Postnatal Maturation of Phrenic Nerve in Children Fernanda J. Teixeira, M D * t , F r a n q e s c o A r a n d a , PhD*.% a n d L a u r e n c e E. Becker, M D *
The phrenic nerve at the pericardial level was examined poslmortem in 17 children, ages 3 days to 8 years. Detailed macroscopic and histologic examination of the central nervous system in all patients disclosed no abnormalities. Quantitative developmental studies demonstrated that myelinated axons doubled in number from birth to age 1 year when a plateau was reached. The main period of growth in diameter of myelinated axons also corresponded to the first year when median diameters increased from 1.75 gm at 3 days of age to 3.0 gm by 8 months of age. Unmyelinated axons also grew significantly in the first 11 months when median diameters reached 1.4 pm. There was no significant increase in axonal diameter at later ages. The slope of the regression line for the number of myelin lameHae on axonal diameters increased with age until 6 months of age, whereas the dispersion around the regression lines decreased in the same period. This finding suggests a direct relationship between myelination and axonal growth. Significant maturation of the phrenic nerve occurs during the first year of life. Teixeira FJ, Aranda F, Becker LE. Postnatal maturation of phrenic nerve in children. Pediatr Neurol 1992;8:450-4.
Introduction The phrenic nerve is a fundamental structure involved in the neural control of respiration because it constitutes the sole motor innervafion of the diaphragm [1]. In 1979, Wrigglesworth and Desai reported that this nerve also controls fetal respiratory movements which are of critical importance for the normal development of the lungs [2]. The development of the phrenic nerve is, therefore, of considerable clinical importance. In 1985, Glenn [3] and Ilbawi et al. [4] reported that phrenic nerve pacing can be used to control a defect in the regulation of alveolar ventilation during sleep in both sudden infant death syndrome and congenital hypoventilation syndrome (Ondine's curse).
From the *Dept. of Pathology (Neuropathology); The Hospital for Sick Children; Toronto, Canada; rDept, of Experimental Neu.ropathology; National Institute of Neurology; Mexico City, Mexico; rDept, of Statistics and Actuarial Science; University of Waterloo; Waterloo, Canada; ~Dept. of Statistics; IIMAS, UNAM; Mexico City, Mexico. tDeceased.
450
PEDIATRIC NEUROLOGY
Vol. 8 No. 6
Although the morphology of the phrenic nerve has been described in detail in 3 studies of animals [5-7], data concerning the normal development of this nerve in humans are scarce. This report describes the morphologic characteristics of the axons and myelin sheaths of the phrenic nerves of children at different ages to establish reference values for comparison with nerves from patients with deficient respiratory control. Methods Preparation of Nerve Specimens. We selected 17 children, ranging in age from 3 days to 8 years, whose illnesses or deaths did not involve the peripheral nervous system. Patients with congenital heart disease were excluded because of the potential neurologic repercussions of any protracted periods of cardiac failure and hypoxemia [8]. The central nervous systems of all children were examined in detail to ensure that there were no gross or microscopic abnormalities. A 1.5 cm segment of the left phrenic nerve was obtained at the pericardial level and made to adhere to a small piece of card, straightened with a minimum of handling, and immersed in 4% formaldehyde and 1% glutaraldehyde in a 200 mOsm phosphate buffer for 3 hours. The specimen then w a s washed in the same buffer, postfixed in 2% osmium tetroxide, dehydrated in acetone, and embedded fiat in Epon. Sections of 1 ~tm w e r e stained with toluidine blue to evaluate the preservation of the nerve and the orientation of its fibers. Sections of 60-90 nm obtained from blocks transverse to the long axis of the nerve were stained with uranyl acetate and lead citrate and examined in a Philips EM 300 transmission electron microscope operating at 60 kV. Number and Size ()["Axons. To determine the number and size of axons, we used electron microscopic morphometry rather than light microscopy for several reasons. The chromatin of Schwann cell nuclei tends to conglomerate against the nuclear envelope a s a postmortem alteration; therefore, nuclei can be misinterpreted as myelinated axons with light microscopy. Myelination can be studied more accurately with electron microscopy because all myelin lameltae can be identif~l and counted. Moreover, morphometry, when used in combination with electron microscopy, will not underestimate the number of small fibers [91. Only those sections cut transversely were used; 50-70 electron micrographs of adjacent, nonoverlapping fields were taken from each nerve and printed at a final magnification of xlO,000. Both myelinated and nonmyelinated axons were measured. Among the nonmyelinated axons, we included all axons not enveloped by myelin (i.e., both unmyelinated fibers and axons yet to be myelinated). Maximum diameters were measured on the prints with the aid of a Zidas image analyzer (Carl Zeiss Co.). The axons were classified into groups according to diameter, corresponding to the interval ranges of 0.1-0.5 gm to 3.0 lain for nonmyelinated axons and 0.1-0.5 ~tm to 10.0 lain for myelinated axons. Histo-
Communications should be addressed to: Dr. Becker; Department of Pathology (Neuropathology); The Hospital for Sick Children; 555 University Avenue; Toronto, Ontario, Canada M5G iX8. Received April 13, 1992; accepted July 17, 1992.
