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Microvascular smooth muscle cell quantitation from scanning electron microscopic preparations.

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THE ANATOMICAL RECORD 216:443-447 (1986)
Microvascular Smooth Muscle CelI Quant itation
From Scanning Electron Microscopic Preparations
VINCENT H. GATTONE 11, BRYAN G. MILLER, AND ANDREW P. EVAN
Departments of Anatomy, The University of Kansas Medical Center,
Kansas City, K S 66103 (VH.G.),Indiana University School of Medicine,
Indianapolis, IN 46223 (B. G.M., A. I? E.)
ABSTRACT
A morphometric method to analyze scanning electron micrographs
(SEMI of microvascular smooth muscle cells (SMC) is validated using intestinal
arterioles. Features easily obtained from these microvasculature preparations are
counted (number of tapers and number of complete wraps in the central 87% of the
vessel, which is one-third of the vessel’s circumference) or measured (vessel diameter
and vessel segment length). These data allow the determination of wraps around
the vessel per SMC, cell length, and cell width, which are not different from either
the values as determined by circumferential examination and quantitation of
the vascular segment or a n alternative SEM quantitation method (Miller et al., 1986).
The method described herein provides a relatively easy way to determine microvascular SMC parameters.
It is difficult to discern microvascular smooth muscle
cell features owing, in part, to the inability to adequately visualize these cells. However, recently developed methods (Uehara and Suyama, 1978; Miller et al.,
1982) have allowed relatively large segments of microvasculature to be visualized by scanning electron microscopy (SEM). Several different vascular beds have
been examined in this way (cerebral: Moore et al., 1985;
intestinal: Fujiwara and Uehara, 1982; Miller et al.,
1986; skeletal muscle: Holley and Fahim, 1983; mammary gland: Fujiwara and Uehara, 1984; and kidney;
Gattone et al., 1984). Using these SEM preparations,
theoretical formulas were derived for quantitating several vascular smooth muscle cell (VSM) parameters
(Gattone et al., 1985). The present study provides support for these theoretical formulas and compares the
data calculated by this method to values obtained by the
procedures of Miller et al. (1986).
MATERIALS AND METHODS
of the vessels to accurately obtain the data on the vascular smooth muscle cells (number and wraps per cell);
2) using the data that are easily obtained from a single
view of a vessel as suggested by Gattone et al. (19851,
namely a) number of tapered ends and b) completely
wrapped VSM segments from the central 87% of the
vessels as well as c) diameter, and d) length of vessel
segment; and 3) measuring VSM cell parameters (length
and width) by the single-view-method of Miller et al.
(1986).
In order to use reliably a single view of a vessel to
gain insight into VSM cell features, certain assumptions
must be made (Gattone et al., 1985): 1)vessels are maximally dilated and cylindrical, 2) smooth muscle cells
form a monolayer, 3) each smooth muscle cell has only
two lateral processes (tapered ends), 4) in the visible
vessel segment, the cells are seen as either a) completely
wrapping (CW) around the vessel or b) a s a tapered end
(T) that only partially encircles the visible segment, 5)
that the VSM cells begin and end randomly around the
vessel. These assumptions all appear basically correct
either by selection (vessels with only a monolayer of
VSM and using vasodilated specimens) or from observation. This does not exclude the possibility of exceptions
occurring in certain microvascular beds (efferent arteriole in kidney, Gattone et al., 1984) or in a disease
process (Moore et al., 1985).
The intestinal microvessels were prepared for SEM by
the method of Miller et al. (1982).The animals, 20 WKY
rats, were anesthetized with sodium pentobarbital, and
abdominal vessels were dilated by intraperitoneal (i.p.)
lidocaine and adenosine. Segments of small intestine
were perfusion-fixed and processed by reinfusion of
blood, buffer-rinsed, muscularis externa was removed,
followed by a two-step digestion process (HC1 followed
Collection of Morphometric Data
by collagenase). The tissue was then processed for
For each vessel segment of a given length CLv) and
examination with a n AMR lOOOA SEM. Segments of
vessel were dissected free and placed “end-on” for diameter (d), the central 87% of the vessel was delinecircumferential examination. Scanning electron micro- ated (Fig. 2). The number of tapered ends (T) and comgraphs of these precisely identified submucosal arteri- plete wraps (CW) through this central region was
oles (from specimens like that illustrated in Fig. 1)were
photographed on Polaroid 55 P/N film.
