dimensional image from each region was then reconstructed with
MetaMorph software. The coverslip was subsequently removed from the
preparation, and the section was then stained with hematoxylin and
eosin and visualized with a Zeiss Axiophot microscope with a 20x air
objective. Images from several regions of the vessel section were then
taken with NIH Image 1.6 software. A comparison of Hoechst 33342– and
hematoxylin and eosin–stained images at the same level of
magnification showed a similar pattern of nuclei. In addition, LSCM
images had a better resolution and allowed individual nuclei to be more
clearly defined (Fig 2).
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The different wall layers (adventitia, media, and intima) were measured
in Z axis. Stacks of 1-µm-thick serial optical slices were
taken in Z axis from the first adventitial to the last
endothelial nuclei. Fig 3A shows a schematic diagram that
illustrates the process of stack capturing in Z axis. Three
of these stacks were captured in different regions along the vessel
length and stored for further analysis with MetaMorph software.
This protocol was identical for fixed or live tissue. Under conditions
used in the present study, Z-axis resolution, given by
the LSCM microscope manufacturer, was 0.5 µm for 40x
oil-immersion objective and 1.2 µm for 40x water-immersion
objective.
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To have reliable and reproducible data, we defined the location of the first images from each layer based on computer measurement of fluorescence intensity. Thus, the first image of the adventitia in the stack was considered to be that which showed the first adventitial nucleus at its maximum intensity. In a similar way, the first images of the media and intima were defined. The last plane of the intima was considered to be that which showed the last endothelial nucleus at its maximum intensity. From these stacks of images, thickness and nuclei number were measured in each coat by image analysis.
In WKY arteries, there was an homogeneous distribution of endothelial cells. However, in SHRSP arteries, the number of endothelial nuclei was variable along the vessel length. Therefore, to have an accurate average number of the endothelial cells in the vessel, 10 images of the intima were also taken from each artery. This approximately covered the entire vessel length, excluding the extremes, which could have been damaged by the cannulae.
Wall CSA was calculated on the basis of the wall and lumen measurements
obtained with VDA or LSCM, using the following formula: CSA=external
CSA-internal CSA, where external CSA=(lumen/2+wall)2
and internal CSA=(lumen/2)2.
To allow comparison of control and remodeled vessels, the following
calculations were performed on the basis of 1-mm-long segments: vessel
volume (in mm3); volume=wall CSA
(mm2)x1 mm; adventitial, medial, and intimal volumes
(volume=respective layer CSA (mm2)x1 mm); total
number of cells (adventitial, smooth muscle, or
endothelial) (cell n=n of nuclei per stackxn of stacks
per vessel volume); luminal surface calculated from vessel diameter
(in mm2); luminal surface=2diameter/2; total number
of endothelial cells was calculated per luminal surface
of 1-mm-long vessel (endothelial cell n=endothelial
cells/mm2xluminal surface).
Statistical and Data Analyses
Results are expressed as mean±SEM, and n denotes the number of
animals used in each experiment. Statistical comparisons were made
using Student's t test for paired or unpaired experiments
and one-way ANOVA for interobserver reliability experiments. A value of
P<.05 was considered significant.
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Results |
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Validation of LSCM for Morphological Analysis
Comparison Between Wire and Perfusion Myograph Measurements
In WKY, comparison between wire-mounted segments set at 0.9d100 and arteries pressurized at their equivalent pressure showed that lumen was significantly larger and wall thickness and wall-to-lumen ratio were significantly smaller in pressurized vessels (Fig 4
). CSAs were similar with
both methods (wire myograph, 27 375±1160 µm2;
perfusion myograph, 25 422±2490 µm2; n=7).
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In SHRSP arteries, there was no significant difference between wire or
perfusion myography measurements of lumen, wall thickness,
wall-to-lumen ratio (Fig 4), or CSA (wire myograph, 42 525±5068
µm2; perfusion myograph, 40 277±4318
µm2).
Comparison Between VDA and LSCM Measurements in Fixed
Arteries
Lumen dimensions were similar when taken with VDA or LSCM in both
WKY (Fig 5A) and SHRSP (data not
shown).