Table 1.
Pt. No.
Number of myelinated and nonmyelinated axons
Age
M/n
M/mm 2
N/mm 2
N/M
1
3 days
2,173
19,936
88,325
4.4
2
5 days
2,329
21,506
117,289
5.4
3
15 days
2,448
19,459
73,257
3.8
4
21/2 mos
3,963
17,230
26,173
1.5
5
4 mos
3,646
18,910
35,451
1.9
6
4 mos
3,879
18,959
29,316
1.5
7
5 mos
3,753
17,458
30,285
1.7
8
51//2 mos
4,281
16,327
28,647
1.7
9
61/2 mos
4,857
16,264
27,704
1.7
10
8 mos
5,014
16,219
25,727
1.6
11
9 mos
4,385
13,702
24,664
1.8
12
11 mos
5,326
14,409
31,768
2.2
13
21 mos
4,992
13,869
26,258
1.9
14
22 mos
5,398
14,297
20,025
1.4
15
4 yrs
5,244
14,133
29,146
2.1
16
6 yrs
5,266
13,165
25,678
1.9
17
8 yrs
5,329
13,653
27,393
2.0
Abbreviations: M/n = Numbers of myelinated axons per transverse section of nerve M/mm2 = Numbers of myelinated axons per square millimeter N/mm2 = Numbers of nonmyelinated axons per square millimeter N/M = Ratio between numbers of nonmyelinated and myelinated axons
grams were obtained for each nerve, as well as individually for each set of myelinated and nonmyelinated axons. A maximum of 350 myelinated and 450 nonmyelinated axons were measured because above these figures, the relative proportion of axons in each interval range did not vary more than 5%. The number of myelinated and nonmyelinated axons/mm 2 was calculated by extrapolation from the density of axons in the area sampled from the electron micrographs. The fascicular area of each nerve was measured with an IBAS-Kontron image analyzer coupled to a light microscope provided with a camera that projected the image of the 1 gm section on a screen. The number of myelinated and nonmyelinated axons per nerve was calculated by extrapolation from the numbers per square millimeter. Correlation Between the Axonal Diameter and Number of Myelin Lamellae. We studied the development of myelination in the phrenic nerves by randomly sampling 95-100 myelinated axons from each nerve. An electron micrograph was taken of each of these samples and printed at a final magnification of x18,000. The number of myelin lamellae was counted with a dissecting microscope, and the diameter of the associated axons was measured with the Zidas image analyzer. For each nerve, the correlation coefficient between the axonal diameters and the number of lamellae was calculated, and scatter diagrams were prepared. Numbers of lamellae at different ages were regressed
on axonal diameters, and regression lines were superimposed on the scatter diagrams. Results
N u m b e r o f Axons. Table 1 lists the n u m b e r s o f m y e l i n ated and n o n m y e l i n a t e d a x o n s p e r t r a n s v e r s e s e c t i o n o f n e r v e and p e r square millimeter. T h e n u m b e r o f m y e l i n ated a x o n s per t r a n s v e r s e s e c t i o n i n c r e a s e d until a b o u t 1 y e a r o f age w h e n it r e a c h e d a plateau, w h e r e a s the d e n s i t y o f m y e l i n a t e d fibers p e r square m i l l i m e t e r d e c r e a s e d in t h e first 9 m o n t h s and stabilized thereafter. T h e n u m b e r o f nonmyelinated axons per square millimeter decreased sharply in the first 2V2 m o n t h s o f life, but d e m o n s t r a t e d little variation at later ages. A x o n a l Diameter. S i z e - f r e q u e n c y h i s t o g r a m s o f the distribution o f m y e l i n a t e d and n o n m y e l i n a t e d a x o n s in all p h r e n i c n e r v e s s t u d i e d w e r e u n i m o d a l . T h e g r o w t h in d i a m e t e r o f m y e l i n a t e d a n d n o n m y e l i n a t e d a x o n s is d e p i c t e d in F i g u r e 1. T h e m e d i a n d i a m e t e r o f the m y e l -
Teixeira et al: Maturation of Phrenic Nerve 451
,i
!
