MICROSCOPY RESEARCH AND TECHNIQUE 34556462 (1996)
Stereological Methods: A New Approach in the Assessment
of Pulmonary Emphysema
B.A.M. HEEMSKERK-GERRITSEN,J.H. DIJKMAN, AND A.A.W. TEN HAVE-OPBROEK
Department of Pulmonology, University of Leiden, 2300 RC Leiden, The Netherlands (BA.M.H.-G.,J.HD., AA.W.TSI.-0.);
Department of Surgery, University of California, Davis, California 95616 (A4.W.T.H.-0.)
KEY WORDS
Stereology, Morphometry, Mouse lungs
ABSTRACT
In order to develop a reliable and sensitive method for studying the development
and progression of pulmonary emphysema, we compared stereological indices with the usual index
for grade of emphysema, i.e., the mean linear intercept (Lm), in elastase-induced emphysema in
mice. The Lm and stereological indices, including volumes of total lung tissue (V(lt)),airspaces
(V,,,), and surface area of alveolar walls
were determined in B-p.m, H&E-stained, parafinembedded lung sections from elastase- (n = 7) or saline-treated (n = 8) mice. The indices were
measured by point counting, using Cavalieri's principle (V(lt)and V(air))or by counting intersections
of alveolar walls with test lines of a known length (S,,, and Lm). Elastase treatment resulted in
a significant increase of Lm and of V(air),both indicating airspace enlargement, and in a significant
decrease of V(lt)and S(alv),
indicating destruction of alveolar walls. Between each of the stereological
indices and the Lm, significant correlations were found when all lungs were included, but not when
the emphysematous lungs were considered separately. We conclude that stereological methods can
be powerful morphometric tools for studying pulmonary emphysema development and progression,
since they give information not only about the grade of airspace enlargement but also about the
grade of destruction of alveolar walls. Based on this unique property, stereological methods also
allow a distinction between pulmonary emphysema and unrelated conditions with dilatation of
airspaces only. 0 1996 Wiley-Liss, Inc.
INTRODUCTION
Pulmonary emphysema is defined as a condition of
the lung characterized by abnormal, permanent enlargement of airspaces distal to the terminal bronchiole, accompanied by destruction of their walls, and
without obvious fibrosis (Snider et al., 1985). Severity
of emphysema in humans and animal models is usually
evaluated by calculating the average distance between
the alveolar walls, i.e., the mean linear intercept (Lm).
The Lm allows a rapid and reliable assessment of pulmonary emphysema. A disadvantage of this method,
however, is that the Lm is only an expression of airspace enlargement, and does not reflect destruction of
the alveolar walls. This means that the Lm cannot be
used to distinguish between pulmonary emphysema
and other unrelated conditions in which there is an
increased airspace dilatation without destruction. In
the aging lung, for example, the Lm is increased without apparent destruction (Escolar et al., 1994; Thurlbeck, 1967a; Verbeken et al.,1992). Other morphometric indices for emphysema, such as the destructive
index (DI) and the number of destroyed alveolar attachments to the airways (AA), which focus on alveolar
wall destruction, are available but have been shown to
be less sensitive in emphysema models (Eidelman et
al., 1990). Therefore, we have been searching for a reliable and sensitive method to quantitate the two major
aspects of emphysema, i.e., airspace enlargement and
destruction of the alveolar walls, for our ongoing studies of emphysema development and progression.
0 1996 WILEY-LISS, INC.
Stereological methods as described by Weibel(1970)
have been shown to be useful tools for estimating surface areas and volumes. Based on the idea that destruction of the alveolar walls leads to a decrease of volume
and surface area of the alveolar walls, and that airspace enlargement leads to an increase of volume of the
airspaces, we hypothesized that these methods may be
powerful tools in the evaluation of grade of emphysema
during the development and progression of this disease. In the present study we investigated the applicability of stereological methods in elastase-induced emphysema in mice, and compared these methods with
the more classical index, the Lm.
MATERIALS AND METHODS
Animals and Intratracheal Instillations
Young adult female, inbred, Swiss-type (CPB-S)
mice (age 2-3 months), weighing 0.03 f 0.002 kg
(mean f standard deviation), were used for the experiments. Emphysema was induced in seven mice by intratracheal instillation of porcine pancreatic elastase
(144 U/mg; Calbiochem, La Jolla, CAI dissolved in saline. The administration took place on days 1 , 5 , and 8,
Received July 9, 1995; accepted August 24, 1995.
