J Appl Physiol 118: 1429–1434, 2015.
First published April 16, 2015; doi:10.1152/japplphysiol.01017.2014.
Regional differences in alveolar density in the human lung are related to lung
height
John E. McDonough,1 Lars Knudsen,2,4 Alexander C. Wright,3 W. Mark Elliott,1 Matthias Ochs,2,4,5
and James C. Hogg1
1
Centre for Heart Lung Innovation, St. Paul’s Hospital, Vancouver, British Columbia, Canada; 2Institute of Functional and
Applied Anatomy, Hannover Medical School, Hannover, Germany; 3Department of Radiology, University of Pennsylvania
School of Medicine, Philadelphia, Pennsylvania; 4Biomedical Research in Endstage and Obstructive Lung Disease Hannover,
Member of the German Center for Lung Research (DZL), Hannover, Germany; and 5Cluster of Excellence REBIRTH,
Hannover, Germany
Submitted 12 November 2014; accepted in final form 10 April 2015
alveoli; pleural pressure gradient; human lung
lung is to remove oxygen from the
surrounding atmosphere and release carbon dioxide back into
it, to meet the high energy demands of large, multicellular
organisms. This requires the separation of blood and air by a
very large, thin surface area that allows oxygen and carbon
dioxide to equally and rapidly diffuse between the alveolar gas
and the blood stream. This exchange is coupled to the movement of air into and out of the lung, which occurs by a
combination of bulk air flow into and out of the conducting
airways of the tracheobronchial tree with diffusion beyond the
terminal bronchioles, into and out of the alveolated tissue.
Each pair of adult human lungs contains ⬃44,000 terminal
bronchioles, which are the last purely conducting airways
present in the lung. Each of these terminal bronchioles supplies
a structural unit of the lung anatomy termed an acinus, which
consists of transitional airways where alveoli first appear,
respiratory bronchioles that contain a mixture of conducting
airway and alveolar openings, and alveolar ducts, where the
THE PRIMARY FUNCTION OF THE
Address for reprint requests and other correspondence: J. C. Hogg, 166-1081
Burrard St., Vancouver, BC, Canada V6Z 1Y6.
http://www.jappl.org
luminal surface is entirely taken up by alveolar openings and
blind-ending alveolar sacs. Investigators have used a variety of
techniques that include lung casts (13) and histological sections
(1) to count the number of alveoli within the normal human
lung. The most recent study of alveolar numbers by Ochs et al.
(21) used an unbiased stereological approach to arrive at an
average of 480 million alveoli within a pair of adult human lungs.
Although these studies provide a global assessment of the
number of alveoli within a human lung, regional variation in
number has not been well studied. This is relevant because
regional ventilation is dependent on the pleural pressure gradient and lung height. This study addresses this question by
examining alveolar numbers throughout the lung. This report
provides structural information derived from images of lung
tissue taken using microcomputed tomography (microCT) that
provide a volumetric image of the tissue sample, allowing for
all individual alveoli to be counted without bias. The localization of these samples within the lung allows for regional
variation of alveolar numbers to be measured.
METHODS AND MATERIALS
Subject demographics. Donor lungs were collected from the Gift of
Life program (Philadelphia, PA) with written informed consent obtained from the next of kin. These lungs were found not to have a
suitable recipient in time for transplant and were, therefore, donated
for research with ethical committee approval. The donor lungs used in
this study consisted of two lungs from nonsmoking subjects and two
from subjects with a history of smoking. Donor age, sex, height, weight,
and smoking history are shown in Table 1.
Lung tissue processing. Lungs were reinflated using a compressed
air source and an underwater seal to first inflate them to total lung
capacity (TLC); they were then maintained at 10 cmH2O on the
deflation limb of the pressure volume curve and then frozen solid by
surrounding the lung in liquid nitrogen vapor. Frozen lungs were then
sent to St. Paul’s Hospital in Vancouver, BC, Canada, where they
were scanned while frozen using a multidetector CT scanner (Sensation 16; Siemens Medical Solutions, Germany) using a volumetric
scanning protocol of 120 kVp and 100 mA. Contiguous images were
reconstructed using slice thickness of 1 mm and a B60f (high spatial
frequency) reconstruction kernel. Lung volume was calculated by
summing the multidetector CT (MDCT) voxels that were lung. Lung
mass was calculated by measuring the lung density calculated from
the X-ray attenuation values of the lung in Hounsfield units and
multiplying it by the lung volume. Following the CT scan, the lungs
were cut into contiguous 2-cm-thick slices in the transverse plane.
