- Email: [email protected]
Cholinergic nerve development of fetal lung in vitro
Cholinergic Nerve Development of Fetal Lung In Vitro By Yasuhide Morikawa, Patricia K. Donahoe, and W. Hardy Hendren 9 The development of the cholinergic or parasympathetic nervous system was studied in embryonic lung dissociated from the central nervous system and placed in organ culture. In vitro development was similar to that seen in vivo. This study demonstrated that migration of neuroblasts from the central nervous system to the trachea takes place before day 12. Neuroblasts differentiate to small Immature ganglia and then to larger more mature ganglia that give off nerve fibers to the submucosa and the epithelium. This isolated lung preparation was used to study neurosUmulators. Thyroxine ('1"4) was found to accelerate nerve fiber growth end differentiation of ganglia in vitro. The neuroepithelial body, an epithelial cell with acetylchoIine-esterase-positive granules, also differentiated in vitro. This cell may fill the morphologic criteria of an Intrapulmonary chemoreceptor. INDEX WORDS: Cholinergic nervous system; sympathethic nervous system; fetal lung.
para-
understand congenital disease states characterized by lability of the autonomic nervous system, growth stimulators and inhibitors have been added to the in vitro system designed for this study to determine their effect on cholinergic maturation of the lung. It is anticipated that this in vitro system can be used to study factors that may influence maturation of the autonomic nervous system of the lung after migration has occurred. MATERIALS AND METHODS Timed pregnant female rats were obtained from Holtzman Laboratories, and fetuses were delivered by cesarean section at day 12, day 13, day 14, and day 17 post coitus. Fetal lungs were harvested on each of these days using microsurgical technique at X 16 magnification.
I n Vivo
ITTLE IS K N O W N about the development of the autonomic nervous system in the embryonic lung. Physiologic studies have indicated that parasympathetic and sympathetic afferent and efferent pathways from vagus and sympathetic trunks mediate respiratory functions. Changes in frequency, depth, and regularity of breathing are initiated in response to stretch, irritation, and chemical changes (CO2, 02, pH) by these pathways. 1'2 As part of a systematic study of the development of the autonomic nervous system of the embryonic lung, we previously described development of the cholinergic or parasympathetic nervous system in the lungs of embryo rats from day 12 of fetal life to the end of gestation at 21 days, using acetylcholine esterase histochemical techniques) Cholinergic development in the embryonic lung is characterized by migration of neuroblasts from the vagus to the carina and subsequently to the third branch point of the trachea (Fig. 1), proximal to distal maturation of ganglia after migration, growth of nerve fibers to the tracheal epithelium from the ganglia, and formation of neuroepithelial cells. The present study, undertaken to determine whether similar development would occur in the embryonic lung dissociated from the central nervous system, demonstrates that development of the cholinergic nervous system in vitro parallels development in vivo. As part of an effort to
L
Journal of Pediatric Surgery,
Vol. 13, No. 6D (December), 1978
The control lungs (i.e., those not exposed to organ culture incubation) were immersed in 10% formol calcium solution (pH 7.2) 4 for 12 hr at 4~ (n = 47). Specimens were then washed in 0.88M g u m sucrose5 and allowed to harden in fresh g u m sucrose at 4~ for 1-3 days, after which they were cut in 8-# sections on a Harris W R C cryostat at -25~ Frozen sections were mounted on precooled 3% gelatinized slides and incubated in 10% formol calcium at 4~ for 5 min to allow the gelatin to solidify. Acetylcholine esterase histochemistry staining was done by the Karnovsky and Roots direct coloring m e t h o & using acetylthiocholine iodide (Sigma) as the substrate and incubating at 37~ for 1 hr. Buffered methyl green was used as a counterstain. Tetraisopropyl phosphoramide ( 8 ) < 10"SM) was added during acetylcholine esterase incubation in order to inhibit nonspecific choline esterase activity]
In Vitro Whole lung with esophagus was dissected from 12-day (n = 44), 13-day (n = 30), and 14-day (n = 32) fetal rats and cultured on agar-coated grids in Falcon 1030 organ culture dishes with 2 ml of medium: C M R L 1066 with 10%
From the Pediatric Surgical Research Laboratory, Division of Pediatric Surgery and Department of Surgery, Massachusetts General Hospital, and Harvard Medical School, Boston, Mass. Presented before the 9th Annual Meeting of the American Pediatric Surgical Association, Hot Springs, Virginia, May 3~6, 1978. Address reprint requests to Patricia K. Donahoe, M.D., Division of Pediatric Surgery, Massachusetts General Hospital, Boston, Mass. 02114. 9 1978 by Grune & Stratton, Inc.
oo22-3468/78/13o7-oo175Ol.OO/O 653
654
MORIKAWA.
DONAHOE,
AN D HENDREN
" ~, ~l'84
12 day 13 doy 14 doy
15 day Fig. 1. Migration of neuroblasts during embryonic development. Dots around bronchial buds indicate the position of neuroblasts at various ages.
fetal calf serum, 200 units penicillin, and 200 ~tg streptomycin (Gibco). The lungs were incubated at 37~ in saturated humidity with 95% air and 5% CO2 (Fig. 2). Selected specimens (n = 13) were observed for rhythmic movements from 1 to 3 days. Incubation was terminated on the remainder Of the specimens after 1 or 2 days for acetylcholine esterase histochemistry. The trachea was dissected from 17-day fetuses (n = 54), and tracheal rings were cut transversely. Cross-sectionrings from the level of the carina were cultured as above for 1 or 2 days. Processingof the in vitro specimens had to be varied slightly because of the increased fragility of the tissue. The initial steps of fixation and rinsing for acetylcholine esterase histochemistry were the same; subsequently, however, the cultured tissue was embedded in agar to make it firmer before sectioning on the cryostat. Acetylcholine esterase histochemistry was carried out on these agar-embedded frozen sections using the Karnovsky and Roots technique, but substrate incubation time with acetylthiocholine iodide had to be increased.
or
Fig. 2. In vitro culture of embryonic lung incubating on agar-coated stainless-steel grid over complete medium.
