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Cell proliferation in the embryonic mouse neocortex following acute maternal alcohol intoxication
Itrr. J. Dd
Ncrmrmci
073657JX/H5$03.Iwl+o.0t) Perpamon Press Ltd. 0 IL)XS ISDN
V<,I.3. No. J. pp. .II l-315. IYXS.
Printed in Great Britain.
CELL PROLIFERATION IN THE EMBRYONIC MOUSE NEOCORTEX FOLLOWING ACUTE MATERNAL ALCOHOL INTOXICATION L. A. KENNEDY and M. J. ELLIOTT Teratology Research Laboratory.
Department of Pharmacology. University of Saskatchewan. Saskatoon. Saskatchewan. Canada S7N OWO (Accepted
4 November 1984)
Abstract-Experiments were conducted to examine the effects of ‘binge’ or acute maternal alcohol intoxication during the early proliferative phase of embryonic brain development. Primiparous mice received ethanol as 0, 10, 15 or 20% (v/v) aqueous solutions by gavage on days 13, 14 and 15 of gestation. Mean daily doses were 0.0, 2.58, 4.03 and 5.40 ml/kg, respectively. There was no alcohol-related reduction in fetal body weight, length or fixed brain weight. Coronal sections (1 km) of the dorsal roof of the lateral ventricles over the optic chiasma were examined from nine embryonic day 15 brains for each treatment group. The ventricular surface index of mitotic figures, the number and distribution of non-surface mitotic figures, and the depth of the cortical roof and its constituent layers were determined. There was no alcohol-related difference in any of these parameters. These results are in contrast to those of a previous experiment using the same mouse strain, in which prolonged or chronic maternal alcohol consumption in the drinking water from days 11 to 19 of pregnancy was associated with a reduction in the surface index, a reduction in the depth of the cortical roof and an increase in the non-surface mitotic figures. These latter changes, however, occurred in the presence of reduced body weight. Our observations suggest that during this particular developmental period (corresponding to the second trimester of human pregnancy) alcohol-related reductions in brain growth parallel restrictions in general body growth. Key words: Alcohol, Mouse, Cell proliferation,
Cortex.
Central nervous system (CNS) dysfunctions in the infants are the most debilitating consequences of maternal alcohol abuse during pregnancy and can occur independently of the craniofacial malformations which are characteristic of fetal alcohol syndrome (FAS). They appear, however, to be closely related to general body growth deficits. 1,2*11*28 Postmortem examination of severely affected infants has revealed microcephaly, reduced numbers of all neural cell populations and abnormal cytoarchitecture.’ Similar observations have been made in experimental animals337323,29*31 and suggest that alcohol-related derangements in the patterns of proliferation and migration of the neural progenitor cells contribute to the pathogenesis of the intellectual and behavioral impairments in FAS children. Teratogens generally exert their effect when exposure occurs during critical periods of development which represent ‘once only’ opportunities for growth.8 Compared to other organ systems, the ontogeny of the mammalian brain is extremely complex and prolonged6 making it highly vulnerable to derangement for extended periods of time. Impairments of CNS function in the offspring of alcohol-consuming women are highly variable both in frequency and severity, probably reflecting the variable patterns of alcohol abuse. The pathogenesis of these dysfunctions, however, is not well understood. It is as yet unclear, for example, whether there are periods of development during which the brain is relatively less vulnerable to alcohol’s teratogenicity than others, whether some processes contributing to brain development are more or less sensitive to disturbance than others, and to what extent secondary or indirect alcohol-related changes in the maternal-placental-fetal organism can contribute at different stages in development. Binge drinking has been linked to FAS in humans and it has been shown experimentally that one brief period of maternal alcohol intoxication during a sensitive period of development in the mouse Address all correspondence and reprint requests to Dr. L. A. Kennedy, Department of Pharmacology. College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N OWO. Abbreviations: SI, surface index of mitotic figures; MFs. mitotic figures; FAS. fetal alcohol syndrome; CNS, central nervous system; CRL. crown-rump length; Ex. embryonic day x: SVZ, subventricular zone; INTZ. intermediate zone: CP, cortical plate; MZ, marginal zone; ANOVA. analysis of variance. DN):&-A
311
312
L. Kennedy and M. Elliott
gastrula (corresponding to week 3 of human gestation) can produce changes in the facial and brain primordia which result in the characteristic FAS midfacial and midline brain abnormalities.‘,2” Alcohol exposure later in development has also been shown to be associated with biochemical and morphological abnormalities in the developing brain~.7,“.‘f’,2”,2s.~’but not with the classical craniofacial characteristics of FAS. In previous experiments,” we found that prolonged or chronic alcohol consumption during the early proliferative period of the fetal brain growth spurt resulted in altered patterns of neural cell proliferation in the fetal mouse neopallium which were detectable in situ and in vitro. In this chronic model, however, the changes were closely associated with fetal body and brain growth deficits. The present investigation was conducted to determine if maternal alcohol intoxication during this same period of brain development resulted in impairment in the recruitment of embryonic neural cells in the absence of general body growth impairment.