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In WKY, wall thickness was significantly greater when measured
with LSCM compared with VDA (Fig 5B). CSA was also significantly larger
when measured with LSCM compared with VDA (CSA LSCM=38 104±2577
µm2, CSA VDA=25 422±2490 µm2; n=5,
P<.05, paired experiments). LSCM measurements of the medial
layer were similar whether measured in the X-Y
(19.8±1.1 µm) or Z (21.3±1.6 µm, n=5, paired
experiments) direction. In addition, these LSCM measurements of media
were similar to the "wall" measurements estimated with perfusion
myography VDA (23.0±0.25 µm, n=5, paired experiments). Similar
results were found in SHRSP rats (data not shown).
Effect of Fixation on WKY Artery Structure
The effect of fixation was tested only in arteries from WKY. VDA
and LSCM measurements were compared.
Fixation produced a small but significant reduction of lumen, which was
similar for the two methods (VDA, 12%; LSCM, 13%) (Fig 5A).
Fixation had no significant effect on wall thickness or CSA when
measured by either method (Fig 5B). With LSCM, it was possible to
distinguish between cell types within the vessel wall by the shape of
the nuclei and therefore to measure the thickness of the different
layers (adventitia, media, and intima) in the Z axis.
Fixation produced no significant difference in the thickness of any of
the layers (Fig 5C).
Accuracy of LSCM Measurements
Measurements in Z axis were calculated with the
computer program on the basis of fluorescence intensities of
stained objects in the image. To determine the reliability of
measurements in X-Y plane (more susceptible to
subjectivity), the samples were blindly taken and the images were
measured by three different observers. Statistical analysis
with one-way ANOVA showed no significant difference between observers
in measurements of lumen (observer A, 369.46 pixels; observer B, 360.8
pixels; and observer C, 354.9 pixels; P=.93) or media
(observer A, 166.7 pixels; observer B, 173.7 pixels; and observer C,
198.2 pixels; P=.33).
Comparison of WKY and SHRSP Mesenteric Artery Morphology
Systolic blood pressure of SHRSP was significantly higher
compared with that of WKY (SHRSP, 164±9.8 mm Hg; WKY,
122±4.3 mm Hg; P<.001).
All three methods showed a significantly smaller lumen and larger wall
thickness and wall-to-lumen ratio in SHRSP compared with WKY (Fig 4 and
Table 1). With LSCM, it was also possible
to distinguish between cell types within the vessel wall on the basis
of the shape of the nuclei and therefore to determine the structure of
the different coats (adventitia, media, and intima) in Z
axis. The changes observed in each layer are described below.
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Changes in the Morphology of the Adventitia
SHRSP mesenteric arteries showed similar adventitial thickness
(Fig 6A, Table 1) and volume (Fig 6B)
compared with WKY arteries. The total number of cells (Figs 3B and 6C)
and density of cells in the adventitia (Fig 6D) were significantly
higher in SHRSP arteries.
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In WKY arteries, there was a clear separation between adventitial and
medial coats; adventitial cells were confined to its layer, and the
maximum depth within the media at which adventitial cells could be
found was 1.45±0.6 µm. However, in SHRSP, cells with an
irregular nuclei shape similar to those of adventitial cells were found
in deep layers of the medial coat; the maximum penetration within the
media of these adventitia-like cells was 14.4±3.9 µm
(P<.01 compared with WKY). This abnormality can be observed
in the three-dimensional reconstruction of the media in Fig 3B
(middle); in SHRSP, elongated SMC nuclei coexist with irregular
adventitia-like nuclei (top), whereas in WKY arteries, only SMC nuclei
were found.
Changes in Morphology of the Media
In SHRSP arteries, medial thickness (Fig 6A, Table 1) and volume
(Fig 6B) were significantly greater compared with WKY vessels. There
was no difference in the total SMC number between WKY and SHRSP (Figs 3B and 6C). The density of SMC in the media was significantly reduced
in SHRSP (Fig 6D).
