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The effects of intrauterine growth retardation on the structural development of cranial nerves (optic, trochlear) in fetal sheep
hr. J. Devl. Neuroscience, Printed in Great Britain.
Vol. 8,
No. 2, pp. 133-141, 1990.
0736-5748/90 $03.00+0.00 Pergamon Press plc @ 1990 ISDN
THE EFFECTS OF INTRAUTERINE GROWTH RETARDATION ON THE STRUCTURAL DEVELOPMENT OF CRANIAL NERVES (OPTIC, TROCHLEAR) IN FETAL SHEEP SANDRA
REES,*~ MALCOLM CLARK, MICHAEL
SNOWDEN
and RICHARD HARDING*
Departments of Physiology* and Mathematics, Monash University, Clayton, Victoria 3168, Australia (Received 12 June 1989; in revised form 29 August 1989; accepted 1 September 1989)
Abstract-A quantitative morphometric study of the development of myelinated fibres in the optic and trochlear nerves has been made in growth-retarded fetal sheep at 140 days gestation (term = 146 days). Intrauterine growth retardation was induced as a result of the reduction of placental mass, by prior removal of placentation sites in six ewes. In the optic nerve (central nervous system) the mean diameter of myelinated fibres was not significantly reduced but the thickness of the myelin sheath relative to axon diameter was disproportionately reduced. In the trochlear nerve (peripheral nervous system) there was a significant reduction of 23% (PCO.01) in the mean diameter of myelinated fibres; however the normal axon:myelin ratio was maintained. The total number of myelinated fibres in the trochlear nerve did not differ between the normal and growth-retarded group, indicating that there was not a greater than normal incidence of cell death during intrauterine growth retardation in the nucleus of the trochlear nerve. The differential effect of intrauterine growth retardation on myelination in the central and peripheral nervous systems suggests that chronic intrauterine deprivation affects oligodendrocyte activity but does not markedly affect the capacity of Schwann cells to produce myelin.
Key words: optic, trochlear, nerve development,
intrauterine growth retardation, axon diameter, myelin
thickness, fetal sheep.
In previous studies on fetal brain development in experimental intrauterine growth retardation (IUGR) produced by reducing placental mass in sheep, it has been shown that a number of parameters are significantly affected. For example, in the cerebellum growth of granule cell dendrites and of the Purkinje cell dendritic tree is reduced,23 in the visual cortex synaptogenesis is reduced,3 in the peroneal nerve axonal development is retarded and conduction velocity is slower than in age-matched control fetuses.24 In the present quantitative morphometric study, the effects of fetal growth retardation on axonal development and myelination in the optic and trochlear nerves were examined. Since the optic nerve is part of the central nervous system (CNS) and the trochlear nerve part of the peripheral nervous system (PNS), it was possible to compare the effects of fetal growth retardation on two fibre tracts, both cranial nerves but in different divisions of the nervous system. Identification of any structural abnormalities that occur in particular areas of the nervous system or amongst specific cell types during experimental growth retardation will assist in understanding the neurological deficits which occur with a greater than normal incidence in growthretarded or small-for-gestational-age children.7~8*“~‘9
EXPERIMENTAL
PROCEDURES
Endometrial caruncles (potential sites of placental attachment) were removed from six nonpregnant Meriono-Border Leicester ewes2*26under halothane anaesthesia. A low midline incision was made in the abdomen and the uterus was exposed. Each horn of the uterus was opened from the cervix to near the uterotubal junction and most of the accessible caruncles (80-95) were removed by cautery. After a minimum of 8 weeks recovery, the ewes were allowed to mate, the day of mating being taken as day zero of pregnancy. Control fetuses were from unoperated ewes of the same flock. t Author to whom correspondence should be addressed. CNS, central nervous system; IGF, insulin-like growth factor; IUGR, intrauterine growth retardation; PNS, peripheral nervous system. Abbreviations:
133
S. Rees et al.
134
At 139-141 days of gestation, the fetuses were delivered by Caesarean section under deep anaesthesia (Pentobarbitone) and perfused via the aorta with 4% paraformaldehyde and 1% glutaraldehyde in 0.1 M phosphate buffer at pH 7.3. The brains and eyes, with attached optic nerves, were removed after 2 hr and immersed in fresh fixative for an additional 24 hr. The trochlear nerve was removed at its point of emergence from the brainstem and a section of optic nerve was taken approximately 5 mm from the point of exit from the eye. These nerves were cut into small pieces (l-2 mm in length) washed in buffer, post fixed in 1% 0~0, overnight and stained en bloc with 2% aqueous uranyl acetate for l-2 hr. The tissue was then dehydrated and embedded in Araldite. Semi-thin sections (0.5 pm) were cut and stained with 1% toluidine blue and 1% sodium tetraborate for light microscopy. Thin sections (70 nm) of these nerves were cut, stained with uranyl acetate and Reynold’s lead citrate and examined in a JEOL 100s electron microscope. Morphometric
analysis
Light microscopy.
