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The distribution of myelinated nerve fibers in the mature opossum esophagus
Journal of the Autonomic Nervous System, 35 (1991) 227-236
227
© 1991 Elsevier Science Publishers B.V. All rights reserved 0165-1838/91/$03.50 JANS 01198
The distribution of myelinated nerve fibers in the mature opossum esophagus Alastair J. MacGilchrist, James Christensen and Gary A. Rick Division of Gastroenterology-Hepatology, Department of Internal Medicine, University of Iowa, Iowa City, Iowa, U.S.A. (Received 8 November 1990) (Revision received and accepted 23 May 1991)
Key words: Catecholamine fluorescence; Esophagus; Extrinsic nerve; Myelin; Myenteric plexus; Opossum; Prematurity Abstract Myelination of nerve fibers could be important in establishing normal esophageal peristalsis.We therefore examined the general distribution and the age of appearance of myelinated nerve fibers in the smooth-muscle part of the esophagus of the American opossum. Tissues stained with thionin and Sudan black B were examined by light microscopy. Other tissues were prepared for electron microscopy and examined in the light microscope in Toluidine blue stained sections as well as at electron microscopy. In mature animals (weight > 2.0 kg, age > 1 year), myelinated nerve fibers, oriented mainly craniocaudally, were most abundant at the striated muscle-smooth muscle junction, and declined in density distally along the organ. They were nearly absent at the esophagogastric junction. They were more abundant in the stomach just below the esophagogastric junction. The myelinated nerve fibers commonly lay within sheathed fascicles that had the appearance of peripheral nerves, like the shunt fascicles of the stomach and colon. In immature animals myelinated fibers did not appear until a weight of about 1 kg was reached, 50 days after weaning and about 150 days after birth. Since the younger animals are presumably swallowing normally, myelination of the extrinsic nerves is not essential for esophageal motor function.
Introduction
Myelinated nerve fibers in the walls of the gastrointestinal tract are extrinsic in origin, probably arising from both craniosacral and thoracolumbar systems [7,8]. Motility in the esophagus critically depends upon the integrity of its extrinsic innervation [1,6]. This makes it of interest to examine the distribution of myelinated nerve
Correspondence: J. Christensen, Division of Gastroenterology-Hepatology, Department of Internal Medicine, University of Iowa Hospitals and Clinics, Iowa City, IA 52242, U.S.A.
fibers in the esophagus, as representative of the distribution of the extrinsic innervation, only a part of which is myelinated. Myelinated fibers in the gut have received rather little attention. They are distributed to the myenteric plexus in the colon by intramural projections of the extrinsic nerves, called 'shunt fascicles' or the ascending nerves [3,15,20], and in the stomach they are similarly concentrated in intramural projections of extrinsic nerves, also called 'shunt fascicles' [2,15,18]. This study was intended to discover if the esophagus contains myelinated nerve fibers, how they are distributed, and at what age they first appear.
228 As a sub-protocol, we examined the question of the age of appearance of myelination because of the fact that esophageal peristalsis in human infants is not fully developed until after birth, esophageal motility being particularly abnormal in premature infants [10-13]. This abnormality is commonly attributed to neuronal immaturity. The appearance of myelinated fibers could be taken as a rough index of the state of maturation of the extrinsic nerves in developing animals. We examined the American opossum, now the standard model for the study of the esophagus because it closely resembles that of man in the proportions of smooth and striated muscle. Also, the delivery of large litters at a very early stage of embryonic development makes the opossum especially suited to developmental studies [9,16,17]. These animals never cease to grow, and they age rapidly, dying at about three years of age with many stigmata of senility. Thus, at one year of age (the age of the usual 2 - 3 kg animal), these animals are about one-third through the natural life-span.
Materials and Methods
Studies in mature animals Whole esophagus preparations One-year-old opossums of either sex, weighing about 2.5 kg, were anesthetized by the intraperitoneal injection of sodium pentobarbital 50 m g / k g . The esophagus and a generous cuff of the stomach were removed, pinned flat with maximal stretch to a rubber pad, and immersed in 10% formalin for 24 h. The mucosa and submucosa were removed and the lower esophageal sphincter, identifiable as a thickened band of circular muscle, was marked. The circular muscle layer was carefully removed under a dissecting microscope to leave the myenteric plexus intact. The preparation was rinsed in distilled water, placed in a saturated solution of Sudan black B in 70% alcohol for 20 min, and then rinsed for 5 min twice in distilled water. It was exposed for 30 min to a buffered solution of thionin (1 ml of a 1% aqueous solution of thionin, 7 g sodium acetate
and 2 ml glacial acetic acid in 1000 ml distilled water) followed by 25 rain in acetate buffer (7 g sodium acetate and 2 ml glacial acetic acid in 1000 ml distilled water). After two 5-rain rinses in distilled water, the organ was fixed to a glass plate with 10% glycerin, and mounted with Aqua-Mount (Lerner Laboratories). The thionin stain allowed us to visualize the ganglia and interganglionic fascicles which could not otherwise be seen clearly, while the Sudan stain showed the myelin sheaths. Transverse lines parallel to the long axis of the lower esophageal sphincter were drawn across the five specimens at 1 cm intervals. The number of myelinated nerve fibers crossing each line was counted. The mean values were compared by one-way analysis of variance. A diagram of the complete distribution of myelinated fibers was made from one such whole-mount by constructing a grid of the whole organ. Each of the 1200 compartments of the grid represented one field of view at a magnification of x 10. All stained fibers and ganglia in each field of view were traced.