Received December 6, 1985; accepted June 17, 1986.
Morphometric data were collected from the same 20 Address reprint requests to Vincent H. Gattone 11, Department of
vessel segments (1 vessel segment per animal) by the Anatomy, The University of Kansas Medical Center, Kansas City, KS
three following methods: 1)using circumferential views 66103.
0 1986 ALAN R. LISS. INC.
444
V.H. GATTONE 11, B.G. MILLER,AND A.P. EVAN
I
-
-_.-
Fig. 1. Low magnification scanning electron micrograph illustrating the submucosal microvasculature of the small intestine. This microvascular bed sits on the outer surface of the
muscularis mucosa. In this specimen most of the venous components have been removed by
dissection. Therefore, the regular branching order shown clearly illustrates the first, second,
and third order (lo,2", and 3") arterioles. ~ 3 0 .
.07 d
determined (Figs. 2, 3). The central 87% of the vessel is
used since this represents the chord for one-third of the
vessel's circumference. The length of the chord is defined as: d sine ?hO which for 120" is 0.866d. The number
of tapered ends (T)is used to estimate the number of
cells since each cell has two ends, and approximately
one-third of these tapered ends should appear in the
central 87% of the vessel; therefore the number of cells
(C) would be
C = 3/2 T.
2
Fig. 2. Diagram of a n arteriole showing the chord (dotted lines) for
one-third of the circumference (1209, which represents 87% of the
vessel diameter. Two smooth muscle cells are seen to wrap around the
vessel and in this 87% quantitation region are noted as tapered ends
(TI or complete wraps (CW). A centerline drawn along this arteriole is
crossed by four smooth muscle cell processes whose average width
would be determined by dividing the vessel segment length by the
number of SMC processes.
(1)
The real number of cells per vessel segment is determined from the circumferential examination of the
vessel.
cell lengths were obtained from circumferential views
by determining the nW&er of times each VSM
wraps around the vessel (w/c)and obtaining an average
for this value for each vessel. From single views, the
average number of wraps per cell (W/C)can be obtained
by determining the total number of wraps (W) divided
445
SMOOTH MUSCLE MORPHOLOGY
Fig. 3. The circumference of a 2" arteriole segment highlighting the same smooth muscle
cell through a series (A-C) of three scanning electron micrographs. The cell outlined is seen in
each view as either a complete wrap (CW) or a tapered end (T)of the cell. Only the central 87%
of the vessel is used to determine the cell process designation (per Fig. 2). X 1,800.
by the number of cells (C). Total wraps (W) would equal
the number of wraps seen in any partial view of the
segment (i.e., the central 87%) since this would be a
representative view of the vessel (e.g., the wraps seen in
the view are continuations of, rather than additional to,
those from adjacent views of the same segment). Therefore,
W=CW+Tw
So W/C
2:
(CW + 0.5T)C.
(4)
Thereafter, cell length (Lc) determined by both the circumferential method and the protocol described herein
would be the number of wraps per cell, times the vessel
circumference:
(5)
Lc = ad(W/C).
(2)
So in single views,
with Tw being tapered ends weighted for the proportion
of the vessel circumference in the area of quantitation
that they transverse and CW being the number of complete wraps. Since it was assumed that the tapered ends
are randomly distributed around the vessel (assumption
5 above) the average taper would go half way across the
viewing area, allowing equation 2 to be simplified to:
W = CW
+
0.5T
(3)
LC = ad(CW
+ 0.5T)/C.
(6)
Miller et al. (1986) determined Lc by actually determining the value of total wraps (WT).
This was done by
measuring the sum total of the complete wraps (Sw) and
sum total of the tapered wraps (%)so that
LC = ad(Sw
+
ST)/C.
(7)
446
V.H. GATTONE II, B.G. MILLER, AND A.P. EVAN
Cell width (Wc) was determined by dividing the vessel
segment length by the total number of wraps (W) using
the formula
wc = Ls/w.
(8)
and the present study used
=
Ls/(CW + 0.5T).
Wraps/cell*
Circumferential
0.87 k 0.06
view value
Calculated
0.94 f 0.09
value
Value by
Miller et al.