Transverse sections (0.5 pm) of the optic and trochlear nerves were projected onto the measuring tablet of a Zeiss MOP-l image analyser. For the optic nerve, the total cross-sectional area was measured. For the trochlear nerve myelinated axons (1000-1500 per animal) in a strip across the middle third of the nerve were analysed (magnification x 1800). Fibre and axon areas were measured and fibre diameter (D), axon diameter (d) and myelin thickness (D-d/2) were calculated. The total number of myelinated axons per nerve was also counted (magnification x 1800). Electronmicroscopy. For the optic nerves, two fields were photographed from the central region of each nerve at an initial magnification of x 2000 and enlarged to x 7500 in printing. The central region was chosen for consistency of sampling since it is known that, at least in the cat ,6 the distribution of fibre diameters across the optic nerve is not random. Measurements, similar to those described above, were made from the electronmicrographs, on 250-300 axons per animal. Electronmicrographs of 10-20 myelinated axons in the control and growth-retarded groups were photographed at random at x 20,000 and enlarged to x 60,000 in printing. The thickness of a single myelin lamella was estimated by dividing the width of the myelin sheath by the number of lamellae. A calibration grid was included with each series of photographs to ensure the correct estimation of magnification. The trochlear nerve was scanned to determine the extent of myelination at this stage of gestation. Statistics. Results are expressed as means ? S.E.M. For the trochlear nerve, the distribution of myelinated fibre diameters was analysed using Analysis of Variance (ANOVA) techniques. For the optic nerve, the total cross-sectional areas and the mean fibre diameters were compared with a Mann-Whitney U-test. For both nerves, the relationship between myelin thickness and axon diameter was investigated by fitting least squares regression lines for each fetus, separately. The significance of any difference in the slopes of the lines between experimental and control groups was tested using the two-tailed Student’s (unpaired) t-test. Differences in brain and body weights between the two groups were also tested with a t-test. RESULTS Fetuses were classified as growth retarded if their body weight was more than 2 S.D. below the mean weight for gestational age of control fetuses. Using this as a criterion there was a reduction of 56% in mean body weight in growth-retarded fetuses (2.18 -+0.4 kg, n = 6) compared with control fetuses (4.9920.3 kg, n= 6; PcO.01). The brain weights were significantly reduced in weight by 21% in growth-retarded fetuses (44.5 + 1.5 g) as compared with controls (56.5 + 1.7 g; P
Myelinated fibre diameters were unimodally distributed with a range of 0.65-3.75 km in control fetuses and 0.60-3.50 p.rn in growth-retarded fetuses. There was a significant reduction of 19% (P= 0.05) in the mean area of a transverse section of optic nerve in the growth-retarded fetuses compared with controls (Table 1). Myelination was defined as two to three complete wraps of
Nerve development
in growth retardation
Fig. 1. Electromicrographs taken from the central region of the optic nerve of a control (A) and a growth-retarded (B) fetus at 140 days gestation. Note that the myelin sheath is thinner for a given axon diameter in the growth-retarded fetus than in the control fetus. Bar = 2 km.
135
136
f . Rem ef al.
Fig. 4. Photomicrographs of transverse sections of the trochlear nerve. Control (A) and growth-retarded (B). Bar = 125 JJ.~. Comparison of myelinated fibres at higher magnification, control (C) and growthretarded (D) shows that there is a reduction in mean fibre diameter in growth retardation. Bar = 30 Frn.
Nerve development in growth retardation Table 1. Measurements
137
on the optic nerve of fetal sheep at 140 days gestation Control
Total cross-sectional area of nerve (mm*) Mean fibre diameter (km) Mean g ratio axon diameter:fibre diameter
Growth-retarded
% Difference
3.24kO.15 1.35 f 0.04
2.63 2 0.25 1.2220.05
-19’ -10
0.80 f 0.01
0.87 -r-O.02
+9**
All values are given as means? S.E.M. For both groups n = 4. *p=o.o5. **p
compact myelin and at this stage of gestation almost all fibres are myelinated (Fig. 1A). In growth retardation the mean diameter of myelinated axons as measured in the central region of the nerve, was reduced by 10% (Table 1) but this was not significant (P= 0.1). The mean width of a single compact myelin lamella was 12.3 r0.3 nm in control and 11.7r0.4 nm in growth-retarded fetuses. For each fetus, myelin thickness was plotted against axon diameter and least squares linear regression lines were fitted to the data points. Plots for a control and a growth-retarded fetus are shown in Fig. 2. As the regression lines were significantly different between animals within each group (P
D71
a 0.6
s
CONTROL
.
i
0.5
GROWTH
RETARDED
= 0.L si
g 0.3 5 z 0.2 f 0.1 w,
5 0.04 0
1
I
1 AXON
DIAMETER
2 (,uM)
I
3
Fig. 2. Plots of axon diameter vs myelin thickness for a control and a growth-retarded fetus at 140 days gestation. Least squares linear regression lines fitted to the data points gave slopes of 0.086 (control) and 0.161 (growth-retarded).
S. Table 2. Measurements
-_-
Rees et al.
on the trochlear
nerve of fetal sheep at 140 days gestation
Control -__
__---.
Total cross-sectional area of nerve (mm’) Total number of myelinated libres Mean fibre diameter (km) Mean g ratio axon diameter: fibre diameter
Growth-retarded
% Difference -
0.202 t 0.004
0.178 k 0.019
- 12
2710-t 127 6.08 i 0.27
2737 + 75 4.71 t 0.31
-1 -23*
0.64 ~fr0.02
0.63 k 0.01
-2
All values are given as means t S.E.M For both groups n =6; ‘P
Trochlear nerve
There was no significant difference in the total cross-sectional area or in the number of myelinated fibres in the trochlear nerve of growth-retarded fetuses compared with control fetuses (Table 2). Ultrastructural examination of the nerve showed that at this stage of development almost all fibres are myelinated. Myelinated fibre diameters in the trochlear nerve were bimodally distributed in both the growth-retarded and control fetuses. The distribution of fibre diameters for nerves from a control and growth-retarded fetus are shown in Fig. 3. The range of myelinated fibres was from 1.5 to 12.6 km in control fetuses and from 1.3 to 11.7 pm in growth-retarded fetuses.