Trans~,erse esophageal rings The esophagus was taken from each of two one-year-old opossums (mean weight 3.8 kg) anesthetized as described above. One of the preparations included the lower esophageal sphincter and the proximal stomach. The organs were distended with iced Krebs solution and immersed for 2 h in a modified Zamboni's solution (4% paraformaldehyde and 0.05% glutaraldehyde in 51)0 ml 0.4 M Sorensen's buffer containing 75 ml saturated picric acid) at 0 o C. Rings cut from the esophagus were divided into 8 radial segments, rinsed for 20 rain three times in phosphate-buffered saline and placed in a 4.5% dext r o s e / p h o s p h a t e buffer solution overnight. After two more rinses in d e x t r o s e / p h o s p h a t e buffer, the segments were immersed for 60 min in a 1 solution of osmic acid in Sorensen's buffer containing 1.5% potassium ferrocyanide. They were dehydrated in a graded series of alcohol and propylene oxide, infiltrated with a 6 0 / 4 0 mixture of Epon and propylene oxide, and embedded in Epon. 0.5 # transverse sections were cut on a Porter-Blum ultramicrotome and stained with
229
Toluidine blue for light-microscopic examination. The myelin sheaths were displayed conspicuously as dark blue-black rings within nerve bundles and ganglia. The nerve bundles with and without myelinated fibers in the plane of the myenteric plexus were counted at 1 cm intervals along the length of the smooth-muscled esophagus. Some of the Epon embedded segments were cut into 100 nm transverse section with an A O / R e i c h e r t ultracut E ultramicrotome. The sections were floated onto copper grids and stained with uranyl acetate for 10 min, and with lead citrate for 8 min. Electron micrographs were made using a Hitachi H-7000 transmission electron microscope. All light microscopy was carried out on a Dialux 22 microscope (Leitz). Light photomicrographs were made using a Vario-Orthomat 2 camera (Leitz). Studies in immature animals
We examined one or two animals at each of various stages of development beginning from
soon after birth. The exact date of birth could not be known in these wild-trapped animals, but the ages of the young could be estimated from published data that relate age to weight (Table I). The young were removed from the pouch and fixed in toto for examination of the esophagus in transverse rings as described above. The sections were taken from the midpoint of the esophagus where the study of the one-year-old animals had previously revealed myelinated fibers to be most numerous. We examined only ring preparations in the immature animals because we expected myelinated fibers to be scarce. In the whole-mount preparation it seems likely that small myelinated fibers could be missed or overlooked when they are sparse, while the ring preparations seem to be much less susceptible to such error. We examined only one level in these animals, the level where myelinated fibers were most abundant in the mature animal, because the question was one of the age of first appearance of myelination. That is, it was a deterministic rather than a quantitative question. Since nearly all myelinated fibers in the
TABLE I
The growth and development of opossum pouch young [9,16,17] Day
Snout-Rump Length (mm)
1 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100
13 17 24 30 33 40 45 53 60 75 100 125 150 180
Weight (g) 0.13 0.40 0.90 1.3 1.7 2.4 3.9 5.4 7.0 13.0 25.0 45.0 -
Comment (days) Fixed to nipple; no hind limbs.
Genital organs are visible (11-17).
Flexes hind toes (43). Spontaneously releases nipple (49). Eyes and mouth begin to open (50). Mouth is fully opened (55-68). Eyes are fully opened (55-72). Wandering begins (70). Testicular descent occurs (77).
80.0 125.0
Weaning begins (87). Interest in solid food begins (96). Weaning completed (96-102).
230
mature animals extend in the cranio-caudad axis, we reasoned that transverse rings should show them best if they were present.
7 E 6
s =
4 3
Results
2 o
0
Whole esophageal preparations in mature animals t~
The thionin-stained ganglia and interganglionic fascicles of the myenteric plexus formed a network overlying the longitudinal muscle layer, showing that the dissection had left the myenteric plexus intact. Nerve fibers stained by the Sudan black B and showing nodes of Ranvier lay within
Fig. 1A. Myelinated nerve fibers (arrows) within a nerve bundle of the myenteric plexus, bypassing a ganglion (G). Asterisks are on or near a few nerve cell bodies. The vertical dark band is overlying circular muscle. Sudan black B and thionin. Bar = 50 #. lB. Electron micrograph of a portion of a nerve bundle. Note the myelin sheath (dense black ring), the perineurium (arrows) and the adjoining blood vessel (asterisk). Bar = 1 #.
l 0
T
5
T 1 T 2r5 3tO T 10 15 20 35 Total No. of Myelinated Fibers
~0
Fig. 2. The number of myelinated fibers at different transverse levels of the esophagus as determined in whole-mounts. Each bar is the number of fibers (mean and SEM) intersecting a transverse line drawn on each of the 4 Sudan-stained whole organ preparations. The lines were drawn at 1 cm intervals, from the upper esophageal body (6 cm) through the esophagogastric (EG) junction (0 cm) to the gastric fundus ( - 1 cm).
the interganglionic nerve bundles (Fig. 1). Many, but not all, nerve bundles contained myelinated fibers, and single myelinated fibers could often be traced over long distances, usually following the cranio-caudad axis. They largely bypassed ganglia, but they remained within the general framework of the myenteric plexus throughout their distributions. Their points of entry from the serosal surface could be seen at many sites along the length of the smooth-muscled segment of the esophagus. The density of myelinated fibers declined steeply along the smooth-muscle segment. They were most numerous in the region of the junction of smooth and striated muscle in the esophageal body. Very few myelinated fibers were found just above the esophagogastric junction or crossing into the stomach. In contrast, many myelinated fibres were found in the proximal stomach just below the lower esophageal sphincter. This distribution can be seen in Fig. 2. The numbers of myelinated fibers and of nerve bundles containing such fibers seen in the whole-mounts are given in Table II. The pattern of distribution of myelinated nerves in the esophagus is presented in Figs. 3A and 3B.
231
7
r:
"..-
s
,,t /
~
A
/
// f
/
/ is
m
B Fig. 3A. Diagram of one of the Sudan-stained preparations. The arrows indicate the level of the transition from striated muscle to smooth muscle. The curved line shows the postition of the esophagogastric junction. The rectangle is the area shown in enlargement in Fig. 3B. All lines are myelinated fibers lying within nerve bundles of the myenteric plexus. Closed circles are the recognized points of entry of myelinated nerves through the longitudinal muscle. (See text for details of construction of diagram.) Bar = 1 cm. 3B. Larger scale portion of diagram indicated on Fig. 3A by the enclosed rectangle. The thick line is the edge of the preparation. Thin lines are myelinated fibers. Closed circles (arrows) are points of entry of myelinated fibers. The solid irregular shapes are ganglia of the myenteric plexus. Bar = 1 mm.
