J Comp Physiol B (1994) 164:206-214

Journal of
Comparative ~.~,~"~
and EnvironPhysiology B mental
Physiology
9 Springer-Verlag 1994

Gas exchange morphometry of the lungs of the tokay,

Gekko gecko L. (Reptilia: Squamata: Gekkonidae)
S.F. Perry*, J. Hein, E. van Dieken
Fachbereich 7 (Biologie) der Carl-von-Ossietzky-Universit/itOldenburg, Postfach 2503, D-26111 Oldenburg, Germany
Accepted: 14 January 1994

Abstract. The tokay lizard (Gekko gecko) possesses singlechambered lungs, each of which is a mirror image of the
other reflected in the midsagittal body plane. When standard techniques are employed for instilling 2% phosphate-buffered glutaraldehyde to three-quarters of the
total lung capacity, neither the left nor the right lung is
consistently larger. Internally, the lungs are characterized
by a row of 11 dorsomedial niches and by honeycomblike (faveolar) gas exchange tissue, which is deeper cranially than caudally. Based upon mean values for all experimental animals, a 100-g tokay would have an overall
anatomical diffusion factor (respiratory surface area divided by the appropriate 'I2ht) of 203 cm2"~m 1-100 g-l,
61% of which is located on the interfaveolar septa. Of the
total septal anatomical diffusion factor, 94% is evenly
divided between the anterior and middle thirds of the
lung, with 6% in the posterior third. The 39% of the
anatomical diffusion factor located on the inner lung wall
is predominantly (76%) in the middle and posterior lung
thirds, with only 24% in the anterior region. These tendencies toward heterogeneous distribution of anatomical
diffusion factor were most pronounced in a 55 g juvenile
animal. In this animal the total anatomical diffusion fax* Present address: Respiratory Research Group, University of
Calgary Medical School, 3330 Hospital Drive NW, Calgary, AB,
T2N 4N1, Canada
Abbreviations: ADF, anatomical diffusion factor; %AR,percentage
of potential respiratory surface area which makes up SAa; DtO2,
diffusing capacity for air-blood tissue barrier; IUR, isotropic uniform randomly distributed; bm, body mass; %P, percentage of lung
volume devoted to parenchyma; SA, potential respiratory surface
area (SL minus the surface area of the trabeculae); SANR,non-respiratory surface area; SAR,respiratory surface area; SL, total internal surface area of the lung; Sv, surface area-to-volume ratio in
parenchyma; zht, harmonic mean thickness of the air-blood tissue
barrier; VL, morphometrically determined volume of both lungs,
fixed at 0.75"VLm;VLm,maximal lung volume, similar to total lung
capacity in mammals; VLr,resting lung volume, similar to functional
residual capacity in mammals; VP, morphometrically determined
volume of parenchyma of both lungs, fixed at three-quarters of VLm
Correspondence to: S.F. Perry

tor/body mass was 3.6 times that of a 197 g adult. This

difference was attributable to a greater body massspecific lung volume and respiratory surface area as well
as to a greater surface-to-volume ratio in the parenchyma
and to a thinner air-blood diffusion barrier in the juvenile
animal.

Key words: Lung - Morphometry - Gas exchange - Diffusing capacity - Gecko, Gekko gecko

Introduction
Geckos represent one of the most successful lizard
families in terms of their distribution and phylogenetic
diversity. Their single-chambered lungs, however, remain
with few exceptions simple in structure (Wiedersheim
1906; Werner 1912; Mahendra 1947; Perry and Duncker
1978, 1980; Btising 1990). The tokay (Gekko gecko), because of its simple lungs, its relatively large size, its ease to
maintain and its availability through commercial sources
has been the subject of physiological and anatomical
studies of the respiratory system (Duncker 1978; Perry
and Duncker 1978; Welsch and Mfiller 1980; Milsom
1984; Milsom and Vitalis 1984). In spite of the popularity
of the tokay as an experimental animal, a quantitative
study of its lungs is lacking.
Perry et al. (1989 a) carried out a detailed descriptive
study of the lungs of an archetypal diplodactyline gecko
Rhacodactylus leachianus. They did not, however,
provide any morphometric data except a single estimate
of the air-blood barrier thickness. Morphometric data
regarding the surface area, air-blood diffusion distance
and the distribution of respiratory surfaces in the lungs of
the tokay could provide not only a morphological reference point for the interpretation of physiological studies,
but also a starting point for further studies on the adaptive capability of simple lungs to environmental stress
factors. The present study is designed to achieve these
goals.