EXPERIMENTAL,
PROCEDURES
Primiparous DBA/lJ mice (age 8-10 weeks, 1.5-19 g body weight) were used, day 1 of gestation being designated as the day on which a copulation plug was found. Dams were housed individually in glass cages with wire mesh tops and Sani-seal corn cob bedding, under controlled environmental conditions (27°C 60-70% relative humidity, 13L 11D photoperiod). Mouse chow and water were available ad libitum. Ethanol was administered by gavage as 0, 10, 15 or 20% (v/v) aqueous solutions on each of days 13, 14 and 15 of gestation. Each dam received two daily treatments of 0.3 ml aqueous alcohol. Alcohol was administered in the first hours of the light schedule (0900 and 1100 h) to minimize the interference of periods of intoxication with activity and feeding. There were at least six pregnant mice in each of the four treatment groups. Maternal weight was recorded on day I and on each treatment day. On day 1.5 (1300 h) dams were killed by cervical dislocation, the uterine horns were opened and the numbers of live, dead and resorbed fetuses recorded. Fetuses were removed, examined for gross external malformation, weighed and measured for crownrump length (CRL). Fetal heads were placed in 3% glutaraldehyde in Sorensen’s buffer. Brains were subsequently removed by severing the connection with the olfactory bulb and with the brain stem behind the cerebellum. They were left overnight in the fixative, weighed and embedded in methacrylate. All brains were weighed after the same length of time in fixative to ensure that differences in dehydration did not influence fixed brain weight. Semi-serial coronal sections (1 km) were cut over the optic chiasma and stained with 1% toluidine blue in saturated Borax solution. Nine embryonic day 15 (E15) brains, obtained from at least four different litters were evaluated from each treatment group. For each embryonic brain, three sections (one section from each of three slides, taken at least 10 Frn apart) were analyzed. Brains were assigned random numbers and the evaluation was conducted in a blind manner. The ventricular surface index of mitotic figures (SI) was determined using an eyepiece micrometer under oil immersion (mag 1000 x). The number of mitotic figures present were counted in an area 250 pm long and 20 km deep starting directly from the dorso-medial angle of the ventricular surface of the dorsal roof and proceeding laterally. Each surface area (250 X 20 km) was comprised of five segments (50 x 20 pm) from medial to lateral and each segment had five divisions (10 X 20 km). The mean SI for each segment was calculated by averaging the number of mitotic figures from the five divisions for the three slides, and mean SI for the five segments was averaged to produce the SI for each hemisphere. The overall SI for each brain was obtained by averaging the SI from the two hemispheres and is thus a measure of the number of mitotic figures per unit area of ventricular surface of the dorsal roof of the neocortex.2h The numbers and the distribution of mitotic figures (MFs) were also examined in the subventricular zone (SVZ), intermediate zone (INTZ), cortical plate (CP) and marginal zone (MZ), using the 250 pm area directly above the surface area used to determine the SI. These nonsurface MFs were plotted on projection microscope diagrams by scanning at 400 X. Both the absolute number of non-surface MFs and the proportional distribution of MFs in the different cortical layers were determined.