232 Total
Transverse esophageal rings in mature animals
•
7
In cross-sections of nerve bundles and ganglia of the myenteric plexus, myelinated fibers were conspicuous from their darkly-stained myelin sheaths. Counts of total numbers of nerve bundles, and of those both with and without myelinated fibers (Table III), confirmed the decline in the number of myelinated fibers in the distal esophagus and at the lower esophageal sphincter, without a corresponding decline in the total number of nerve bundles (Fig. 4). Some nerve bundles (both with and without myelinated fibers) possessed an enveloping sheath or perineurium. At the light microscopic level, these sheaths could not always be clearly distinguished from surrounding connective tissue, and so no formal counts of such ensheathed nerve bundles were made. The electron micrographs provided further evidence of the presence of myelin sheaths around some axons, and also confirmed the existence of a perineurium encasing the nerve bundles (Fig. 1B), corresponding to the sheath-like structures seen at the light microscopic level. Blood vessels closely accompanied these nerve bundles.
Myelinated
I
6 "g
5 4
3
l m
0
,
,
0
lrO
,
2TO
~
r
r
~
30
40 No. of Nerve Bundles
r
•
50
•
60
Fig. 4. The total number of nerve bundles and the number of nerve bundles containing myelinated fibers at different levels of the esophagus. Each bar represents the sum of the number of nerves seen in all 8 portions of a plastic-embedded, Toluidine blue-stained transverse ring. The rings were counted at 1 cm intervals from the upper esophageal body (6 cm) to just above the esophagogastric junction (1 cm).
Transverse rings in developing animals We examined one animal at each of a series of body weights that covered the whole period of postnatal development, 0.72, 8.08, 9.20, 16.11,
TABLE II
Distribution o f myelinated nerves in the esophageal myenteric plexus." whole mounts stained with Sudan black B Distance proximal to esophagogastric junction (cm)
Esophagus
Stomach
6
5
4
3
2
1
(/
- 1
28 14 53 30 31.3 4.2
32 10 52 28 30.5 8.6
22 15 48 19 26.0 7.5
26 20 36 20 25.5 3.8
14 18 33 5 17.5 5.8
4 24 13 14 13.8 4.1
4 4 7 4 4.8 0.8
25 3 40 12 20.0 8.1
7 8 22 11 12.0 3.4
8 11 20 4 10.8 3.4
2 8 8 9 6.8 1.6
3 2 5 3 3.3 0.6
13 3 17 t0 10.8 2.9
Total number of myelinated fibers Opossum 1 2 3 4 mean SE
Number of nerve bundles containing one or more myelinated .fibers Opossum 1 2 3 4 mean SE
13 7 23 18 15.3 3.6
15 6 21 15 14.3 3.1
8 10 19 10 11.8 2.5
Analysis of variance gives P values of 0.10 and 0.09 respectively for these distributions. The lack of significance at the 5% level results from the small numbers studied and the large inter-animal variation. Each opossum shows a reduction in the number of myelinated fibers at the esophagogastric junction.
233 TABLE III Distribution of myelinated nerves in the esophageal myenteric plexus: 0.5 # sections of transverse rings stained with Toluidine blue
Distance proximal to esophagogastricjunction (cm)
Esophagus 6 5
4
3
2
1
0
- 1
Stomach
53
54
54
48
43
45
-
-
21
25
21
20
9
7
-
-
40 70
46 86
39 44
42 40
21 11
16 12
-
-
-
-
61
-
-
46
55
148
-
-
23
-
-
8
3
22
-
-
38 90
-
-
17 24
5 4
15 37
Opossum 1
Total number of nerve bundles Number of nerve bundles with myelinated fibers % of nerve bundles with myelinated fibers Total number of myelinated fibers Opossum 2
Total number of nerve bundles Number of nerve bundles with myelinated fibers % of nerve bundles with myelinated fibers Total number of myelinated fibers
The preparation for Opossum 1 did not include the esophagogastric junction. In Opossum 2 the esophagogastric junction was studied in detail, with one ring from the mid-esophageal body included for comparison. A reduction in the number of myelinated fibers without any corresponding reduction in the total number of nerve bundles in the distal esophagus and at the esophagogastric junction is seen.
60.90, 66.93, 70.34, 200.00, 302.00, 450.00, 590.00 a n d 1010.00 g. A t t h e y o u n g e s t ages (i.e. 0.72 a n d 8.08 g), s t r u c t u r e s i d e n t i f i a b l e as g a n g l i a c o u l d b e d i s c e r n e d , a n d t h e s e p a r a t i o n of the m u s c l e into two layers was a l r e a d y a p p a r e n t . W i t h d e v e l o p ment, ganglia became more conspicuous. Myelin s h e a t h s w e r e not f o u n d at any s t a g e until t h e last, at 1010.00 g, c o r r e s p o n d i n g to an age o f 50 days a f t e r w e a n i n g . In t h e a n i m a l at this age, w e f o u n d only o n e myelin s h e a t h p r o f i l e in the full circumference of the esophagus.
