Vol. 279, Issue 4, H1591-H1599, October 2000
Skin microvascular adaptations during maturation and aging of
hairless mice
Brigitte
Vollmar,
Martin
Morgenthaler,
Michaela
Amon, and
Michael D.
Menger
Institute for Clinical and Experimental Surgery, University of
Saarland, D-66421 Homburg/Saar, Germany
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ABSTRACT |
Using intravital fluorescence microscopy in the ears of
hairless mice, we determined skin microvascular adaptations during the
process of aging from juvenile to adult and senescent life (6-78
wk). Despite an increase of ear area within the first 36 wk,
the number and branching pattern of both arteriolar and venular microvessels remained constant during the whole life period. Both arterioles and venules exhibited an increase in length, diameter, and
intervascular distance up to the age of 36 wk. With the increase of the
size of the ears, the observation that cutaneous capillary density
remained unchanged implied new capillary formation. During aging to 78 wk, capillary density in the ears was reduced to ~40%. Functional
analysis revealed an appropriate hyperemic response to a 2-min period
of ischemia during late juvenile and adult life, which, however, was
markedly reduced during senescence. Thus, except for capillaries, there
is no indication for age-related new vessel formation. The process of
aging from adult to senescent life does not cause any significant
remodeling but is associated with a decrease of nutritive perfusion and
a functional impairment to respond to stimuli such as ischemia.
growth; tortuosity; elongation; intervascular distance; capillary
density
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INTRODUCTION |
INCREASE OF TISSUE
MASS during juvenile maturation may require distinct changes of
the microvasculature aiming at adequate blood flow for the enlarged
tissue. In general, angiogenic processes, i.e., new vessel
formation, but also remodeling and elongation of existing microvessels,
are potential ways that the microvasculature may meet the needs of
increasing tissue mass from juvenile to adult life (17,
18). During senescence, nutritional needs of organs might again
change when the onset of functional decline occurs. Detailed
information on age-related adaptations of the microvasculature is
necessary for the understanding of development and aging of an
organism. However, so far there are no studies dealing with changes in
vascular architecture of any individual tissue during the whole life
span of an animal. This is, at least in part, because of the fact that
the analysis of microvascular development essentially requires the
study of identical regions of tissue over time and that most of the
animal models do not satisfy this requirement and do not permit
quantitative analysis without surgery and invasive handling of tissue.
With the present intravital fluorescence microscopic study, we
introduce an experimental approach in the hairless mouse that allowed
us to repetitively assess the skin microvasculature over the extended
time period of an animal's life without the need of surgical
preparation. The mouse ear is accessible with minimal handling and
provides a fingerprint-like angioarchitecture, making any vessel
identification redundant. The major purpose was to determine both
vascular adaptations of the skin during growth of mice from juvenile to
adult and during aging from adulthood to senescent life, respectively.
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METHODS |
Animals.
In conformity with the guiding principles for research involving
animals, eight male homozygous (hr/hr) mice
(3, 7) purchased from Charles River Laboratories
(Sulzfeld, Germany) were used in this study. The animals were
housed in single cages at a temperature of 22-24°C at a relative
humidity of 60-65% with a 12:12-h day-night cycle. The animals
were allowed free access to drinking water and standard laboratory chow
(Altromin, Lage, Germany). At the first experimental time point, mice
were 6 wk old. This age was selected so that the animals, although
clearly immature, were well beyond weaning (~3 wk) and were free of
neonatal characteristics. At 6 wk of age, the mouse ear, which consists of a single layer of cartilage sandwiched between two full dermal layers of skin (overall thickness 300 µm), is completely naked (3). Blood supply is provided by three vascular bundles,
entering the ear at its base and forming an interconnecting capillary
network at the periphery.
Intravital fluorescence microscopy.