Changes in Morphology of the Intima
Intima thickness and volume were similar in both rat strains (Fig 6A and 6B), but the luminal surface was reduced in SHRSP (Table 2). In SHRSP mesenteric arteries, there
was a significant reduction in the total number of
endothelial cells (Fig 6C); however, the density of
endothelial cells per mm2 (Table 2)
remained unchanged (ie, the cell number was reduced in proportion of
the decrease in luminal surface). In SHRSP segments, the cells were not
homogeneously distributed along the artery (data not
shown). In SHRSP, there was a significant reduction in nuclei area, and
the shape of the endothelial nuclei was variable;
however, the mean of the shape factor was not statistically different
between strains (Table 2, Fig 3B).
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Discussion |
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The aim of this study was to make a direct quantitative analysis of the cellular basis of vascular remodeling in hypertension using a method based on LSCM in intact vessels and compare it with previous methodology.
LSCM provides a rapid and accurate new method for determining the morphology of whole resistance arteries in live and fixed conditions. It correlates well with the more limited data available from wire and perfusion myography.
LSCM has several advantages over previous methodology for morphological measurements. First, it provides a higher resolution of image. This allows for straightforward measurements at the cellular level with image analysis software, including automated counting of different types of cells as well as determination of several cell parameters such as nuclear shape, orientation, and area. Second, it can be performed in intact arteries without the need for histological section. Third, it gives objective and bias-free data on basic dimensions. Therefore, LSCM represents an important advantage over labor-intensive histological procedures; even in improved methods such as the three-dimensional dissector, the tissue has to be sectioned and photographed, and pictures must be assessed by eye.20
In addition, LSCM showed differences that had not been found previously in the cellular characteristics of adventitial, medial, and intimal coats of mesenteric resistance arteries from SHRSP compared with WKY. This finding provides new and potentially important information on the remodeling process in hypertension and other vascular diseases that is relevant to understanding the pathogenesis and developing new therapeutic approaches.
Remodeling of SHRSP Mesenteric Resistance Arteries
It is well established that hypertension is associated with
structural changes in the resistance vasculature, such as reduced lumen
and increased wall-to-lumen ratio. These changes can be associated with
net growth of the vessel or rearrangement of wall material
(remodeling).3 This previous concept of remodeling has been
redefined, and remodeling is now considered to be a more complex
process that may include growth as well as rearrangement and either
reduction or increase in vessel lumen.21 Our results
suggest that mesenteric resistance arteries from SHRSP present
hypertrophic inward remodeling, resulting from a reduced vessel lumen
and an increased wall volume. We use "hypertrophic" here to refer
to tissue volume, not to cell size.
There is controversy about the nature of structural vascular changes associated with genetic and experimental hypertension in rats. Some reports show that the media of small mesenteric arteries from spontaneously hypertensive rats is thicker due to an increased number of SMCs.22 On the contrary, in New Zealand genetically hypertensive23 and renal hypertensive24 rats, the changes in wall growth are not due to SMC hyperplasia. Our results with LSCM, in which the integrity of wall structure is preserved, also point in this latter direction. In SHRSP mesenteric arteries, the volume of the media was greater by 32% compared with WKY, without changes in the number of SMCs. Within a given length of artery, there is an increase in the average volume occupied by each SMC and its associated extracellular space/matrix. Because in the present study we measured the number of nuclei (hence number of cells) per unit volume, we cannot distinguish whether there is an increase in cell volume or in extracellular space/matrix, both of which have been proposed before.23 Rearrangement of SMCs was found in the cerebral vasculature of SHRSP,25 so rearrangement of a constant number and size of SMCs within a larger coat volume remains an attractive possibility.