Na-L-thyroxine (Sigma) was added to the organ culture media (CMRL 1066) in concentrations of 100, 10, or 1 g/ml: The 17-dayfetal carinal r~ngswere then incubated in organ culture for 24 hr (n = 6), and acetylcholineesterase histochemistry was performed. Colchicine(Calbiochem) was added to the organ culture media in concentrations of 1mM, 0.5mM, or 0.1 mM.9The 17-daycarinal rings were incubated in organ culture for 48 hr (n = 12), and acetylcholine esterase histochemistry was performed.
RESULTS T h e 12-day e m b r o n i c lung dissociated from the central nervous system had no detectable acetylcholine esterase activity except in the vagus nerve (Fig. 3A). However, after 2 days of i n c u b a t i o n in vitro, neuroblasts could be detected in the trachea at the level of the carina, dispersed a m o n g the indifferent m e s e n c h y m e (Fig. 3B). These cells were indistinguishable from other m e s e n c h y m a l cells, except that they took the acetylcholine esterase stain. After 3 days in organ culture, r h y t h m i c contraction of the first b r o n c h i a l b r a n c h was seen in all specimens. A t 13 days the embryonic lung had lightly staining neuroblasts in the m e s e n c h y m e s u r r o u n d i n g the trachea. After 1 day of in vitro i n c u b a t i o n (Fig. 4), neuroblasts could be seen as far distal as the first bronchial branch. R h y t h m i c m o v e m e n t was seen at the end of the second day of i n c u b a t i o n in 2 of 4 cultures, and it became evident in all specimens after 3 days of culture. A t day 14, smooth muscle b e g a n to differentiate, and neuroblasts enlarged a n d stained more intensely. After 1 day in culture, smooth muscle differentiated further, neuroblasts migrated to the third b r o n c h i a l b r a n c h , a n d m o v e m e n t could be observed (Fig. 5). D u r i n g day 15 a n d 16 both smooth muscle and cartilage formed. Neuro-
CHOLINERGIC NERVE DEVELOPMENT IN FETAL LUNG
655
Fig. 3. (A) Twelve-day fetal trachea (T) and esophagus (Es). Acetylcholine esterase activity is seen in the vagus nerve (V) (X 360); (B) Twelve-day fetal trachea (T) and 2-day incubation. Neuroblasts (Nb) migrate from vagus (V) to mesenchyme around trachea
(• 18o).
656
MORIKAWA,
Fig. 4.
Thirteen-day fetal bronchus (first branch) and 1-day incubation. Neuroblaeta (Nb) surround the epithelium (X200).
DAYS IN ORGAN CULTURE AGE OF EMBRYO (doys)
12
13
o
1
2
3
neuroblost ( - )
nb
(-)
nb (+)
movement ( - )
m (-)
m (+)
(+)
nb ( + )
m (_+)
m (+)
nb
(_+)
m
(--)
nb
nb
(+)
nb
m
(_+)
m (+)
m (+)
7,14
DONAHOE, AND HENDREN
(+)
7
4- MaturationalAge i~
^f ~mk.yo J_ days in culture)
Fig. 5. Progressive development o f rhythmic movement o f the first bronchial branch of 12- to 14-day embryonic lung in organ culture. Embryonic lung was dissociated from the spinal cord and placed in culture from 1 to 3 days until a maturaUonal age of 15 days was reached. Movement was detected after smooth muscle had d i f f e r e n t i a t e d and neuroblasts had migrated.
blasts took a tangential position outside the smooth muscle and differentiated into ganglia. 3 Seventeen-day fetal trachea was characterized by well-developed cartilage, smooth muscle, and cilia formation. Ganglia enlarged, but no nerve fibers could be seen. After 1 day of incubation, ganglion cells outside the smooth muscle differentiated more fully, but nerves still could not be seen. Neuroepithelial cells, however, were clearly demonstrated (Fig. 6). After 2 days of incubation in vitro, in addition to generalized growth characterized by cartilage and smooth muscle development, acetylcholine-esterasepositive nerve fibers could be seen extending from the extramuscular ganglion cells to the submucosa and thence to the epithelium. Small submucosal ganglia were also seen. Since the changes of cholinergic nerve development were most marked in the 1- to 2-day interval after 17 days, this in vitro system was used to test the effects of growth inhibitors and
CHOLINERGIC NERVE DEVELOPMENT IN FETAL LUNG
657
Fig. 6. Seventeen-day fetal trachea after 1-day incubation. Neuroepithelial cells, which contain acetylcholine esterase granules, are found among the epithelial cells that line the airway.
stimulators (Fig. 7): 100 and 10 #g/ml of Na-L-thyroxine (T4) resulted in severe toxicity; however, after 1 day of incubation with l#g/ml, ganglia were observed to mature and nerves to grow from the ganglia to the epithelium submucosalaganglia were also observed (Fig. 8B). This accelerated development was not observed in controls to which no thyroxine was added (Fig. 8A). Colchicine at lmM and 0.5raM was toxic to the culture; 0.1 mM colchicine was effective in selectively blocking differentiation of ganglia and completely inhibiting nerve fiber growth (Fig. 9A and B). The neuroepithelial body, however, could be detected despite inhibition of both ganglionic and nerve fiber development. 17 + 1 day in culture G
DISCUSSION Maturation of the parasympathetic nervous system can occur in vitro ]~ and can be accurately
monitored using acetylcholine esterase histochemical techniques. Acetylcholine-esterasepositive neuroblasts cannot be seen in in viva embryonic lung until day 13 of gestation;3 however, if 12-day embryo lung (the youngest lung that can be successfully dissected from the central nervous system and sectioned on the cryostat) is placed in culture, neuroblasts can be detected 2 days later, indicating clearly that migration to the dissociated lung occurred prior to day 12 of gestation. Despite the lack of continued stimulus from the central nervous
17 -I- 2 days in culture G Nf
~M
CONTROL 17 day
~
"
embryo STIMULATOR tracheal ring / - - ~ ~ S { THYROXIN M * ~Ipg/ml)
( COLCHICINE0.1 mM)
. ~'Nf e .~~ff/ \ ~ ,
Fig. 7. Seventeen-day fetal tracheal rings exposed to in vitro incubation with thyroxine or colchicine. Thyroxine accelerated nerve fiber growth and ganglionic differentiation. Colchicine delayed this development, when compared with upper controls.