S.F. Perry et al.: Gecko lung morphometry

207

Materials and methods

Animals and preparative methods. Seven tokays, ranging in bm from
55.1 to 196.8 g (mean 104.9 g) were used. The animals were maintained, two or three to a terrarium, on a 12/12-h light-dark cycle for
approximately 6 months before sacrifice. A 40-W light bulb in each
terrarium provided a local source of heat and illumination. Daytime
temperatures varied between 24 and 28 ~ with nighttime temperature being approximately 5 ~ lower. The relative humidity was
maintained near 70%. In addition to fresh water, which was always
available, the tokays were offered crickets, grasshoppers and freshly-killed newborn mice ad libitum twice weekly. Three additional
animals provided corroborating qualitative light and electron-microscopic data.
The tokays were weighed, then killed with an overdose of sodium pentobarbital (Nembutal; 35 m g - k g 1, i.p.). VLm and VLr were
determined in situ using a pressure-volume apparatus according to
the method of Perry and Duncker (1978), and the lungs were fixed
at 0.75. VLm with cold (4 ~ phosphate-buffered glutaraldehyde (to
2.0% in 0.1% mol.1-1 buffer, pH 7.3, 300 mosmol.1-1) by instillation
through the glottis. After 2 h the thorax was carefully opened and
the lungs were removed intact. The displacement volumes of both
lungs together and of each separately were determined according to
the method of Scherle (1970). Either the right or the left lung was air
dried at 10 cm H20 pressure for macroscopic observation while the
other lung was further processed for morphometry.
The lung that was further processed was cut transversely into six
slices of equal thickness and three small samples for light and electron microscopy were removed from the dorsomedial, dorsolateral
and ventral aspects of the caudal surface of each slice. These samples were placed in labelled vials to indicate the animal, left or right
lung, slice, and location on the slice. They were individually exposed
to 1% OsO4 in 0.1 mol.l ~ cacodylate buffer (pH 7.4) for 2 h at 0 ~
Following a buffer rinse and change to 0.05 mol-1-1 maleate buffer
(pH 5), the tissue was en bloc contrasted in 0.5% maleate-buffered
uranyl acetate, alcohol dehydrated (Weibel 1970/1971) using propylene oxide as an intermediary, and embedded in Epon synthetic
resin such that the pleural surface rested on the bottom of the
embedding mold.
After removal of the samples for electron microscopy, the remaining tissue was postfixed in Bouin's fluid and following dehydration through graded alcohols and xylene, embedded in paraffin.
The paraffin blocks were then planed using a sliding microtome
until whole lung profiles were obtained. The cut surfaces of the
blocks were stained using alcoholic iodine and methylene blue solutions (Perry 1981 a). These paraffin preparations served as a basis for
the determination of lung surface areas and volumes.

Morphomewic methods. The stereological point and intersection

counting procedures for reptilian lungs have been described in detail elsewhere (Perry 1978, 1981, 1983). We therefore emphasize details relevant only to the present study as well as new methods
employed for the determination of the air-blood barrier thickness.
The ultimate reference volume for all point and intersection
counts is the VL of the lung, but the morphometric results are
obtained in paraffin-embedded preparations. In order to correct for
shrinkage due to paraffin embedding, a volumetric shrinkage factor
(s3) was calculated as the volume of the embedded lung determined
by the Cavalieri principle (Michel and Cruz-Orive 1989) divided by
the displacement volume. The linear shrinkage factor for correction
of the surface area-to-volume ratio was $3033. NO correction for
shrinkage was introduced for Epon-embedded material (Weibel
1970/1971).
Paraffin-embedded, stained lung tissue was employed for the
determination of parenchymal volume as well as for tissue volume
and surface area determinations at magnifications of 16:1 and 65:1,
respectively. For these purposes, a modified Merz test system (Merz
1967) was inserted as a film copy in the eyepiece of a stereomicroscope (Zeiss SV8 with a PLS fl00 fiat field objective) and the
paraffin preparations were illuminated from below by reflected light
from a fibre-optic source. Volume proportions of capillaries and

Fig. 1. Setup for determining barrier thicknesses and respiratory

surface area on electronmicrographic negatives. A Eyepiece
graticule for selecting direction of orientation lines. B Set of orientation lines. C Negative of low power electron micrograph on light
box. D Stereomicroscope with drawing tube. E Negative of test line
system composed of random quarter-cycloids on light box

lung tissue including the proportion of trabecular and non-trabecular smooth muscle, were determined using 1 gm thick Epon sections
observed at a magnification of 500:1 using a Reichert Visopan
projecting microscope and an isodiametric point array as a test
system.
Volumes per unit reference volume and surface areas per unit
reference volume were recorded as points per unit reference area
and intersections per unit reference area, respectively. They were
converted to the desired parameter according to standard stereological principles: Pp = Vv, 2IL = Sv. The indices, P, V, I, and S
refer to the point count, volume, intersection count and surface area
of the entities to be measured, and the subscripts P, V, and L refer
to the point count, volume, and line length of the reference unit,
respectively.
Low power (400x) electron micrographs (Zeiss EM 109) were
used for determining "~htas well as the proportion of the potentially
respiratory surface area that is important for gas exchange (%AR).
Since all lung samples had a pleural surface (the center of the lung
is a tissue-free central lumen) and this surface rested at the bottom
of the embedding mold, it was possible to make both "semithin"
(1 gin) and ultrathin (8~90 nm) sections which conformed in their
orientation to Baddeley et al.'s (1986) definition of vertical sections.
Accordingly the sections were made perpendicular to the pleural
surface of the lung but were randomly oriented with respect to its
long and transverse axes.
For each sample, two independent sections separated by at least
1 mm of block depth were measured. Ten fields on each grid were
chosen by first determining at scanning power (150x) the total number (n) of grid squares that contained technically measurable tissue,
then dividing this number by 10. Beginning with the first measurable grid square, electron micrographs at 400x were prepared at
every n/10th square.
The electron micrographic negatives (Fig. 1C) were placed on
the transillumination stage of the stereomicroscope described
above, which was now fitted with a drawing tube. Each 10x eyepiece
contained at the level of the focal plane diaphragm a film copy of a
different specially designed eyepiece graticule. The left eyepiece
graticule (Fig. 1A) was a scale from 1 to 97, the unit spacing repre-