t
2
i :! |,.,,g,...~,,,l| o
1
2
3
4
6 7 ~(YEARS)
Figure 1. Growth in diameter of myelinated (solid line) and nonrayelinated axons (broken line) according to age.
inated axon increased from 1.75 ~tm at 3 days of age up to 3 lam by 8 months. At later ages, there was little variation in the value of the medians. In a similar way, the diameter of nonmyelinated axons increased up to 11 months when it reached a maximum median of 1.4 ttm.
t~
o
& e l (YEARS) Figure 2. The sum of the areas of all fasciculi present in the transverse sections of normal phrenic nerves increases with age and reaches a maximum by the second year of life.
452 PEDIATRICNEUROLOGY Vol.8 No. 6
Size o f F a s c i c u l a r Area. Figure 2 discloses the size of the fascicular areas in transverse sections of phrenic nerves as they changed with age. The main periled ~t" growth corresponded to the first 2 years of life. D e v e l o p m e n t o f M v e l i n a t i o n . Scatter diagrams for children, ages 21/2, 4, 51/2, and 20 months arc illustrated in Figures 3A-3D, respectively. The change in the rate of development of myelination from 21/2-5 V2 months of age and the stability of the regression lines of the scatter diagrams for subsequent ages are evident. The major change occurred in the first 6 months; afterwards, the correlation between the number of lamellae and the axonal diameter was stable.
Discussion
The first year of an infant's life was the main period of growth in the size of myelinated axons of the phrenic nerves in the children we studied. In spite of the increase in numbers of myelinated axons in the nerve, the number of axons per square millimeter diminished because of the growth in the diameter of the fibers. Myelination was well-developed by the age of 6 months, but correlation coefficients continued to increase until the second year, indicating that myelin lamellae continued to be layered around axons. The increase in size of the nerve fasciculi up to 2 years of age may have been caused partially by the expansion of the endoneurial connective tissue. In 1967, Ekholm reported that there was an important difference in the rate of myelination of axons in different nerves in animals [10]. For example, in the sural nerve of newborn cats, the proportion of myelinated to unmyelinated axons does not exceed 30% as compared to adult values. In their 1979 study of the kitten, Marlot and Duron observed that these values were 80% for the phrenic nerve and 10% for the vagus nerve at birth [7]; by the 30th postnatal day, these values increased to 100% and 40%, respectively. In the phrenic nerves of the children we studied, 43% of the fibers were myelinated at birth; maximum myelination was reached only by the first year of life. The rate of growth of the axonal diameter also differs, depending on the nerve. Marlot and Duron reported that the rate of growth of the vagal nerve fibers (0.2 ~tm/day) was greater than that of the phrenic axons (0.1 ~trrdday) [7]. By the 30th postnatal day, the former reached 100% of the adult development, whereas phrenic axons reached only 30% of the adult values. Although they mostly used light microscopy and included the myelin sheaths in axonal diameter measurements [7], the findings of Marlot and Duron about the development of axonal nerves in kittens are comparable to our study of the axonal diameters only in children. Our present data demonstrate that the mean diameter of myelinated axons at birth was 60% of that of adult values; by 8 months it reached 100%. It is possible that the motor innervation of the diaphragm is of such primary importance for survival that the phrenic
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nerve has to be almost completely myelinated at birth. It is difficult, however, to relate morphologic to functional observations. In 1976, Duron and Marlot reported that in kittens the Hering-Breuer reflex, mediated by vagal myelinated axons, was potent at birth, although only a small proportion of axons was myelinated at this age [5]. Although it is difficult to determine accurately the number of nonmyelinated axons because the number was extrapolated from a direct count performed on a projected image of a portion of the nerve, in the present study, nonmyelinated axons appeared to be numerous at birth and maintained a proportion to myelinated fibers of 4-5:1. With the myelination of the axons, this proportion fell to 2:1 by the age of 4 months and did not vary significantly at later ages. This proportion is similar to that observed in other species, such as that of the rat (1.7 to 1.1:1) [6] and that of the kitten (1.9:1) [7]. Our results also suggest that the period of growth in the diameter of unmyelinated axons is short, and that by 11 months, the median diameter is very similar to that found in older children. At this stage,
myelinated axons are not developing from unmyelinated axons. This finding suggests that the phrenic nerve is relatively mature at birth; nevertheless, significant maturation occurs during the first year of life.