CONTROL 0 GROWTH RETARDED
l
I
00
I
10
20
I
I,
30
4.0
1
5.0
FIBRE
60
(
,
,
,
70
8.0
9.0
100
DIAMETER
,
110
1
,
120
130
(PM)
Fig. 3. The frequency distribution of myelinated fibre diameters in the trochlear nerve of a control and a growth-retarded fetal sheep at 140 days gestation. Fibre diameters are bimodally distributed in both cases but in the growth-retarded fetus there is a greater frequency of small (<5 wrn) diameter fibres and a reduced frequency of larger ( > 5 km) diameter fibres.
A standard two way ANOVA revealed significant differences in mean fibre diameter between animals within the growth-retarded and control groups (P~O.001). Because of the bimodal distribution of fibre diameters, the normal F-test for differences between groups was of limited validity. Therefore, a permutation testI (which makes no assumption about the form of the distribution) based on mean fibre diameter per animal was used. The 23% reduction in mean fibre diameter due to IUGR was significant, judged by this distribution-free test (PCO.01). The effect of growth retardation on fibre diameter is illustrated in Fig. 4. Linear regression analysis of myelin thickness vs axon diameter showed that there was no significant difference in this relationship
Nerve development in growth retardation
139
between control and growth-retarded fetuses as judged by a f-test on the slopes of the lines (0.164 -C0.01 IUGR vs 0.158 + 0.01 control; P> 0.1). There was no difference between the groups in the mean value of the g ratio. DISCUSSION In this quantitative study the effects of IUGR on the structural development of the optic and trochlear nerves in fetal sheep at 140 days gestation have been examined. An interesting finding has been that myelin formation in relation to axonal development is disproportionately reduced in the CNS but not in the PNS. At this stage of gestation, virtually all of the axons in the trochlear nerve are myelinated. Thus, the observation that in both groups the number of myelinated fibres was similar indicates that in IUGR the incidence of cell death in the trochlear nucleus was not greater than normal. However, the mean diameter of myelinated fibres was significantly reduced and there was a deficit in large diameter fibres. In a previous studyz4 on the peroneal, another peripheral nerve, it was also found that the growth of myelinated (and unmyelinated) fibres was retarded in IUGR. In both studies myelin thickness was appropriate for axonal diameter,’ indicating that Schwann cells, the myelinforming cells in the PNS, are not significantly affected in growth retardation. It must be borne in mind, however, that Schwann cells have been shown to influence axonal growth’ and thus the reduction in radial growth of axons in IUGR could indicate that to some extent, Schwann cells have been affected by growth retardation. Factors likely to have an important influence on axonal growth are nutrition and thyroid hormones. Growth-retarded sheep are known to be hypoxic, hypoglycaemicz6 and to have low levels of circulating thyroid hormone. l2 Both malnutrition4*31 and hypothyroidism4T2s have been shown to reduce fibre growth in the sciatic nerve of the developing rat. In thyroid deficiency, it has been suggested that this might be related to a reduction in the formation of microtubules.” In malnutrition4,13 it has been reported that the deposition of myelin is disproportionately inhibited in relation to the radial growth of the axon; however, in hypothyroidism4*2s the normal axon:myelin ratio is maintained. By comparison, in the optic nerve (CNS) radial growth of axons has not been significantly affected by IUGR, but myelination, in relation to axonal expansion, has been disproportionately affected, suggesting a reduction in the capacity of oligodendrocytes to generate myelin. As the widths of myelin lamellae were similar in growth-retarded and control fetuses, the deficit appears to be in the number of lamellae formed. This effect on myelination has been confirmed for other areas of the CNS in the accompanying study on the growth-retarded guinea pig.21 Hence these results support the observation13 that axonal expansion cannot be the sole factor controlling the rate of growth of the myelin sheath. In seeking an explanation for the effect of IUGR on myelination in the CNS several factors must be taken into account. Recently, it has been shown that insulin-like growth factor I (IGF I) activity can be detected in all areas of the CNS29 and receptors for this peptide have now been identified in the fetal mammalian brain. 1s,28The role of IGFs in brain development are not yet certain but it is interesting that McMorris et al. 2o have shown that IGF I is a potent inducer of oligodendrocyte development, at least in vitro. IGF I levels in plasma are reduced in ovine IUGRr4 and it has also been shown that net somatomedin-like activity is low in small-forgestational age neonates. lo The cause of this low level might be the hypoglycaemia associated with growth restriction in both the sheep26 and in man,16 as the nutritional status has been shown to be one of the primary factors regulating plasma IGF I concentration. 22,28It is possible that the CNS myelin deficiency we have demonstrated in IUGR might be partly due to a deficiency in circulating IGF I levels. Other factors which are known to be involved in IUGR’2,26 might also play a part. Studies have shown that in malnourished rats the myelination of axons in the optic nerves*30*32is reduced. Contrary to our findings, however, Sima3’ reported that axonal expansion was more affected than was the myelin sheath development. In this study, it was not possible to determine the functional consequences of the abnormally thin myelin in the CNS and the reduced axonal diameter in the PNS. In a previous study on the peroneal nerve,24 a deficit of large diameter axons resulted in a slowing of conduction velocity. on 89-B