658
Fig. 8. Seventeen-day tracheal ring in vitro exposed to 1 /~g/ml thyroxine for 24 hr. Ganglia (G) differentiate and nerve fibers (Nf) appear (B). Nervous development is accelerated in comparison with that observed in control tracheal rings (A).
system in later cultures, neuroblasts differentiate into ganglia from which nerve fibers, previously undetected, grow to the submucosa and epithelium. Rhythmic contractile movements, limited primarily to the region of the first bronchial branch, were observed in 12-day, 13-day, and 14-day specimens cultured for 3 days, 2 days, or 1 day in the organ culture system, when they attained a maturational age (embryonic age + age in culture) of 15 days. The appearance of movement correlated with the condensation of mesenchyme and differentiation of smooth muscle (Fig. 5). Nerve fibers were not evident at this time; therefore it seems probable that this peristaltic movement is propagated by musclemuscle interaction rather than by nerve-muscle interaction. Dramatic changes were observed in the 17day tracheal ring during 1 and 2 days of culture. Immature ganglia enlarged and took on the morphology characteristic of the mature adult ganglion cell. Tangential to the smooth-muscle layer, large multinucleated cells with intense
MORIKAWA, DONAHOE, AND HENDREN
Fig. 9. Seventeen-day tracheal rings in vitro exposed to 0.1mM colchicine for 48 hr. hours. Nervous development is inhibited (B) in comparison with that seen in control tracheal rings (A), in which ganglia (G) differentiate and nerve fibers (Nf) appear.
acetylcholine esterase activity in their cytoplasm appeared, possibly from condensation of smaller neuroblasts. These ganglia gave nerve fibers to the smooth muscle and to the submucosa. Smaller ganglia appeared in the submucosa and in turn gave off nerve fibers that traveled both circumferentially in the submucosa and to the epithelium. The function of these epithelial nerve fibers is not completely understood, although it is speculated that they may play some role in mucus secretion or ciliary movement. Colchicine, by blocking binding of a and subunit proteins via guanine nucleotide and thus preventing polymerization of tubulin, inhibits the formation of microtubules, a predominant organelle in nerve fibers, n Overall toxicity was produced by l m M and 0.5mM concentrations; however, 0.1mM selectively blocked nerve fiber formation and differentiation of ganglia. It is of interest that despite its effect on these nervous structures, neuroepithelial cells developed under these conditions, indicating, as expected, that another developmental mechanism is involved.
CHOLINERGIC NERVE DEVELOPMENT IN FETAL LUNG
659 AchE + gronules
02
Fig. 10. Diagram of proposed intrapulmonary chemoreceptor. We speculate that changes in the O= or CO= in the airway affect acetylcholine esteraae (Ach E + ) granules, which in turn signal the firing of afferent neurons to the central nervous system.
--'~'-
~~
-
efferent
t~_
~afferent \-Nerves
Thyroxine is known to affect the growth, development, and metabolism of virtually all tissues of higher organisms. Its specific effect on nervous elements as studied in the rat central nervous system in vivo is obscure, but it appears to accelerate differentiation after proliferation has occurred. 12"13 Hamburgh 14 observed that thyroxine was required to maintain acetylcholine esterase activity in rat brain tissue in vivo. It seemed reasonable, therefore, to test thyroxine as a neurostimulator in this cholinergic lung developmental system. At a concentration of 1 #g/ml, thyroxine stimulated both ganglia and nerve fibers. It is of interest that thyroxine could not increase lamellar body formation in tissue culture of type II penumocytes of 18-day fetal rat lung, an effect readily seen with steroids. 8 The neurostimulatory affect observed in the present experiment was quite specific, more than a generalized growth stimulatory effect, and it deserves further study. The neuroepithelial cells with positive acetylcholine esterase granules in their cytoplasm may
fit the morphologic criteria for an intrapulmonary chemoreceptor. ~5'~6 We speculate that changes in CO2, 02, or pH in the airway may initiate nerve fiber activity via acethylcholineesterase-positive granules or dense core vesicles (Fig. 10). Recent physiologic experiments have suggested the presence of chemoreceptors in the isolated airways of birds ~7 and mammals. ~s'~9 However, the morphologic evidence for the existence of these chemoreceptors in the lung has not been clearly established. The neuroepithelial cells seen in this experiment differentiate in vitro and therefore can be singled out for more intense study. The combination of ultrastructural techniques with acetylcholine esterase histochemistry may yield more information about this particular cell. It is known that infants with aborted sudden infant death syndrome are less responsive to elevated intraalveolar CO 2 levels, z~ It is interesting to speculate that diminished sensitivity or diminished response of these neuroepithelial cells may play a part in the etiology of neonatal sudden infant death syndrome.