For the analysis of the cutaneous microcirculation of the ear, the mice
were intraperitoneally anesthetized with a mixture of ketamine (90 mg/kg body wt) and xylazine (25 mg/kg body wt) and placed prone on a
Plexiglas pad. After injection of 0.15 ml 5%
fluorescein-isothiocyanate (FITC)-labeled dextran (molecular weight
150,000; Sigma Chemical, St. Louis, MO) via a tail vein, the ear to be
investigated was gently extended over a microscopic slide embedded into
the pad and covered with an oxygen-impermeable plastic wrap. The
anesthetized mice were then placed with the Plexiglas pad under an
intravital Leitz Orthoplan microscope (Leitz, Wetzlar, Germany). The
epi-illumination microscopic setup included a 100-W mercury lamp and a
Ploemopak illuminator equipped with an I2 blue filter (450- to 490-nm excitation, >580-nm emission wavelength). Microscopic images
were recorded by a charge-coupled device video camera (CF8/1FMC, Kappa,
Gleichen, Germany) and recorded on videotape (Panasonic AG-7350-SVHS,
Matsushita, Tokyo, Japan) for subsequent offline evaluation.
Analysis of skin microvasculature.
The microscopic procedure for analysis of skin microvasculature was
performed at a constant room temperature of 23°C. Contrast enhancement for visualization of the cutaneous vasculature with the
individual microvascular segments was provided by FITC-dextran (mol wt
150,000), which binds neither to endothelial cells nor to individual
blood cells and which does not extravasate under physiological
conditions. The dye remains for ~4 h within the intravascular space
and is cleared from the circulation by both the liver and the kidney.
Leitz objectives [×2.5, NA (numerical aperture) = 0.08; ×10,
NA = 0.3; and ×20 long distance (L), NA = 0.32]
were used in the recordings and produced a total magnification of
approximately ×100, ×400, and ×800 at the video monitor. With the
×2.5 objective, the extended ear was scanned in a meandering manner through the entire longitudinal and transverse lengths to
monitor the whole area as well as to count the number of arterioles (first, second, third, and fourth order) and venules (first, second, third, fourth, and fifth order). At the first time point of observation (6 wk), distinct tissue regions of interest, which had an easily identifiable branching pattern of either arterioles or venules, were
selected and video printouts were made during videography using the
×10 objective. Within those tissue regions, functional capillary
density, i.e., the length of red blood cell-perfused capillaries per
observation area (cm/cm2), was monitored and analyzed by
means of the ×20 L objective. The video prints were initially marked
to indicate the exact locations for measurements of length, diameter,
tortuosity, and distances between the individual microvessels.
All parameters were analyzed offline with a computer-assisted image
analysis system (CapImage, Zeintl Software, Heidelberg, Germany)
(10), including planimetric assessment of ear area from
the ×100 recordings by tracing along the boundaries of each ear
section with a computerized digitizing tablet. From the ×400 recordings, functional capillary density, tortuosity of arterioles and
venules, arteriolar and venular diameters, red blood cell velocity, and
arteriolar and venular vessel segment length were assessed. Red blood
cell velocity was determined by means of the line-shift method
(modified "frame-to-frame" measurement, CapImage) (10), including the Baker-Wayland factor (= 1.6) for
consideration of the parabolic profile of blood in microvessels
(1). Arterioarteriolar and venovenular distances were
assessed between respective vessels of the three vascular bundles,
which supply the ear and run in parallel to each other. Volumetric
blood flow (VO) was assessed in arterioles and venules from
red blood cell velocity (V) and vessel cross-sectional area
(
· r2) according to the equation of
Gross and Aroesty (6), i.e., VO = V · · r2,
assuming a cylindrical vessel shape. After selection of two individual
branch points, the tortuosity of both arteriolar and venular vessels
was calculated from the ratio of the actual path length and the
straight line, i.e., the shortest length between the two branch points.
The individual vessel segment length was determined by the straight
line distance between two successive landmarks determined by either the
merging of venules or the branching of arterioles.
After the first observation time point at 6 wk of age, the animals were
repetitively studied in 6-wk intervals. At each observation time point,
both ears were examined, and the exact same tissue regions were found
by comparison of the microvascular branching pattern of the tissue with
the video prints. Although positive identification of the previously
recorded tissue regions was verified by a second investigator, the
branching pattern is unique and so consistent over time that there was
never a problem with mistaken identity. The experimental protocol,
including scanning/recording of ear area and of the preselected tissue
sites, was repeated as described above. The animals were kept under a
heat lamp to maintain body temperature until they recovered from
anesthesia. One animal died because of anesthesia at 30 wk of age.