140
S. Rees et al.
Myelin thickness is another factor that influences conduction velocity3’ and it has been argued that if the ratio of axon diameter to fibre diameter (g ratio) falls outside an optimal range (approximately 0.6-0.7) conduction will be less than maxima1.27,33 For the optic nerve, as reported for other developing fibre tracts34 the g ratio fell outside this range. It is interesting to note that the values for the control nerves are closer to the optimal range than the values for the nerves from IUGR fetuses, suggesting that conduction velocity might be lower in the growth-retarded fetus. In the trochlear nerve where myelination is more advanced, the g ratio has already reached the optimal value and so is unlikely to bear unfavourably on conduction velocity. Thus we conclude from this and the earlier study24 that IUGR leads to interference with the radial growth of axons in the PNS but does not specifically affect the deposition of myelin. In the CNS on the other hand, the myelin sheath relative to the axon diameter is disproportionately reduced. Acknowledgements-We are grateful to Mrs Jane Ng, MS Maria Bisignano, MS Ann Martsi and Mr Peter Angus for technical assistance, to Mr David Finkelstein and Dr George Kotsanas for assistance with computer analysis of the data and to Mrs Sally Williams for typing the manuscript. This work was supported by a grant from the National Health and Medical Research Council of Australia.
REFERENCES
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Aguayo A. J., Bray G. M. and Perkins C. S. (1979) Axon-Schwann cell interactions in peripheral and central nervous system transplants. Ann. N. Y. Acad. Sci. 317, 512-531, Alexander G. (1964) Studies on the placenta of the sheep (Ovis a&s L). Effects of reduction in the number ot caruncles. J. Reprod. Fert. 7, 309-322. Bisignano M. and Rees S. (1988) The effects of intrauterine growth retardation on synaptogenesis and mitochondrial formation in the cerebral and cerebellar cortices of fetal sheep. ht. J. devl Neurosci. 6, 453-460. Clos J. and LeGrand J. (1970) Influence de la deficience thyroidienne et de la sous-alimentation sur la croissance et la myelinisation des fibres nerveuses du nerf sciatique chez le jeune rat blanc. Etude au microscope electronique. Brain Res. 22, 285-291. Conradi N. C., Sjostrom A., Karlsson B. and Sourander P. (1985) Functional development of the visual system in normal and protein deprived rats. II. Morphometric and biochemical studies in adult optic nerves. Actu physiol. stand. 125,277-283. Donovan A. (1967) The nerve fibre composition of the cat optic nerve. J. Anat., Land. 101, 1-I 1. Dunn H. G., Crichton J. U., Grumau R. V. E., Kara B., Burney A., McCormick A. Q., Robertson A. M. and Schulzer M. (1980) Neurological, psychological and educational sequelae of low birth weight. Bruin Devl. 2, 57-67. Fitzhardinee P. and Steven E. (1972) The small-for-date infant. II. Neuroloeical and intellectual seauellae. Paediatrics-SO, 50-57.
Friede R. L. and Samorajski T. (1967) Relation between the number of myelin lamellae and axon circumference in fibres of vagus and sciatic nerves of mice. J. camp. Neurol. 130, 223-232. 10. Gluckman P. D. and Brinsmead M. W. (1976) Somatomedin in cord blood: Relationship to gestational age and birth size. J. clin. Edocr. Metab. 43, 1378-1381. 11. Gross S. J., Kosmetatos N., Grimes C. T. and Williams M. L. (1978) Newborn head size and neurological status. Am. 9.
J. Dis. Child 12, 753-756.
12. Harding J. E., Jones C. T. and Robinson J. S. (1985) Studies on experimental growth retardation in sheep. The effects of a small placenta in restricting transport for growth of the fetus. J. devl Physiol. 7, 427-442. 13. Hedley-Whyte E. T. (1973) Myelination of rat sciatic nerve: Comparison of nundernutrition and cholesterol biosynthesis inhibition. J. Neuropath. exp. Neurol. 32, 284-302. 14. Jones C. T., Gu W., Harding J. E., Price D. A. and Parer J. Y. (1988) Studies on the growth of fetal sheep. Effect of surgical reduction in placental size or experimental manipulation of uterine blood flow on plasma sulphation promoring activity on the concentration of insulin like growth factors I and II. J. devl Physiol. 16, 179-189: 15. Lesniak M. A.. Hill J. M.. Kiess W.. Roieski M.. Pert C. B. and Roth J. (1988) Receptors for insulin-like growth factors I & II: Autoradiographic locahsatibn in rat’brain and comparison to receptors fo;insulin. Endocrinology 123, 2089-2099.