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Alcohol and neural cell proliferation
The total depth of the dorsal roof, as well as the thickness of its constituent layers was determined in the same sections exactly 250 km from the dorsomedial angle of the neocortex. To adjust for curvature in the dorsal roof, the measurements were taken at 90” to a line bissecting this angle and at a point 250 pm distant from the angle. Two measurements per hemisphere were taken using these points and the mean depths were calculated for each brain. A nested Analysis of Variance (ANOVA) adjusted for unequal sample sizes was used to test for treatment-related differences in fetal body weight, brain weight and CRL. A one-way ANOVA was used to assess the treatment-related difference in SI, numbers and distribution of non-surface MFs and the cortical depth. RESULTS When 0.3 ml of 0, lo,15 or 20% (v/v) aqueous ethyl alcohol was administered to pregnant mice twice daily on days 13,14 and 15 of gestation the mean daily dose of absolute alcohol per dam was 0.0,2.58,4.03 and 5.40 ml/kg body weight, respectively. There was no alcohol-related difference in the number of live, dead, malformed or resorbed embryos using this protocol, and there were no significant (P~0.05) deficits in general body growth or fixed brain weight (Table 1) Table
1. The effects of acute maternal Maternal dose (ml/kg/day)
Treatment group
alcohol
intoxication
Fetal mortality (%)’
on fetal growth
Fetal body weight(g)
Fetal CRL (cm)
Fetal brain weight (mg)
Control (N=7;n=44)
0.0
18.7
0.202
I .03
14.45
10% (N=7;n=42)
2.58
27.1
0.194
I .os
14.94
15% (N=6;n=44)
4.03
13.3
0. I80
0.99
13.62
20% (N=6;n=38)
5.40
22.5
0.20
0.99
14. I7
Significance
-
F= 0.28 NS
F= 0.83 NS
sites x 100.
The data obtained from the examination of the fetal neocortex was no significant treatment-related difference (P>O.O5) in the of the neocortex nor was there any treatment-related difference ments comprising the total area of ventricular surface examined Table 2. The effects of acute maternal
alcohol intoxication
on the proliferative
SI
Total number of non-surface MFs
vz+svz
INTZ
CP+MZ
Control (N=5;n=9)
1.94 (0.41)
322.11 (32.13)
94.89 (1.85)
4.22 (1.81)
0.89 (0.87)
10% (N=6;n=9)
1.96 (0.18)
347.33 (23.19)
94.22 (2.57)
5.33 (2.26)
15% (N=4;n=9)
1.94 (0.18)
301.67 (38.22)
95.33 (2.36)
20% (N=4;n=9)
1.76 (0.37)
317.89 (48.06)
Significance
F= 0.76 NS
F= 2.14 NS
Treatment
Distribution
F=0.55 NS
F= 2.38 NS
All measured are presented as group means. N = number of dams; n = total number of fetuses. *Resorptions plus dead fetuses + total number of implantation
group
and development
is summarized in Table 2. There SI of the dorsal ventricular roof in the SI of the individual seg(data not presented). There was
neural cells and the depth of the fetal mouse neocortex
of MFs (%)
Depth of neocortex INTZ
CP+MZ
Total
103.0 (21.73)
60.56 (15.25)
37.67 (10.24)
201.22 (36.89)
0.44 (0.50)
97.56 (9.18)
67.11 (25.86)
45.22 (19.31)
211.20 (45.15)
3.78 (1.99)
0.89 (0.87)
98.89 (13.12)
61.67 (12.52)
40.78 (8.32)
201.33 (10.73)
94.89 (2.33)
4.11 (2.02)
1.0 (0.47)
99.0 (9.84)
55.44 (24.65)
43.0 (20.06)
197.44 (42.45)
F= 0.319 NS
F= 0.887 NS
F= 0.971 NS
F=0.215 NS
F=0.440 NS
F=0.351 NS
F=0.209 NS
All measures are presented as group means, 1 S.D. in brackets. N= number of litters sampled; n = number of fetal brains evaluated.