Between the ages of 66 and 72 wk, all but two mice died, which
survived until the experimental time point of 78 wk.
Additional experiments in animals of young (~12 wk, n = 6), mid (~42 wk, n = 6), and old age (~72 wk,
n = 6) were performed to analyze the functional
capacity of vessels to show reactive hyperemic response after a short
period of ischemia in dependency to age. For this purpose we induced
complete ischemia to the ear by clamping the ear at its base for 2 min
followed by reopening of the vessel clamp and reperfusion of the ear.
Normal physiological vascular response comprises an immediate, but
transient, hyperemic response, i.e., acute vasodilation with increase
of blood flow velocity. We analyzed the reactive hyperemic response by
intravital microscopic assessment of arteriolar microvessel diameters
(µm) and blood flow velocities (µm/s) at baseline and during the
first minutes on postischemic reperfusion. Moreover, we analyzed tissue PO2 using a flexible polyethylene microcatheter
Clark type PO2 device (Licox System, GMS,
Kiel-Mielkendorf, Germany) that was positioned beneath the
oxygen-impermeable foil on the animals' ears. This allowed the
LICOX probe to integrate local tissue
PO2 values over the tissue area in contact with
the 5-mm-long PO2-sensitive area near the
catheter tip without interference with the ambient air. Online
temperature compensation was performed by a temperature probe (type K
thermocouple probe; LICOX System, GMS) that was also positioned between
the skin surface and the oxygen-impermeable foil. Average tissue
oxygenation was continuously monitored during baseline and postischemic reperfusion.
Statistical analysis.
All values are given as means ± SE (n, number of
animals; N, number of vessels). After proving the assumption
of normality, comparison between the experimental time points (6, 36, and 66 wk) was performed by the Student's t-test, including
corrections of the 
-error according to the Bonferroni probabilities
for repeated measurements (SigmaStat, Jandel, San Rafael, CA).
Intergroup comparison was performed by using one-way analysis of
variance followed by the appropriate post hoc multiple pairwise
comparison test. To assess the correlation between the area of the ears
and both vessel segment length and intervascular distance, regression
analysis was performed (SigmaStat, Jandel). The criterion for
significance was taken to be P < 0.05.
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RESULTS |
At the first experimental time point, the 6-wk-old animals had a
mean body wt of 22.8 ± 0.2 g (Table
1), which significantly (P < 0.01) increased by 35 ± 5% by the age of
36 wk (Table 1). No significant weight gain or loss was observed during
the second half of the observation time period up to the age of 78 wk
(Table 1). Initially, the average area of the mice ears was 39.8 ± 1.3 mm2, which significantly increased by 26 ± 1%
to a mean area of 50.5 ± 2.3 mm2 (P < 0.01) between 6 and 36 wk of age. The 1.35 ± 0.05 times increase in body weight compared with the 1.26 ± 0.01 times
increase of the area of the ears indicates that surface area of the ear does not proportionally increase as much as whole body mass during the
late juvenile maturation phase and early adult life. Within the time
period of 36 to 78 wk of age, the ear area was found to be slightly
reduced in the range of 116-122% of the initial size (Table 1).
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Table 1.
Body weight, area, and cutaneous functional capillary density of ears
of 8 hairless mice during their growth from late juvenile to adult
and senescent life (6-78 wk)
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During growth, adulthood, and senescence, the number of both arterioles
and venules as well as the number and pattern of vessel branches
remained essentially constant (Table 2,
Fig. 1). Moreover, the angle between
branching arterioles and merging venules did not change over the time
period of 6 to 78 wk of age (Fig. 1). However, within the life span
from juvenile to early adult, assessment of the individual vascular
segment lengths revealed an elongation of vessels. In both arteriolar
and venular vessels with diameters in the range of 10-100 µm, a
marked (P < 0.01) increase of the segment length
between successive branching points was found with no significant
difference in the extent of elongation between either arteriolar and
venular or smaller and larger vessels, respectively (Figs.
2 and 6, A and B).
