/. Embryo!, exp. Morph. Vol. 48, pp. 177-183, 1978
^77
Printed in Great Britain © Company of Biologists Limited 1978
Acetylcholinesterase- and norepinephrinecontaining nerves in developing rat lung
By AL-WALID I. EL-BERMANI 1 AND
EUNICE I. BLOOMQUIST 2
From the Departments of Anatomy and Physiology,
Tufts University School of Medicine, Boston
SUMMARY
The lungs of rat fetuses at various stages of gestation and lungs of infant rats were examined
histochemically for acetylcholinesterase (AChE) and norepinephrine (NE). No AChE is
present in the fetal lungs until 15 days of gestation. At this stage a number of large round
cells appear which stain heavily for AChE. These cells disappear by the 18th day of development and at 18 days no AChE-positive structures are demonstrable within the lung. The
large AChE-positive cells are of similar size and distribution tofluorescentcells which become
apparent after treatment of the mothers with L-DOPA. At 20 days, the day before delivery,
a diffuse AChE reaction appears in the walls of large branches of intrapulmonary bronchi.
At 20 days, also, sparse NE-containing nerves are present near the hilum and extend along
bronchial arteries into the lung. Not until birth do AChE-containing nerves appear in
intrathoracic structures. These are vagal preganglionic and postganglionic fibers near the
trachea, bronchi, and esophagus. AChE-positive ganglion cells are present in the walls of
extrapulmonary bronchi at birth, and perimuscular nerve plexuses containing AChE are
also present in the bronchial walls. NE-containing nerves are visible in several divisions of
the bronchial artery at birth. Three days postnatally, AChE-containing nerves have not yet
invaded intrapulmonary structures, but at this stage the adult pattern of adrenergic innervation is present. By the fifth postnatal day, sparse AChE-positive nerves are associated with
intrapulmonary bronchi, and rats 9 days old present the adult pattern of cholinesterasecon tain ing nerves.
INTRODUCTION
The adult lung contains large amounts of specific acetylcholinesterase (AChE)
associated with cholinergic parasympathetic preganglionic and postganglionic
nerves (El-Bermani, McNary & Bradley, 1970; El-Bermani & Grant, 1975).
These parasympathetic nerves are distributed to the bronchi and bronchioles
in rat and monkey (El-Bermani & Grant, 1975), where they regulate mucus
secretion and airway diameter (Nadel, 1977). Norepinephrine (NE)-containing
nerves are associated with vascular structures and are occasionally found in
bronchial and bronchiolar walls (El-Bermani, 1978; El-Bermani et ah 1970).
1
Author's address: Department of Anatomy, Tufts University School of Medicine, 136
Harrison Avenue, Boston, Massachusetts 02111, U.S.A.
2
Author's address: Department of Physiology, Tufts University School of Medicine, 136
Harrison Avenue, Boston, Massachusetts 02111, U.S.A.
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AL-WALID I. EL-BERMANI AND E. I. BLOOMQUIST
Physiologic studies in lamb fetuses which have shown that bilateral section of
cervical vago-sympathetic trunks has no effect on pulmonary vascular resistance
suggest lack of autonomic innervation to the pulmonary circulation in the fetus
(Emery, 1969). The present studies were undertaken to trace the development of
AChE- and NE-containing nerves in fetal and infant rat lung.
MATERIALS AND METHODS
Individual rat fetuses were removed by Cesarean section from their mothers
at 10, 15, 18 and 20 days of gestation and rapidly frozen in dry ice. Remaining
fetuses were allowed to progress to term to corroborate the gestational times.
A total of 20 litters were used for these studies. Young rats 3, 5, and 9 days old
were dissected and their thoraces rapidly frozen in dry ice. Frozen sections of
fetal or neonatal thoraces 15 /im thick were cut in a cryostat and stained either
for AChE by the El-Badawi & Schenk (1966) modification of the method of
Karnovsky & Root (1964) or subjected to formaldehyde condensation after
freeze drying for the fluorescent demonstration of catecholamines (Falck, 1962).