Frozen core samples were collected throughout the lung using a
14-mm-diameter cork borer. The location of samples taken was noted
8750-7587/15 Copyright © 2015 the American Physiological Society
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McDonough JE, Knudsen L, Wright AC, Elliott WM, Ochs M,
Hogg JC. Regional differences in alveolar density in the human lung
are related to lung height. J Appl Physiol 118: 1429 –1434, 2015. First
published April 16, 2015; doi:10.1152/japplphysiol.01017.2014.—
The gravity-dependent pleural pressure gradient within the thorax
produces regional differences in lung inflation that have a profound
effect on the distribution of ventilation within the lung. This study
examines the hypothesis that gravitationally induced differences in
stress within the thorax also influence alveolar density in terms of the
number of alveoli contained per unit volume of lung. To test this
hypothesis, we measured the number of alveoli within known volumes
of lung located at regular intervals between the apex and base of four
normal adult human lungs that were rapidly frozen at a constant
transpulmonary pressure, and used microcomputed tomographic imaging to measure alveolar density (number alveoli/mm3) at regular
intervals between the lung apex and base. These results show that at
total lung capacity, alveolar density in the lung apex is 31.6 ⫾ 3.4
alveoli/mm3, with 15 ⫾ 6% of parenchymal tissue consisting of
alveolar duct. The base of the lung had an alveolar density of 21.2 ⫾
1.6 alveoli/mm3 and alveolar duct volume fraction of 29 ⫾ 6%. The
difference in alveolar density can be negated by factoring in the
effects of alveolar compression due to the pleural pressure gradient at
the base of the lung in vivo and at functional residual capacity.
1430
MicroCT Counts of Alveolar Number
Table 1. Subject demographics
Sex
Age
Height, m
Weight, kg
Pack years
Donor 1
Donor 2
Donor 3
Donor 4
Male
51
1.79
80
39
Male
62
1.79
107
24
Male
59
1.7
61.8
Nonsmoker
Male
43
1.82
82
Nonsmoker
Means ⫾ SE
53.8 ⫾ 4.3
1.78 ⫾ 0.03
82.7 ⫾ 9.3
31.5 ⫾ 7.5
(n ⫽ 2)
McDonough J et al.
To compare alveolar density using microCT measurements and
histological measurements, tissue embedded in JB4 from one subject
(donor 3) was sectioned at a thickness of 3 ␮m for alveolar counting
using disector look-up sections with a disector height of 9 ␮m and a
counting frame area of 281,073 ␮m2 (21).
Statistical analysis. Data were analyzed by linear regression and
Pearson’s correlation. The lung tissue samples between the upper
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on photographs of slices before and after sampling with these locations marked on the MDCT scan.
MicroCT sample processing. Frozen core samples were processed
for microCT imaging by first fixing the samples in a combination of
1% glutaraldehyde in acetone at ⫺80°C for 2 h followed by 2 h at
⫺20°C, and then overnight at 4°C. Samples were then washed with
acetone followed by contrast staining of the sample using 1% osmium
tetroxide in acetone for 1 h. This was followed by three washes with
acetone and several washes with anhydrous ethanol. Samples were
then dried using the critical point of liquid CO2 (Automegasamdri915B Series B Critical-Point Dryer; Tousimis, Rockville, MD). Dried
specimens were scanned using a GE eXplore Locus SP microCT
scanner (GE Healthcare, Waukesha, WI) at the University of Pennsylvania. The following scanning protocol provided 16.24-␮m isotropic voxel resolution and 460-1,000 contiguous microCT images per
lung tissue sample: peak X-ray tube voltage 80 kVp, current 80 ␮A,
3 s exposure time, 500 views at 0.4° increments (short scan), 1⫻1
pixel binning, and an average of four scans. Following microCT
scanning, select samples were embedded in plastic resin (JB4; Polysciences, Warrington, PA) for tissue sectioning.