This report was prepared with the assistance of Medical Publications, The Hospital for Sick Children, Toronto, Ontario, Canada.
References
[1] Moore KL. Clinically oriented anatomy. In: The abdomen. Baltimore: Williams and Wilkins, 1980;256. [2] Wigglesworth JS, Desai R. Effect on lung growth of cervical cord section in the rabbit fetus. Early Hum Dev 1979;3:51-65. [3] Glenn WWL. Pacing the diaphragm in infants. Ann Thorac Surg 1985;40:319-20. [4] llbawi MN, Idriss FS, Hunt CE, Brouillette RT, DeLeon SY. Diaphragmatic pacing in infants: Techniques and results. Ann Thorac
Surg 1985;40:323-9.
Teixeira et al: Maturation of Phrenic Nerve
453
[5] Duron B, Marlot D. Evolution des reflexes de Hering-Breuer au cours de la periode neo-natale chez le charon. In: Duron B, ed. Respiratory centres and afferent systems. Paris: Institut National de la Sante et de la Recherche Medic',de (INSERM), 1976;281-6. [6] Langford LA, Schmidt RE An electron microscopic analysis of the left phrenic nerve in the rat. Anat Rec 1983;205:207-13. [7] Marlot D, Duron B. Postnatal maturation of phrenic, vagus, and intercostal nerves in the kitten. Biol Neonate 1979;36:264-72. [8] Naeye RL. Pathologists' role in SIDS research: The unfinished task. In: Tildon JT, Roeder LM, Steinschneider A, eds. Sudden infant death syndrome. New York: Academic Press, 1983;161-7.
454
PEDIATRIC NEUROLOGY
Vol. 8 No. 6
[9] Bronson R"[\ Bishop Y, Hedley-Whyte ET. A c
The phrenic nerve at the pericardial level was examined poslmortem in 17 children, ages 3 days to 8 years. Detailed macroscopic and histologic examination of the central nervous system in all patients disclosed no abnormalities. Quantitative developmental studies demonstrated that myelinated axons doubled in number from birth to age 1 year when a plateau was reached. The main period of growth in diameter of myelinated axons also corresponded to the first year when median diameters increased from 1.75 gm at 3 days of age to 3.0 gm by 8 months of age. Unmyelinated axons also grew significantly in the first 11 months when median diameters reached 1.4 pm. There was no significant increase in axonal diameter at later ages. The slope of the regression line for the number of myelin lameHae on axonal diameters increased with age until 6 months of age, whereas the dispersion around the regression lines decreased in the same period. This finding suggests a direct relationship between myelination and axonal growth. Significant maturation of the phrenic nerve occurs during the first year of life. Teixeira FJ, Aranda F, Becker LE. Postnatal maturation of phrenic nerve in children. Pediatr Neurol 1992;8:450-4.
Introduction The phrenic nerve is a fundamental structure involved in the neural control of respiration because it constitutes the sole motor innervafion of the diaphragm [1]. In 1979, Wrigglesworth and Desai reported that this nerve also controls fetal respiratory movements which are of critical importance for the normal development of the lungs [2]. The development of the phrenic nerve is, therefore, of considerable clinical importance. In 1985, Glenn [3] and Ilbawi et al. [4] reported that phrenic nerve pacing can be used to control a defect in the regulation of alveolar ventilation during sleep in both sudden infant death syndrome and congenital hypoventilation syndrome (Ondine's curse).
From the *Dept. of Pathology (Neuropathology); The Hospital for Sick Children; Toronto, Canada; rDept, of Experimental Neu.ropathology; National Institute of Neurology; Mexico City, Mexico; rDept, of Statistics and Actuarial Science; University of Waterloo; Waterloo, Canada; ~Dept. of Statistics; IIMAS, UNAM; Mexico City, Mexico. tDeceased.