208
senting equal intervals along a horizontally oriented cycloid arc.
The right eyepiece graticule (Fig. 1B) was a set of 6 or 11 orientation
lines, the choice dictated by the user's preference. A negative film
copy of a linear test system composed of random quarter segments
of horizontally oriented cycloids was placed on a light box and its
image transmitted to the microscope by means of the drawing tube.
The total magnification of the microscope was 8x relative to the
drawing tube image. Thus, including magnification by the electron
microscope, 1 mm in the drawing tube image actually represented
0.31 gm on the lung.
In practise both the pleural side of the electron micrographic
negative and the abscissa of the left eyepiece graticule (Fig. 1A)
faced the observer. A two-digit number (e.g., 34 or 03) was selected
from a random numbers table and the right eyepiece graticule
(Fig. 1B) was oriented parallel to a line connecting the origin (lower
left) and the chosen value on the right margin of the superimposed
image of the left eyepiece graticule. Then, the air-blood barrier
thickness (cord length) was measured parallel to the orientation
lines in the eyepiece graticule using a flexible ruler with greatest
accuracy at the low end of the scale (Perry 1981). With proper
surface illumination the image of the ruler was clearly superimposed
upon the linear test system. The starting point of each cord-length
measurement was given by the intersection of a cycloid arc with a
potentially respiratory surface and the end point was the intersection of the measurement cord with a capillary surface. The cord
length measurements were tallied according to increment midpoint.
For three tokays a total of 4650 individual cord lengths were measured.
For each set of fields comprising a single sample, Tht was calculated as two-thirds of the harmonic mean cord length (Weibel and
Knight 1964). In addition, ~ht and %AR were calculated separately
for the inner surface of the lung wall and for the interfaveolar septa.
The most proximal and most distal fields for a given septum, identified by the presence of trabecular smooth muscle or the pleural
wall, respectively, were also recorded separately.
On electron micrographs, the intersection point with a potentially respiratory lung surface (SA) was defined as pertaining to a
"respiratory" lung surface (SAR),if a randomly oriented, straight test
line connected it with a capillary surface. A point on a non-respiratory lung surface (SANR),was connected by a randomly oriented test
line to another point on the lung surface. The number of intersections with respiratory surface (IAI~.) divided by the total number of
intersections with respiratory and non-respiratory surfaces (/AR
-}- IANR)gave the proportion of respiratory surface in the potentially respiratory part of the lung. %AR is this proportion multiplied by
100. Cases in which the test line exited the frame of the photograph
or contacted the pleural surface before intersecting with another
lung or capillary surface did not enter into the calculation of %AR.
Data from paraffin preparations were tallied separately for dorsomedial, dorsolateral, ventromedial and ventrolateral quadrants of
each slice according to the methods of Perry (1983) and appropriately corrected for shrinkage (see above). For presentation, the corrected values were combined to yield volumes, surface densities and
surface areas for the anterior, middle and posterior thirds of the
lung as well as for dorsal and ventral or for medial and lateral
half-lungs. In addition, the surface area of the interfaveolar septa
and of the inner surface of the lung wall were tabulated separately.
Data from semithin sections and electron microscopic preparations were not recorded from quadrants but only from whole slices.
They were combined with the paraffin data to yield ADF values for
the lung wall and the lung septa for the anterior, middle and posterior thirds of the lung according to the methods of Perry (1981b).
ADF values were calculated as SAR'~ht-~ for the lung wall and
interfaveolar septa for anterior, middle and posterior lung regions
separately, then summed to yield values for larger regions or for
whole lungs.

S.F. Perry et al.: Gecko Iung morphometry

Results

Descriptive anatomy
The lungs are single c h a m b e r e d a n d possess a r o w of 11
dorsomedial niches (Fig. 2A,B). The apex of the lung in
G. gecko tends to f o r m a small, conical p r o t u b e r a n c e
(Fig. 2A) that is b r o a d l y contiguous with the general lung
cavity. The internal lung surface is raised in h o n e y c o m b fashion, forming a typical faveolar p a r e n c h y m a
[Fig. 2A,B; D u n c k e r (1978)] in which the contained air
spaces (faveoli) are deeper t h a n they are broad. The interfaveolar septa (Fig. 2B,D) which separate adjacent faveoli, bear p r e d o m i n a n t l y a double capillary net, a l t h o u g h
connections across the interfaveolar septum are seen
(Fig. 2E). The central leaflet of the septa contain in addition to collagen, a small a m o u n t of non-vascular s m o o t h
muscle, which tends to be oriented from top to b o t t o m in
the septa. The latter are dynamically s u p p o r t e d at their
free ends by trabeculae (Fig. 2C), which contain a core of
s m o o t h muscle, and often bear a clearance-type epitheliu m (Fig. 2C).