16. Lugo G. and Cassady G. (1971) Intrauterine growth retardation, clinicopathologic findings in 233 consecutive infants. Am. J. Obstet. Gynecol. 109, 615422. 17. Marc C. and Rabit A. (1985) Microtubules and neurofilaments in the sciatic nerve times of the developing rat: effects of thyroid deficiency. ht. J. devl Neurosci. 3, 353-358. 18. Maritz J. S. (1981) Distribution Free Statistical Methods. Chapman & Hall, London. 19. Matilainen R., Heinonen K. and Siren-Tiusanen H. (1988) Effect of intrauterine growth retardation (IUGR) on the psychological performance of preterm children at school age. J. Child Psychol. Psychiat. 29, 601-609. 20. McMorris F. A., Smith T. J., De Salvo S. and Furlanetto R. W. (1986) Insulin like growth factor Ilsomatomedin C: A potent inducer of oligodendrocyte development. Proc. natl. Acad. Sci., U.S.A. 83,822-826. 21. Nitsos I. and Rees S. (1989) The effects of intrauterine growth retardation on the development of neuroglia in fetal guinea pigs: An immunohistochemical and ultrastructural study. Inc. J. devl. Neurosci. (in press). 22. Phillips L. V. and Young H. S. (1976) Nutrition and somatomedin. I. Effect of fasting and refeeding on serum somatomedin activity and cartilage growth activity in rats. Endocrinology 99, 304-314. 23. Rees S. and Harding R. (1988) The effects of intrauterine growth retardation on the development of the Purkinje cell dendritic tree in the cerebellar cortex of fetal sheep. Inl. J. devl Neurosci. 6, 461-469.
Nerve development in growth retardation 24. Rees S., Proske U. and Harding R. (1989) Conduction
25. 26. 27. 28. 29. 30. 31. 32. 33. 34.
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velocity and fibre diameter of the peraneai nerve in narmaf and growth retarded fetal sheep. Neurosci. Lens 99,157~163. Reier P. .I. and Hughes A. F. (1972) An effect of neanate radiathyroidectamy upon non~myelinated axons and associated Schwann cells during maturation of mause sciatic nerves. Bruin Res. 41, 263-282. Robinson J. S., Kingston E. J., Jones C. T. and Tborburn G. D. (1979) Studies on experimental grawth retardation in sheep. The effect of removal of endometrial caruncles aa fetal size and metabdism. j. devl Physioi. 1, 379-398. Rushton W. (1953) A theary of the effects of @we size in medullated nerve. 1. P&szX 115, 101-122. Sara V. R., Hait K., Mizaki M., Frykiund L., Christensen N. and Wetterberg L. (1983) The ontogenesis of somatomedin and insulin receptors on the human fetus. f. clin. Invest. 71, 1084-1094. Sara V. R. 1Uvnas-Moberg K., Uvnls B., Hall K., Wetterberg L., Poslonec B. and Goiny M. (1982) The distribution of somatomedins in the nervous system of the cat and their release following neural stimulation. Actuphysiol. scond. 115,467-470. Sima A. (1974) Relation between the number of myelin lamellae and axon circumference in fibres and ventral and dorsal roots and optic nerve in normal, undemou~shed, and rehabilitated rats. Ac~~~hys~~. stand. 5kppL 410,3-38. Sima A. (1974) Studies on tibre size in devefoping sciatic nerve and spinal roots in normal, undernourished and rehabilitated rats. Actu pItysir_&stand. Suppt. 406, 5-54. Sima A. and Saurander P. (1974) The effect of early undernutrition on the calibre spectrum of the rat optic nerve. Acta Newpath., Bell. 28, 152-160. Smith R. S. and Roles Z. J, (1970) Myelinated nerve fibres: computed effect of myelin thickness on conduction velocity. Am. J. Physiof. 219, 12564258. Vejsada R, , Palecek J., Hnik P. and Soukup T. (1985) Postnatal development of conduction velocity and fibre size in the rat tibial nerve. ftrr. 1. devf ~e~~~scj~ 3,585595. Waxman S. G. (1980) Determinants of conduction velocity in mye~inated nerves. MUS& and Nerve 3, f41450.
Vol. 8,
No. 2, pp. 133-141, 1990.
0736-5748/90 $03.00+0.00 Pergamon Press plc @ 1990 ISDN
THE EFFECTS OF INTRAUTERINE GROWTH RETARDATION ON THE STRUCTURAL DEVELOPMENT OF CRANIAL NERVES (OPTIC, TROCHLEAR) IN FETAL SHEEP SANDRA
REES,*~ MALCOLM CLARK, MICHAEL
SNOWDEN
and RICHARD HARDING*
Departments of Physiology* and Mathematics, Monash University, Clayton, Victoria 3168, Australia (Received 12 June 1989; in revised form 29 August 1989; accepted 1 September 1989)
Abstract-A quantitative morphometric study of the development of myelinated fibres in the optic and trochlear nerves has been made in growth-retarded fetal sheep at 140 days gestation (term = 146 days). Intrauterine growth retardation was induced as a result of the reduction of placental mass, by prior removal of placentation sites in six ewes. In the optic nerve (central nervous system) the mean diameter of myelinated fibres was not significantly reduced but the thickness of the myelin sheath relative to axon diameter was disproportionately reduced. In the trochlear nerve (peripheral nervous system) there was a significant reduction of 23% (PCO.01) in the mean diameter of myelinated fibres; however the normal axon:myelin ratio was maintained. The total number of myelinated fibres in the trochlear nerve did not differ between the normal and growth-retarded group, indicating that there was not a greater than normal incidence of cell death during intrauterine growth retardation in the nucleus of the trochlear nerve. The differential effect of intrauterine growth retardation on myelination in the central and peripheral nervous systems suggests that chronic intrauterine deprivation affects oligodendrocyte activity but does not markedly affect the capacity of Schwann cells to produce myelin.