450
PEDIATRIC NEUROLOGY
Vol. 8 No. 6
Although the morphology of the phrenic nerve has been described in detail in 3 studies of animals [5-7], data concerning the normal development of this nerve in humans are scarce. This report describes the morphologic characteristics of the axons and myelin sheaths of the phrenic nerves of children at different ages to establish reference values for comparison with nerves from patients with deficient respiratory control. Methods Preparation of Nerve Specimens. We selected 17 children, ranging in age from 3 days to 8 years, whose illnesses or deaths did not involve the peripheral nervous system. Patients with congenital heart disease were excluded because of the potential neurologic repercussions of any protracted periods of cardiac failure and hypoxemia [8]. The central nervous systems of all children were examined in detail to ensure that there were no gross or microscopic abnormalities. A 1.5 cm segment of the left phrenic nerve was obtained at the pericardial level and made to adhere to a small piece of card, straightened with a minimum of handling, and immersed in 4% formaldehyde and 1% glutaraldehyde in a 200 mOsm phosphate buffer for 3 hours. The specimen then w a s washed in the same buffer, postfixed in 2% osmium tetroxide, dehydrated in acetone, and embedded fiat in Epon. Sections of 1 ~tm w e r e stained with toluidine blue to evaluate the preservation of the nerve and the orientation of its fibers. Sections of 60-90 nm obtained from blocks transverse to the long axis of the nerve were stained with uranyl acetate and lead citrate and examined in a Philips EM 300 transmission electron microscope operating at 60 kV. Number and Size ()["Axons. To determine the number and size of axons, we used electron microscopic morphometry rather than light microscopy for several reasons. The chromatin of Schwann cell nuclei tends to conglomerate against the nuclear envelope a s a postmortem alteration; therefore, nuclei can be misinterpreted as myelinated axons with light microscopy. Myelination can be studied more accurately with electron microscopy because all myelin lameltae can be identif~l and counted. Moreover, morphometry, when used in combination with electron microscopy, will not underestimate the number of small fibers [91. Only those sections cut transversely were used; 50-70 electron micrographs of adjacent, nonoverlapping fields were taken from each nerve and printed at a final magnification of xlO,000. Both myelinated and nonmyelinated axons were measured. Among the nonmyelinated axons, we included all axons not enveloped by myelin (i.e., both unmyelinated fibers and axons yet to be myelinated). Maximum diameters were measured on the prints with the aid of a Zidas image analyzer (Carl Zeiss Co.). The axons were classified into groups according to diameter, corresponding to the interval ranges of 0.1-0.5 gm to 3.0 lain for nonmyelinated axons and 0.1-0.5 ~tm to 10.0 lain for myelinated axons. Histo-
Communications should be addressed to: Dr. Becker; Department of Pathology (Neuropathology); The Hospital for Sick Children; 555 University Avenue; Toronto, Ontario, Canada M5G iX8. Received April 13, 1992; accepted July 17, 1992.
Table 1.
Pt. No.
Number of myelinated and nonmyelinated axons
Age
M/n
M/mm 2
N/mm 2
N/M
1
3 days
2,173
19,936
88,325
4.4
2
5 days
2,329
21,506
117,289
5.4
3
15 days
2,448
19,459
73,257
3.8
4
21/2 mos
3,963
17,230
26,173
1.5
5
4 mos
3,646
18,910
35,451
1.9
6
4 mos
3,879
18,959
29,316
1.5
7
5 mos
3,753
17,458
30,285
1.7
8
51//2 mos
4,281
16,327
28,647
1.7
9
61/2 mos
4,857
16,264
27,704
1.7
10
8 mos
5,014
16,219
25,727
1.6
11
9 mos
4,385
13,702
24,664
1.8
12
11 mos
5,326
14,409
31,768
2.2
13
21 mos
4,992
13,869
26,258
1.9
14
22 mos
5,398
14,297
20,025
1.4
15
4 yrs
5,244
14,133
29,146
2.1
16
6 yrs
5,266
13,165
25,678
1.9
17
8 yrs
5,329
13,653
27,393
2.0
Abbreviations: M/n = Numbers of myelinated axons per transverse section of nerve M/mm2 = Numbers of myelinated axons per square millimeter N/mm2 = Numbers of nonmyelinated axons per square millimeter N/M = Ratio between numbers of nonmyelinated and myelinated axons