vz+svz
(urn)
314
L. Kennedy and M. Elliott
no significant difference between treatment groups either in the total numbers of non-surface MFs counted in the area of the dorsal roof immediately above the ventricular surface area or in the numbers of MFs in the SVZ, INTZ, or CP plus MZ. It can also be seen that there was no difference in their distribution throughout the different cortical layers. Finally, there was no difference in the total depth of the dorsal roof or in the depth of the individual cortical layers. DISCUSSION Fetal alcohol syndrome (FAS) is comprised of a non-specific cluster of minor malformations which vary in the frequency and severity of expression and which can have other etiologies. Although the direct effects of alcohol undoubtedly account for some of the deleterious effects on embryofetal development, it is becoming increasing clear that there are many indirect ways in which maternal alcohol intoxication can alter the in utero environment on which normal growth and development is dependent. 13,” Examples of indirect mechanisms of alcohol-related teratogenesis include primary and/or secondary undernutrition,24%30 hypothermia,” placental dysfunction’4,‘X,2’ and endocrine disturbances.4.‘h.‘2 Although the pathogenesis of alcoholrelated developmental abnormalities is not clearly understood, it is evident from epidemiological and experimental studies that the risk to embryofetal development increases with increasing alcohol consumption and in the presence of other alcohol-related maternal risk facfors.15,‘8.‘9.27 In previous experiments using the same mouse strain and a chronic model, we found that prolonged maternal alcohol consumption during the early proliferative phase of the mouse brain growth spurt (Ell-E18) resulted in changes in the proliferative cells of the embryonic neocortex which were detectable in vitro and in situ. These changes, however, were seen in association with significant reductions in brain weight and general body growth deficits.‘” The present investigations were conducted to determine whether similar changes in the proliferative neural cells would be seen using a ‘binge’ or acute model of alcohol abuse during the same developmental period. Preliminary trials using a single dose of 0.2 ml of 0, 10, 15 or 20% (v/v) EtOH resulted in blood alcohol levels of 0, 55, 98 and 125 mg/dl in non-pregnant adult mice 30 min later. Higher peak levels would be expected in the present experiments with two doses of 0.3 ml of the ethanol solutions administered 2 h apart. Based on the severity and duration of locomotor impairment the ucute model resulted in higher peak blood alcohol levels than were achieved in the chronic model, but the periods of intoxication were shorter and the daily doses were lower. There were no alcohol-related differences in prenatal mortality or morbidity in either model. In contrast to the chronic model, the acute model did not result in impairment in embryofetal body or brain growth, nor were there any detectable alcohol-related changes in the proliferative neural cells. In this investigation, the ventricular SI was used as an indicator of neural stem cell proliferation, the number and distribution of non-surface MFs were used as indicators of the replication and migration of the early glioblasts, and the thickness of the layers of the dorsal roof was used as a general indicator of cellular growth, differentiation and organization. Although the actual number of cells studied may be relatively small, this area of the developing neocortex has been used extensively to study neural cell proliferation, and the behavior of these cells may be considered representative of other populations of cortical progenitor cells. During the treatment period used in this study, neuroblast replication is at a peak, glial replication is rising to a peak which occurs postnatally and progenitor cells are migrating outward to their final location.h*8726Our results indicate that acute maternal alcohol intoxication during the early proliferative phase of the mouse brain growth spurt (corresponding to the second trimester of human pregnancy) is not associated with any lasting impairment in the recruitment of neural precursor cells in the absence of general body growth impairment. Acknowledgements-We gratefully acknowledge the financial support of the Saskatchewan Health Research Board and the University of Saskatchewan. Mrs. M. Matheson did the typing. Thanks to Dr. P. Flood and M. Diocee for the use of their JB4 microtome. This work has been presented at the Western Pharmacological Society Meetings in Rena, Nevada. January 1984, and the 27th Annual Meeting of Canadian Federation of Biological Societies, Saskatoon, Saskatchewan, June 1984.
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Alcohol and neural cell proliferation