MicroCT image analysis. Five lung cores from each of the four
donors were randomly selected from lung apex to base for analysis. A
random-number generator was also used to determine the starting
slice number of the microCT image stack for placement of the region
of interest to measure the number of alveoli. A 4⫻4 grid was placed
on the image and a random-number generator was used to select a
counting frame within lung parenchyma (i.e., reference volume) free
from blood vessels or airways to be measured. Measurements were
made on 10 consecutive 16.24-␮m-thick images using a field of view
of 0.1 cm2; this equals a sampling volume of 1.624 mm3. The alveolar
openings were visually identified on the basis of anatomical formation
of pockets within the three-dimensional volume examined (Fig. 1).
In other words, the disappearance of a bridge between the free edges
of septal walls within the counting frame was counted (15, 21),
representing a unidirectional disector. These openings were counted
and divided by the disector volume of tissue examined to determine
the number of alveoli per cubic millimeter of lung. An average of four
fields evenly distributed throughout the core was used to calculate the
number of alveoli per cubic millimeter in each core. To calculate the
total number of alveoli per lung, the volume fraction of parenchymal
tissues vs. blood vessels and airways was also measured by point
counting. The volume fraction of alveolar ducts was also measured by
a point-counting method from three fields per core in the apex and
base cores for each lung.
The number of alveoli per terminal bronchiole was calculated by
dividing the total number of alveoli in the lung by the total number of
terminal bronchioles in the lung as calculated from a previous study
(19). Acinus volume was estimated by dividing the number of alveoli
per terminal bronchiole by the number of alveoli per cubic millimeter
of lung parenchyma to equal a cubic millimeter per terminal bronchiole. The mean linear intercept (Lm) has a direct linear relationship
with airspace size of the alveoli and alveolar ducts (5, 24) and was
measured from images captured at 20 regularly spaced intervals
within the microCT scans of each sample using a previously validated
grid of test lines projected onto the image and a custom macro
(ImagePro Plus; MediaCybernetics, Silver Spring, MD).
•
Fig. 1. A microcomputed tomographic image of normal human lung parenchyma in an upper lung (A) and lower lung (B) region with several alveolar
openings indicated by arrows. Alveoli were counted at the point at which they
opened in the series of 10 images that were examined.
J Appl Physiol • doi:10.1152/japplphysiol.01017.2014 • www.jappl.org
MicroCT Counts of Alveolar Number
(apex) lungs was compared with samples from the lower (base) lungs
by a Student’s t-test. All numbers are expressed as means ⫾ SE.
RESULTS
DISCUSSION
The number of alveoli within the human lung has been the
focus of study by multiple groups over the past several decades. Weibel and Gomez (31) published one of the earliest
reports of lung structure, and in five subjects (3 men, 2 women,
age range 8 –74 yr) reported 296 ⫾ 11 million alveoli within a
single lung. Subsequent studies have used similar techniques
and found variations in the number of alveoli. Dunnill (4)
reported 286 million alveoli in a single lung of a 55-yr-old
woman; Angus and Thurlbeck (1) examined 42 lungs from 32
subjects (age range 19 – 85 yr) and reported 375 ⫾ 18 million
alveoli per lung (range, 212– 605 million). These classic studies counted alveoli on two-dimensional histological sections
1431
McDonough J et al.
and used a mathematical model that made assumptions about
the shape of the alveoli to determine the final number. These
assumptions have since been shown to underestimate the number of ducts within the tissue sections (12).
More recently, a stereological approach was developed to
allow the counting of discrete structures within tissue samples
without prior assumptions on their shape and size (27). This
approach uses two histological sections with a certain distance
and a counting frame to define a volume of parenchyma
(disector volume) with the number of alveolar openings within
this volume counted. Using this new method on six adult
human lungs (2 men, 4 women, aged 18 – 41 yr), Ochs et al.
(21) reported 240 ⫾ 36 million alveoli per single lung (range,
137–395 million). Accurately counting the number of alveoli
according to their openings requires that each alveolus does not
close completely. This appears to be the case in the lung,
because even at very low lung pressures, alveolar openings
remain (20). However, further studies may be required to
determine the validity of this assumption.