Discussion
T h e i n t r a m u r a l m y e l i n a t e d fibers o f t h e e s o p h agus differ little f r o m t h o s e f o u n d in the s t o m a c h a n d colon. In t h o s e organs, t h e m y e l i n a t e d fibers run c r a n i o - c a u d a d for long d i s t a n c e s within the s h u n t fascicles, s h e a t h e d nerve t r u n k s t h a t follow a s t r a i g h t c o u r s e for long d i s t a n c e s in t h e s a m e p l a n e o f t h e m y e n t r i c plexus, b u t distinct f r o m it, b y p a s s i n g g a n g l i a [2,3]. T h e m y e l i n a t e d fibers d e p a r t f r o m s h u n t fascicles in those o r g a n s into t h e g a n g l i o n a t e d plexus a n d e x t e n d only a s h o r t dis-
t a n c e b e f o r e losing t h e myelin s h e a t h . T h e myelin a t e d fibers o f t h e e s o p h a g u s a r e m o r e u n i f o r m l y d i s t r i b u t e d within the f r a m e w o r k o f t h e m y e n teric plexus t h a n t h e y a r e in t h e s t o m a c h a n d colon. Still, they a r e o f t e n f o u n d in fascicles having a p e r i n e u r i u m (like t h e s h u n t fascicles of t h e s t o m a c h a n d colon, a n d like extrinsic nerves) r a t h e r t h a n in t h e u n s h e a t h e d i n t e r g a n g l i o n i c fascicles. Like t h e m y e l i n a t e d fibers in t h e shunt fascicles of the s t o m a c h a n d colon, they e x t e n d over long distances, p r i n c i p a l l y in t h e c r a n i o caudad direction. N e i t h e r the exact s o u r c e n o r the d e s t i n a t i o n of t h e s e m y e l i n a t e d n e r v e fibers can b e s t a t e d with certainty. T h e fact t h a t the sites w h e r e they p i e r c e t h e l o n g i t u d i n a l layer to e n t e r t h e p l a n e o f the m y e n t e r i c plexus a r e r a t h e r evenly d i s t r i b u t e d suggests that t h e y arise m a i n l y from the vagal e s o p h a g e a l plexus. M y e l i n a t e d fibers lose t h e i r myelin s h e a t h s an u n d e f i n a b l e d i s t a n c e short o f t h e i r t e r m i n a l s , h e n c e t h e r e m u s t be d o u b t a b o u t their precise destination. T h e d i s t r i b u t i o n d e n s i t y o f m y e l i n a t e d fibers shows a striking g r a d i e n t f r o m t h e e s o p h a g e a l b o d y to t h e e s o p h a g o g a s t r i c j u n c t i o n . This p a r a l -
234 lels a g r a d i e n t in the density of intrinsic n e u r o n s [4,5]. T h e virtual a b s e n c e of m y e l i n a t e d fibers crossing the e s o p h a g o g a s t r i c j u n c t i o n i n d i c a t e s that the m y e n t e r i c plexus o f the e s o p h a g u s is not an i m p o r t a n t source of the a b u n d a n t m y e l i n a t e d fibers of the stomach. Thus, g a s t r o - s p h i n c t e r i c reflexes [19] m u s t m a k e use o f n o n m y e l i n a t e d r a t h e r t h a n m y e l i n a t e d pathways. P r e s u m a b l y , the a b u n d a n t m y e l i n a t e d nerve fibers o f the s t o m a c h arise from the vagal trunks a n d e n t e r the wall of the s t o m a c h close to the cardia. T h e m a p of m y e l i n a t e d fibers from o n e m a t u r e animal shown in Fig. 3 A i n d i c a t e s that myelin a t e d fibers a r e not uniformly d i s t r i b u t e d in the c i r c u m f e r e n c e of the e s o p h a g u s at any single level. F u r t h e r m o r e , the d a t a from the transverse rings in T a b l e III suggest t h a t t h e r e may be c o n s i d e r a b l e v a r i a t i o n f r o m one a n i m a l to ano t h e r in the p r o p o r t i o n o f m y e l i n a t e d fibers at any single level. This variability s u p p o r t s the idea that m y e l i n a t i o n of extrinsic nerve fibers is not a critical d e t e r m i n a n t of n o r m a l peristalsis in the s m o o t h muscle o f the e s o p h a g u s . T h e d e v e l o p m e n t of m y e l i n a t i o n in the extrinsic nerves to t h e o p o s s u m e s o p h a g u s has not even b e g u n at the time o f w e a n i n g , n o r d o e s it begin until long a f t e r the a n i m a l s have b e c o m e fully i n d e p e n d e n t a n d a r e c o n s u m i n g a n o r m a l solid diet. Thus, a l t h o u g h the extrinsic i n n e r v a t i o n o f the e s o p h a g u s is clearly not fully d e v e l o p e d in the w e e k s a f t e r w e a n i n g (in t h a t it is not m y e l i n a t e d ) , e s o p h a g e a l m o t o r function m u s t be essentially normal. This late m y e l i n a t i o n of nerves to the e s o p h a g u s is consistent with the late m y e l i n a t i o n of vagal fibers o b s e r v e d by Krous, J o r d a n , W e n a n d F a r b e r [14] who f o u n d m y e l i n a t e d fibers (in the cervical vagus of t h e d e v e l o p i n g o p o s s u m ) to have a c h i e v e d only 11% of a d u l t values by the age of 50 days. T h e s e cervical vagal fibers, of course, w e r e d e s t i n e d for the w h o l e vagal distribution. O u r study indicates that vagal m y e l i n a t e d fibers begin to r e a c h the e s o p h a g u s only after m o r e t h a n a b o u t 150 days of age, when the animal is half-grown. T h e d e v e l o p m e n t of m y e l i n a t e d fibers in the h u m a n s m o o t h - m u s c l e e s o p h a g u s is not known. If it p a r a l l e l s t h e d e v e l o p m e n t in t h e o p o s s u m , t h e n the a b s e n c e o f m y e l i n a t i o n is not an e x p l a n a t i o n
for the e s o p h a g e a l m o t o r a b n o r m a l i t i e s of p r e m a ture infants. M y e l i n a t i o n can be t a k e n as a morp h o l o g i c a l index of m a t u r a t i o n of a system of nerves. If so, then t h e s e results do not s u p p o r t t h e i d e a that i m m a t u r i t y of the extrinsic nerve supply to the e s o p h a g u s is the e x p l a n a t i o n for the a b n o r m a l e s o p h a g e a l motility of infants.
Acknowledgements This w o r k was s u p p o r t e d by R e s e a r c h G r a n t D K 11242 a n d C o r e C e n t e r G r a n t D K 34986 from the N a t i o n a l Institutes of H e a l t h .