REFERENCES 1. Abel E. L. (1980) Fetal alcohol syndrome: behavioral teratology. Psychol. Bull. 87, 2%50. 2. Abel E. L. and Greizerstein H. B. (1980) Growth and development in animals prenatally exposed to alcohol. In Fetal Alcohol Syndrome, Vol. 3, Animal Studies (ed. Abel E. L.), pp. 39-58. CRC Press, Boca Raton. 3. Bauer-Moffett C. and Altman J. (1977) The effect of ethanol chronically administered to pre-weanling rats on cerebellar development. A morphological study. Brain Res. 119, 249-268. 4. Castells S., Mark F.. Abaci F. and Schwartz E. (1981) Growth retardation in fetal alcohol syndrome. Unresponsiveness to growth-promoting hormones. Devl Pharmac. Ther. 3, 232-241. 5. Clarren S. K.. Alvord C. A. Jr., Sumi S. M., Streissguth A. P. and Smith D. W. (1978) Brain malformations related to prenatal exposure to ethanol. J. Pediatr. 92, 64-67. 6. Cowan M. (1979) The development of the brain. Sci. Am. 241, 112-133. 7. Diaz J. and Samson H. H. (1980) Impaired brain growth in neonatal rats exposed to ethanol. Science, Wish. 208, 751-753. 8. Dobbing J. (1974) Human brain development and its vulnerability. In Biologic and Clinical Aspects ofBrain Development, Mead Johnson Symposium on Perinatal and Developmental Medicine, No. 6, pp. 3-14. Mead Johnson and Co., Evansville. 9. Druse M. J. and Hofteig J. H. (1977) The effect of chronic maternal alcohol consumption on the development of CNS myelin subfractions in rat offspring. Drug Ale. Depend. 2, 421-429. 10. Hammer R. P. and Scheibel A. B. (1981) Morphologic evidence for a delay of neuronal maturation in fetal alcohol exposure. Exp. Neurobiol. 74, 587-596. 11. Hanson J. W., Jones K. L. and Smith D. W. (1976) Fetal alcohol syndrome: experience with 41 patients. J.A.M.A. 235, 14581460. 12. Henderson G. I., Hoyumpa A. M.. Rothschild M. A. and Schenker S. (1980) Effect of ethanol and ethanol-induced hypothermia on protein synthesis in pregnant and fetal rats. Alcoholism 4, 165-177. 13. Henderson G. I., Patwardhan R. V., Hoyumpa A. M. Jr. and Schenker S. (1981) Fetal alcohol syndrome: overview of pathogenesis. Neurobehav. Toxicol. Teratol. 3, 73-80. 14. Henderson G. I., Patwardhan T. V., McLeroy S. and Schenker S. (1982) Inhibition of placental amino acid uptake in rats following acute and chronic ethanol exposure. Alcoholism 6, 495-504. 15. Kaminski M.. Fumeau C. and Schwartz D. (1978) Alcohol consumption in pregnant women and the outcome of pregnancy. Alcoholism 2, 155-163. 16. Kennedy L. A. and Bhaumick B. (1983) Placental pathology and changes in placental binding of [‘25I] basic somatomedin (B-SM) associated with alcohol-related fetal growth deficits. Proc. Can. West Sot. Reprod. Biol. 4, 23
(Abstract). 17. Kennedy L. A. (1984) The pathogenesis of brain abnormalities in the fetal alcohol syndrome: an integrating hypothesis. Teratology 29, 363-368. 18. Kennedy L. A. (1984) Changes in the term mouse placenta associated with maternal alcohol consumption and fetal growth deficits. Am. J. Obstet. Gynec. 149, 518-522. 19. Kennedy L. A., Elliott M. J. and Laverty W. H. (1984) Reductions in the plating efficiency of the fetal neural precursor cells following maternal alcohol consumption. Int. J. Devl Neurosci. 2, 43746. 20. Leichter J. and Lee M. (1982) Method of ethanol administration as a confoundine factor in studies of fetal alcohol syndrome. Life Sci. 31, 221-227. 21. Mukherjee A. B. and Hodgen G. D. (1982) Maternal ethanol exposure induces transient impairment of umbilical circulation and fetal hypoxia in monkeys. Science. Wash. 218. 7C&702. 22. Rawatt A. (1975) Ribosomal protein synthesis in the fetal and neonatal rat brain as influenced by maternal alcohol consumption. Res. Comm. Chem. Path. Pharmac. 12, 723-732. 23. Samson H. H. and Diaz J. (1982) Effects of neonatal ethanol exposure on brain development in rodents. In Fetal Alcohol Syndrome, Vol. 3, Animal Studies (ed. Abel E. L.), pp. 131-150. CRC Press, Boca Raton. 24. Shaw S. and Lieber C. S. (1979) Effects of ethanol on nutritional status. In Human Nutrition: Metabolic and Clinical Application (ed. Hodges R. E.), pp. 293-398. Plenum Press, New York. 25. Shoemaker W. J., Baetge G., Azad K., Sapin V. and Bloom F. E. (1983) Effect of prenatal alcohol exposure on amine and peptide neurotransmitter systems. In Drugs and Hormones in Brain Development, Monographs in Neuroscience, Vol. 9, p. 130. 26. Smart I. H. M. (1973) Proliferative characteristics of the ependymal layer during the early development of the mouse neocortex: a pilot study based on recording the number, location and plane of cleavage of mitotic figures. J. Anat. 116, 67-91. 27. Sokol R. J., Miller S. I. and Reed G. (1980) Alcohol during pregnancy: an epidemiological study. Alcoholism 4, 135145. 28. Streissguth A. P., Sandernan-Dwyer S., Martin J. C. and Smith D. W. (1980) Teratogenic effects of alcohol in
humans and laboratory animals. Science. Wash. 209. 353-361. 29. Sulik K. K.. Lauder J: M. and Dehart D. B. (1984)‘Brain malformations ethanol
administration.
Inr. J. Devl Neurosci.
in prenatal
mice following
acute maternal
2, 203-214.