Key words: optic, trochlear, nerve development,
intrauterine growth retardation, axon diameter, myelin
thickness, fetal sheep.
In previous studies on fetal brain development in experimental intrauterine growth retardation (IUGR) produced by reducing placental mass in sheep, it has been shown that a number of parameters are significantly affected. For example, in the cerebellum growth of granule cell dendrites and of the Purkinje cell dendritic tree is reduced,23 in the visual cortex synaptogenesis is reduced,3 in the peroneal nerve axonal development is retarded and conduction velocity is slower than in age-matched control fetuses.24 In the present quantitative morphometric study, the effects of fetal growth retardation on axonal development and myelination in the optic and trochlear nerves were examined. Since the optic nerve is part of the central nervous system (CNS) and the trochlear nerve part of the peripheral nervous system (PNS), it was possible to compare the effects of fetal growth retardation on two fibre tracts, both cranial nerves but in different divisions of the nervous system. Identification of any structural abnormalities that occur in particular areas of the nervous system or amongst specific cell types during experimental growth retardation will assist in understanding the neurological deficits which occur with a greater than normal incidence in growthretarded or small-for-gestational-age children.7~8*“~‘9
EXPERIMENTAL
PROCEDURES
Endometrial caruncles (potential sites of placental attachment) were removed from six nonpregnant Meriono-Border Leicester ewes2*26under halothane anaesthesia. A low midline incision was made in the abdomen and the uterus was exposed. Each horn of the uterus was opened from the cervix to near the uterotubal junction and most of the accessible caruncles (80-95) were removed by cautery. After a minimum of 8 weeks recovery, the ewes were allowed to mate, the day of mating being taken as day zero of pregnancy. Control fetuses were from unoperated ewes of the same flock. t Author to whom correspondence should be addressed. CNS, central nervous system; IGF, insulin-like growth factor; IUGR, intrauterine growth retardation; PNS, peripheral nervous system. Abbreviations:
133
S. Rees et al.
134
At 139-141 days of gestation, the fetuses were delivered by Caesarean section under deep anaesthesia (Pentobarbitone) and perfused via the aorta with 4% paraformaldehyde and 1% glutaraldehyde in 0.1 M phosphate buffer at pH 7.3. The brains and eyes, with attached optic nerves, were removed after 2 hr and immersed in fresh fixative for an additional 24 hr. The trochlear nerve was removed at its point of emergence from the brainstem and a section of optic nerve was taken approximately 5 mm from the point of exit from the eye. These nerves were cut into small pieces (l-2 mm in length) washed in buffer, post fixed in 1% 0~0, overnight and stained en bloc with 2% aqueous uranyl acetate for l-2 hr. The tissue was then dehydrated and embedded in Araldite. Semi-thin sections (0.5 pm) were cut and stained with 1% toluidine blue and 1% sodium tetraborate for light microscopy. Thin sections (70 nm) of these nerves were cut, stained with uranyl acetate and Reynold’s lead citrate and examined in a JEOL 100s electron microscope. Morphometric
analysis
Light microscopy.
Transverse sections (0.5 pm) of the optic and trochlear nerves were projected onto the measuring tablet of a Zeiss MOP-l image analyser. For the optic nerve, the total cross-sectional area was measured. For the trochlear nerve myelinated axons (1000-1500 per animal) in a strip across the middle third of the nerve were analysed (magnification x 1800). Fibre and axon areas were measured and fibre diameter (D), axon diameter (d) and myelin thickness (D-d/2) were calculated. The total number of myelinated axons per nerve was also counted (magnification x 1800). Electronmicroscopy. For the optic nerves, two fields were photographed from the central region of each nerve at an initial magnification of x 2000 and enlarged to x 7500 in printing. The central region was chosen for consistency of sampling since it is known that, at least in the cat ,6 the distribution of fibre diameters across the optic nerve is not random. Measurements, similar to those described above, were made from the electronmicrographs, on 250-300 axons per animal. Electronmicrographs of 10-20 myelinated axons in the control and growth-retarded groups were photographed at random at x 20,000 and enlarged to x 60,000 in printing. The thickness of a single myelin lamella was estimated by dividing the width of the myelin sheath by the number of lamellae. A calibration grid was included with each series of photographs to ensure the correct estimation of magnification. The trochlear nerve was scanned to determine the extent of myelination at this stage of gestation. Statistics. Results are expressed as means ? S.E.M. For the trochlear nerve, the distribution of myelinated fibre diameters was analysed using Analysis of Variance (ANOVA) techniques. For the optic nerve, the total cross-sectional areas and the mean fibre diameters were compared with a Mann-Whitney U-test. For both nerves, the relationship between myelin thickness and axon diameter was investigated by fitting least squares regression lines for each fetus, separately. The significance of any difference in the slopes of the lines between experimental and control groups was tested using the two-tailed Student’s (unpaired) t-test. Differences in brain and body weights between the two groups were also tested with a t-test. RESULTS Fetuses were classified as growth retarded if their body weight was more than 2 S.D. below the mean weight for gestational age of control fetuses. Using this as a criterion there was a reduction of 56% in mean body weight in growth-retarded fetuses (2.18 -+0.4 kg, n = 6) compared with control fetuses (4.9920.3 kg, n= 6; PcO.01). The brain weights were significantly reduced in weight by 21% in growth-retarded fetuses (44.5 + 1.5 g) as compared with controls (56.5 + 1.7 g; P
Myelinated fibre diameters were unimodally distributed with a range of 0.65-3.75 km in control fetuses and 0.60-3.50 p.rn in growth-retarded fetuses. There was a significant reduction of 19% (P= 0.05) in the mean area of a transverse section of optic nerve in the growth-retarded fetuses compared with controls (Table 1). Myelination was defined as two to three complete wraps of
Nerve development
in growth retardation
Fig. 1. Electromicrographs taken from the central region of the optic nerve of a control (A) and a growth-retarded (B) fetus at 140 days gestation. Note that the myelin sheath is thinner for a given axon diameter in the growth-retarded fetus than in the control fetus. Bar = 2 km.