(1986)
Miller et al. (1986)used
Wc
TABLE 1. Morphometric data on vascular smooth
muscle cells’ (mean & SEM)
(10)
Cell length*
(um)
Cell width*
(urn)
96 f 3.0
1.98 f 0.07
108 k 6.5
2.07 f 0.06
109 k 7.0
2.07 f 0.05
‘n = 20 vessel segments, one vessel per animal.
* P > .05 for difference between any of the values.
An alternative way to determine the mean celI width
(Wc) is to divide the vessel length (Lv)by the number of
wraps (Wm) that cross a line drawn longitudinally vessel segment). Therefore, no special equipment is
through the center of the vessel (centerline method), i.e., needed in order t o obtain VSM data such as cell length
(Lc) and width (Wc) from single views of vessels. The
(11) method of Miller et al. (1986) requires the measurement
Wc = Lv/Wm.
of the sum total lengths of the complete wraps (SW)and
tapered wraps (ST),in addition to the diameter, vessel
Comparison of Methods
length and number of tapered ends. These data would
The values calculated by the formulas for the method require more time and effort, especially if a computer
described herein were compared in a paired fashion morphometry system was not available to aid in obtain(paired t test) to values obtained for the same parame- ing these values. Otherwise, these two methods give
ters by both the circumferential examination of the ves- virtually identical values both of which are not different
sel (or centerline method for width) and the method of (P > .05) from the values obtained for these vessel
segments as determined by examining the entire cirMiller et al. (1986).
cumference. When using single SEM views of microvesRESULTS
sels, both VSM quantitation methods provide a
An example of the kind of intestinal vessel used in considerable time savings over either examining the
this study is shown in Figure 3. These series of three entire circumference with SEM (Miller et al., 1986) or
SEM micrographs of the same vessel show how a single reconstruction of smooth muscle cells by serial reconcell can be seen to encircle the vessel and be viewed as struction with either light microscopy (Friedman et al.,
either forming a complete wrap or as tapered ends of a 1971; Walmsley et al., 1982) or transmission electron
cell. The morphometric data in the vascular smooth microscopy (Carlson et al., 1982; Todd et al., 1983; Todd
muscle cells are shown in Table 1. The standard of and Manard, 1985). The only drawback to the singlereference for the data obtained in the present study were view SEM approach is that the values obtained would
the values obtained by the circumferential view method need to be corrected for tissue shrinkage in SEM prepaor centerline method for cell widths. The calculated val- ration in order to obtain the “real” values. However, as
ues obtained for SMC features using the formulas de- long as a uniform preparation method is used, compararived by the present study are not different (P > .05, tive analysis should be possible with the data directly
paired t test) from those obtained by the circumferential obtained. The single-view methods provide an entirely
method or the single-view method of Miller et al. (1986). new way to approach microvascular VSM quantitation
These calculated values were also virtually identical and provide more meaningful data than typical methods
with the individual values obtained by the method of such as lumerdwall ratio. This type of information could
be very useful in helping delineate the arteriolar smooth
Miller et al. (1986).
muscle changes associated with disease states such as
DISCUSSION
hypertension. The sensitivity of the present method in
The present study supports, using real vessels, the detecting smooth muscle cell changes awaits future
basic theoretical formulas for VSM quantitation previ- studies on diseases or conditions that are known to have
ously described by Gattone et al. (1985). The data of the altered smooth muscle cells.
present study also favorably compares to that obtained
ACKNOWLEDGMENTS
by a closely related method (Miller et al., 1986)when we
This
study
was
funded in part from a grant through
utilized the same vessels.
Using either the method described herein or that of the American Heart Association, Indiana Affiliate and
Miller et al. (19861, the values for several VSM parame- National Institute of Health PHS grants HL 32409 and
ters can be accurately determined from arterioles. It HL 37602. The authors wish to thank Doris Lineweaver
should be noted that there are small but important and Stacie Allen for typing the manuscript.
operational differences between these two methods as
far as the data required to calculate the parameters. The
LITERATURE CITED
method, herein, requires only two measured features
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scopic studies of cat mesenteric arterioles: A structure-function
two counted features (the number of complete wraps,
analysis. Microvasc. Res., 24~123-141.
CW, and tapered ends, T, from the central 87% of the Fujiwara, T., and Y. Uehara (1982) Scanning electron microscopical
SMOOTH MUSCLE MORPHOLOGY
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