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235 12 Gryboski, J.D., Gastrointestinal function in the infant and young child, Clinics in Gastroenterology, 6 (1977) 253-265. 13 Gryboski, J.D., Thayer, Jr., W.R., and Spiro, H.M., Esophageal motility in infants and children, Pediatrics, 31 (1963) 382-395. 14 Krous, H.F., Jordan, J., Wen, J., and Farber, J.P., Developmental morphometry of the vagus nerve in the opossum, Brain Res., 20 (1985) 155-159. 15 Kumar, D. and Phillips, S.F., Human myenteric plexus: confirmation of unfamiliar structures in adults and neonates, Gastroenterology, 96 (1989) 1021-1028. 16 Petrides, G.A., Sex and age determination in the opossum, J. Mammal., 30 (1949) 364-378. 17 Reynolds, H.C., Studies on reproduction in the opossum (Didelphis virginiana virginiana). In R.M. Eakin, H. Kirby,
A.H. Miller, and C. Stern, (Eds.), University of California Publications in Zoology, University of California Press, Berkeley, 1954, pp. 223-283. 18 Schabadasch, A., Die Nerven des Magens der Katze, Z. Zellforschung. Mikrosk. Anat., 10 (1930) 254-319. 19 Schulze-Delrieu, K., Percy, W.H., Ren, J., Shirazi, S. and von Derau, K., Evidence for inhibition of opossum LES through intrinsic gastric nerves, Am. J. Physiol., 256 (1989) G198-G205. 20 Stach, W., Uber die in der dickdarmwand azendieren Nerven des Plexus pelvinus und die Grenze des vagalen and sakralparasympathetischen Innervation, Z. Mikrosk. Anat. Forsch, 84 (1971) 65-90.
227
© 1991 Elsevier Science Publishers B.V. All rights reserved 0165-1838/91/$03.50 JANS 01198
The distribution of myelinated nerve fibers in the mature opossum esophagus Alastair J. MacGilchrist, James Christensen and Gary A. Rick Division of Gastroenterology-Hepatology, Department of Internal Medicine, University of Iowa, Iowa City, Iowa, U.S.A. (Received 8 November 1990) (Revision received and accepted 23 May 1991)
Key words: Catecholamine fluorescence; Esophagus; Extrinsic nerve; Myelin; Myenteric plexus; Opossum; Prematurity Abstract Myelination of nerve fibers could be important in establishing normal esophageal peristalsis.We therefore examined the general distribution and the age of appearance of myelinated nerve fibers in the smooth-muscle part of the esophagus of the American opossum. Tissues stained with thionin and Sudan black B were examined by light microscopy. Other tissues were prepared for electron microscopy and examined in the light microscope in Toluidine blue stained sections as well as at electron microscopy. In mature animals (weight > 2.0 kg, age > 1 year), myelinated nerve fibers, oriented mainly craniocaudally, were most abundant at the striated muscle-smooth muscle junction, and declined in density distally along the organ. They were nearly absent at the esophagogastric junction. They were more abundant in the stomach just below the esophagogastric junction. The myelinated nerve fibers commonly lay within sheathed fascicles that had the appearance of peripheral nerves, like the shunt fascicles of the stomach and colon. In immature animals myelinated fibers did not appear until a weight of about 1 kg was reached, 50 days after weaning and about 150 days after birth. Since the younger animals are presumably swallowing normally, myelination of the extrinsic nerves is not essential for esophageal motor function.
Introduction
Myelinated nerve fibers in the walls of the gastrointestinal tract are extrinsic in origin, probably arising from both craniosacral and thoracolumbar systems [7,8]. Motility in the esophagus critically depends upon the integrity of its extrinsic innervation [1,6]. This makes it of interest to examine the distribution of myelinated nerve
Correspondence: J. Christensen, Division of Gastroenterology-Hepatology, Department of Internal Medicine, University of Iowa Hospitals and Clinics, Iowa City, IA 52242, U.S.A.
fibers in the esophagus, as representative of the distribution of the extrinsic innervation, only a part of which is myelinated. Myelinated fibers in the gut have received rather little attention. They are distributed to the myenteric plexus in the colon by intramural projections of the extrinsic nerves, called 'shunt fascicles' or the ascending nerves [3,15,20], and in the stomach they are similarly concentrated in intramural projections of extrinsic nerves, also called 'shunt fascicles' [2,15,18]. This study was intended to discover if the esophagus contains myelinated nerve fibers, how they are distributed, and at what age they first appear.
228 As a sub-protocol, we examined the question of the age of appearance of myelination because of the fact that esophageal peristalsis in human infants is not fully developed until after birth, esophageal motility being particularly abnormal in premature infants [10-13]. This abnormality is commonly attributed to neuronal immaturity. The appearance of myelinated fibers could be taken as a rough index of the state of maturation of the extrinsic nerves in developing animals. We examined the American opossum, now the standard model for the study of the esophagus because it closely resembles that of man in the proportions of smooth and striated muscle. Also, the delivery of large litters at a very early stage of embryonic development makes the opossum especially suited to developmental studies [9,16,17]. These animals never cease to grow, and they age rapidly, dying at about three years of age with many stigmata of senility. Thus, at one year of age (the age of the usual 2 - 3 kg animal), these animals are about one-third through the natural life-span.
Materials and Methods
Studies in mature animals Whole esophagus preparations One-year-old opossums of either sex, weighing about 2.5 kg, were anesthetized by the intraperitoneal injection of sodium pentobarbital 50 m g / k g . The esophagus and a generous cuff of the stomach were removed, pinned flat with maximal stretch to a rubber pad, and immersed in 10% formalin for 24 h. The mucosa and submucosa were removed and the lower esophageal sphincter, identifiable as a thickened band of circular muscle, was marked. The circular muscle layer was carefully removed under a dissecting microscope to leave the myenteric plexus intact. The preparation was rinsed in distilled water, placed in a saturated solution of Sudan black B in 70% alcohol for 20 min, and then rinsed for 5 min twice in distilled water. It was exposed for 30 min to a buffered solution of thionin (1 ml of a 1% aqueous solution of thionin, 7 g sodium acetate
and 2 ml glacial acetic acid in 1000 ml distilled water) followed by 25 rain in acetate buffer (7 g sodium acetate and 2 ml glacial acetic acid in 1000 ml distilled water). After two 5-rain rinses in distilled water, the organ was fixed to a glass plate with 10% glycerin, and mounted with Aqua-Mount (Lerner Laboratories). The thionin stain allowed us to visualize the ganglia and interganglionic fascicles which could not otherwise be seen clearly, while the Sudan stain showed the myelin sheaths. Transverse lines parallel to the long axis of the lower esophageal sphincter were drawn across the five specimens at 1 cm intervals. The number of myelinated nerve fibers crossing each line was counted. The mean values were compared by one-way analysis of variance. A diagram of the complete distribution of myelinated fibers was made from one such whole-mount by constructing a grid of the whole organ. Each of the 1200 compartments of the grid represented one field of view at a magnification of x 10. All stained fibers and ganglia in each field of view were traced.