Controls for the histochemical reactions for AChE and NE were frozen sections
of adult lung subjected simultaneously to the same procedure. Sections were
counterstained with hematoxylin for identification of pulmonary structures.
RESULTS
10 days. The lung appears as groups of small cells arranged in columns which
radiate in many directions. No specific AChE reaction can be found nor can the
non-specific cholinesterase (ChE) reaction be seen on lowering the concentration
of inhibitor (ISO-OMPA). Occasionally random clusters of small cells with
intense counter-staining of the nuclei are seen. No catecholamine fluorescence
is demonstrable.
75 days. The number of cells is increased and the presence of two types of
cells becomes apparent. A large number of homogeneous cells lightly stained
for AChE form a background for a few randomly appearing larger cells which
are positively stained for AChE. Canalization between the smaller cells appears
in some areas but there is no consistent relation between the canalized areas
and the darkly stained AChE cells (Fig. 1). One to two hours after administration of L - D O P A to mothers 15-16 days pregnant, highly fluorescent cells
with a similar size and pattern to the AChE-containing cells become apparent
in the fetuses. The fluorescence of these cells is of the same wavelength as that
of the catecholamines. Even with the L-DOPA treatment, no fluorescent nerves
are apparent at this stage (Fig. 2).
18 days. The lungs at this stage demonstrate increased canalization and
reorganization of the cellular elements. A large number of bronchial buddings
are present at various stages of organization. No AChE reaction in the lung is
AChE- and NE-containing nerves in rat lung
179
Fig. 1. Lung from a 15-day rat fetus showing scattered large intensely AChEpositive cells among smaller lightly stained cells.
Fig. 2. Lung from a 15-day rat fetus whose mother was injected intraperitoneally
2 h prior to Cesarean section with L-DOPA. Scattered cells with the characteristic
fluorescence of catecholamines are demonstrated and are of similar size to the
AChE-containing cells of Fig. 1.
Fig. 3. Thorax from an 18-day rat fetus treated to demonstrate AChE. The intensely
AChE-positive cells of the 15-day fetus are no longer visible in the lungs. No AChE
nerves are visible in the lung (L), although other thoracic structures such as skeletal
muscle (arrow) react for AChE.
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A L - W A L I D I. EL-BERMANI AND E. I. BLOOMQUIST
Fig. 4. Newborn rat lung treated to demonstrate AChE. AChE-containing perimuscular nerve plexuses are visible in the bronchial wall. At this stage they extend
only as far as the major bronchi. Intrapulmonary bronchial walls (Br) show an
increased diffuse AChE reaction at this age (arrow).
Fig. 5. Newborn rat lung treated to demonstrate catecholamine fluorescence. NEcontaining nerves are visible in the bronchial artery walls for 4-5 divisions.
Fig. 6. Lung from a 5-day-old rat treated to demonstrate AChE. Fine AChEcontaining nsrves (arrow) can be seen associated with the intrapulmonary bronchi
(Br) in relation to the muscular layer (M).
AChE- and NE-containing nerves in rat lung
181
apparent now despite the presence of the reaction in other thoracic tissues such
as skeletal muscle (Fig. 3). The intensely AChE-positive cells of an earlier age
(15 days) have disappeared. No NE-containing nerves are detectable at this
stage, but with L-DOPA treatment of the mothers a few fluorescent cells similar
to those present at 15 days are visible.
20 days. At this period most bronchi can be seen to have definite mucosal
layers. Specific AChE-staining occurs in the walls of larger branches of intrapulmonary bronchi, but this staining reaction is diffuse and lacks the morphologic localization characteristics of nerves. NE-containing nerves are associated
with hilar structures and extend along some bronchial arteries for a short
distance into the lungs.