REFERENCES
1o Bartoli A, Cross BA, Guz A, et al: The effect of carbon dioxide in the airways and alveoli on ventilation, a vagal reflex studied in the dog. J Physiol (Lond) 240:91-109, 1974 2. Fillenz M, Widdicombe JG: Receptors of the lungs and airways, in Neil E (ed): Handbook of Sensory Physiology, vol 3. Berlin, Springer, 1972, pp 81-112 3. Morikawa Y, Donahoe PK, Hendren WH: Cholinergic nerve development in fetal lung. Dev Biol (in press) 4. Baker, JR: Principles of Biological Microtechnique, A
Study of Fixation and Dyeing. London, Methuen, 1958, pp 111-118 5. Holt S J, Hicks RM: Studies on formalin fixation for electron microscopy and cytochemical staining purpose. J Biophys Biochem Cytol 11:31-45, 1961 6. Karnovsky M J, Roots L: A "direct-coloring" thiocholine method for cholinesterase. J Histochem Cytochem 12:219-221, 1964 7. EI-Badawi A, Schenk EA: Histochemical methods for separate consecutive and simultaneous demonstration of
660
MORIKAWA, DONAHOE, AND HENDREN
acetylcholinesterase and norepinephrine in cryostat sections. J Histoehem Cytochem 15:580-588, 1967 8. Adamson YR, Bowden DH: Reaction of cultured adult and fetal lung to prednisolone and thyroxine. Arch Pathol 99:80-85, 1975 9. Thoa NB, Wooten GF, Axelrod J, et al: Inhibition of release of dopamine-/3-hydroxylase and norepinephrine from sympathetic nerves by colchicine, vinblastine or cytochalasin-B. Proe Natl Aead Sci USA 69:520-522, 1972 10. |to Y, Donahoe PK, Hendren WH: Differentiation of intramural ganglia in the dissociated rectosigmoid of the rat. J Pediatr Surg 12:969 976, 1977 11. Bucher NLR: Microtubules. N Engl J Med 287:195197, 1972 12. Balazs R: Biochemical effects of thyroid hormones in the developing brain, in Pease DC (ed): Cellular Aspects of Neural Growth and Differentiation. Berkeley, University of California Press, 1971 13. Schapiro S, Vukowich K, Globus A: Effects of neonatal thyroxin and hydrocortisone administration on the development of dendritic spines in the visual cortex of rats. Exp Neurol 40:286-296, 1973 14. Hamburgh M, Flexner LB: Effect of hypothyroidism
and hormone therapy on enzyme activities of the developing cerebral cortex of the rat. XXI. Biochemical and physiological differentiation during morphogenesis. J Neurochem 1:279-288, 1957 15. Laweryns JM, Cokelaere M, Theunynck P: Neuroepithelial bodies in the respiratory mucosa of various mammals. Z Zellforsch 135:569-592, 1972 16. Laweryns JM, Cokelaere M: Hypoxia-sensitive neuro-epithelial bodies intrapulmonary secretory neuroreceptors, modulated by the central nervous system. Z Zellforsch 145:521-540, 1973 17. Scheid P, Slama H, Gatz RN, et al: Intrapulmonary CO 2 receptors in the duck: III. Functional localization. Respir Physiol 22:123-136, 1974 18. Marsland DW, Callahan BJ, Shannon DC: The afferent vagus and regulation of breathing in response to inhaled CO2 in awake newborn lambs. Biol Neonate 27:102107, 1975 19. Fagenholtz SA, O'Connell K, Shannon DC: Chemoreceptor function and sleep state in apnea. Pediatrics 58:3136, 1976 20. Shannon DC, Kelly DH, O'Connell K: Abnormal regulation of ventilation in infants at risk for sudden infant death sundrome. N Engl J Med 297:747-570, 1977
Discussion J. Folkman (Boston): T h e a u t h o r s have offered us two exciting ideas to think about. O f p a r a m o u n t i m p o r t a n c e is the a t t e m p t to localize the c h e m o r e c e p t o r s in the lung, and if the neuroepithelial cell is a c a n d i d a t e , then it is entirely possible in the future t h a t one could see neonatologists using thyroxine as they now use steroids for the induction of s u r f a c t a n t in the lung. T h e second idea is linked with the field of developmental biology. A very c o m m o n p a t t e r n is emerging: in the e a r l y embryo, nerve cells m a k e long journeys and arrive at a p p r o p r i a t e t a r g e t organs. O n l y after they arrive do the other cell types begin their m a t u r a t i o n , i.e., cartilage, bone. If the neuroblasts do not arrive, one result m a y be H i r s c h s p r u n g ' s disease. But w h a t h a p p e n s if the neuroblasts arrive late? Is there a n y p e n a l t y ? Is differentiation d e l a y e d ? For example, if in a s a l a m a n d e r you a m p u t a t e the leg, the leg regenerates; if you section the sciatic nerve, the leg does not regenerate. This raises a question: Do the a u t o n o m i c neuroblasts, in fact, p l a y an i m p o r t a n t development role in organs such as the lung j u s t as the p e r i p h e r a l nerves do in limb development in lower v e r t e b r a t e s ? The techniques of Dr. M o r i k a w a and his colleagues
m a y be helpful in the future to e x a m i n e this question. K. Pringle (Chicago): I wonder if the authors have thought of looking at the status of these neuroepithelial cells in the hypoplastic lung of a d i a p h r a g m a t i c hernia to see w h e t h e r this is p e r h a p s a clue to the distressing incidence of late development of fatal respiratory distress in these patients. P. Donahoe (closure)." W e u n d e r t o o k these studies s t i m u l a t e d by the frustration t h a t we as p e d i a t r i c surgeons all face in w a t c h i n g infants w h o m we h a v e a d e q u a t e l y r e p a i r e d w i t h d i a p h r a g m a t i c hernias die because of i m m a t u r e lungs, and by the children with sudden infant d e a t h s y n d r o m e who a r e believed to have autonomic lability or i m m a t u r i t y o f unknown etiology. C u r r e n t thinking is t h a t the a u t o n o m i c deficit is central. O u r a p p r o a c h is t h a t the autonomic lability m a y be a distal deficit due to failure of m i g r a t i o n or failure of differentiation of autono m i c ganglia a f t e r migration. W e looked at possible ways of a c c e l e r a t i n g distal differentiation in vitro and chose thyroxine because o f evidence that this h o r m o n e s t i m u l a t e s differentiation of nerve cells.