Morphometric results
The quantitative d a t a presented in the text are n o r m a l ized per 100 g bm, which lies within the range of n o r m a l
b m for this species, whereas those in Figs. 3-5 and in
Table 1 are normalized per kg b m to facilitate c o m p a r i son with values from other studies. Conversion is m a d e
by m o v i n g the decimal point and has not been allometrically calculated.
The m e a n (_+ SE) c o m b i n e d displacement volume of
b o t h lungs calculated for a 100-g t o k a y is 22.2 + 4.3 ml, of
which 2.6_+0.4 ml, or 12.1%, is p a r e n c h y m a . Within the
p a r e n c h y m a l c o m p a r t m e n t , 2.3 ml is air and the remaining 0.3 ml (11.0%) is tissue. Thus, only 1.3% of the lung
volume is tissue. Of this tissue, 57% makes up potential
gas exchange tissue of the interfaveolar septa and the
lung wall, whereas the remaining 43 % is a p p r o x i m a t e l y
evenly divided between large b l o o d vessels and trabeculae.
Within the septa, 70% of the volume is occupied by
capillaries, less than 1% is non-vascular s m o o t h muscle
and 30% is connective tissue and other tissue c o m p o nents. The trabeculae, however, are c o m p o s e d of 16%
capillaries, 65% s m o o t h muscle, and 19% connective tissue and other c o m p o n e n t s .
Thus, the lungs of a 100-g t o k a y would contain
0.12 ml capillary blood, 0.11 ml of which is located on the
septa and the inner lung wall. N o n - v a s c u l a r s m o o t h muscle is located p r e d o m i n a n t l y in the trabeculae
(39 gl.100 g l ) , with only 0.6 pl.100 g 1 in the septa.
Juvenile tokays (Fig. 3A: C12 and C3) tend to have
larger lungs per unit b m than do m a t u r e animals (C2,
C5). This tendency is reflected in the bm-specific pare n c h y m a l volume, whereas the per cent p a r e n c h y m a
( 1 0 % - 1 5 % of total lung volume) remains relatively stable as b m increases from 55 to 197 g (Fig. 3A). Since the
surface a r e a - t o - v o l u m e ratio in the p a r e n c h y m a (mean

S.F. Perry et al.: Gecko lung morphometry

209

Fig. 2. Structure of the tokay lung. A Dried, inflated left lung. View
of medial inner surface (upper) and of lateral inner surface (reflected
down). Ant, Mid and Pos indicate anterior, middle and posterior
lung regions, respectively. Scale bar = 1 cm. B Transverse section
through the anterior third of the lung. Dorsal, up; lateral, left.
Stained paraffin preparation. Scale bar = 1 ram. C Transverse section through a primary trabecula, showing its core of smooth muscle and its ciliated epithelium. 1-gm Epon section, toluidine blue.
Scale bar = 25 gin. D Transverse section through the basal portion
of an interfaveolar septum. 1-ttm Epon section, toluidine blue. Scale

bar = 25 gm. E Transverse section through a small trabecula and

associated septum in the posterior lung region. Note the lack of
ciliated epithelium on the trabecula and the presence of a single
capillary net on the lung wall and on the surface of the vein, which
typically occupies the corner of the air space. 1-gin Epon section,
toluidine blue. Scale bar = 25 gm. Symbols: a, apex; b, bronchial
orifice; by, large blood vessel; c, capillary; ci, ciliated epithelium; cl,
central lumen; din, dorsal mesopneumonium; f, faveolar airspace;
m, smooth muscle; n, niche; s, interfaveolar septum; t, trabecula; vm,
ventral mesopneumonium; w, lung wall

78.3 cm -1, range 71.8-86.7 cm 1) also remains stable over

the m e a s u r e d range of bm, the decrease in SI~ per unit b m
reflects the decrease in bin-specific p a r e n c h y m a l volume
(Fig. 3B), decreasing from 291.5 cm2.100 g-1 in the 55-g
t o k a y to 138.4 cm2.100 g-1 in the 197-g specimen.
In specimens of b m 55, 99, and 197 g, the %AR is
84.36%, 67.51% and 69.33%, respectively. These values
have been surface-area weighted to a c c o u n t for the different %AR values on septa and walls. The large difference

between the smallest specimen and the two larger specimens is reflected in the twofold difference in SAR between
these two groups (Fig. 3C). The similarly weighted, overall m e a n ~ht for the same three tokays (Fig. 3C) is similar
in the two smaller animals (0.87 g m and 0.73 gm) and
greater in the larger one (1.08 gm). A l t h o u g h the SI~ per
unit b m in the smallest t o k a y is twice that of the largest,
the physiologically m o r e relevant parameter, A D F . b m -1,
is 3.6 times greater in the smallest specimen than in the