Trans~,erse esophageal rings The esophagus was taken from each of two one-year-old opossums (mean weight 3.8 kg) anesthetized as described above. One of the preparations included the lower esophageal sphincter and the proximal stomach. The organs were distended with iced Krebs solution and immersed for 2 h in a modified Zamboni's solution (4% paraformaldehyde and 0.05% glutaraldehyde in 51)0 ml 0.4 M Sorensen's buffer containing 75 ml saturated picric acid) at 0 o C. Rings cut from the esophagus were divided into 8 radial segments, rinsed for 20 rain three times in phosphate-buffered saline and placed in a 4.5% dext r o s e / p h o s p h a t e buffer solution overnight. After two more rinses in d e x t r o s e / p h o s p h a t e buffer, the segments were immersed for 60 min in a 1 solution of osmic acid in Sorensen's buffer containing 1.5% potassium ferrocyanide. They were dehydrated in a graded series of alcohol and propylene oxide, infiltrated with a 6 0 / 4 0 mixture of Epon and propylene oxide, and embedded in Epon. 0.5 # transverse sections were cut on a Porter-Blum ultramicrotome and stained with
229
Toluidine blue for light-microscopic examination. The myelin sheaths were displayed conspicuously as dark blue-black rings within nerve bundles and ganglia. The nerve bundles with and without myelinated fibers in the plane of the myenteric plexus were counted at 1 cm intervals along the length of the smooth-muscled esophagus. Some of the Epon embedded segments were cut into 100 nm transverse section with an A O / R e i c h e r t ultracut E ultramicrotome. The sections were floated onto copper grids and stained with uranyl acetate for 10 min, and with lead citrate for 8 min. Electron micrographs were made using a Hitachi H-7000 transmission electron microscope. All light microscopy was carried out on a Dialux 22 microscope (Leitz). Light photomicrographs were made using a Vario-Orthomat 2 camera (Leitz). Studies in immature animals
We examined one or two animals at each of various stages of development beginning from
soon after birth. The exact date of birth could not be known in these wild-trapped animals, but the ages of the young could be estimated from published data that relate age to weight (Table I). The young were removed from the pouch and fixed in toto for examination of the esophagus in transverse rings as described above. The sections were taken from the midpoint of the esophagus where the study of the one-year-old animals had previously revealed myelinated fibers to be most numerous. We examined only ring preparations in the immature animals because we expected myelinated fibers to be scarce. In the whole-mount preparation it seems likely that small myelinated fibers could be missed or overlooked when they are sparse, while the ring preparations seem to be much less susceptible to such error. We examined only one level in these animals, the level where myelinated fibers were most abundant in the mature animal, because the question was one of the age of first appearance of myelination. That is, it was a deterministic rather than a quantitative question. Since nearly all myelinated fibers in the
TABLE I
The growth and development of opossum pouch young [9,16,17] Day
Snout-Rump Length (mm)
1 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100
13 17 24 30 33 40 45 53 60 75 100 125 150 180
Weight (g) 0.13 0.40 0.90 1.3 1.7 2.4 3.9 5.4 7.0 13.0 25.0 45.0 -
Comment (days) Fixed to nipple; no hind limbs.
Genital organs are visible (11-17).
Flexes hind toes (43). Spontaneously releases nipple (49). Eyes and mouth begin to open (50). Mouth is fully opened (55-68). Eyes are fully opened (55-72). Wandering begins (70). Testicular descent occurs (77).
80.0 125.0
Weaning begins (87). Interest in solid food begins (96). Weaning completed (96-102).
230
mature animals extend in the cranio-caudad axis, we reasoned that transverse rings should show them best if they were present.
7 E 6
s =
4 3
Results
2 o
0
Whole esophageal preparations in mature animals t~
The thionin-stained ganglia and interganglionic fascicles of the myenteric plexus formed a network overlying the longitudinal muscle layer, showing that the dissection had left the myenteric plexus intact. Nerve fibers stained by the Sudan black B and showing nodes of Ranvier lay within
Fig. 1A. Myelinated nerve fibers (arrows) within a nerve bundle of the myenteric plexus, bypassing a ganglion (G). Asterisks are on or near a few nerve cell bodies. The vertical dark band is overlying circular muscle. Sudan black B and thionin. Bar = 50 #. lB. Electron micrograph of a portion of a nerve bundle. Note the myelin sheath (dense black ring), the perineurium (arrows) and the adjoining blood vessel (asterisk). Bar = 1 #.
l 0
T
5
T 1 T 2r5 3tO T 10 15 20 35 Total No. of Myelinated Fibers
~0
Fig. 2. The number of myelinated fibers at different transverse levels of the esophagus as determined in whole-mounts. Each bar is the number of fibers (mean and SEM) intersecting a transverse line drawn on each of the 4 Sudan-stained whole organ preparations. The lines were drawn at 1 cm intervals, from the upper esophageal body (6 cm) through the esophagogastric (EG) junction (0 cm) to the gastric fundus ( - 1 cm).
the interganglionic nerve bundles (Fig. 1). Many, but not all, nerve bundles contained myelinated fibers, and single myelinated fibers could often be traced over long distances, usually following the cranio-caudad axis. They largely bypassed ganglia, but they remained within the general framework of the myenteric plexus throughout their distributions. Their points of entry from the serosal surface could be seen at many sites along the length of the smooth-muscled segment of the esophagus. The density of myelinated fibers declined steeply along the smooth-muscle segment. They were most numerous in the region of the junction of smooth and striated muscle in the esophageal body. Very few myelinated fibers were found just above the esophagogastric junction or crossing into the stomach. In contrast, many myelinated fibres were found in the proximal stomach just below the lower esophageal sphincter. This distribution can be seen in Fig. 2. The numbers of myelinated fibers and of nerve bundles containing such fibers seen in the whole-mounts are given in Table II. The pattern of distribution of myelinated nerves in the esophagus is presented in Figs. 3A and 3B.