This increase in length over time ceased at the age of ~36-42 wk
(early to late adult life). In parallel, volumetric blood flow
increased comparably in both arterioles and venules only from early
lifetime up to the age of 36 wk (Table 3). During the subsequent life time
period, volumetric blood flow in arterioles and venules remained almost
constant (Table 3), but a moderate shortening of vessel segment lengths
occurred, probably due to an age-related tissue shrinkage (Fig. 2).
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Table 2.
Changes in number of arterioles and venules in the exact same tissue
regions of ears of 8 hairless mice during their growth from late
juvenile to adult and senescent life (6-78 wk)
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Fig. 1.
Intravital microscopic images of an identical tissue
region of a mouse ear at 6 (A), 36 (B), 54 (C), and 66 (D) wk of age. Contrast enhancement
was achieved by intravenous injection of fluorescein-isothiocyanate
(FITC)-dextran (mol wt 150,000). Note the strict maintenance of number
and pattern of vessel branches during the animal's life span
(A-D). Magnification ×15.
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Fig. 2.
Arteriolar (A) and venular (B)
segment lengths (percentage of values at 6 wk of age) within the ears
of 8 hairless mice during their growth from juvenile to adult
(6-36 wk) and senescent life (up to 78 wk). Identical ear tissue
regions were repetitively studied using intravital fluorescence
microscopy for the analysis of age-related adaptations of the skin
microcirculation. Exact locations for the measurement of vascular
segment lengths were marked on the initially made video prints of the
preselected tissue regions. Vessels were grouped in accordance to their
diameter at the first observation time point of 6 wk of age (100%).
Arterioles (<30 µm), N = 8; arterioles (>30 m),
N = 7; venules (<40 µm), N = 24; venules
(>40 µm), N = 20. Values are means ± SE.
* P < 0.05, ** P < 0.01 vs. 6 wk; ## P < 0.01 vs. 36 wk.
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Table 3.
Volumetric blood flow in arterioles and venules of ears of 8 hairless
mice during their growth from late juvenile to adult and senescent
life (6-78 wk)
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The age-related elongation of vessels, which can be interpreted as
longitudinal growth, was associated with a significant (P < 0.01) increase of the intervascular distances,
i.e., the arterioarteriolar and the venovenular distances, by ~10%
within the first 36 wk of age (Figs. 3
and 6, A and B). This transverse dispersion of
arteriolar and venular vessels slightly decreased during late adulthood
and senescence to 78 wk of age (Fig. 3).
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Fig. 3.
Arterioarteriolar and venovenular distances (percentage
of values at 6 wk of age) within the ears of 8 hairless mice during
their growth from juvenile to adult (6-36 wk) and senescent life
(up to 78 wk). Identical ear tissue regions were repetitively studied
using intravital fluorescence microscopy for the analysis of
age-related adaptations of the skin microcirculation. Exact locations
for the measurement of intervascular distances were marked on the
initially made video prints of the preselected tissue regions. Values
are means ± SE. * P < 0.05, ** P < 0.01 vs. 6 wk; ## P < 0.01 vs. 36 wk.
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Regression analysis revealed significant (P < 0.01)
correlations between the increase of the ear area and both the
longitudinal growth, as given by the vascular segment lengths, and the
transverse growth, as given by the intervascular distances (Figs.
4 and
5).
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Fig. 4.
Regression analysis between mean values of mice ear area
and mean values of both the arteriolar (top) and the venular
segment lengths (bottom). With intravital fluorescence
microscopy, data were assessed by repetitive analysis of 8 hairless
mice during their growth from late juvenile to adult and senescent life
(6-78 wk). Values are means ± SE; r, regression
coefficient.
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Fig. 5.
Regression analysis between mean values of mice ear area
and mean values of both the arterioarteriolar (top) and
venovenular distances (bottom). With intravital fluorescence
microscopy, data were assessed by repetitive analysis of 8 hairless
mice during their growth from late juvenile to adult and senescent life
(6-78 wk). Values are means ± SE.
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In addition to the increase in lengths, venules with a diameter <40
µm became tortuous, with ratios of the actual path length to the
straight line length increasing from 1.12 ± 0.02 at 6 wk to
1.17 ± 0.03 at 36 wk of age (P < 0.01) with no
further change from late adult to senescent life (1.16 ± 0.06)
(Figs. 6, C and D,
and 7). In contrast, venules with
diameters >40 µm did not increase in tortuosity (Figs. 6,
C and D, and 7). Moreover, arterioles (diameter
range: 15.0-52.5 µm) failed to exhibit a tortuous path at either
of the mice ages studied (Figs. 6, C and D, and
7).