210

>

S.F. Perry et al.: Gecko lung morphometry

400

80

C12

A_

300

LUNG VOLUME (VL)

n
4o1

C5

200 -'.

lOO

20

OO

4,

.
[~

% PARENCHYMA (% P)
[J

!If

I E+__--l ( [ l l

I.'I=m , ~/ - - I ~ | ,

C12

~-'280
~240

B
TOTAL SURF. AREA (SL)

(~ 200

1.6

O)
-~ 120

..C5

SURF. AREA/VOLUME RATIO (Sv)

e~_~NCHYMoAL VOL.(VR)

40

O) 3.4

1.2
0.8

80

2.4
2.0 "~

=--- ~6o

>

2.8

50

70

90

110

130

0.4
150

170

190

C12

3.0

3.4
3.0

.:1 2.6

2.6

2.2

2.2

~3 1.8

<

1.8 "~

1.4

1.4

1.0

1.0

o 0.6
n-

0.6

50

b9

70

90

110

130

150

170

190

BODY MASS (g)

Fig. 3. Combined values for bm-specific lung volumes, surface areas

and A D F as a function of bin. Barrier thicknesses (~ht) are not
bm-specific. Where Tht values for septa and lung walls have been
combined, they have been weighted in proportion to the respective
surface areas. A Lung volume, parenchymal volume,% parenchyma. B Volume, surface area and surface area-to-volume ratio of
parenchyma. C Respiratory surface area, Tht and A D F

largest one (Figs. 3B, 3C; Table 1); this is because ADF
calculation is based on SAR rather than on SL and also
contains the reciprocal of rht.
For a regional comparison of lung volumes, surfaces
and barrier thicknesses, the data for individual animals
rather than for group means are presented in graphical
form. Inspection of Figs. 4 and 5 reveals that the regional
distribution patterns within lungs are similar in spite of
their grossly different absolute values.
Since the division of the lung into dorsal and ventral
or medial and lateral halves, or into anterior, middle and
posterior thirds was arbitrary, the total volumes of these
regions (Fig. 4A) reflect the accuracy of the technique as
much as the anatomy of the lung. It is interesting to note,
however, that only the anterior region, with its narrow
apical portion, differs strongly from the others. As illustrated in Fig. 4B, % P is nearly the same in dorsal, ventral, medial and lateral half-lungs but decreases sharply
as one progresses from anterior to posterior. Figure 4C
combines graphically Figs. 4A and 4B. The distribution
of parenchymal volumes in the transverse plane remains
homogeneous. With respect to the long axis of the lung,
however, the lung volume is greatest where the % P is
least (anterior region) and least where the %P is greatest
(posterior region). Thus, the parenchymal volume tends
to be somewhat more homogeneously distributed than is
the lung volume or the %P. The general pattern of parenchymal volume distribution in all specimens is: middle
> anterior > posterior.
The surface area-to-volume ratio exclusive of trabeculae (72.9-t-3.3 cm -1) varies little from lung to lung and is
also similar in different lung regions (Fig. 4D). An exception is the posterior region, where it increases dramatically due to the very low septa; i.e., the inside surface area of
the lung wall is very large relative to the small parenchymal volume.
The mean pulmonary surface area exclusive of trabeculae calculated for a 100-g tokay is 197.1 _+42.3 cm 2. Inspection of Fig. 4E reveals that the bm-specific pulmonary surface area is greatest in the middle third of the

Table 1. Morphometric comparison of the lungs of tokays with those of other reptiles
Parameter

Symbol

Units

Body weight
Total lung volume
Parenchymal volume
Parenchymal volume
Total parenchymal surface
area
Respiratory surface area
Effective surface-to-volume
ratio in parenchyma
Respiratory surface area
Harmonic mean thickness
of tissue barrier
Anatomical diffusion factor

W
VL
%P
VP
SA

kg
m l . k g -1
% of VL
ml-kg 1
103.cmZ.kg -1

%AR
% of SA
SAR/VP cm2-cm 3

Teju a Lacerta b Snake ~ Monitor a Turtle d Crocodile e sin. Tokay lg. Tokay
0.72 0,02
84.4 94.8
26.0 34.6
22.0 32.8
3.76 6.07
78,2 70.0
133.6 129.6

SAR
~ht

103.cm 2"kg-1
1 0 - 4 - c m (=glTl)

2.94
0.46

4.25
0.53

ADF

103. cm a. g m - ~. k g - ~
(10- 7cm" k g - 1)

6.39

8.01

1.00
0.44
97.5 306.7
28.7
83.2
2.00
6.13
101
-

88.6
65.3

1
262.3
44.9
117.8
2.49

3.59
113.1
42.0
39.7
1.38

0.055
348.5
10.8
37.8
2.78

0.197
164.6
11.7
19.3
1.38

82.7
18.0

50.0
15.3

84.3
63.4

69.3
36.4

0.46

5.43
0.65

2.06
0.50

0.66
1.4

2.40
0.87

0,95
1.08

4.4

8.35

4.12

0.63

3.27

0.90

a Tupinambis nigropunctatus, Varanus exanthematicus; Perry (1983). b Lacerta spp.; Cragg (1975). c Pituophis melanoleucus; Stinner (1982).
d Pseudemys seripta; Perry (1978). e CrocodyIus niloticus; Perry (1990).