30. Tabakoff B., Nobel E. P. and Warren K. R. (19791 Alcohol, nutrition and the brain. In Nutrition and the Brain, Vol.4 leds 1 and -~-------R. --- -. ---- Wurtman J. J.), pp.‘159-213. Raven Press, New York. \ ~-Wurtman 31. Volk B. (1977) Delayed cerebe :llar ._~ histopenesis ~~~_~___..__.in “Embryofetal alcohol syndrome.” Acta Neuropath., Berl. 39, 157-
163. 32. Wunderlich S. M., Baliga S. and Munro H. N. (1979) Rat placental protein synthesis and peptide secretion in relation to malnutrition from protein deficiency in alcohol administration. J. Nutr. 109, 1534-1541.
Ncrmrmci
073657JX/H5$03.Iwl+o.0t) Perpamon Press Ltd. 0 IL)XS ISDN
V<,I.3. No. J. pp. .II l-315. IYXS.
Printed in Great Britain.
CELL PROLIFERATION IN THE EMBRYONIC MOUSE NEOCORTEX FOLLOWING ACUTE MATERNAL ALCOHOL INTOXICATION L. A. KENNEDY and M. J. ELLIOTT Teratology Research Laboratory.
Department of Pharmacology. University of Saskatchewan. Saskatoon. Saskatchewan. Canada S7N OWO (Accepted
4 November 1984)
Abstract-Experiments were conducted to examine the effects of ‘binge’ or acute maternal alcohol intoxication during the early proliferative phase of embryonic brain development. Primiparous mice received ethanol as 0, 10, 15 or 20% (v/v) aqueous solutions by gavage on days 13, 14 and 15 of gestation. Mean daily doses were 0.0, 2.58, 4.03 and 5.40 ml/kg, respectively. There was no alcohol-related reduction in fetal body weight, length or fixed brain weight. Coronal sections (1 km) of the dorsal roof of the lateral ventricles over the optic chiasma were examined from nine embryonic day 15 brains for each treatment group. The ventricular surface index of mitotic figures, the number and distribution of non-surface mitotic figures, and the depth of the cortical roof and its constituent layers were determined. There was no alcohol-related difference in any of these parameters. These results are in contrast to those of a previous experiment using the same mouse strain, in which prolonged or chronic maternal alcohol consumption in the drinking water from days 11 to 19 of pregnancy was associated with a reduction in the surface index, a reduction in the depth of the cortical roof and an increase in the non-surface mitotic figures. These latter changes, however, occurred in the presence of reduced body weight. Our observations suggest that during this particular developmental period (corresponding to the second trimester of human pregnancy) alcohol-related reductions in brain growth parallel restrictions in general body growth. Key words: Alcohol, Mouse, Cell proliferation,
Cortex.
Central nervous system (CNS) dysfunctions in the infants are the most debilitating consequences of maternal alcohol abuse during pregnancy and can occur independently of the craniofacial malformations which are characteristic of fetal alcohol syndrome (FAS). They appear, however, to be closely related to general body growth deficits. 1,2*11*28 Postmortem examination of severely affected infants has revealed microcephaly, reduced numbers of all neural cell populations and abnormal cytoarchitecture.’ Similar observations have been made in experimental animals337323,29*31 and suggest that alcohol-related derangements in the patterns of proliferation and migration of the neural progenitor cells contribute to the pathogenesis of the intellectual and behavioral impairments in FAS children. Teratogens generally exert their effect when exposure occurs during critical periods of development which represent ‘once only’ opportunities for growth.8 Compared to other organ systems, the ontogeny of the mammalian brain is extremely complex and prolonged6 making it highly vulnerable to derangement for extended periods of time. Impairments of CNS function in the offspring of alcohol-consuming women are highly variable both in frequency and severity, probably reflecting the variable patterns of alcohol abuse. The pathogenesis of these dysfunctions, however, is not well understood. It is as yet unclear, for example, whether there are periods of development during which the brain is relatively less vulnerable to alcohol’s teratogenicity than others, whether some processes contributing to brain development are more or less sensitive to disturbance than others, and to what extent secondary or indirect alcohol-related changes in the maternal-placental-fetal organism can contribute at different stages in development. Binge drinking has been linked to FAS in humans and it has been shown experimentally that one brief period of maternal alcohol intoxication during a sensitive period of development in the mouse Address all correspondence and reprint requests to Dr. L. A. Kennedy, Department of Pharmacology. College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N OWO. Abbreviations: SI, surface index of mitotic figures; MFs. mitotic figures; FAS. fetal alcohol syndrome; CNS, central nervous system; CRL. crown-rump length; Ex. embryonic day x: SVZ, subventricular zone; INTZ. intermediate zone: CP, cortical plate; MZ, marginal zone; ANOVA. analysis of variance. DN):&-A
311
312
L. Kennedy and M. Elliott
gastrula (corresponding to week 3 of human gestation) can produce changes in the facial and brain primordia which result in the characteristic FAS midfacial and midline brain abnormalities.‘,2” Alcohol exposure later in development has also been shown to be associated with biochemical and morphological abnormalities in the developing brain~.7,“.‘f’,2”,2s.~’but not with the classical craniofacial characteristics of FAS. In previous experiments,” we found that prolonged or chronic alcohol consumption during the early proliferative period of the fetal brain growth spurt resulted in altered patterns of neural cell proliferation in the fetal mouse neopallium which were detectable in situ and in vitro. In this chronic model, however, the changes were closely associated with fetal body and brain growth deficits. The present investigation was conducted to determine if maternal alcohol intoxication during this same period of brain development resulted in impairment in the recruitment of embryonic neural cells in the absence of general body growth impairment.