135
136
f . Rem ef al.
Fig. 4. Photomicrographs of transverse sections of the trochlear nerve. Control (A) and growth-retarded (B). Bar = 125 JJ.~. Comparison of myelinated fibres at higher magnification, control (C) and growthretarded (D) shows that there is a reduction in mean fibre diameter in growth retardation. Bar = 30 Frn.
Nerve development in growth retardation Table 1. Measurements
137
on the optic nerve of fetal sheep at 140 days gestation Control
Total cross-sectional area of nerve (mm*) Mean fibre diameter (km) Mean g ratio axon diameter:fibre diameter
Growth-retarded
% Difference
3.24kO.15 1.35 f 0.04
2.63 2 0.25 1.2220.05
-19’ -10
0.80 f 0.01
0.87 -r-O.02
+9**
All values are given as means? S.E.M. For both groups n = 4. *p=o.o5. **p
compact myelin and at this stage of gestation almost all fibres are myelinated (Fig. 1A). In growth retardation the mean diameter of myelinated axons as measured in the central region of the nerve, was reduced by 10% (Table 1) but this was not significant (P= 0.1). The mean width of a single compact myelin lamella was 12.3 r0.3 nm in control and 11.7r0.4 nm in growth-retarded fetuses. For each fetus, myelin thickness was plotted against axon diameter and least squares linear regression lines were fitted to the data points. Plots for a control and a growth-retarded fetus are shown in Fig. 2. As the regression lines were significantly different between animals within each group (P
D71
a 0.6
s
CONTROL
.
i
0.5
GROWTH
RETARDED
= 0.L si
g 0.3 5 z 0.2 f 0.1 w,
5 0.04 0
1
I
1 AXON
DIAMETER
2 (,uM)
I
3
Fig. 2. Plots of axon diameter vs myelin thickness for a control and a growth-retarded fetus at 140 days gestation. Least squares linear regression lines fitted to the data points gave slopes of 0.086 (control) and 0.161 (growth-retarded).
S. Table 2. Measurements
-_-
Rees et al.
on the trochlear
nerve of fetal sheep at 140 days gestation
Control -__
__---.
Total cross-sectional area of nerve (mm’) Total number of myelinated libres Mean fibre diameter (km) Mean g ratio axon diameter: fibre diameter
Growth-retarded
% Difference -
0.202 t 0.004
0.178 k 0.019
- 12
2710-t 127 6.08 i 0.27
2737 + 75 4.71 t 0.31
-1 -23*
0.64 ~fr0.02
0.63 k 0.01
-2
All values are given as means t S.E.M For both groups n =6; ‘P
Trochlear nerve
There was no significant difference in the total cross-sectional area or in the number of myelinated fibres in the trochlear nerve of growth-retarded fetuses compared with control fetuses (Table 2). Ultrastructural examination of the nerve showed that at this stage of development almost all fibres are myelinated. Myelinated fibre diameters in the trochlear nerve were bimodally distributed in both the growth-retarded and control fetuses. The distribution of fibre diameters for nerves from a control and growth-retarded fetus are shown in Fig. 3. The range of myelinated fibres was from 1.5 to 12.6 km in control fetuses and from 1.3 to 11.7 pm in growth-retarded fetuses.
CONTROL 0 GROWTH RETARDED
l
I
00
I
10
20
I
I,
30
4.0
1
5.0
FIBRE
60
(
,
,
,
70
8.0
9.0
100
DIAMETER
,
110
1
,
120
130
(PM)
Fig. 3. The frequency distribution of myelinated fibre diameters in the trochlear nerve of a control and a growth-retarded fetal sheep at 140 days gestation. Fibre diameters are bimodally distributed in both cases but in the growth-retarded fetus there is a greater frequency of small (<5 wrn) diameter fibres and a reduced frequency of larger ( > 5 km) diameter fibres.