S.F. Perry et al.: Gecko lung morphometry

211

REGIONAL DISTRIBUTION OF LUNG VOLUME

03
03
<

180

AI~

~;

160

/'l

I~

140

"7

a POTENTIALLY RESPIRATORY SURFACE AREAS

-r

120
U j ~91 0 0

80
"J
O

60

>

40

20
O-

7
2

,..a

PERCENT PARENCHYMA IN LUNG REGIONS

<

40

C12

C3

C2

CS
INN3

1.4

1.2
1.0

3o

LU >
o.

HARMONIC MEAN THICKNESS OF AIR-BLOOD

TISSUE BARRIER

020

0.8

e-- 0.6

L'.,, 0.4
0.2

03

REGIONAL DISTRIBUTION OF PARENCHYMA

ANATOMICAL DIFFUSION FACTOR

1.4

2o
"T

g
_i .~

1.2

~"
"T,

1.0

0.8

0.6

14. 0.4
r",

<~

0.2.

m
Sepia Wall
Anterior

11.

14o S U R F A C E - T O - V O L U M E RATIO IN PARENCHYMA

0
W<:

120
100

,...J

0>~. 80
u~ -JE~ 60
0
<
U,n~

40
2O

03/'3

03
o3
<C

1.6

O~
n~c~

1.(1

,,<,z:

0.6

IA

u~
03

0.2
0

Sepia waI
Posterior

Fig. 5. Combination of potentially respiratory surface areas (excluding trabeculae) and harmonic mean thickness of the air-blood tissue
barrier (%,) in three tokays. Data are grouped according to location
on either the interfaveolar septa or the lung wall as well as in the
anterior, middle or posterior lung region. A Distribution of potentially respiratory (SA) and respiratory ( S A R ) and nonrespiratory
(SANR) surface area. Only SA measured in specimen C 3. B Distribution of zht. Not measured in specimen C 3. C Distribution of
anatomical diffusion factor (ADF --- SAR/%t)

REGIONAL DISTRIBUTION OF SURFACE AREA

-..E

rr

Sepia Wal
Middle

DORS~

VENTRAL

MEDIAL

POSTERIOR
ANTERIOR
M~DDLE
LATERAL

Fig. 4. Volumes and surface areas in different regions of the lungs of

four tokays. A Distribution of total tung volume. B Per cent parenchyma in different lung regions, C Distribution of parenchymal
volume. D Distribution of surface area-to-volume ratio in parenchyma. E Inside surface area of lung

lung, followed by the anterior and then by the posterior

thirds. The m o d e of surface distribution (Fig. 5A) is also
quite different from region to region. In the anterior region the septa have approximately three times as much
surface area as does the lung wall, in the middle region
only 1.5 times as much, and in the posterior region the
lung wall presents a greater surface area than do the
septa.
A total of 517 measurements of % A a at proximal and
distal extremes of the interfaveolar septa revealed only a
tendency toward a greater value in the proximal location
(proximal: 79.4-t- 2.5; distal 69.7-t- 9.9). C o m p a r i n g the
septa with the lung wall (Fig. 5A), in general the septal
%AP, is greater than the corresponding value for the lung
wall: septum, 7 8 . 0 + 1 . 9 % ; wall, 68.4-t-10.8%. Extreme
values were 92% on the septa in the posterior region of
the specimen C12 (55 g) and 29% on the lung wall in the
same lung region of specimen C2 (99 g).

2t2
Similarly, 742 measurements of %t at the proximal
and distal extremes of the interfaveolar septa revealed no
significant gradient along the septa (proximal: 0.90 jam;
distal: 0.92 gin). Comparing the septa with the lung wall,
~Thton the septa tends to vary inversely with the relative
thickness of the parenchymal layer. Thus it is thin in
the anterior and middle regions (0.74 _+0.14gm and
0.92_+ 0.20 jam, respectively) and thick (1.25 __0.05 jam) in
the posterior region. In general the air-blood barrier is
thinner on the lung wall than on the septa of the same
lung region. It is thinnest in the anterior region
(0.68 +0.19 jam) and thicker in the middle and posterior
regions (0.85 _+0.13 jam and 0.86 _+0.02 jam, respectively).
The mean A D F calculated for a 100-g tokay is
202.8cm2"jam -I but showed extreme variation (cf.
Fig. 3C) as indicated by a standard error (119.2) that is
greater than half the mean value. A mean of 61% of the
ADF is located on the septa; the remainder is on the lung
wall (Fig. 5C). Trabeculae have been excluded. Of the
A D F on the interfaveolar septa, 94% is approximately
evenly divided between the anterior and middle regions
and only 6% is in the posterior region. The distribution
of lung wall ADF is quite different: only 24% is located
in the anterior region, the remaining 76% being evenly
divided between the middle and posterior regions. The
55-g specimen (C12) showed the strongest tendency toward heterogeneity of lung structure. In the anterior region, 77% of the A D F is on the septa; in the posterior
region, 9%. Comparable values for the 197-g specimen
are 79% and 55% for anterior and posterior regions,
respectively.
Discussion