grams were obtained for each nerve, as well as individually for each set of myelinated and nonmyelinated axons. A maximum of 350 myelinated and 450 nonmyelinated axons were measured because above these figures, the relative proportion of axons in each interval range did not vary more than 5%. The number of myelinated and nonmyelinated axons/mm 2 was calculated by extrapolation from the density of axons in the area sampled from the electron micrographs. The fascicular area of each nerve was measured with an IBAS-Kontron image analyzer coupled to a light microscope provided with a camera that projected the image of the 1 gm section on a screen. The number of myelinated and nonmyelinated axons per nerve was calculated by extrapolation from the numbers per square millimeter. Correlation Between the Axonal Diameter and Number of Myelin Lamellae. We studied the development of myelination in the phrenic nerves by randomly sampling 95-100 myelinated axons from each nerve. An electron micrograph was taken of each of these samples and printed at a final magnification of x18,000. The number of myelin lamellae was counted with a dissecting microscope, and the diameter of the associated axons was measured with the Zidas image analyzer. For each nerve, the correlation coefficient between the axonal diameters and the number of lamellae was calculated, and scatter diagrams were prepared. Numbers of lamellae at different ages were regressed
on axonal diameters, and regression lines were superimposed on the scatter diagrams. Results
N u m b e r o f Axons. Table 1 lists the n u m b e r s o f m y e l i n ated and n o n m y e l i n a t e d a x o n s p e r t r a n s v e r s e s e c t i o n o f n e r v e and p e r square millimeter. T h e n u m b e r o f m y e l i n ated a x o n s per t r a n s v e r s e s e c t i o n i n c r e a s e d until a b o u t 1 y e a r o f age w h e n it r e a c h e d a plateau, w h e r e a s the d e n s i t y o f m y e l i n a t e d fibers p e r square m i l l i m e t e r d e c r e a s e d in t h e first 9 m o n t h s and stabilized thereafter. T h e n u m b e r o f nonmyelinated axons per square millimeter decreased sharply in the first 2V2 m o n t h s o f life, but d e m o n s t r a t e d little variation at later ages. A x o n a l Diameter. S i z e - f r e q u e n c y h i s t o g r a m s o f the distribution o f m y e l i n a t e d and n o n m y e l i n a t e d a x o n s in all p h r e n i c n e r v e s s t u d i e d w e r e u n i m o d a l . T h e g r o w t h in d i a m e t e r o f m y e l i n a t e d a n d n o n m y e l i n a t e d a x o n s is d e p i c t e d in F i g u r e 1. T h e m e d i a n d i a m e t e r o f the m y e l -
Teixeira et al: Maturation of Phrenic Nerve 451
,i
!
t
2
i :! |,.,,g,...~,,,l| o
1
2
3
4
6 7 ~(YEARS)
Figure 1. Growth in diameter of myelinated (solid line) and nonrayelinated axons (broken line) according to age.
inated axon increased from 1.75 ~tm at 3 days of age up to 3 lam by 8 months. At later ages, there was little variation in the value of the medians. In a similar way, the diameter of nonmyelinated axons increased up to 11 months when it reached a maximum median of 1.4 ttm.
t~
o
& e l (YEARS) Figure 2. The sum of the areas of all fasciculi present in the transverse sections of normal phrenic nerves increases with age and reaches a maximum by the second year of life.
452 PEDIATRICNEUROLOGY Vol.8 No. 6
Size o f F a s c i c u l a r Area. Figure 2 discloses the size of the fascicular areas in transverse sections of phrenic nerves as they changed with age. The main periled ~t" growth corresponded to the first 2 years of life. D e v e l o p m e n t o f M v e l i n a t i o n . Scatter diagrams for children, ages 21/2, 4, 51/2, and 20 months arc illustrated in Figures 3A-3D, respectively. The change in the rate of development of myelination from 21/2-5 V2 months of age and the stability of the regression lines of the scatter diagrams for subsequent ages are evident. The major change occurred in the first 6 months; afterwards, the correlation between the number of lamellae and the axonal diameter was stable.