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Fig. 6.
Intravital microscopic images of preselected tissue
regions of 2 mouse ears at 6 wk (A and C) and
identical tissue regions at 36 wk of age (B and
D). Contrast enhancement was achieved by intravenous
injection of FITC-dextran (mol wt 150,000). Note the longitudinal
growth, i.e., elongation of both arteriolar and venular vessels with
their transverse dispersion (A vs. B) and the
marked increase of venular tortuosity (C vs. D)
during maturation from juvenile to adult life. Magnification ×15.
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Fig. 7.
Tortuosity of arterioles (A) and venules
(B) (percentage of values at 6 wk of age) within the ears of
8 hairless mice during their growth from juvenile to adult and
senescent life (6-78 wk). Identical ear tissue regions were
repetitively studied using intravital fluorescence microscopy for the
analysis of age-related adaptations of the skin microcirculation. Exact
locations of beginning and end points for the measurement of vessel
tortuosity were marked on the initially made video prints of the
preselected tissue regions. Venules were grouped in vessel classes of
<40 µm (N = 24) and >40 µm (N = 20) according to their initial diameter at the first observation time
point of 6 wk of age. Arterioles: diameter range 15.0-52.5 µm,
N = 15. Values are means ± SE,
** P < 0.01 vs. 6 wk.
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During maturation, adulthood, and aging to senescence, analysis of
vessel diameters that were assessed at specific locations along the
arterioles and venules showed a progressive (P < 0.01) increase in arterioles with initial diameters <20 µm, whereas larger
arterioles, i.e., those with diameters >20 µm, remained in the range
of 100 and 120% of their diameters at 6 wk of age. Venules of either
diameter, <40 µm and >40 µm, exhibited an inconsistent pattern of
dilatation up to a maximum of 120% within the life span of 6 to 78 wk
(Table 4).
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Table 4.
Diameter changes of arteriolar and venular microvessels of ears of 8 hairless mice during their growth from late juvenile to adult and
senescent life (6-78 wk)
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The quantitative assessment of the functional capillary density
revealed an initial mean value of 74.5 ± 3.7cm/cm2.
During the life span from 6 to 36 wk, density of capillaries was
maintained (Table 1), which, in regard to the increased ear size,
implies the recruitment or new formation of microvessels. In contrast,
with aging up to 78 wk, functional capillary density progressively
decreased to 43.2 ± 4.3% (P < 0.01; Table 1).
As a functional parameter, reactive hyperemic response to short
ischemia of the ear was tested in relation to the animals' age. By
intravital fluorescence microscopy, we found an increase of arteriolar
diameters during initial postischemic reperfusion, however, without any
differences between the groups (Fig. 8). Of note, arteriolar blood flow velocity, which ranged between 515 and
1,180 µm/s at baseline, increased on reperfusion up to 120%
of baseline values in young animals, whereas the mid-aged animals were
only able to reconstitute blood flow velocity to preischemic baseline
values. Strikingly, old animals failed to show a hyperemic response
inasmuch as blood flow velocity remained significantly lowered during
reperfusion (65 ± 9% of baseline values; Fig. 8).
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Fig. 8.
Arteriolar diameters (top), arteriolar blood
flow velocities (middle), and cutaneous tissue
PO2 (bottom) at baseline (open bars)
and during the initial period of reperfusion (solid bars) after a 2-min
period of complete ischemia of the ear. Analyses were performed in
animals of young (~12 wk, n = 6), mid (~42 wk,
n = 6), and old age (~72 wk, n = 6).
Values are means ± SE. * P < 0.05 vs. old-age
animals; # P < 0.05, ## P < 0.01 vs. baseline.
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Analysis of tissue PO2 revealed a slight
difference in cutaneous oxygenation already at baseline conditions in
that animals with higher age showed a tendency toward lower tissue
PO2 values. Differences between the three
groups became more evident during the initial reperfusion period:
although young and mid-aged animals exhibited a marked hyperemic
response, reflected by an increase of cutaneous
PO2 to 202 and 193% of preischemic baseline
values, hyperemic response in old animals was found limited in extent with a PO2 of only ~150% of baseline (Fig.