All previous studies on resistance artery vascular morphology have been focused on the changes in the media, probably due to the difficulty in quantifying adventitia and intima. With LSCM, which allows the study of these layers, we have been able to demonstrate almost doubling of adventitial cell number in the mesenteric vasculature of SHRSP rats compared with WKY. Similar alteration has been previously reported in coronary arteries after balloon injury, suggesting that adventitia can play an important role in the process of repair.5 Adventitial thickness remained unchanged, and therefore this layer did not contribute to the overall increase in wall volume; however, the increase in adventitial cell number suggests that this layer might contribute to the changes in wall architecture and thus play an active role in the remodeling process. The finding of irregularly shaped nuclei in the medial layer is also interesting. One possible explanation is that these nuclei correspond to adventitial cells; if that were the case, this would imply a migration of these cells from the outermost part of the vessel into the media and further supports the hypotheses of an active role of the adventitia in the remodeling process. An second explanation is that these nuclei correspond to disoriented SMCs, similar to the alteration found in the SHRSP cerebral vasculature, in which there was a rearrangement of SMCs in X-Y plane.25 However, the spherical shape of the nuclei found in the medial coat in the present study supports the hypotheses of a change in orientation in Z axis rather than in X-Y plane. A third alternative would be that those irregular nuclei correspond to a different phenotype of SMCs.
Endothelium is potentially an important element in the genesis and development of remodeling.2 It is an important determinant in vivo of the lumen dimensions and overall vessel structure; structural changes in vessel diameter due to changes in blood flow are dependent on an intact endothelium.26 In addition, the endothelium is in contact with circulating substances, including mitogens, and therefore may be the first to undergo structural changes in abnormal situations. In SHRSP, the total number of endothelial cells was reduced by half compared with WKY arteries. However, when the number of endothelial cells was expressed per mm2, there was no difference between strains due to the reduction in luminal surface in SHRSP arteries. Thus, this change in the endothelium could reflect a primary change as part of the remodeling process associated with hypertension, but it could also indicate a secondary adaptation of the endothelium to the narrower lumen, such as to maintain the density of endothelial cells.
Another notable feature was the heterogeneous distribution of endothelial cells. Some regions of the luminal surface from SHRSP arteries were almost devoid of endothelial nuclei, whereas others had normal numbers of cells. In SHRSP arteries, the surface areas of the individual nuclei were also significantly smaller, and the nuclear shape was variable. It is possible that these abnormalities could be associated with endothelial dysfunction such as the impairment of endothelium-dependent relaxations, which has been described in hypertension.27,28
Differences Between Wire and Perfusion Myography
In a few studies, wire and perfusion myography have
compared.29–31 Like these previous reports, our study
shows a larger lumen, smaller wall thickness, and smaller wall-to-lumen
ratio for perfusion compared with wire myography. The gold standard
usually used in wire myography, defined by Mulvany and
Halpern,7 and on which we have based the conditions for our
study, is the theoretical internal circumference that the artery should
have if relaxed and under a transmural pressure of 100 mm Hg: the
actual measurements on which this calculation is based are taken with
the wires just under tension. In contrast, in the pressure myograph,
the vessel morphology is estimated directly with a VDA. It has been
argued that part of the difference may thus lie in the theoretical
assumptions necessary for the wire myography, exacerbated perhaps by
other physical differences such as the axial distension with increasing
pressure that occurs in the perfusion myograph, but not experienced on
a wire myograph.30
Our objective was to validate our basic myography technique against the known standards. This was accomplished in relation to both the differences between the two established methods and the gross "remodeling" in the hypertensive model shown by changes in lumen, wall thickness, and wall-to-lumen ratio, which was demonstrated by either method. We then chose the perfusion myograph, a more physiological method, to apply LSCM because the measurements are directly comparable with the VDA system: the comparison is based on real measurements of the same physical situation using different measuring instruments and allows the maintenance of the vessel shape at different distending pressures.31
Differences Between LSCM and Perfusion Myography
Using tissues from WKY, we found significantly larger wall
thickness and CSA measurements with LSCM, under both live and fixed
conditions. This was not due to a general calibration problem of the
LSCM because lumen measurements were similar when taken with both
methods, provided the conditions were the same (live or fixed). This is
also supported by the similarity of LSCM measurements of the media
whether taken on the X-Y plane or the Z axis. We