CHOLINERGIC NERVE DEVELOPMENT IN FETAL LUNG
The hypothesis that the epithelial cell with positive acetylcholine esterase activity in its cytoplasmic granules may be an intrapulmonary chemoreceptor is important. These granules may, indeed, correspond to those cytoplasmic synaptic vesicles seen at the electron microscopic level by Laweryns. If, indeed, this proves to be
661
true, then this cell may provide a morphologic prototype for a peripheral chemoreceptor. There has been a great deal of physiologic evidence for the existence of intrapulmonary chemoreceptors, but no morphologic documentation. Perhaps this cell may fit the criteria.
para-
understand congenital disease states characterized by lability of the autonomic nervous system, growth stimulators and inhibitors have been added to the in vitro system designed for this study to determine their effect on cholinergic maturation of the lung. It is anticipated that this in vitro system can be used to study factors that may influence maturation of the autonomic nervous system of the lung after migration has occurred. MATERIALS AND METHODS Timed pregnant female rats were obtained from Holtzman Laboratories, and fetuses were delivered by cesarean section at day 12, day 13, day 14, and day 17 post coitus. Fetal lungs were harvested on each of these days using microsurgical technique at X 16 magnification.
I n Vivo
ITTLE IS K N O W N about the development of the autonomic nervous system in the embryonic lung. Physiologic studies have indicated that parasympathetic and sympathetic afferent and efferent pathways from vagus and sympathetic trunks mediate respiratory functions. Changes in frequency, depth, and regularity of breathing are initiated in response to stretch, irritation, and chemical changes (CO2, 02, pH) by these pathways. 1'2 As part of a systematic study of the development of the autonomic nervous system of the embryonic lung, we previously described development of the cholinergic or parasympathetic nervous system in the lungs of embryo rats from day 12 of fetal life to the end of gestation at 21 days, using acetylcholine esterase histochemical techniques) Cholinergic development in the embryonic lung is characterized by migration of neuroblasts from the vagus to the carina and subsequently to the third branch point of the trachea (Fig. 1), proximal to distal maturation of ganglia after migration, growth of nerve fibers to the tracheal epithelium from the ganglia, and formation of neuroepithelial cells. The present study, undertaken to determine whether similar development would occur in the embryonic lung dissociated from the central nervous system, demonstrates that development of the cholinergic nervous system in vitro parallels development in vivo. As part of an effort to
L
Journal of Pediatric Surgery,
Vol. 13, No. 6D (December), 1978
The control lungs (i.e., those not exposed to organ culture incubation) were immersed in 10% formol calcium solution (pH 7.2) 4 for 12 hr at 4~ (n = 47). Specimens were then washed in 0.88M g u m sucrose5 and allowed to harden in fresh g u m sucrose at 4~ for 1-3 days, after which they were cut in 8-# sections on a Harris W R C cryostat at -25~ Frozen sections were mounted on precooled 3% gelatinized slides and incubated in 10% formol calcium at 4~ for 5 min to allow the gelatin to solidify. Acetylcholine esterase histochemistry staining was done by the Karnovsky and Roots direct coloring m e t h o & using acetylthiocholine iodide (Sigma) as the substrate and incubating at 37~ for 1 hr. Buffered methyl green was used as a counterstain. Tetraisopropyl phosphoramide ( 8 ) < 10"SM) was added during acetylcholine esterase incubation in order to inhibit nonspecific choline esterase activity]
In Vitro Whole lung with esophagus was dissected from 12-day (n = 44), 13-day (n = 30), and 14-day (n = 32) fetal rats and cultured on agar-coated grids in Falcon 1030 organ culture dishes with 2 ml of medium: C M R L 1066 with 10%
From the Pediatric Surgical Research Laboratory, Division of Pediatric Surgery and Department of Surgery, Massachusetts General Hospital, and Harvard Medical School, Boston, Mass. Presented before the 9th Annual Meeting of the American Pediatric Surgical Association, Hot Springs, Virginia, May 3~6, 1978. Address reprint requests to Patricia K. Donahoe, M.D., Division of Pediatric Surgery, Massachusetts General Hospital, Boston, Mass. 02114. 9 1978 by Grune & Stratton, Inc.
oo22-3468/78/13o7-oo175Ol.OO/O 653
654
MORIKAWA.
DONAHOE,
AN D HENDREN
" ~, ~l'84
12 day 13 doy 14 doy
15 day Fig. 1. Migration of neuroblasts during embryonic development. Dots around bronchial buds indicate the position of neuroblasts at various ages.
fetal calf serum, 200 units penicillin, and 200 ~tg streptomycin (Gibco). The lungs were incubated at 37~ in saturated humidity with 95% air and 5% CO2 (Fig. 2). Selected specimens (n = 13) were observed for rhythmic movements from 1 to 3 days. Incubation was terminated on the remainder Of the specimens after 1 or 2 days for acetylcholine esterase histochemistry. The trachea was dissected from 17-day fetuses (n = 54), and tracheal rings were cut transversely. Cross-sectionrings from the level of the carina were cultured as above for 1 or 2 days. Processingof the in vitro specimens had to be varied slightly because of the increased fragility of the tissue. The initial steps of fixation and rinsing for acetylcholine esterase histochemistry were the same; subsequently, however, the cultured tissue was embedded in agar to make it firmer before sectioning on the cryostat. Acetylcholine esterase histochemistry was carried out on these agar-embedded frozen sections using the Karnovsky and Roots technique, but substrate incubation time with acetylthiocholine iodide had to be increased.
or
Fig. 2. In vitro culture of embryonic lung incubating on agar-coated stainless-steel grid over complete medium.
Na-L-thyroxine (Sigma) was added to the organ culture media (CMRL 1066) in concentrations of 100, 10, or 1 g/ml: The 17-dayfetal carinal r~ngswere then incubated in organ culture for 24 hr (n = 6), and acetylcholineesterase histochemistry was performed. Colchicine(Calbiochem) was added to the organ culture media in concentrations of 1mM, 0.5mM, or 0.1 mM.9The 17-daycarinal rings were incubated in organ culture for 48 hr (n = 12), and acetylcholine esterase histochemistry was performed.