8).
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DISCUSSION |
These experiments show that neither maturation nor aging
significantly modifies the overall vascular branching pattern of ear
microvasculature. Microvascular dimensions simply expand in length to
suit the increased dimensions of mice ears. There is no indication for
age-related new vessel formation, except the fact that unchanged
functional capillary density implies formation of new capillaries,
because the ear dimensions expanded. Enlargement of feeding and
draining vessels as well as maintenance of vascular density at the
capillary level seem to be the compensatory mechanisms to match
perfusion to the ear growth up to the early adult life. In addition to
a substantial reduction of functional capillary density, there is no
particular remodeling of the skin microvasculature in response to aging
to senescent life.
Methodological considerations.
The hairless mouse initially develops a full hair coat, but, at the age
of 10-14 days, begins to lose hair on the face and legs. This
continues until the whole head is bare. Shedding proceeds in a
cephalocaudal direction until the animal is completely naked by ~21
days of age (3). This pertinent hair loss guarantees perfect visualization of the microvasculature of the ear, which, per
se, does not require any surgical preparation. Thus concerns regarding
the effect of surgical procedures, such as laparotomy and
exteriorization of the intestine (15, 17), on the normal growth of the animal as well as of the tissue of interest are negligible in the present study. Moreover, the fingerprint-like angioarchitecture of the skin does not require an artificial vessel marking, as must be necessarily performed using india ink dots for
identification of intestinal segments of interest at a subsequent time point (15). Taken together, effects of surgery and
tissue manipulation on age-related adaptations of the skin
microvasculature can be considered minimal in this study, contrasting
the methodological approach in previous studies analyzing the
intestinal microvasculature in identical rats at 10 (late juvenile
life) and 20 wk of age (early adult life) (17). Moreover,
the present study extends previous studies (15, 17) in
that repeated analysis of identical microvessels can be performed over
time periods of several months up to 1.5 years (78 wk), thereby also
covering the period from late adulthood to senescence. The fact that
all measurements are confined to identical vessels throughout the whole
experimental time period reduces potential variation of data due to
intraindividual biological heterogeneity to a minimum and can be
considered as a major advantage compared with longitudinal studies in
animals of different ages (11, 13). Moreover, the present
study encompasses the analysis of the whole microvascular network,
including arterioles, capillaries, and venules, and does not focus only
on the capillary arrangement (13) and the proximal or
distal parts of the arteriolar tree (15, 17).
When measuring cutaneous capillarization, we used the functional
capillary density as a measure of length of perfused capillaries per
area of observation, contrasting parameters such as capillary path and
segment lengths typically obtained in the skeletal muscle tissue
(5, 13). The characteristic honeycomb-like arrangement of
capillaries within the skin makes it inappropriate to define a
capillary in accordance with the commonly used criteria as a vessel
with its origin at the last division of the terminal arteriole and its
terminus either at the point where it converges with another capillary
to form a venule or where it joins a venule directly (4,
8). However, the herein presented parameter of functional capillary density estimates only those vessels actively serving for
nutritive perfusion, rather than the maximum (anatomic) number of
capillaries available for blood flow.
Age-related adaptations of the skin microvasculature.
By comparison of the same vascular areas of mice ears at all ages
studied, it is evident that during both maturation from late juvenile
to adult life and aging to senescent life, no new arterioles or venules
arise; therefore, only growth of previously formed blood vessels takes
place in the investigated ears. It is well known that the vasculature
is not a static but is a dynamic structure and can remodel in the face
of a variety of physiological/pathophysiological challenges, including
growth and senescence. However, in contrast to the heart, which is
extensively studied in terms of postnatal growth and age-related
adaptation of its blood vessels (9, 12), the skin
microvascular growth has not been investigated yet. Despite the fact
that the present study does not provide insight into mechanisms
underlying the microvascular network adaptation to development and
aging, it is the first to offer a detailed description of the
morphological nature of skin microvascular changes during a whole life
span. In line with previous studies in the intestine (15),
we observed that the absolute number of vessels as well as the general
pattern of vessel arrangement within the defined tissue areas was fixed
over the late juvenile growth phase as well as in early and late
adulthood. We additionally failed to demonstrate remarkable remodeling
of the skin microvasculature during aging to senescence, except for the
drastic reduction of the functional capillary density. The unique and
time-consistent branching pattern as well as the maintenance of clear
microscopic visualization of the microvasculature of the ear over time
allowed a 100% probability of finding the individual microvessels in
virtually every animal. Thus, in terms of geometry, our data are
consistent with observations by others that the microvascular bed
appears to be preformed at birth (2).