The present study used microCT images to count the number
of alveoli within the lungs of four male subjects age 43– 62 yr,
and showed an average number of 80 ⫾ 10 million alveoli per
lung (range, 68 –111 million) or 160 million per pair of lungs,
which is less than what has been reported in previous studies.
Several factors may explain the smaller number of alveoli
reported in this study. One is that our study used microCT
images with a voxel resolution of 16.24 ␮m, whereas previous
studies used histological sections. The higher resolution of
histology may allow smaller and less obvious alveoli to be
counted. Also, a reduction in the number of alveoli has been
suggested to occur in humans, as was shown in a study that
found increased mean linear intercept with age (29). This may
be why fewer alveoli were counted in the subjects in the
present study; they were older, with an average age of 53.8 yr
compared with 28.5 yr in the study by Ochs et al. (21).
Measurements of alveolar density in JB4 tissue blocks from
one subject showed that both of these factors had an effect on
alveolar density. In that subject (donor 3), alveolar density was
found to be twice as dense on tissue sections than it was when
microCT was used. In addition, alveolar density was much
lower in the subjects in the present study compared with those
in the study by Ochs et al. (21), suggesting that age-related
effects on alveolar density do indeed occur (Fig. 3).
Despite fewer alveoli being counted in these lungs, acinar
volumes were similar to those reported previously. Acinar
volume was calculated by dividing the total number of alveoli
per terminal bronchiole by the number of alveoli per volume to
Table 2. Lung and airway measurements
CT lung volume, ml
CT lung mass, g
Vv parenchymal tissues
No. of alveoli/mm3
No. of alveoli/lung, ⫻106
(per lung pair)
No. of terminal bronchioles/lung
No. of alveoli/terminal bronchiole
Acinus volume, mm3
Donor 1
Donor 2
Donor 3
Donor 4
2,826
323
0.92
26.2
68.0
(136.0)
21,306
3,191
133
2,959
308
0.93
25.6
70.1
(140.2)
12,472
5,618
237
3,227
359
0.90
24.3
71.0
(142.0)
31,343
2,264
103
3,992
339
0.82
33.9
111.3
(222.6)
23,898
4,656
167
CT, computed tomography, Vv, volume density.
J Appl Physiol • doi:10.1152/japplphysiol.01017.2014 • www.jappl.org
Means ⫾ SE
3,251 ⫾ 261
332 ⫾ 11
0.89 ⫾ 0.02
27.5 ⫾ 2.2
80.1 ⫾ 10.4
(160.2 ⫾ 20.8)
22,255 ⫾ 3,893
3,932 ⫾ 747
160 ⫾ 29
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Total lung volume, lung mass, and number of alveoli measured per lung, terminal bronchioles per lung, and alveoli per
terminal bronchiole are shown in Table 2. The percentage of
parenchymal lung tissue vs. blood vessels and airways for
calculating total number of alveoli within the lungs was measured to be 89 ⫾ 2%.
Ranking lung samples from the apex to base of the lung
shows a significant correlation between lung height and alveolar density, with a greater number of alveoli per cubic millimeter of lung present in the upper regions compared with lower
regions. Correlation of alveolar number to lung height is equal
to y ⫽ ⫺2.5015x ⫹ 34.983 (R ⫽ 0.567, P ⫽ 0.0091), where y
equals number of alveoli and x is lung height (range, 1-5; 1 ⫽
lung apex, 5 ⫽ lung base). In the lung apex, alveolar density is
31.6 ⫾ 3.4 alveoli per mm3, in the lung base alveolar density
is 21.2 ⫾ 1.6 alveoli per mm3 (Fig. 2A). A comparison of
samples from lung apex to lung base shows a significant
difference (P ⬍ 0.05). Despite an alveolar density gradient that
occurs through the lung, there was no change in airspace size
at TLC from apex to base when compared using the Lm values
for lung height (Fig. 2B). The volume fraction of alveolar ducts
in the parenchymal lung tissue was increased in the lung base
compared with the lung apex (29 ⫾ 6% vs. 15 ⫾ 6%, P ⬍
0.05).
Alveolear density was found to be approximately twice as
great in JB4 sections (53 alveoli/mm3 in the lung apex and 48
alveoli/mm3 in the lung base) compared with that using microCT (26 alveoli/mm3 and 22 alveoli/mm3, respectively) on
the same tissue blocks from this subject.