RESULTS T h e 12-day e m b r o n i c lung dissociated from the central nervous system had no detectable acetylcholine esterase activity except in the vagus nerve (Fig. 3A). However, after 2 days of i n c u b a t i o n in vitro, neuroblasts could be detected in the trachea at the level of the carina, dispersed a m o n g the indifferent m e s e n c h y m e (Fig. 3B). These cells were indistinguishable from other m e s e n c h y m a l cells, except that they took the acetylcholine esterase stain. After 3 days in organ culture, r h y t h m i c contraction of the first b r o n c h i a l b r a n c h was seen in all specimens. A t 13 days the embryonic lung had lightly staining neuroblasts in the m e s e n c h y m e s u r r o u n d i n g the trachea. After 1 day of in vitro i n c u b a t i o n (Fig. 4), neuroblasts could be seen as far distal as the first bronchial branch. R h y t h m i c m o v e m e n t was seen at the end of the second day of i n c u b a t i o n in 2 of 4 cultures, and it became evident in all specimens after 3 days of culture. A t day 14, smooth muscle b e g a n to differentiate, and neuroblasts enlarged a n d stained more intensely. After 1 day in culture, smooth muscle differentiated further, neuroblasts migrated to the third b r o n c h i a l b r a n c h , a n d m o v e m e n t could be observed (Fig. 5). D u r i n g day 15 a n d 16 both smooth muscle and cartilage formed. Neuro-
CHOLINERGIC NERVE DEVELOPMENT IN FETAL LUNG
655
Fig. 3. (A) Twelve-day fetal trachea (T) and esophagus (Es). Acetylcholine esterase activity is seen in the vagus nerve (V) (X 360); (B) Twelve-day fetal trachea (T) and 2-day incubation. Neuroblasts (Nb) migrate from vagus (V) to mesenchyme around trachea
(• 18o).
656
MORIKAWA,
Fig. 4.
Thirteen-day fetal bronchus (first branch) and 1-day incubation. Neuroblaeta (Nb) surround the epithelium (X200).
DAYS IN ORGAN CULTURE AGE OF EMBRYO (doys)
12
13
o
1
2
3
neuroblost ( - )
nb
(-)
nb (+)
movement ( - )
m (-)
m (+)
(+)
nb ( + )
m (_+)
m (+)
nb
(_+)
m
(--)
nb
nb
(+)
nb
m
(_+)
m (+)
m (+)
7,14
DONAHOE, AND HENDREN
(+)
7
4- MaturationalAge i~
^f ~mk.yo J_ days in culture)
Fig. 5. Progressive development o f rhythmic movement o f the first bronchial branch of 12- to 14-day embryonic lung in organ culture. Embryonic lung was dissociated from the spinal cord and placed in culture from 1 to 3 days until a maturaUonal age of 15 days was reached. Movement was detected after smooth muscle had d i f f e r e n t i a t e d and neuroblasts had migrated.
blasts took a tangential position outside the smooth muscle and differentiated into ganglia. 3 Seventeen-day fetal trachea was characterized by well-developed cartilage, smooth muscle, and cilia formation. Ganglia enlarged, but no nerve fibers could be seen. After 1 day of incubation, ganglion cells outside the smooth muscle differentiated more fully, but nerves still could not be seen. Neuroepithelial cells, however, were clearly demonstrated (Fig. 6). After 2 days of incubation in vitro, in addition to generalized growth characterized by cartilage and smooth muscle development, acetylcholine-esterasepositive nerve fibers could be seen extending from the extramuscular ganglion cells to the submucosa and thence to the epithelium. Small submucosal ganglia were also seen. Since the changes of cholinergic nerve development were most marked in the 1- to 2-day interval after 17 days, this in vitro system was used to test the effects of growth inhibitors and
CHOLINERGIC NERVE DEVELOPMENT IN FETAL LUNG
657
Fig. 6. Seventeen-day fetal trachea after 1-day incubation. Neuroepithelial cells, which contain acetylcholine esterase granules, are found among the epithelial cells that line the airway.
stimulators (Fig. 7): 100 and 10 #g/ml of Na-L-thyroxine (T4) resulted in severe toxicity; however, after 1 day of incubation with l#g/ml, ganglia were observed to mature and nerves to grow from the ganglia to the epithelium submucosalaganglia were also observed (Fig. 8B). This accelerated development was not observed in controls to which no thyroxine was added (Fig. 8A). Colchicine at lmM and 0.5raM was toxic to the culture; 0.1 mM colchicine was effective in selectively blocking differentiation of ganglia and completely inhibiting nerve fiber growth (Fig. 9A and B). The neuroepithelial body, however, could be detected despite inhibition of both ganglionic and nerve fiber development. 17 + 1 day in culture G
DISCUSSION Maturation of the parasympathetic nervous system can occur in vitro ]~ and can be accurately
monitored using acetylcholine esterase histochemical techniques. Acetylcholine-esterasepositive neuroblasts cannot be seen in in viva embryonic lung until day 13 of gestation;3 however, if 12-day embryo lung (the youngest lung that can be successfully dissected from the central nervous system and sectioned on the cryostat) is placed in culture, neuroblasts can be detected 2 days later, indicating clearly that migration to the dissociated lung occurred prior to day 12 of gestation. Despite the lack of continued stimulus from the central nervous
17 -I- 2 days in culture G Nf
~M
CONTROL 17 day
~
"
embryo STIMULATOR tracheal ring / - - ~ ~ S { THYROXIN M * ~Ipg/ml)
( COLCHICINE0.1 mM)
. ~'Nf e .~~ff/ \ ~ ,
Fig. 7. Seventeen-day fetal tracheal rings exposed to in vitro incubation with thyroxine or colchicine. Thyroxine accelerated nerve fiber growth and ganglionic differentiation. Colchicine delayed this development, when compared with upper controls.