The present methods for paraffin preparations have been

dealt with in previous publications (Perry 1981b, 1983)
but those for morphometric determination of "l~ht and
%AR have been modified in order to allow unbiased
comparison of the inner lung wall and the septa that
separate adjacent faveoli (interfaveolar septa) in the parenchyma. The use of perpendicular sections represents an
immense time saving over I U R sections by reducing the
number of ultrathin sections that must be made and analyzed. However, it also introduces a systematic error because the barrier thicknesses measured on the perpendicularly sectioned lung wall are randomized only in two
dimensions, whereas all other surfaces are cut randomly
in three dimensions.
Baddeley et al. (1986) showed that if the direction of
measurements is distributed according to the occurrence
of equidistant points along a cycloid arc, the systematic
underestimation of distances on the perpendicularly sectioned surface can be corrected. That was the purpose of
the specially designed eyepiece graticules. In addition, cycloid arcs were used for selection of the measuring points
for the air-blood diffusion barrier. The probability of
sampling a radially oriented surface in a "vertical" section using an I U R test line system is less than that of
sampling the circumferentially oriented lung wall. By
definition, a cycloid with a height of 1 unit has a horizon-

S.F. Perry et al.: Gecko lung morphometry

tal width of re/2 units (Baddeley et al. 1986). Thus, the
probability of intersecting a surface oriented perpendicularly to the long axis of the cycloid is greater than is that
of intersecting a horizontally oriented surface such as the
lung wall. The use of a test system composed of randomly
generated cycloid arcs thus assures that barrier thickness
measurements are made on surfaces of all orientations in
vertical sections with equal probability.
Structurally speaking, tokay lungs are similar to the
single-chambered lungs of the New Caledonian giant
gecko Rhacodactylus leachianus, which have been described in detail (Perry et al. 1989). A salient internal
feature of the tokay lung is the lack of a transversely
oriented ridge which divides the medial surface of the
Rhacodactylus lung at the level of the bronchial opening
into apical and main lung lobes. The number of dorsomedial niches and the decrease in the depth of the faveoli as
one proceeds posteriorly are similar in these two species.
The unequal location of the mesopneumonia on the outer surfaces of the right and left lungs, noted by Milsom
and Vitalis (1984), is corroborated by the present study.
Bfising (1990) noted little variability in this character in
his comparison of the lungs and trachea among 26
gekkonid species.
The descriptive ultrastructure of the pulmonary epithelium does not differ from that previously reported
(Welsch and Mfiller 1980) and will therefore not be discussed further. Similarly, the present VLof 22.2 ml. 100 g-i
is in good agreement with 23 ml.100 g-~ based on static
pressure volume curves in this species (Perry and Duncker 1978) with 27 ml.100 g-1 in R. leachianus. These gecko
lungs are approximately ten times a large as rat lungs on
a bm basis, but compared with the lungs of other reptiles
they occupy the middle of the range (Perry and Duncker
1978).
Quantitatively, the interfaveolar septa show an interesting coincidence of three factors that indicate a high
degree of specialization for gas exchange: in those regions in which the proportion of parenchyma per unit
lung volume is greatest, the air-blood barrier is also
thinnest and the %AR is greatest. There is no a priori
reason for assuming an anatomical interdependence
among these characters.
A surprising result is the relatively great importance
of the lung wall as a potential site of gas exchange in the
tokay lung. More than 40% of the total non-trabecular
pulmonary surface area is located there, and since the
combined "Chtand %AR values are only slightly less advantageous for gas exchange than on the septa, 39% of
the ADF is found on the lung wall.
The efficiency of the lung as a gas exchange organ
increases with the simplicity of its structure. Thus, the
diffusing capacity of salamander skin (Gatz et al. 1975)
and through the pleural membrane of the dog lung (Magnussen et al. 1974) closely approximate the morphometrically calculated maximum value, whereas the lungs of the
turtle, teju, and dog lie progressively more distant from
this ideal (Perry 1978, 1983; Weibel et al. 1983). The relatively great potential for gas exchange of the inner lung
wall in the tokay suggests that two different gas exchange
strategies may exist: a gas/gas diffusion dominated type