Address reprint requests to B.A.M. Heemskerk-Gerritaen,Respiratory Biology
Group, Departmentof Anatomy and Embryology, University of Leiden, P.O.Box
9602, 2300 RC Leiden, The Netherlands.
STEREOLOGICAL METHODS TO ASSESS EMPHYSEMA
each time as a single dose of 1.2 mgkg body weight
(BW). The control group consisted of eight mice, which
received a similar volume of only saline according to
the same schedule (days 1,5,and 8).
Intratracheal instillations were done as described
previously (Otto-Verberne et al., 1992). Briefly, the
mice were anesthetized by C02 asphyxiation. Animals
were held in an almost upright position by suspending
them on their front teeth. To visualize the trachea, the
tongue was extended from the mouth using forceps,
and the light tip of a flexible cold light fibr-optic was
placed against the skin at throat level. By looking into
the throat, the white cartilaginous rings of the trachea
could be seen in an otherwise transparent pink surrounding. As soon as the mice started to gasp for
breath, the trachea was quickly intubated with a 20gauge blunt needle attached to an automatic Hamilton
syringe (CR-700),(Hamilton Company, Reno, Nevada)
and 50 pl of elastase or saline were instilled into the
lungs. The mice were kept in an upright position for a
few seconds until they regained consciousness.
Morphometric Evaluation
After 8 weeks, the mice were sacrificed by indenting
the cerebellum and exsanguinated by cutting the vena
cava. The lungs with the trachea were excised carefully. After intubation of the trachea, the lungs were
inflated with Bouin's fixative at a pressure of 20 cm
HzO for 3 hours, resulting in stable fixation of both
saline- and elastase-treated lungs. Left and right lungs
were separated, and the air was removed using a vacuum pump. The lungs were stored in the same fixative
overnight, dehydrated, and embedded in paraffin.
Five-pm sections were cut and stained with hematoxylin and eosin (H&E).
The Lm was calculated according to Dunnill (1962)
and Thurlbeck (1967b),using the light microscope with
a x 4 0 objective lens and a x 8 ocular lens, the latter
lens containing a crossed hairline of known length. For
each left lung ( = one lobe) and each right lung ( = four
lobes), three sections obtained from different levels
were examined. Per section we evaluated 10 randomlyselected fields. Lm was calculated using the equation:
Lm = (N x l)SI(pm),
where N is the number of times the crossed hairline is
placed on the lung, 1 is the length of the hairline, and
81is the total number of intersections counted. Lm was
calculated for left and right lungs separately. Lm for
the two lungs represents the mean of left and right
lung Lm values.
Total lung volume was estimated as described previously (Gundersen et al., 1988;Michel and Cruz-Orive,
1988),using Cavalieri's principle. Briefly, systematic
parallel sections from different levels a known distance
"t" apart were used to estimate the volume unbiasedly
and efficiently by point counting. To do this, a test system with 40.57 test points per cm2was superimposed at
random on each section of lung. We counted the points
coinciding with the lung, i.e., airspaces and nonairspaces. To estimate total volume of the lung we used
the equation:
V(,-)
=
557
t x A x 8P1(cm'),
where V(l-) is the total lung volume, t is the distance
between the sections, A is the area represented by one
point, and SPl is the total number of points coinciding
with the lung. The V(lung)was calculated for the left
and right lungs separately. The Vlung)for the two
lungs represents the sum of the lek and right lung
V(lun) values.
Volume density and absolute volume of total lung
tissue (defined here as all lung tissue without air
spaces, i.e., parenchyma, blood vessels, airways, lobular septa, and pleura) were estimated in the sections
mentioned above, using a multipurpose test system
with 0.91 test points per cm2, and test lines with a
length of 0.269cm per test point and an Olympus BH-2
microscope with a projection arm (Olympus Optical Co.
Ltd., Tokyo, Japan). Volume density of the total lung
was determined by dividing the total
tissue (Vv
number o i points coinciding with lung tissue by the
total number of points coinciding with the reference
space (i.e., the entire lung), as in the equation:
where ZP1,is the total number of points coinciding with
lung tissue, and 8P1is the total number of points coinciding with the entire lung. The VV(lt,lung)was calculated for the left and right lungs separately. The
VV(lt,lun for the two lungs represents the mean of the
left anckright lung Vv(lt,lun ) values.