suggest that the difference observed in wall measurements between
methods is due to an underestimation of wall thickness when measured
with the perfusion myograph VDA. Adventitia is more translucent than
the media, and it is difficult to detect when the vessel is brightly
illuminated for optimal visualization of the lumen. As seen in Fig 1A, it is likely that much of the adventitia and intima are not included in
the "wall" measurement taken with the VDA. Therefore, what has been
defined as "wall" in previous studies probably was mainly media,
resulting in underestimation of adventitia and
endothelium. In addition, the terminology used in the
literature to define "wall" measurements is confusing, reflecting
the difficulty of clearly measuring different layers. Some studies use
the term "wall thickness,"7,29,31,32 others use
"media thickness,"24,33,34 and one established that
"media" was clearly discernible but considered it
"wall."30
In the images captured with LSCM in the X-Y plane, the adventitia and endothelium were more clearly discernible than with VDA. Nevertheless, we considered the images to be inadequate for providing an exact measurement of these layers because the outer limits of adventitia and intima were not always clearly shown. Conversely, the stacks of sharp nuclei, obtained at sequential Z-axis depths, allow the construction of accurate three-dimensional images. The ability to distinguish between cell types with the use of nuclear dyes14,15 allows visualization and separate measurement within the different layers in the wall. The limiting factor for LSCM measurements is the quality of the images, which is in turn dependent on the thickness of the specimen. That means that the thickness of the wall represents a limitation of the technique, and the present version of this method for intact pressurized vessels is restricted to resistance arteries or thinner vessels. Another important factor is the capacity of the different cell nuclei to capture the dye; this is always less for the endothelial nuclei, even when the dye is applied intraluminally.
Effect of Fixation
We considered it important to determine the effect of fixation on
lumen and wall dimensions for three reasons. First, the ability to use
fixed tissue greatly facilitates the experimental process because it
can be stored for later analysis, particularly when combined
with histochemical methods, which are not practicable in live tissue.
Second, the use of water-immersion lenses, which are necessary for live
tissue measurements, limits the quality of image because they generally
are not available with Plan Apochromatic corrections and cannot be made
with a high NA. This is an essential feature, especially for
Z-axis measurements, because the axial resolution rises with
the square of the NA.10 Third, fixation allows the use of a
wider range of dyes, including membrane-impermeable dyes such as
propidium iodide, that can be detected with a red-green filter with a
very good signal.19
Fixatives are known to shrink tissues. Formaldehyde, the fixative used in our study, is superior to others, minimizing this problem, especially when prepared in saline solution.35,36 In our experimental conditions, the adventitia, media, and intima thickness were not affected by this fixative. However, lumen demonstrated a small reduction, an effect that was equally observed with VDA or LSCM measurements. This effect could be partially due to a slight contraction of the vessel produced by the change in temperature (from the PSS at 37°C to the formal-saline solution at room temperature). This temperature-induced contraction could be further minimized by heating the fixative to 37°C before its addition to the perfusion bath.37
In conclusion, LSCM uncovered previously unknown aspects of the remodeling of the vascular wall in SHRSP rats, a genetic model of hypertension. This included changes in adventitia, a layer that has not been previously assessed in studies of small vessel physiology. LSCM is a reliable method with which to measure morphology of intact resistance arteries in live and fixed conditions, and it has several advantages over previous methodology. Limitations of the technique are the development of fluorescent dyes and the improvement of objective lenses and of software for image analysis. We are confident that the further development of LSCM for measurements of vascular morphology will help us to gain knowledge about the structure of resistance arteries and its changes during remodeling in different pathological conditions.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by grants from the British Heart Foundation (PG 95123 and FS93025), Medical Research Council Clinical Research Initiative in Heart Failure (PG 9307850), and Wellcome Trust (045924/Z/95). Dr González was a recipient of a short-term fellowship PR95–392 from DGICYES, Spain. We are grateful to Craig Daly for his advice with LSCM and Dr Ian Montgomery for his help with histology.
Received June 9, 1997; first decision June 27, 1997; accepted June 27, 1997.
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References |
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|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
-
Wong LCW, Langille BL. Developmental remodeling of
the internal elastic lamina of rabbit arteries: effect of blood flow.
Circ Res. 1996;78:799–805.
[Abstract/Free Full Text] -
Gibbons GH, Dzau VJ. The emerging concept of vascular
remodeling. N Engl J Med. 1994;330:1431–1438.