S.F. Perry et al.: Gecko lung morphometry

in the faveolar parenchyma and a convection dominated
type on the inner lung wall, where the exchange surfaces
are easily accessible to moving air. The combination of
these two models in the same lung could help balance the
ventilation/perfusion ratio at all sustained levels of activity.
A second surprising result is the apparent bm-dependent structural change in the lung. Although %P and Sv
remain relatively constant among all specimens, the bmspecific lung volume and surface area decrease logarithmically with increasing bm (Fig. 3). In addition, Fig. 5
reveals that the heterogeneity of distribution of respiratory surfaces and of ADF is most pronounced in the
smaller specimens. Although these results remain tentative due to the small number of animals involved, the
physiological implications merit consideration.
On the basis of the greater ADF, one would therefore
expect that small, juvenile specimens would be capable of
achieving greater bm-specific 02 consumption rates. The
more extensive, sac-like regions of the juvenile animal, on
the other hand, would result in a highly compliant lung
and a low pulmonary component of the elastic work of
breathing (Perry and Duncker 1980). Thus, the juvenile
animal would be expected not only to achieve but also to
maintain a higher bin-specific 02 consumption rate than
an adult specimen. This hypothesis remains to be tested
physiologically.
The ADF value represents only the anatomical "hardware" for gas exchange and indicates little about the actual state of perfusion and ventilation of the respiratory
surfaces. In the Nile crocodile (Glass and Johansen 1979),
blood flow to the lung varies with the ventilatory state,
increasing during breathing episodes and decreasing during the non-ventilatory period, and in the red-eared turtle
the absorption of radioactive xenon gas is fastest in the
parenchyma-rich, central lung region (Spragg et al. 1980).
Although these data indicate a great variation in the ventilation/perfusion state of reptilian lungs, in light of the
anatomical differences between multichambered and single-chambered lungs mentioned above, their direct relevance to the tokay is questionable.
On inner lung wall "Chtis consistently less than on the
interfaveolar septa and tends to remain thin in spite of
decreasing parenchymal thickness in the posterior region. The %AR, while variable in the posterior region, is
not significantly less there than in other regions. While
these results are in keeping with those for the singlechambered lungs of the teju (Tupinambis nigropunctatus)
and the colubrid snake Pituophis melanoleucus (Perry
1983; Stinner 1981) they are in contrast to those for other
reptiles. The parenchyma-poor regions of the multichambered lungs of the savanna monitor (Varanus exanthematicus), the red-eared turtle (Trachemys = Pseudemys
scripta elegans) and the Nile crocodile (Crocodylus niloticus) show a relatively large "~ht, a large %AR or both,
respectively (Perry 1978, 1983; 1990.)
The relatively low degree of specialization of the lung
parenchyma for gas exchange in the tokay lung vis-/t-vis
the single-chambered teju lung (Table 1) is corroborated
by a comparison of the non-vascular smooth muscle in
the two species. The purpose of non-vascular smooth

213
muscle is presumably to enhance ventilation of deep faveolar surfaces during non-ventilatory periods (Perry et al.
1989b), whereby the septal component collapses the faveoli by reducing their depth and the trabecular component
restores them to their resting depth. Although smooth
muscle occupies approximately the same proportion of
the pulmonary tissue in both species (teju 15%, tokay
18%), in the teju it is equally divided between trabeculae
and septa (Perry 1983), whereas in the tokay 98.5% of the
smooth muscle is in the trabeculae.
Comparatively speaking, the tokay has the lowest
ADF per unit bm of any reptile measured to date, except
the Nile crocodile (Table 1). The lung volume per unit bm
is greater than that of the more active lizards Lacerta and
Tupinambis, but the surface area-to-volume ratio is lower
and "l~htis greater in the tokay. Lacerta and Tupinambis
also lack the extensive, saccular posterior lung region.
Multiplying ADF by Krogh's diffusion constant
for lung tissue at 20 ~ gives a Dto2 of 56.6 ml.min -I
-100 g-torr -~. Assuming a resting metabolic rate similar
to that of the European gecko Tarentola mauritanica
(Nielsen 1961), of 55ml.min 1-100gl, the calculated
ratio of 0 2 consumption per unit Dto2 is 1.03 torr. This
value is very close to that for the resting turtle (Pseudemys
= Trachemys scripta) of 1.25 torr and also falls within the
range observed in small mammals [mouse, 1.57 torr; rat,
0.51 torr; guinea pig, 1.07 torr; for references see Perry
(1978)]. While this value has the same units as intrapulmonary driving pressure, it is important to realize, as
most elegantly expounded by Weibel (1984) that there is
not a single diffusion barrier but a number of them, finally ending at the mitochondrial inner membrane. The
above numerical ratio serves only as an index of the degree of exploitation of the pulmonary "hardware." Considered in this context, the ADF and consequently the
Dto2 of the tokay lung, while low compared with that of
other reptiles on the basis of bm, is well matched to the
modest metabolic requirements of a gecko.
The large lung volume relative to the ADF in the
tokay may serve other than a respiratory function. Vocalization is important in many gecko species including G.
gecko. A large air reserve could benefit the vocal repertoire and the high compliance of the lung and body walls
could allow the lungs to serve as a resonance chamber
during calling.

Acknowledgements. The authors gratefully acknowledge the technical assistance of Ms Sabine Willig and Ms Anke M/iller. Without
their help and the financial assistance of the Deutsche Forschungsgemeinschaft (Pe 276/5-1) completion of this project would not
have been possible.

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