Absolute volume of the total lung tissue (V(lt))was
calculated by multiplying volume density with total
lung volume (V(lung)),
as in the equation:
V(lt) = VV(lt,lung) x
3
V(1ung)(cm )*
The Vat) was calculated for the left and right lungs
separately. The V for the two lungs represents the
sum of the left anrright lung VOt)values.
Volume density of the airspaces and absolute volume
of the airspaces (VV(air,lun) and V air), respectively) was
determined as described &r total iung tissue, using the
equations:
ZPaifiPl, and
V(air) = VV(air,lung) x V(1ung) (cm3),
VV(air,lung) =
where SP, is the total number of points coinciding
with airspaces. The VV(air,lun) and the V(air)were calculated for the left and rigfit lungs separately. The
VV(air,lunglfor the two lungs represents the mean of the
left and nght lung VV(air,lung)
values, and the V
for
the two lungs represents the sum of the left a n H g h t
lung V(air)values.
For estimation of surface area of the alveolar walls
(S(alv)),
the same test system and microscope were used.
We counted the intersections (I) of the test lines with
the alveolar walls. To estimate the S(alv)we used the
equation:
558
B.A.M. HEEMSJSERK-GERRITSENET AL.
where S(alv)is the total absolute surface area of the
alveolar walls, p/l is the ratio of test point number to
test line length for the test system used, M is the final
magnification, XI is the total number of intersections
between alveolar surfaces and test lines on the sections
counted, 8Pl is the total number of test points in the
lung (tissue and airspaces)on the sections counted, and
V(lun) is the total volume of the lung. The S(alv)was
calcu7ated for the left and right lungs separately. The
S(alv)for the two lungs represents the sum of the left
and right lung S(alvvalues.
In order to test tke reproducibility of both the conventional and stereological methods, all parameters
were measured a second time, with an interim of 6
weeks.
Data Analysis
*
Results are expressed as mean
Standard Deviation. Differences between the saline- and elastasetreated groups were analyzed statistically by using the
nonparametric Mann-Whitney test. Correlations between morphometric indices and between first and second measurements were made by linear regression. P
< 0.05 was considered significant.
RESULTS
Morphology
Representative micrographs of mouse lung tissue
sections are shown in Figure 1. Comparison of salinetreated animals (Fig. 1A) and elastase-treated animals
(Fig. 1B,C) showed the presence of centrilobular (Fig.
1B)or panlobular (Fig. 1C)emphysematous lesions in
the latter group of animals.
Morphometry
Results of the various morphometric measurements
and calculations for the saline- and elastase-treated
groups are listed in Table 1.Elastase treatment had no
impact on the body weight of the mice. In salinetreated mice the mean body weight was 0.031 f 0.003
kg, whereas the mean body weight in elastase-treated
mice was 0.03 f 0.002 kg. For elastase-treated animals
the V lung) showed a significant increase compared to
the sahne-treated mice (P < 0.05). Decrease of volume
of total lung tissue (i.e., the VY(lt,lung)
and the V(lt))in
elastase-treated animals was significant (P < 0.005 in
both cases). Accordingly, the volume of the airspaces in
the lungs of elastase-treated animals increased significantly (P < 0.005 for the Vv alrzlung),and P < 0.05 for
the V(air)).T h e S(alv)decrease6 significantly (P < 0.01)
in elastase-treated mice. The Lm in these animals was
significantly higher (P < 0.005). The most intriguing
data are visualized in Figure 2. The significant
elastase-induced decrease of volume of total lung tissue is shown in Figure 2A (Vv(lt,lung))
and Figure 2B
(V(lt)).The significant increase of volume of the airspaces in elastase-treated animals is shown in Figure
2C (VV(airlung)) and Figure 2D (V(air)).Figure 2E shows
the significant elastase-induced increase in S(alv),and
the significant increase in Lm values in elastasetreated animals is shown in Figure 2F. As shown in
Table 2, first and second measurements of all parame-
Fig. 1. Micrographs of mouse lung tissue sections, showing salinetreated lungs with normal architecture (A), and elastase-treated
lungs with centrilobular (B)or panlobular (C) emphysematous lesions. H&E staining. Bars, 150 pm.
ters observed were significantly correlated (P 5 0.002
in all cases).