Arterioles and venules increased up to 1.12-fold in length, which is
not in proportion to the 1.26-fold increase in ear area. However,
arterioarteriolar and venovenular distances also increased up to
1.12-fold, thus with the consequence that the product of growth in
length and width equals the total ear growth. This mathematical aspect
is reflected by the second-order regression analysis with significant
correlations between ear area and both the longitudinal growth, i.e.,
the arteriolar and venular segment lengths, and the transverse growth,
as given by the intervascular distances, and implies that both
elongation and dispersion of existing vessels are the main features of
skin microvasculature to suit the dimensions of an enlarging tissue.
Microvascular growth and development through the enlargement of
preformed vessels is in agreement with results obtained in the rat
cremaster (18) and skeletal muscle (11, 14)
as well as in the rat intestine (15). Despite the fact that the microvascular network geometry of the mice ear skin is not
absolutely comparable to that of rat intestine or cremaster and
skeletal muscle with its arcade and bridging arterioles (11, 15), the similarity in elongational growth implies common
underlying mechanisms of vascular aging of tissue.
The most striking effects of age-related adaptations of the skin
microvasculature occurred during the late juvenile growth phase and
early adult life. Compared with immature mice, the adult mice exhibited
a greater mean ear area, mean arteriolar and venular segment length and
volumetric blood flow, mean arterioarteriolar and venovenular distance,
and mean venular tortuosity. Except for the venular tortuosity, all
other parameters showed a slight decrease in mice from late adulthood
to senescence. During aging, the increase of venular tortuosity seems
to reflect a disproportional increase in length, in comparison with the
ear growth, rather than a shrinkage/shortening of surrounding tissue.
Tissue shrinkage, in turn, might be causative for the simultaneous
reduction of ear size together with the dimensions of its supplying
microvascular network during the time period from 36 wk of age to
senescence. In addition to these "passive" adaptations of cutaneous
arterioles and venules to the age-related changes of tissue mass, there
is evidence for a need-oriented response of nutritive blood supply, i.e., recruitment and/or new formation of capillaries, during the late
juvenile and early adult growth phase, thereby maintaining the
functional capillary density and, thus, oxygen supply to tissue despite
the increase of tissue area. This is in line with and extends previous
studies by Unthank and co-workers (16, 17), who suggested
capillary angiogenesis when comparing intercapillary distances for the
longitudinal muscle layers of the rat intestine in 20- (17) and 5-wk-old rats (16). The marked
reduction of cutaneous functional capillary density with aging to
senescence might also be interpreted in that age significantly modifies
this microvascular parameter in relation to reduced oxygen demands. In
addition to this morphological aspect, functional analysis of
microvascular reactivity by studying the hyperemic response on short
ischemia additionally revealed a restricted capacity of the
microcirculation to produce adequate postischemic reactive hyperemia in
old vs. young and early adult animals.
| |
ACKNOWLEDGEMENTS |
The study was supported in part by a grant of the Deutsche
Forschungsgemeinschaft (Me 900/1-3 and 1-4). B. Vollmar is the recipient of a Heisenberg-Stipendium of the Deutsche
Forschungsgemeinschaft (Vo 450/6-1).
| |
FOOTNOTES |
Address for reprint requests and other correspondence: B. Vollmar, Institute for Clinical and Experimental Surgery, Univ. of
Saarland, D-66421 Homburg/Saar, Germany (E-mail:
exbvol{at}med-rz.uni-sb.de).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 14 July 1999; accepted in final form 2 May 2000.
| |
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INTRODUCTION