Newborn. In newborn rats AChE is present in vagal preganglionic and postganglionic fibers near the esophagus, trachea, and bronchi. Some ganglion
cells containing AChE can be seen in the walls of extrapulmonary bronchi near
the AChE-containing nerves. At the tracheo-bronchial junction (the carina)
AChE-positive perimuscular nerve plexuses similar to the postganglionic
parasympathetic nerves in the adult are visible (Fig. 4). There is an increase
in the diffuse AChE reaction in the intrapulmonary bronchial walls. NEcontaining nerves are demonstrable through four tofivedivisions of the bronchial
artery (Fig. 5).
Postnatal. In lungs from 3-day-old animals there are no AChE-containing
intrapulmonary nerves or plexuses, but the adrenergic nerves present essentially
the adult pattern (El-Bermani, 1978). On the fifth postnatal day, sparse AChEcontaining nerves can be seen associated with the intrapulmonary bronchi
(Fig. 6). Animals 9 days old show an AChE-containing innervation similar to
that seen in the adult rat (El-Bermani, 1973).
DISCUSSION
The present results indicate that AChE-containing nerves do not extend to
the intrapulmonary structures of the rat until 5-9 days after birth, and no
sympathetic innervation is present within the rat lung until just before birth.
Since AChE is always associated with cholinergic nerves in the adult (El-Bermani
& Grant, 1975), it seems probable that parasympathetic innervation of the rat
lung is lacking during prenatal and early postnatal life. These findings provide
anatomical support for physiological studies in lamb fetuses which showed that
bilateral section of cervical vago-sympathetic trunks has no effect on pulmonary
vascular resistance suggesting lack of autonomic innervation to the pulmonary
circulation in the fetus (Emery, 1969).
The AChE-containing nerves which do appear in the lung parenchyma after
5 postnatal days arise from AChE-positive fibers in the vagus and ganglia near
the hilum. These nerves gradually extend to extrapulmonary thenintrapulmonary
structures forming characteristic preganglionic fibers and postganglionic
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AL-WALID I. EL-BERMANI AND E. I. BLOOMQUIST
plexuses as they enter a given pulmonary tissue. The sympathetic nerves enter
the lung with the bronchial arteries and follow the bronchial arteries to invest
other pulmonary structures.
The significance of the large AChE-positive cells which are present at 15 days
of gestation and disappear by 18 days is unclear. They bear no consistent
morphological position relative to the canals which are forming at this period
of development. It is unlikely that they represent parasympathetic ganglion
cells invading the pulmonary tissue since their AChE reaction disappears
3 days before birth. Since the AChE-containing cells have a similar distribution to those which become apparent after L - D O P A treatment, it is possible
that they represent amine precursor uptake and decarboxylation cells (APUD
cells). These cells are known to be present in the adult respiratory epithelium and are characterized ultrastructurally by small (100 nm) dense-core
cytoplasmic granules. They have the ability to incorporate L-DOPA and to
transform it to a fluorogenic monoamine. Whatever the origin or fate of these
large cells we have observed in the developing rat lung is, their transient AChEpositive reaction apparently represents a specific stage in their maturation.
Our findings in the developing rat lung contrast with those of Hung (1978),
who studied developing lung in the mouse, a species with a gestation period
similar to that of the rat. While we have not observed adrenergic nerves in the
rat lung until after 18 gestational days and have not observed cholinesterasecontaining nerves before birth, Hung has reported that in 15-day mouse fetuses
nerves are present in trachea, bronchi and large bronchioles. According to
Hung (1978), autonomic ganglia are also present in mouse lungs on the 15th
day and single nerve fibers extend into connective tissue between developing
airways. Fluorescent adrenergic nerves appear in the mouse lung by the 15th
day, and cholinergic nerves are present in 18-day-old fetuses. The disparity in
our results may be due to specific differences relating to the timing of development of the pulmonary innervation in the two species. More likely, though, it
relates to general differences in the timing of development of the lung between
the two species. Hung (1978) describes well-developed airways in 18-day fetuses
and identifiable bronchi and bronchioles at 15 days. General development of
the rat lung lags well behind this schedule according to our observations.
This research was supported by N.I.H. Grant no. 20548 from the National Heart, Lung,
and Blood Institute.
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{Received 15 May 1978)