In Table 3, various morphologic indices were compared with the Lm. Correlation coefficients were given
for all lungs examined (n = 151,and for emphysematous lungs separately (n = 7). Some of these correlations are illustrated in Figure 3. The value of the
559
STEREOLOGICAL METHODS TO ASSESS EMPHYSEMA
TABU 1 . Lkta of lung mrphometry obtained in elastase-treated and saline-treated
m u s e lungs by stereologic and conventional methods'
~~~
~~~
BW (kg)
Stereologic methods
VOUng)b m 3 )
VV<lt.lung) (96)
V,lt) (mm3)
VV,.Iung)
(%)
Saline (n = 8)
0.031 2 0.003
Elastase (n = 7)
0.03 2 0.002
372.21 f 72.91
25.40 2 3.60
93.56f 17.05
74.60 2 3.60
278.65 61.21
459.10 2 80.82
483.12 2
12.05 2
56.72 2
87.95 2
426.41 2
306.492
*
Vmr) (mm3)
S(d")(cm2)
Conventional methods
Lm (um)
~~~
38.60 2 1.65
~
~
~~
106.05
2.88
15.88
2.88
97.94
100.11
78.802 30.98
~
Statistical
Analvsis
NS
P < 0.05
P < 0.005
P < 0.005
P < 0.005
P < 0.05
P < 0.01
P < 0.005
~
'BW,body weight; V(lug), total lung volume; VV~~,J,,,,~),
volume density of total lung tissue; V,l,),
volume of total lung tissue; VV(~~,J-). volume density of airspaces; V(ab), volume of airspaces;S,,,),
surface area of alveolar walls; Lm,mean linear intercept; NS, not sigmficant (P 2 0.05).
TABU 2. Correlations between first and second
measurements of all pammeters obtained in
elnstase-treated and saline-treated m u s e lungs by
stereologic and conventional methods'
Parameter
All lungs examined (n = 15)
r = 0.993 (P = 0.000)
r = 0.826 (P = 0.000)
r = 0.729(P = 0.002)
r = 0.826 (P = 0.000)
r = 0.928 (P = 0.000)
r = 0.768 (P = 0.001)
'Lm, mean linear intercept;Vv(lt I
volume density of
v,,,), volume*Y'total lung tissue;
volume density of a i r s p m ; V oy), volume of airs e , (alv), surface area of alveolar wai'i
total lung tissue;
VV(rirly
VV(lt,lun) and the V(lt)(Fig. 3A) decreased significantly
(P = OfbOO and P = 0.005, respectively), with an increasing Lm in all lungs examined, In connection with
) and the Vcair)
(Fig. 3B)
this the value of the VV(air,lun
increased significantly (P = 6.000 and P = 0.002 respectively), with an increasing Lm in all lungs examined. The s(+)(Fig. 3C)showed a negative, significant
correlation unth the Lm (P = 0.004). When elastasetreated animals were considered separately, the correlations were not so pronounced. With the exceptions of
the VV(lt,lunand the VV(air,lung),
no significant correlations were found between the stereologic indices and
the Lm in elastase-treated lungs. Because of the minimal divergence of the Lm in saline-treated mice, it
was hardly possible to study the correlations between
the Lm and the other indices in these animals.
In Figure 4, the V(air)was plotted against the V(lt)
(Fig. 4A) and against the Scalv)
(Fig. 4B). A Significant
correlation was found between the V(air)and the V(lt in
saline-treated animals (F = 0.852, P = 0.007). $he
other correlations were not found to be significant.
DISCUSSION
In the present study we compared in elastase-induced emphysema in mice a conventional, though fast
and reliable, method for measuring the grade of emphysema, namely calculation of the mean linear intercept, with stereological methods. To the best of our
knowledge, this is the first time that the latter methods
have been applied to emphysema.
As presently shown, intratracheal instillation of
elastase in mouse lungs results in a characteristic panlobular or centrilobular emphysema, a finding consistent with previous reports (Otto-Verberne et al., 1992;
Valentine et al., 1983). Although not developed for
measuring grade of emphysema, stereological methods
appear to be reproducible, sensitive, and efficient. They
correlate with the Lm. However, a significant advantage of stereological methods over the Lm is that they
permit measurements of both airspace enlargement
and destruction of alveolar walls, even simultaneously,
which means that they yield a more complete picture of
the grade of emphysema without consuming more labor time of the investigator. Our findings that in
elastase-treated animals the total lung tissue volume
(Vat)) as well as the surface area of the alveolar walls
(S(alv))
is decreased indicates destruction of the alveolar
walls. Airspace enlargement in elastase-treated animals is shown by an increase of volume of the airspaces
(V(air9.