EXPERIMENTAL,
PROCEDURES
Primiparous DBA/lJ mice (age 8-10 weeks, 1.5-19 g body weight) were used, day 1 of gestation being designated as the day on which a copulation plug was found. Dams were housed individually in glass cages with wire mesh tops and Sani-seal corn cob bedding, under controlled environmental conditions (27°C 60-70% relative humidity, 13L 11D photoperiod). Mouse chow and water were available ad libitum. Ethanol was administered by gavage as 0, 10, 15 or 20% (v/v) aqueous solutions on each of days 13, 14 and 15 of gestation. Each dam received two daily treatments of 0.3 ml aqueous alcohol. Alcohol was administered in the first hours of the light schedule (0900 and 1100 h) to minimize the interference of periods of intoxication with activity and feeding. There were at least six pregnant mice in each of the four treatment groups. Maternal weight was recorded on day I and on each treatment day. On day 1.5 (1300 h) dams were killed by cervical dislocation, the uterine horns were opened and the numbers of live, dead and resorbed fetuses recorded. Fetuses were removed, examined for gross external malformation, weighed and measured for crownrump length (CRL). Fetal heads were placed in 3% glutaraldehyde in Sorensen’s buffer. Brains were subsequently removed by severing the connection with the olfactory bulb and with the brain stem behind the cerebellum. They were left overnight in the fixative, weighed and embedded in methacrylate. All brains were weighed after the same length of time in fixative to ensure that differences in dehydration did not influence fixed brain weight. Semi-serial coronal sections (1 km) were cut over the optic chiasma and stained with 1% toluidine blue in saturated Borax solution. Nine embryonic day 15 (E15) brains, obtained from at least four different litters were evaluated from each treatment group. For each embryonic brain, three sections (one section from each of three slides, taken at least 10 Frn apart) were analyzed. Brains were assigned random numbers and the evaluation was conducted in a blind manner. The ventricular surface index of mitotic figures (SI) was determined using an eyepiece micrometer under oil immersion (mag 1000 x). The number of mitotic figures present were counted in an area 250 pm long and 20 km deep starting directly from the dorso-medial angle of the ventricular surface of the dorsal roof and proceeding laterally. Each surface area (250 X 20 km) was comprised of five segments (50 x 20 pm) from medial to lateral and each segment had five divisions (10 X 20 km). The mean SI for each segment was calculated by averaging the number of mitotic figures from the five divisions for the three slides, and mean SI for the five segments was averaged to produce the SI for each hemisphere. The overall SI for each brain was obtained by averaging the SI from the two hemispheres and is thus a measure of the number of mitotic figures per unit area of ventricular surface of the dorsal roof of the neocortex.2h The numbers and the distribution of mitotic figures (MFs) were also examined in the subventricular zone (SVZ), intermediate zone (INTZ), cortical plate (CP) and marginal zone (MZ), using the 250 pm area directly above the surface area used to determine the SI. These nonsurface MFs were plotted on projection microscope diagrams by scanning at 400 X. Both the absolute number of non-surface MFs and the proportional distribution of MFs in the different cortical layers were determined.