•
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MicroCT Counts of Alveolar Number
•
McDonough J et al.
volume of 182.8 mm3. Another study (16) measured two acini
and showed acinar volume at 140 mm3 and 104 mm3. A third
study (10) measured the volume of six acini and found an
average of 185 ⫾ 78 mm3 (range 88.1–306.2 mm3). More
recently, investigators used synchrotron radiation microCT
imaging to reconstruct the alveolar structure, a technique
similar to that used in the present study. Their study examined
12 samples from a single lung and showed an acinar volume of
131.3 ⫾ 29.2 mm3 (range 92.5–171.3 mm3) (17). Finally, one
other study (23) used lung casts to measure four acini in two
subjects and reported volumes of 8.7 mm3 and 1.3 mm3 for a
26-year old woman, and 30.9 and 14.2 mm3 for an elderly man.
However, these values are significantly below the values reported by all other groups and are likely the result of inadequate infiltration of the casting polymer into the alveolar
regions. Although the present study did not directly measure
acinar volume, our calculated acinar volume of 160 ⫾ 29 mm3
(Table 2) was within the range of values reported previously.
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Fig. 2. A: the number of alveoli per cubic millimeter of lung was averaged from
four fields of view from each core with five cores of lung tissue per lung
arranged from apex to base from four normal human lungs. No difference in
alveolar density was found between the lungs from two donors with a history
of smoking (donors 1 and 2) and two donors who did not smoke (donors 3 and
4). These data show a marked reduction in the number of alveoli per cubic
millimeter in the base of the lung compared with the apex, and this trend was
observed in all four lungs examined. B: the mean linear intercept (Lm) was
measured from the apex to the base of four normal human lungs to obtain an
average airspace size. There was no difference in Lm measurements from the
apex to the base of these lungs.
equal the volume per terminal bronchiole. Because each acinus
is associated with one terminal bronchiole, the calculated
volume is equal to acinar volume and was measured to be
160 ⫾ 29 mm3. Several studies have used serial reconstruction
of individual acini to measure volume; one of the earlier
studies (11) examined a single acinus and showed an acinar
Fig. 3. Comparison of alveolar structure in a young subject [age 28 yr, from the
study by Ochs et al. (21)] (A) and from an older subject in the present study
(donor 3, age 59 yr) (B). Greater alveolar density can be observed in the
younger subject, which may account for differences in alveolar counts between
studies. Both images were captured at the same magnification (⫻5).
J Appl Physiol • doi:10.1152/japplphysiol.01017.2014 • www.jappl.org
MicroCT Counts of Alveolar Number
McDonough J et al.
1433
Studies of frozen dogs have shown that due to the pleural
pressure gradient, the lung at functional residual capacity
(FRC) is equal to 80 –90% TLC in the upper lung regions and
only 30 – 40% TLC in the lower lung regions (14). In humans,
it has been shown that at FRC, regional lung volume at the
apex is 65% of TLC and 45% of TLC at the base (28). At TLC,
alveoli are a consistent size throughout the lung (7) with the
decrease in lung volume from TLC to FRC being due to a
decrease in the alveolar size, which results in an increase in
alveolar density (i.e., the same number of alveoli within a
smaller lung volume). As such, we can estimate the regional
alveolar density at FRC by dividing the calculated alveolar
density at TLC by the percent of FRC lung volume over TLC
lung volume. This calculation provides values of 48.6 ⫾ 10.3
alveoli per mm3 in the upper lung compared with 47.1 ⫾ 7.1
alveoli per mm3 in the lower lung. Interestingly, on the basis of
these equations, in a normally breathing person with upright
lungs at FRC, alveolar density appears to be equal between the
upper and lower lung regions.
Increased alveolar density is associated with a decrease in
the volume fraction of alveolar ducts in the parenchymal
tissues. Because mean linear intercept is a measure of airspace
size that includes measurements of both alveolar and ductal
airspace, the combination of increased alveolar duct and fewer
alveoli is likely why no change in Lm was noted between the
upper and lower lung. How the alveoli are arranged around the
alveolar ducts and how the ducts branch within different
regions of the lung can change the numerical density of alveoli
without a concomitant change in the tissue density of the lung.