Another advantage of stereological methods is that
they are more objective: by taking parallel sections
from different levels a known distance apart, sampling
is done in an unbiased and efficient way (Gundersen
and Jensen, 1987). Furthermore, this way of sampling
provides a clear picture of the entire lung, which means
that local emphysema will not be easily overlooked. In
our opinion this may be important, especially since it is
known that centrilobular emphysema in human lungs
is more frequently observed in the upper lobes (Saetta
et al., 1985);we also found centrilobular emphysema in
our experimental mouse model (as well as panlobular
emphysema) (current study; Otto-Verberne et al.,
1992).
The present study does not provide arguments for
rejecting the Lm. The small spreading of Lm in salinetreated animals and the lack of overlap of measurements might suggest that the Lm represents a
morphometric index that discriminates easily between
saline- and elastase-treated lungs. We expect, however,
that normal or saline-treated lungs may also show
some natural variations. As demonstrated by the
B.A.M. HEEMSKERK-GERRITSENET AL.
560
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100 Y v (nir,lunp) ( 8 )
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400 300 200 -
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Elastase
Fig. 2. A Volume density of total lung tissue (VV,lt.,wrp))
in salineand elastase-treated animals. Each symbol represents the value for a
single animal. Horizontal lines represent group means. B Absolute
,
as in A. C:Volume density
volume of total lung tissue (V,,,,)displayed
of airspaces (VV(air,lung)),
displayed as in A. D Absolute volume of
0
0
0
+
8
Saline
Elastase
63
airspaces (V,,,), displayed as in A. E: Surface area of alveolar walls
(S,,,,,), displayed as in A. F: Mean linear intercept (Lm),displayed as
in A. A-F, *P< 0.01, **P< 0.05,***P< 0.005 (nonparametric MannWhitney test).
561
STEREOLOGICAL METHODS TO ASSESS EMPHYSEMA
T A B U 3. Correlations between m r p h m e t r i c pammeters obtained
in elastase-treated and saline-treated m u s e lungs by stereologic and
conventional methods'
Group parameters
r
Lm vs. V,,,,
r
Lm vs. VV(air,une,
Lm ~ 8 V<eir)
.
r
Lm vs. S,,,)
Lm vs. Vv,,,,w)
All lungs examined
Emphysematous lungs
(n = 15)
(n = 7)
= -0.815 (P = 0.000) r = -0.936 (P = 0.002)
= -0.678 (P = 0.005) r = -0.480 (NS)
r = 0.815 (P = 0.000) r = 0.936 (P = 0.002)
r = 0.737 (P = 0.002) r = 0.545 (NS)
= -0.690 (P = 0.004) r = -0.577 (NS)
'Lm,mean linear intercept; VV,,,lunb, volume density of total lung tissue; V(lt)
volume density of airspaw; V(ar,, volvolume of total lung tissue; V&
ume of airspaces; S,l,
surface &a of alveolar walls; NS, not significant (P 2
0.05).
30
\
0
l@O
2@@
greater spreading, stereological indices in particular
may be very suitable for detecting such natural variations. At first glance, the overlap of measurements of
SJalv),
V(air),and especially of V,,,) may give the impression that it is difficult to make the distinction between
saline- and elastase-treated lungs in the stereological
approach. However, it is important to realize that one
of the components of the V,,,) and of the V(air is the
volume of the total lung (V(lmg)).It is clear tkat the
increase of the V(lung)in elastase-treated animals leads
to an underestimation of the decrease of the V(lt)and to
an overestimation of the increase of the V(air).When
the volume densities of the total lung tissue and the
airspaces (Vv(lt,lung)
and VV(air,lun
)) are considered, the
differences between saline-treate8 and elastase-treated
animals are definitely more pronounced. Furthermore,
IS0
when combinations of stereological indices are used,
e.g., V(lt) F d V(+,).(Fig. 4A), and S dv)and V(air)(Fig.