Discussion
The first year of an infant's life was the main period of growth in the size of myelinated axons of the phrenic nerves in the children we studied. In spite of the increase in numbers of myelinated axons in the nerve, the number of axons per square millimeter diminished because of the growth in the diameter of the fibers. Myelination was well-developed by the age of 6 months, but correlation coefficients continued to increase until the second year, indicating that myelin lamellae continued to be layered around axons. The increase in size of the nerve fasciculi up to 2 years of age may have been caused partially by the expansion of the endoneurial connective tissue. In 1967, Ekholm reported that there was an important difference in the rate of myelination of axons in different nerves in animals [10]. For example, in the sural nerve of newborn cats, the proportion of myelinated to unmyelinated axons does not exceed 30% as compared to adult values. In their 1979 study of the kitten, Marlot and Duron observed that these values were 80% for the phrenic nerve and 10% for the vagus nerve at birth [7]; by the 30th postnatal day, these values increased to 100% and 40%, respectively. In the phrenic nerves of the children we studied, 43% of the fibers were myelinated at birth; maximum myelination was reached only by the first year of life. The rate of growth of the axonal diameter also differs, depending on the nerve. Marlot and Duron reported that the rate of growth of the vagal nerve fibers (0.2 ~tm/day) was greater than that of the phrenic axons (0.1 ~trrdday) [7]. By the 30th postnatal day, the former reached 100% of the adult development, whereas phrenic axons reached only 30% of the adult values. Although they mostly used light microscopy and included the myelin sheaths in axonal diameter measurements [7], the findings of Marlot and Duron about the development of axonal nerves in kittens are comparable to our study of the axonal diameters only in children. Our present data demonstrate that the mean diameter of myelinated axons at birth was 60% of that of adult values; by 8 months it reached 100%. It is possible that the motor innervation of the diaphragm is of such primary importance for survival that the phrenic
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nerve has to be almost completely myelinated at birth. It is difficult, however, to relate morphologic to functional observations. In 1976, Duron and Marlot reported that in kittens the Hering-Breuer reflex, mediated by vagal myelinated axons, was potent at birth, although only a small proportion of axons was myelinated at this age [5]. Although it is difficult to determine accurately the number of nonmyelinated axons because the number was extrapolated from a direct count performed on a projected image of a portion of the nerve, in the present study, nonmyelinated axons appeared to be numerous at birth and maintained a proportion to myelinated fibers of 4-5:1. With the myelination of the axons, this proportion fell to 2:1 by the age of 4 months and did not vary significantly at later ages. This proportion is similar to that observed in other species, such as that of the rat (1.7 to 1.1:1) [6] and that of the kitten (1.9:1) [7]. Our results also suggest that the period of growth in the diameter of unmyelinated axons is short, and that by 11 months, the median diameter is very similar to that found in older children. At this stage,
myelinated axons are not developing from unmyelinated axons. This finding suggests that the phrenic nerve is relatively mature at birth; nevertheless, significant maturation occurs during the first year of life.
This report was prepared with the assistance of Medical Publications, The Hospital for Sick Children, Toronto, Ontario, Canada.
References
[1] Moore KL. Clinically oriented anatomy. In: The abdomen. Baltimore: Williams and Wilkins, 1980;256. [2] Wigglesworth JS, Desai R. Effect on lung growth of cervical cord section in the rabbit fetus. Early Hum Dev 1979;3:51-65. [3] Glenn WWL. Pacing the diaphragm in infants. Ann Thorac Surg 1985;40:319-20. [4] llbawi MN, Idriss FS, Hunt CE, Brouillette RT, DeLeon SY. Diaphragmatic pacing in infants: Techniques and results. Ann Thorac
Surg 1985;40:323-9.
Teixeira et al: Maturation of Phrenic Nerve
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[5] Duron B, Marlot D. Evolution des reflexes de Hering-Breuer au cours de la periode neo-natale chez le charon. In: Duron B, ed. Respiratory centres and afferent systems. Paris: Institut National de la Sante et de la Recherche Medic',de (INSERM), 1976;281-6. [6] Langford LA, Schmidt RE An electron microscopic analysis of the left phrenic nerve in the rat. Anat Rec 1983;205:207-13. [7] Marlot D, Duron B. Postnatal maturation of phrenic, vagus, and intercostal nerves in the kitten. Biol Neonate 1979;36:264-72. [8] Naeye RL. Pathologists' role in SIDS research: The unfinished task. In: Tildon JT, Roeder LM, Steinschneider A, eds. Sudden infant death syndrome. New York: Academic Press, 1983;161-7.
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Vol. 8 No. 6
[9] Bronson R"[\ Bishop Y, Hedley-Whyte ET. A c