[Free Full Text] -
Baumbach GL, Heistad DD. Remodelling of cerebral
arterioles in chronic hypertension. Hypertension. 1989;13:968–972.
[Abstract/Free Full Text] -
Deng LY, Schiffrin E. Morphologic and functional
alterations of mesenteric small resistance arteries in the early renal
hypertension in the rat. Am J Physiol. 1991;261:H1171–1177.
[Abstract/Free Full Text] -
Shi Y, Pienick M, Fard A, O'Brien J, Mannion JD,
Zalewski A. Adventitial remodeling after coronary
arterial injury. Circulation. 1996;93:340–348.
[Abstract/Free Full Text] - Mulvany MJ, Halpern W. Mechanical properties of vascular smooth muscle cells in situ. Nature (Lond). 1976;260:617–619.[Medline] [Order article via Infotrieve]
-
Mulvany MJ, Halpern W. Contractile properties of small
arterial resistance vessels in spontaneously hypertensive
and normotensive rats. Circ Res. 1977;41:19–26.
[Free Full Text] - Halpern W, Osol G, Coy GS. Mechanical behaviour of pressurised in vitro prearterial vessels determined with a video system. Ann Biomed Eng. 1984;12:463–479.[Medline] [Order article via Infotrieve]
- Halpern W, Kelley M. In vitro methodology for resistance arteries. Blood vessels. 1991;28:245–251.[Medline] [Order article via Infotrieve]
- Inoué S. Foundations of confocal scanned imaging in light microscopy. In: Pawley JB, ed. The Handbook of Biological Confocal Microscopy. New York, NY: Plenum Press; 1995:1–17.
- Stelzer EHK, Wacker I, De Mey JR. Confocal fluorescence microscopy in modern cell biology. Cell Biol. 1991;2:145–152.
- King R, Delaney PM. Confocal microscopy in pharmacological research. TIPS. 1994;15:275–279.
- Daly CJ, Gordon JF, McGrath JC. The use of fluorescent nuclear dyes for the study of blood vessel structure and function: novel applications of existing techniques. J Vasc Res. 1992;29:41–48.[Medline] [Order article via Infotrieve]
- Arribas SM, Daly CJ, Gordon JF, McGrath JC. Confocal myography: applications for the study of resistance arteries. In: Halpern W, Bevan JA, Brayden J, Dustan H, Nelson M, Osol G, eds. The Resistance Arteries: Integration of Regulatory Pathways. Piscataway, NJ: Humana Press; 1994:259–264.
- McGrath JC, Arribas SM, Daly CJ, Gordon JF. Confocal microscopy for structure and real-time pharmacology in blood vessels. J Human Hypertens. 1995;9:645–647.[Medline] [Order article via Infotrieve]
- Dominiczak AF, McLaren Y, Kusel JR, Ball DL, Goodfriend TL, Bohr DF, Reid JL. Lateral diffusion and fatty acid composition in vascular smooth muscle membrane from stroke prone spontaneously hypertensive rats. Am J Hypertens. 1993;6:1003–1008.[Medline] [Order article via Infotrieve]
-
Carew TE, Vaishnav RN, Patel DJ. Compressibility
of the arterial wall. Circ Res. 1968;23:61–68.
[Abstract/Free Full Text] - Daly CJ, Gordon JF, McGrath JC. Fluorescence image analysis methods for the study of smooth muscle cell interaction within the wall of small blood vessels. In: Mulvany MJ, Aalkjer C, Haegerty AM, Nyborg NCB, Strandgaard S, eds. Resistance Arteries: Structure and Function. Amsterdam, Netherlands: Elsevier Science Publishers; 1991:339–343.
- Ockleford CD, Hsi B, Wakely J, Badley RA, Whyte A, Pagefaulk W. Propidium iodide is a nuclear marker in immunofluorescence, I: use with tissue and cytoskeleton studies. J Immunol Methods. 1981;43:261–267.[Medline] [Order article via Infotrieve]
- Baandrup U, Gundersen HJG, Mulvany MJ. Is it possible to solve the problem: hypertrophy/hyperplasia of smooth muscle cells in the vessel wall of hypertensive subjects? Prog Appl Microcirc. 1985;8:122–128.