231
7
r:
"..-
s
,,t /
~
A
/
// f
/
/ is
m
B Fig. 3A. Diagram of one of the Sudan-stained preparations. The arrows indicate the level of the transition from striated muscle to smooth muscle. The curved line shows the postition of the esophagogastric junction. The rectangle is the area shown in enlargement in Fig. 3B. All lines are myelinated fibers lying within nerve bundles of the myenteric plexus. Closed circles are the recognized points of entry of myelinated nerves through the longitudinal muscle. (See text for details of construction of diagram.) Bar = 1 cm. 3B. Larger scale portion of diagram indicated on Fig. 3A by the enclosed rectangle. The thick line is the edge of the preparation. Thin lines are myelinated fibers. Closed circles (arrows) are points of entry of myelinated fibers. The solid irregular shapes are ganglia of the myenteric plexus. Bar = 1 mm.
232 Total
Transverse esophageal rings in mature animals
•
7
In cross-sections of nerve bundles and ganglia of the myenteric plexus, myelinated fibers were conspicuous from their darkly-stained myelin sheaths. Counts of total numbers of nerve bundles, and of those both with and without myelinated fibers (Table III), confirmed the decline in the number of myelinated fibers in the distal esophagus and at the lower esophageal sphincter, without a corresponding decline in the total number of nerve bundles (Fig. 4). Some nerve bundles (both with and without myelinated fibers) possessed an enveloping sheath or perineurium. At the light microscopic level, these sheaths could not always be clearly distinguished from surrounding connective tissue, and so no formal counts of such ensheathed nerve bundles were made. The electron micrographs provided further evidence of the presence of myelin sheaths around some axons, and also confirmed the existence of a perineurium encasing the nerve bundles (Fig. 1B), corresponding to the sheath-like structures seen at the light microscopic level. Blood vessels closely accompanied these nerve bundles.
Myelinated
I
6 "g
5 4
3
l m
0
,
,
0
lrO
,
2TO
~
r
r
~
30
40 No. of Nerve Bundles
r
•
50
•
60
Fig. 4. The total number of nerve bundles and the number of nerve bundles containing myelinated fibers at different levels of the esophagus. Each bar represents the sum of the number of nerves seen in all 8 portions of a plastic-embedded, Toluidine blue-stained transverse ring. The rings were counted at 1 cm intervals from the upper esophageal body (6 cm) to just above the esophagogastric junction (1 cm).
Transverse rings in developing animals We examined one animal at each of a series of body weights that covered the whole period of postnatal development, 0.72, 8.08, 9.20, 16.11,
TABLE II
Distribution o f myelinated nerves in the esophageal myenteric plexus." whole mounts stained with Sudan black B Distance proximal to esophagogastric junction (cm)
Esophagus
Stomach
6
5
4
3
2
1
(/
- 1
28 14 53 30 31.3 4.2
32 10 52 28 30.5 8.6
22 15 48 19 26.0 7.5
26 20 36 20 25.5 3.8
14 18 33 5 17.5 5.8
4 24 13 14 13.8 4.1
4 4 7 4 4.8 0.8
25 3 40 12 20.0 8.1
7 8 22 11 12.0 3.4
8 11 20 4 10.8 3.4
2 8 8 9 6.8 1.6
3 2 5 3 3.3 0.6
13 3 17 t0 10.8 2.9
Total number of myelinated fibers Opossum 1 2 3 4 mean SE
Number of nerve bundles containing one or more myelinated .fibers Opossum 1 2 3 4 mean SE
13 7 23 18 15.3 3.6
15 6 21 15 14.3 3.1
8 10 19 10 11.8 2.5
Analysis of variance gives P values of 0.10 and 0.09 respectively for these distributions. The lack of significance at the 5% level results from the small numbers studied and the large inter-animal variation. Each opossum shows a reduction in the number of myelinated fibers at the esophagogastric junction.
233 TABLE III Distribution of myelinated nerves in the esophageal myenteric plexus: 0.5 # sections of transverse rings stained with Toluidine blue
Distance proximal to esophagogastricjunction (cm)
Esophagus 6 5
4
3
2
1
0
- 1
Stomach
53
54
54
48
43
45
-
-
21
25
21
20
9
7
-
-
40 70
46 86
39 44
42 40
21 11
16 12
-
-
-
-
61
-
-
46
55
148
-
-
23
-
-
8
3
22
-
-
38 90
-
-
17 24
5 4
15 37
Opossum 1
Total number of nerve bundles Number of nerve bundles with myelinated fibers % of nerve bundles with myelinated fibers Total number of myelinated fibers Opossum 2
Total number of nerve bundles Number of nerve bundles with myelinated fibers % of nerve bundles with myelinated fibers Total number of myelinated fibers
The preparation for Opossum 1 did not include the esophagogastric junction. In Opossum 2 the esophagogastric junction was studied in detail, with one ring from the mid-esophageal body included for comparison. A reduction in the number of myelinated fibers without any corresponding reduction in the total number of nerve bundles in the distal esophagus and at the esophagogastric junction is seen.
60.90, 66.93, 70.34, 200.00, 302.00, 450.00, 590.00 a n d 1010.00 g. A t t h e y o u n g e s t ages (i.e. 0.72 a n d 8.08 g), s t r u c t u r e s i d e n t i f i a b l e as g a n g l i a c o u l d b e d i s c e r n e d , a n d t h e s e p a r a t i o n of the m u s c l e into two layers was a l r e a d y a p p a r e n t . W i t h d e v e l o p ment, ganglia became more conspicuous. Myelin s h e a t h s w e r e not f o u n d at any s t a g e until t h e last, at 1010.00 g, c o r r e s p o n d i n g to an age o f 50 days a f t e r w e a n i n g . In t h e a n i m a l at this age, w e f o u n d only o n e myelin s h e a t h p r o f i l e in the full circumference of the esophagus.