658
Fig. 8. Seventeen-day tracheal ring in vitro exposed to 1 /~g/ml thyroxine for 24 hr. Ganglia (G) differentiate and nerve fibers (Nf) appear (B). Nervous development is accelerated in comparison with that observed in control tracheal rings (A).
system in later cultures, neuroblasts differentiate into ganglia from which nerve fibers, previously undetected, grow to the submucosa and epithelium. Rhythmic contractile movements, limited primarily to the region of the first bronchial branch, were observed in 12-day, 13-day, and 14-day specimens cultured for 3 days, 2 days, or 1 day in the organ culture system, when they attained a maturational age (embryonic age + age in culture) of 15 days. The appearance of movement correlated with the condensation of mesenchyme and differentiation of smooth muscle (Fig. 5). Nerve fibers were not evident at this time; therefore it seems probable that this peristaltic movement is propagated by musclemuscle interaction rather than by nerve-muscle interaction. Dramatic changes were observed in the 17day tracheal ring during 1 and 2 days of culture. Immature ganglia enlarged and took on the morphology characteristic of the mature adult ganglion cell. Tangential to the smooth-muscle layer, large multinucleated cells with intense
MORIKAWA, DONAHOE, AND HENDREN
Fig. 9. Seventeen-day tracheal rings in vitro exposed to 0.1mM colchicine for 48 hr. hours. Nervous development is inhibited (B) in comparison with that seen in control tracheal rings (A), in which ganglia (G) differentiate and nerve fibers (Nf) appear.
acetylcholine esterase activity in their cytoplasm appeared, possibly from condensation of smaller neuroblasts. These ganglia gave nerve fibers to the smooth muscle and to the submucosa. Smaller ganglia appeared in the submucosa and in turn gave off nerve fibers that traveled both circumferentially in the submucosa and to the epithelium. The function of these epithelial nerve fibers is not completely understood, although it is speculated that they may play some role in mucus secretion or ciliary movement. Colchicine, by blocking binding of a and subunit proteins via guanine nucleotide and thus preventing polymerization of tubulin, inhibits the formation of microtubules, a predominant organelle in nerve fibers, n Overall toxicity was produced by l m M and 0.5mM concentrations; however, 0.1mM selectively blocked nerve fiber formation and differentiation of ganglia. It is of interest that despite its effect on these nervous structures, neuroepithelial cells developed under these conditions, indicating, as expected, that another developmental mechanism is involved.
CHOLINERGIC NERVE DEVELOPMENT IN FETAL LUNG
659 AchE + gronules
02
Fig. 10. Diagram of proposed intrapulmonary chemoreceptor. We speculate that changes in the O= or CO= in the airway affect acetylcholine esteraae (Ach E + ) granules, which in turn signal the firing of afferent neurons to the central nervous system.
--'~'-
~~
-
efferent
t~_
~afferent \-Nerves
Thyroxine is known to affect the growth, development, and metabolism of virtually all tissues of higher organisms. Its specific effect on nervous elements as studied in the rat central nervous system in vivo is obscure, but it appears to accelerate differentiation after proliferation has occurred. 12"13 Hamburgh 14 observed that thyroxine was required to maintain acetylcholine esterase activity in rat brain tissue in vivo. It seemed reasonable, therefore, to test thyroxine as a neurostimulator in this cholinergic lung developmental system. At a concentration of 1 #g/ml, thyroxine stimulated both ganglia and nerve fibers. It is of interest that thyroxine could not increase lamellar body formation in tissue culture of type II penumocytes of 18-day fetal rat lung, an effect readily seen with steroids. 8 The neurostimulatory affect observed in the present experiment was quite specific, more than a generalized growth stimulatory effect, and it deserves further study. The neuroepithelial cells with positive acetylcholine esterase granules in their cytoplasm may
fit the morphologic criteria for an intrapulmonary chemoreceptor. ~5'~6 We speculate that changes in CO2, 02, or pH in the airway may initiate nerve fiber activity via acethylcholineesterase-positive granules or dense core vesicles (Fig. 10). Recent physiologic experiments have suggested the presence of chemoreceptors in the isolated airways of birds ~7 and mammals. ~s'~9 However, the morphologic evidence for the existence of these chemoreceptors in the lung has not been clearly established. The neuroepithelial cells seen in this experiment differentiate in vitro and therefore can be singled out for more intense study. The combination of ultrastructural techniques with acetylcholine esterase histochemistry may yield more information about this particular cell. It is known that infants with aborted sudden infant death syndrome are less responsive to elevated intraalveolar CO 2 levels, z~ It is interesting to speculate that diminished sensitivity or diminished response of these neuroepithelial cells may play a part in the etiology of neonatal sudden infant death syndrome.