4B),an easier distinction between the two treatments
0
200
is possible. This latter phenomenon is interesting because it allows distinction between emphysematous
and nonemphysematous control lungs on the basis of
the combination of airspace enlargement and destruction of the alveolar walls. Based on their all-round
properties, stereological methods represent valuable
tools for solving differential diagnostic questions.
We are aware of the fact that the V(!t) also covers
nonparenchymal tissue, i.e., tissue that is not directly
involved in the pathogenesis of emphysema (i.e., blood
vessels, airways, and pleura). However, because
elastase disrupts the elastic fiber network, especially
in the alveolar walls (Snider et al., 1986), and because
the volume of nonparenchymal lung tissue in centriacinar and panacinar emphysematous lungs is not different from that in normal lungs in humans (Cardoso
et al., 1993), we assume that a difference in the V,l,)
reflects mainly a difference in the parenchymal tissue,
and not a difference in the nonparenchymal tissue.
As expected, in normal lungs the VCair)
is related to
the S(dv)and to the V An increase of volume of the
airspaces is attended by an increase of volume of the
total lung tissue and the surface area of the alveolar
Fig. 3. A: Relationship between mean linear intercept (Lm) and
walls. In emphysematous lungs this relationship is volume
of total lung tissue (V,,,,)in saline-treated (0)and elastaseclearly disturbed. An increase of airspace volume is treated (0) animals. Broken line is the regression line for all lungs
attended neither by an increase nor by a decrease of examined ( r = -0.678, P = 0.005). Continuous line is the regression
line for elastaee-treated animals (r = -0.480, P not significant (NS)).
total lung tissue volume or surface area of the alveolar B:
Relationship between Lm and volume of airspaces (V,,,,,),diswalls. However, this does not necessarily imply that played
as in A. Correlation coefficients are 0.737 (P = 0.002) for all
the main event in elastase-induced emphysema is air- lungs examined, and 0.545 (NS)for elastase-treated animals. C: Re-
I@
lationship between Lm and surface area of alveolar walls (S,,,,), displayed as in A. Correlationcoefficients are -0.690 (P= 0.004)for all
lungs examined, and -0.577 (NS)for elastase-txeated animals.
562
1so
B.A.M. HEEMSKERK-GERFUTSEN ET AL.
V(ll) (1.1.3)
I
I
120
O
0
a
'
0
N
a
'
0
90
components of the definition of pulmonary emphysema,
destruction of alveolar walls and enlargement of
airspaces. Therefore, they are also valuable tools for
differential diagnostic purposes. We conclude that the
stereological indices are not directly more sensitive
than the Lm; they can, however, definitely contribute
to a better insight into the entire pathogenesis of pulmonary emphysema. For this reason we employ stereological methods in all our current studies, which focus
on emphysema development and progression and parenchymal regeneration in mice and in humans.
Ii.e.,
0
60
30
0
0
100
2.0
300
400
so0
600
V(air) (mm3)
ACKNOWLEDGMENTS
The authors are indebted to J.J.M. Boex for technical
assistance, to Dr. J. Hermans for statistical advice, and
to J. Lens for photographic assistance. This work was
supported by a grant from the Netherlands Asthma
Foundation.
REFERENCES
400
I
I
0
100
200
300
400
so0
600
V(air) (mm3)
Fig. 4. A Relationship between volume of airspaces (V,,,,,) and
volume of total lung tissue (V(ltJ in saline-treated (0)and elastasetreated ( 0 ) animals. Continuousline is the regression line for elastasetreated animals (r = 0.613,P = 0.106).Broken line is the regression
line for saline-treated animals (r = 0.452,P = 0.309).B Relationship
between V,, and surface area of alveolar walls
displayed as
in A. Correlation coefficientsare 0.852 (P = 0.007)for salinetreated
animals, and 0.314 (P= 0.493)for elastase-treated animals.
space enlargement. Since the accumulation of points of
emphysematous lungs in general is shifted to the right
and downwards (Fig. 4A,B), it is clear that there is
airspace enlargement (i.e., increased airspace volume),
accompanied by destruction of the alveolar walls (i.e.,
decreased total lung tissue volume and decreased surface area of the walls).
In summary, we studied the possibility of using existing stereological methods in evaluations of grade of
elastase-induced emphysema in mice, and compared
the results with classical calculation of the Lm. Stereological methods are as easy to perform; however, contrary to the Lm method, they address the two major
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