- Mulvany MJ, Baumbach GL, Aalkajaer Ch, Heagerty AM. Vascular remodelling. Hypertension. 1996;28:505–506.
-
Owens GK, Schwartz SM, McCanna M. Evaluation of medial
hypertropy in resistance vessels of spontaneously hypertensive rats.
Hypertension. 1988;11:198–207.
[Abstract/Free Full Text] - Ledingham JM, Millar JA. Stereological studies on mesenteric resistance artery structure in New Zealand genetically hypertensive and control rats. Clin Exp Pharmacol. 1993;20:359–361.
-
Kosgaard N, Mulvany MJ. Cellular
hypertrophy in mesenteric resistance vessels from renal
hypertensive rats. Hypertension. 1988;12:162–167.
[Abstract/Free Full Text] -
Arribas SM, Gordon GF, Daly CJ, Dominiczak AF, McGrath
JC. Confocal microscopic characterization of a lesion in a cerebral
vessel of the stroke-prone spontaneously hypertensive rat.
Stroke. 1996;27:1118–1123.
[Abstract/Free Full Text] -
Langille BL, O'Donnell F. Reductions in
arterial diameter produced by chronic decreases in blood
flow are endothelium-dependent. Science. 1986;231:405–407.
[Abstract/Free Full Text] - Benett A, Hillier C, Thurston H. Endothelium-dependent relaxation in resistance arteries from spontaneously hypertensive rats: effect of long term treatment with perindopril, quinapril, hydralazyne or amlodipine. J Hypertens. 1996;14:389–397.[Medline] [Order article via Infotrieve]
- Shiffrin EL. Correction of remodeling and function of small arteries in human hypertension by cylazapril, an angiotensin I-converting enzyme inhibitor. J Cardiovasc Pharmacol. 1996;27(suppl 2):S13–S18.
- Lew MJ. Wall stress and wall to lumen ratios differ between pressurised and myograph mounted arteries. In: Mulvany MJ, Aalkjer C, Haegerty AM, Nyborg NCB, Strandgaard S, eds. Resistance Arteries: Structure and Function. Amsterdam, Netherlands: Elsevier Science Publishers; 1991:259–264.
- Lew ML, Angus JA. Wall thickness to lumen diameter ratios of arteries from SHR and WKY: comparison of pressurised and wire-mounted preparations. J Vasc Res. 1992;29:435–442.[Medline] [Order article via Infotrieve]
-
Falloon BJ, Stephens N, Tulip JR, Heagerty AM.
Comparison of small artery sensitivity and morphology in pressurized
and wire-mounted preparations. Am J Physiol. 1995;268:H670–H678.
[Abstract/Free Full Text] -
Haegerty AM, Aalkajaer C, Bund SJ, Korsgaard N, Mulvany
MJ. Small artery structure in hypertension: dual process of remodeling
and growth. Hypertension. 1993;21:391–397.
[Free Full Text] -
Mulvany MJ, Hansen PK, Aalkajaer C. Direct evidence
that the greater contractility of resistance vessels of
spontaneously hypertensive rats is associated with a narrower lumen, a
thicker media and a greater number of smooth muscle cell layers.
Circ Res. 1978;43:854–864.
[Abstract/Free Full Text] -
Bund SJ, West KP, Heagerty AM. Effects of protection
from pressure on resistance artery morphology and reactivity in
spontaneous hypertensive and Wistar-Kyoto rats. Circ Res. 1991;68:1230–1240.
[Abstract/Free Full Text] - Baker JR. Introduction to fixation. In: Mellanby K, ed. Cytological Technique, The Principles Underlying Routine Methods. London, UK: Methuen and Co; 1966:14–30.
- Drury RAB, Wallington EA. Fixation. Carleton's Histological Technique. London, UK: Oxford University Press; 1967:33–47.
- Fox CH, Johnson FB, Whitung J, Roller PP. Formaldehyde fixation. J Histochem Cytochem. 1985;33:845–853.[Medline] [Order article via Infotrieve]
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