REFERENCES
1o Bartoli A, Cross BA, Guz A, et al: The effect of carbon dioxide in the airways and alveoli on ventilation, a vagal reflex studied in the dog. J Physiol (Lond) 240:91-109, 1974 2. Fillenz M, Widdicombe JG: Receptors of the lungs and airways, in Neil E (ed): Handbook of Sensory Physiology, vol 3. Berlin, Springer, 1972, pp 81-112 3. Morikawa Y, Donahoe PK, Hendren WH: Cholinergic nerve development in fetal lung. Dev Biol (in press) 4. Baker, JR: Principles of Biological Microtechnique, A
Study of Fixation and Dyeing. London, Methuen, 1958, pp 111-118 5. Holt S J, Hicks RM: Studies on formalin fixation for electron microscopy and cytochemical staining purpose. J Biophys Biochem Cytol 11:31-45, 1961 6. Karnovsky M J, Roots L: A "direct-coloring" thiocholine method for cholinesterase. J Histochem Cytochem 12:219-221, 1964 7. EI-Badawi A, Schenk EA: Histochemical methods for separate consecutive and simultaneous demonstration of
660
MORIKAWA, DONAHOE, AND HENDREN
acetylcholinesterase and norepinephrine in cryostat sections. J Histoehem Cytochem 15:580-588, 1967 8. Adamson YR, Bowden DH: Reaction of cultured adult and fetal lung to prednisolone and thyroxine. Arch Pathol 99:80-85, 1975 9. Thoa NB, Wooten GF, Axelrod J, et al: Inhibition of release of dopamine-/3-hydroxylase and norepinephrine from sympathetic nerves by colchicine, vinblastine or cytochalasin-B. Proe Natl Aead Sci USA 69:520-522, 1972 10. |to Y, Donahoe PK, Hendren WH: Differentiation of intramural ganglia in the dissociated rectosigmoid of the rat. J Pediatr Surg 12:969 976, 1977 11. Bucher NLR: Microtubules. N Engl J Med 287:195197, 1972 12. Balazs R: Biochemical effects of thyroid hormones in the developing brain, in Pease DC (ed): Cellular Aspects of Neural Growth and Differentiation. Berkeley, University of California Press, 1971 13. Schapiro S, Vukowich K, Globus A: Effects of neonatal thyroxin and hydrocortisone administration on the development of dendritic spines in the visual cortex of rats. Exp Neurol 40:286-296, 1973 14. Hamburgh M, Flexner LB: Effect of hypothyroidism
and hormone therapy on enzyme activities of the developing cerebral cortex of the rat. XXI. Biochemical and physiological differentiation during morphogenesis. J Neurochem 1:279-288, 1957 15. Laweryns JM, Cokelaere M, Theunynck P: Neuroepithelial bodies in the respiratory mucosa of various mammals. Z Zellforsch 135:569-592, 1972 16. Laweryns JM, Cokelaere M: Hypoxia-sensitive neuro-epithelial bodies intrapulmonary secretory neuroreceptors, modulated by the central nervous system. Z Zellforsch 145:521-540, 1973 17. Scheid P, Slama H, Gatz RN, et al: Intrapulmonary CO 2 receptors in the duck: III. Functional localization. Respir Physiol 22:123-136, 1974 18. Marsland DW, Callahan BJ, Shannon DC: The afferent vagus and regulation of breathing in response to inhaled CO2 in awake newborn lambs. Biol Neonate 27:102107, 1975 19. Fagenholtz SA, O'Connell K, Shannon DC: Chemoreceptor function and sleep state in apnea. Pediatrics 58:3136, 1976 20. Shannon DC, Kelly DH, O'Connell K: Abnormal regulation of ventilation in infants at risk for sudden infant death sundrome. N Engl J Med 297:747-570, 1977
Discussion J. Folkman (Boston): T h e a u t h o r s have offered us two exciting ideas to think about. O f p a r a m o u n t i m p o r t a n c e is the a t t e m p t to localize the c h e m o r e c e p t o r s in the lung, and if the neuroepithelial cell is a c a n d i d a t e , then it is entirely possible in the future t h a t one could see neonatologists using thyroxine as they now use steroids for the induction of s u r f a c t a n t in the lung. T h e second idea is linked with the field of developmental biology. A very c o m m o n p a t t e r n is emerging: in the e a r l y embryo, nerve cells m a k e long journeys and arrive at a p p r o p r i a t e t a r g e t organs. O n l y after they arrive do the other cell types begin their m a t u r a t i o n , i.e., cartilage, bone. If the neuroblasts do not arrive, one result m a y be H i r s c h s p r u n g ' s disease. But w h a t h a p p e n s if the neuroblasts arrive late? Is there a n y p e n a l t y ? Is differentiation d e l a y e d ? For example, if in a s a l a m a n d e r you a m p u t a t e the leg, the leg regenerates; if you section the sciatic nerve, the leg does not regenerate. This raises a question: Do the a u t o n o m i c neuroblasts, in fact, p l a y an i m p o r t a n t development role in organs such as the lung j u s t as the p e r i p h e r a l nerves do in limb development in lower v e r t e b r a t e s ? The techniques of Dr. M o r i k a w a and his colleagues
m a y be helpful in the future to e x a m i n e this question. K. Pringle (Chicago): I wonder if the authors have thought of looking at the status of these neuroepithelial cells in the hypoplastic lung of a d i a p h r a g m a t i c hernia to see w h e t h e r this is p e r h a p s a clue to the distressing incidence of late development of fatal respiratory distress in these patients. P. Donahoe (closure)." W e u n d e r t o o k these studies s t i m u l a t e d by the frustration t h a t we as p e d i a t r i c surgeons all face in w a t c h i n g infants w h o m we h a v e a d e q u a t e l y r e p a i r e d w i t h d i a p h r a g m a t i c hernias die because of i m m a t u r e lungs, and by the children with sudden infant d e a t h s y n d r o m e who a r e believed to have autonomic lability or i m m a t u r i t y o f unknown etiology. C u r r e n t thinking is t h a t the a u t o n o m i c deficit is central. O u r a p p r o a c h is t h a t the autonomic lability m a y be a distal deficit due to failure of m i g r a t i o n or failure of differentiation of autono m i c ganglia a f t e r migration. W e looked at possible ways of a c c e l e r a t i n g distal differentiation in vitro and chose thyroxine because o f evidence that this h o r m o n e s t i m u l a t e s differentiation of nerve cells.
CHOLINERGIC NERVE DEVELOPMENT IN FETAL LUNG
The hypothesis that the epithelial cell with positive acetylcholine esterase activity in its cytoplasmic granules may be an intrapulmonary chemoreceptor is important. These granules may, indeed, correspond to those cytoplasmic synaptic vesicles seen at the electron microscopic level by Laweryns. If, indeed, this proves to be
661
true, then this cell may provide a morphologic prototype for a peripheral chemoreceptor. There has been a great deal of physiologic evidence for the existence of intrapulmonary chemoreceptors, but no morphologic documentation. Perhaps this cell may fit the criteria.