Discussion
T h e i n t r a m u r a l m y e l i n a t e d fibers o f t h e e s o p h agus differ little f r o m t h o s e f o u n d in the s t o m a c h a n d colon. In t h o s e organs, t h e m y e l i n a t e d fibers run c r a n i o - c a u d a d for long d i s t a n c e s within the s h u n t fascicles, s h e a t h e d nerve t r u n k s t h a t follow a s t r a i g h t c o u r s e for long d i s t a n c e s in t h e s a m e p l a n e o f t h e m y e n t r i c plexus, b u t distinct f r o m it, b y p a s s i n g g a n g l i a [2,3]. T h e m y e l i n a t e d fibers d e p a r t f r o m s h u n t fascicles in those o r g a n s into t h e g a n g l i o n a t e d plexus a n d e x t e n d only a s h o r t dis-
t a n c e b e f o r e losing t h e myelin s h e a t h . T h e myelin a t e d fibers o f t h e e s o p h a g u s a r e m o r e u n i f o r m l y d i s t r i b u t e d within the f r a m e w o r k o f t h e m y e n teric plexus t h a n t h e y a r e in t h e s t o m a c h a n d colon. Still, they a r e o f t e n f o u n d in fascicles having a p e r i n e u r i u m (like t h e s h u n t fascicles of t h e s t o m a c h a n d colon, a n d like extrinsic nerves) r a t h e r t h a n in t h e u n s h e a t h e d i n t e r g a n g l i o n i c fascicles. Like t h e m y e l i n a t e d fibers in t h e shunt fascicles of the s t o m a c h a n d colon, they e x t e n d over long distances, p r i n c i p a l l y in t h e c r a n i o caudad direction. N e i t h e r the exact s o u r c e n o r the d e s t i n a t i o n of t h e s e m y e l i n a t e d n e r v e fibers can b e s t a t e d with certainty. T h e fact t h a t the sites w h e r e they p i e r c e t h e l o n g i t u d i n a l layer to e n t e r t h e p l a n e o f the m y e n t e r i c plexus a r e r a t h e r evenly d i s t r i b u t e d suggests that t h e y arise m a i n l y from the vagal e s o p h a g e a l plexus. M y e l i n a t e d fibers lose t h e i r myelin s h e a t h s an u n d e f i n a b l e d i s t a n c e short o f t h e i r t e r m i n a l s , h e n c e t h e r e m u s t be d o u b t a b o u t their precise destination. T h e d i s t r i b u t i o n d e n s i t y o f m y e l i n a t e d fibers shows a striking g r a d i e n t f r o m t h e e s o p h a g e a l b o d y to t h e e s o p h a g o g a s t r i c j u n c t i o n . This p a r a l -
234 lels a g r a d i e n t in the density of intrinsic n e u r o n s [4,5]. T h e virtual a b s e n c e of m y e l i n a t e d fibers crossing the e s o p h a g o g a s t r i c j u n c t i o n i n d i c a t e s that the m y e n t e r i c plexus o f the e s o p h a g u s is not an i m p o r t a n t source of the a b u n d a n t m y e l i n a t e d fibers of the stomach. Thus, g a s t r o - s p h i n c t e r i c reflexes [19] m u s t m a k e use o f n o n m y e l i n a t e d r a t h e r t h a n m y e l i n a t e d pathways. P r e s u m a b l y , the a b u n d a n t m y e l i n a t e d nerve fibers o f the s t o m a c h arise from the vagal trunks a n d e n t e r the wall of the s t o m a c h close to the cardia. T h e m a p of m y e l i n a t e d fibers from o n e m a t u r e animal shown in Fig. 3 A i n d i c a t e s that myelin a t e d fibers a r e not uniformly d i s t r i b u t e d in the c i r c u m f e r e n c e of the e s o p h a g u s at any single level. F u r t h e r m o r e , the d a t a from the transverse rings in T a b l e III suggest t h a t t h e r e may be c o n s i d e r a b l e v a r i a t i o n f r o m one a n i m a l to ano t h e r in the p r o p o r t i o n o f m y e l i n a t e d fibers at any single level. This variability s u p p o r t s the idea that m y e l i n a t i o n of extrinsic nerve fibers is not a critical d e t e r m i n a n t of n o r m a l peristalsis in the s m o o t h muscle o f the e s o p h a g u s . T h e d e v e l o p m e n t of m y e l i n a t i o n in the extrinsic nerves to t h e o p o s s u m e s o p h a g u s has not even b e g u n at the time o f w e a n i n g , n o r d o e s it begin until long a f t e r the a n i m a l s have b e c o m e fully i n d e p e n d e n t a n d a r e c o n s u m i n g a n o r m a l solid diet. Thus, a l t h o u g h the extrinsic i n n e r v a t i o n o f the e s o p h a g u s is clearly not fully d e v e l o p e d in the w e e k s a f t e r w e a n i n g (in t h a t it is not m y e l i n a t e d ) , e s o p h a g e a l m o t o r function m u s t be essentially normal. This late m y e l i n a t i o n of nerves to the e s o p h a g u s is consistent with the late m y e l i n a t i o n of vagal fibers o b s e r v e d by Krous, J o r d a n , W e n a n d F a r b e r [14] who f o u n d m y e l i n a t e d fibers (in the cervical vagus of t h e d e v e l o p i n g o p o s s u m ) to have a c h i e v e d only 11% of a d u l t values by the age of 50 days. T h e s e cervical vagal fibers, of course, w e r e d e s t i n e d for the w h o l e vagal distribution. O u r study indicates that vagal m y e l i n a t e d fibers begin to r e a c h the e s o p h a g u s only after m o r e t h a n a b o u t 150 days of age, when the animal is half-grown. T h e d e v e l o p m e n t of m y e l i n a t e d fibers in the h u m a n s m o o t h - m u s c l e e s o p h a g u s is not known. If it p a r a l l e l s t h e d e v e l o p m e n t in t h e o p o s s u m , t h e n the a b s e n c e o f m y e l i n a t i o n is not an e x p l a n a t i o n
for the e s o p h a g e a l m o t o r a b n o r m a l i t i e s of p r e m a ture infants. M y e l i n a t i o n can be t a k e n as a morp h o l o g i c a l index of m a t u r a t i o n of a system of nerves. If so, then t h e s e results do not s u p p o r t t h e i d e a that i m m a t u r i t y of the extrinsic nerve supply to the e s o p h a g u s is the e x p l a n a t i o n for the a b n o r m a l e s o p h a g e a l motility of infants.
Acknowledgements This w o r k was s u p p o r t e d by R e s e a r c h G r a n t D K 11242 a n d C o r e C e n t e r G r a n t D K 34986 from the N a t i o n a l Institutes of H e a l t h .
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