A standard two way ANOVA revealed significant differences in mean fibre diameter between animals within the growth-retarded and control groups (P~O.001). Because of the bimodal distribution of fibre diameters, the normal F-test for differences between groups was of limited validity. Therefore, a permutation testI (which makes no assumption about the form of the distribution) based on mean fibre diameter per animal was used. The 23% reduction in mean fibre diameter due to IUGR was significant, judged by this distribution-free test (PCO.01). The effect of growth retardation on fibre diameter is illustrated in Fig. 4. Linear regression analysis of myelin thickness vs axon diameter showed that there was no significant difference in this relationship
Nerve development in growth retardation
139
between control and growth-retarded fetuses as judged by a f-test on the slopes of the lines (0.164 -C0.01 IUGR vs 0.158 + 0.01 control; P> 0.1). There was no difference between the groups in the mean value of the g ratio. DISCUSSION In this quantitative study the effects of IUGR on the structural development of the optic and trochlear nerves in fetal sheep at 140 days gestation have been examined. An interesting finding has been that myelin formation in relation to axonal development is disproportionately reduced in the CNS but not in the PNS. At this stage of gestation, virtually all of the axons in the trochlear nerve are myelinated. Thus, the observation that in both groups the number of myelinated fibres was similar indicates that in IUGR the incidence of cell death in the trochlear nucleus was not greater than normal. However, the mean diameter of myelinated fibres was significantly reduced and there was a deficit in large diameter fibres. In a previous studyz4 on the peroneal, another peripheral nerve, it was also found that the growth of myelinated (and unmyelinated) fibres was retarded in IUGR. In both studies myelin thickness was appropriate for axonal diameter,’ indicating that Schwann cells, the myelinforming cells in the PNS, are not significantly affected in growth retardation. It must be borne in mind, however, that Schwann cells have been shown to influence axonal growth’ and thus the reduction in radial growth of axons in IUGR could indicate that to some extent, Schwann cells have been affected by growth retardation. Factors likely to have an important influence on axonal growth are nutrition and thyroid hormones. Growth-retarded sheep are known to be hypoxic, hypoglycaemicz6 and to have low levels of circulating thyroid hormone. l2 Both malnutrition4*31 and hypothyroidism4T2s have been shown to reduce fibre growth in the sciatic nerve of the developing rat. In thyroid deficiency, it has been suggested that this might be related to a reduction in the formation of microtubules.” In malnutrition4,13 it has been reported that the deposition of myelin is disproportionately inhibited in relation to the radial growth of the axon; however, in hypothyroidism4*2s the normal axon:myelin ratio is maintained. By comparison, in the optic nerve (CNS) radial growth of axons has not been significantly affected by IUGR, but myelination, in relation to axonal expansion, has been disproportionately affected, suggesting a reduction in the capacity of oligodendrocytes to generate myelin. As the widths of myelin lamellae were similar in growth-retarded and control fetuses, the deficit appears to be in the number of lamellae formed. This effect on myelination has been confirmed for other areas of the CNS in the accompanying study on the growth-retarded guinea pig.21 Hence these results support the observation13 that axonal expansion cannot be the sole factor controlling the rate of growth of the myelin sheath. In seeking an explanation for the effect of IUGR on myelination in the CNS several factors must be taken into account. Recently, it has been shown that insulin-like growth factor I (IGF I) activity can be detected in all areas of the CNS29 and receptors for this peptide have now been identified in the fetal mammalian brain. 1s,28The role of IGFs in brain development are not yet certain but it is interesting that McMorris et al. 2o have shown that IGF I is a potent inducer of oligodendrocyte development, at least in vitro. IGF I levels in plasma are reduced in ovine IUGRr4 and it has also been shown that net somatomedin-like activity is low in small-forgestational age neonates. lo The cause of this low level might be the hypoglycaemia associated with growth restriction in both the sheep26 and in man,16 as the nutritional status has been shown to be one of the primary factors regulating plasma IGF I concentration. 22,28It is possible that the CNS myelin deficiency we have demonstrated in IUGR might be partly due to a deficiency in circulating IGF I levels. Other factors which are known to be involved in IUGR’2,26 might also play a part. Studies have shown that in malnourished rats the myelination of axons in the optic nerves*30*32is reduced. Contrary to our findings, however, Sima3’ reported that axonal expansion was more affected than was the myelin sheath development. In this study, it was not possible to determine the functional consequences of the abnormally thin myelin in the CNS and the reduced axonal diameter in the PNS. In a previous study on the peroneal nerve,24 a deficit of large diameter axons resulted in a slowing of conduction velocity. on 89-B
140
S. Rees et al.
Myelin thickness is another factor that influences conduction velocity3’ and it has been argued that if the ratio of axon diameter to fibre diameter (g ratio) falls outside an optimal range (approximately 0.6-0.7) conduction will be less than maxima1.27,33 For the optic nerve, as reported for other developing fibre tracts34 the g ratio fell outside this range. It is interesting to note that the values for the control nerves are closer to the optimal range than the values for the nerves from IUGR fetuses, suggesting that conduction velocity might be lower in the growth-retarded fetus. In the trochlear nerve where myelination is more advanced, the g ratio has already reached the optimal value and so is unlikely to bear unfavourably on conduction velocity. Thus we conclude from this and the earlier study24 that IUGR leads to interference with the radial growth of axons in the PNS but does not specifically affect the deposition of myelin. In the CNS on the other hand, the myelin sheath relative to the axon diameter is disproportionately reduced. Acknowledgements-We are grateful to Mrs Jane Ng, MS Maria Bisignano, MS Ann Martsi and Mr Peter Angus for technical assistance, to Mr David Finkelstein and Dr George Kotsanas for assistance with computer analysis of the data and to Mrs Sally Williams for typing the manuscript. This work was supported by a grant from the National Health and Medical Research Council of Australia.
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