Theoretically, the arrangement of alveoli around the alveolar
ducts would also affect lung tissue mechanics. Whereas animal
studies have shown a difference in pressure-volume curves in
upper vs. lower lobes of monkey lungs (22), this difference was
not found in humans (26).
The major limitation of this study is the small sample size of
only four subjects. However, small sample sizes are found in
most studies that have examined alveolar number or acinus
size. Acinar size was not directly measured in this study;
rather, it was only estimated using total number of alveoli and
total number of terminal bronchioles in the lung. In addition,
this study did not examine the branching structure of the
alveolar ducts within the acinus, which may explain the regional variability in alveolar numbers.
This regional variation may also account for why certain
diseases that involve the parenchyma may affect either the
upper or lower lung regions. For example, in centrilobular
emphysema, destruction of the parenchymal tissues tends to
favor the upper lung regions. Also, it is hypothesized that
idiopathic pulmonary fibrosis is due to a collapse of the alveoli
onto the ducts and to occur primarily in the base of the lung
(18). In summary, the novel finding of the present study is that
regional variation of alveolar density is correlated with lung
height, with greater alveolar density found in the lung apex
compared with the lung base in fully inflated excised human
lungs.
GRANTS
Fig. 4. Comparison of acinar size to body weight in several animal models.
Relationship shows a power distribution following the equation: acinar size
(mm3) ⫽ 0.02 ⫻ [body weight (g)]0.77 with an R2 of 0.95.
Support for this study was provided by the British Columbia Lung Association, and by the Thoracic Imaging Network of Canada, which is sponsored
by the Canadian Institute for Health Research.
J Appl Physiol • doi:10.1152/japplphysiol.01017.2014 • www.jappl.org
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In addition to human studies, acinar structure has also been
quantified in several animal models. Rodriguez et al. (25)
examined both rat and rabbit acini and found an average size of
1.86 and 3.46 mm3, respectively. More recently, using microCT techniques, studies have examined the lungs of mouse
and rat models. In the C57BL/6 mouse model, Vasilescu et al.
(30) quantified 10 acini in mice at age 12 wk and 12 acini in
mice at age 91 wk and found an average acinar size of 0.18 ⫾
0.08 mm3 (range 0.07– 0.31 mm3) in the younger mice and
0.36 ⫾ 0.15 mm3 (range 0.13– 0.68 mm3) in the older mice.
Barré et al. (2) examined eight 60-day-old Wistar rats and
measured an acinar size of 1.16 ⫾ 0.138 mm3. This is similar
to values reported by Haberthür et al. (9) when they measured
43 acini in three 60-day-old Sprague-Dawley rats (1.148 ⫾
0.322 mm3). Interestingly, plotting these values of acinar size
in the various species against their respective body weights
shows a power distribution with an exponent of 0.77 (Fig. 4).
Further studies measuring acinar size in other animal species
will be required to determine whether this relationship remains
true.
Variation in lung physiology (i.e., blood flow and ventilation) is known to correlate with lung height. This is thought to
be due to the effects of gravity on the lung, which create a
pleural pressure gradient from the lung apex to the base (3).
However, at least one study (8) has suggested that regional
heterogeneity is not entirely dependent on gravity. Despite the
fact that the pleural pressure gradient is well described, few
studies have attempted to measure regional variation in lung
anatomical structures. One study (6) examined regional differences in the structure of dog lungs and found a small increase
in the alveolar surface density in the dorsal vs. ventral regions
of these lungs. Another study (7) measured alveolar size in
upright dog lungs and noted an increase in alveolar size in the
lung apex compared with the base. Because dogs are naturally
in the horizontal position, the normal orientation for the lung is
from dorsal to ventral position as opposed to humans, who are
upright, and in whom the lung is oriented from apex to base.
•
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MicroCT Counts of Alveolar Number
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
J.E.M. and J.C.H. conception and design of research; J.E.M., L.K., A.C.W.,
and W.M.E. performed experiments; J.E.M. analyzed data; J.E.M. interpreted
results of experiments; J.E.M., L.K., and M.O. prepared figures; J.E.M. drafted
manuscript; J.E.M., L.K., M.O., and J.C.H. edited and revised manuscript;
J.E.M. and J.C.H. approved final version of manuscript.
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