313
Alcohol and neural cell proliferation
The total depth of the dorsal roof, as well as the thickness of its constituent layers was determined in the same sections exactly 250 km from the dorsomedial angle of the neocortex. To adjust for curvature in the dorsal roof, the measurements were taken at 90” to a line bissecting this angle and at a point 250 pm distant from the angle. Two measurements per hemisphere were taken using these points and the mean depths were calculated for each brain. A nested Analysis of Variance (ANOVA) adjusted for unequal sample sizes was used to test for treatment-related differences in fetal body weight, brain weight and CRL. A one-way ANOVA was used to assess the treatment-related difference in SI, numbers and distribution of non-surface MFs and the cortical depth. RESULTS When 0.3 ml of 0, lo,15 or 20% (v/v) aqueous ethyl alcohol was administered to pregnant mice twice daily on days 13,14 and 15 of gestation the mean daily dose of absolute alcohol per dam was 0.0,2.58,4.03 and 5.40 ml/kg body weight, respectively. There was no alcohol-related difference in the number of live, dead, malformed or resorbed embryos using this protocol, and there were no significant (P~0.05) deficits in general body growth or fixed brain weight (Table 1) Table
1. The effects of acute maternal Maternal dose (ml/kg/day)
Treatment group
alcohol
intoxication
Fetal mortality (%)’
on fetal growth
Fetal body weight(g)
Fetal CRL (cm)
Fetal brain weight (mg)
Control (N=7;n=44)
0.0
18.7
0.202
I .03
14.45
10% (N=7;n=42)
2.58
27.1
0.194
I .os
14.94
15% (N=6;n=44)
4.03
13.3
0. I80
0.99
13.62
20% (N=6;n=38)
5.40
22.5
0.20
0.99
14. I7
Significance
-
F= 0.28 NS
F= 0.83 NS
sites x 100.
The data obtained from the examination of the fetal neocortex was no significant treatment-related difference (P>O.O5) in the of the neocortex nor was there any treatment-related difference ments comprising the total area of ventricular surface examined Table 2. The effects of acute maternal
alcohol intoxication
on the proliferative
SI
Total number of non-surface MFs
vz+svz
INTZ
CP+MZ
Control (N=5;n=9)
1.94 (0.41)
322.11 (32.13)
94.89 (1.85)
4.22 (1.81)
0.89 (0.87)
10% (N=6;n=9)
1.96 (0.18)
347.33 (23.19)
94.22 (2.57)
5.33 (2.26)
15% (N=4;n=9)
1.94 (0.18)
301.67 (38.22)
95.33 (2.36)
20% (N=4;n=9)
1.76 (0.37)
317.89 (48.06)
Significance
F= 0.76 NS
F= 2.14 NS
Treatment
Distribution
F=0.55 NS
F= 2.38 NS
All measured are presented as group means. N = number of dams; n = total number of fetuses. *Resorptions plus dead fetuses + total number of implantation
group
and development
is summarized in Table 2. There SI of the dorsal ventricular roof in the SI of the individual seg(data not presented). There was
neural cells and the depth of the fetal mouse neocortex
of MFs (%)
Depth of neocortex INTZ
CP+MZ
Total
103.0 (21.73)
60.56 (15.25)
37.67 (10.24)
201.22 (36.89)
0.44 (0.50)
97.56 (9.18)
67.11 (25.86)
45.22 (19.31)
211.20 (45.15)
3.78 (1.99)
0.89 (0.87)
98.89 (13.12)
61.67 (12.52)
40.78 (8.32)
201.33 (10.73)
94.89 (2.33)
4.11 (2.02)
1.0 (0.47)
99.0 (9.84)
55.44 (24.65)
43.0 (20.06)
197.44 (42.45)
F= 0.319 NS
F= 0.887 NS
F= 0.971 NS
F=0.215 NS
F=0.440 NS
F=0.351 NS
F=0.209 NS
All measures are presented as group means, 1 S.D. in brackets. N= number of litters sampled; n = number of fetal brains evaluated.
vz+svz
(urn)
314
L. Kennedy and M. Elliott
no significant difference between treatment groups either in the total numbers of non-surface MFs counted in the area of the dorsal roof immediately above the ventricular surface area or in the numbers of MFs in the SVZ, INTZ, or CP plus MZ. It can also be seen that there was no difference in their distribution throughout the different cortical layers. Finally, there was no difference in the total depth of the dorsal roof or in the depth of the individual cortical layers. DISCUSSION Fetal alcohol syndrome (FAS) is comprised of a non-specific cluster of minor malformations which vary in the frequency and severity of expression and which can have other etiologies. Although the direct effects of alcohol undoubtedly account for some of the deleterious effects on embryofetal development, it is becoming increasing clear that there are many indirect ways in which maternal alcohol intoxication can alter the in utero environment on which normal growth and development is dependent. 13,” Examples of indirect mechanisms of alcohol-related teratogenesis include primary and/or secondary undernutrition,24%30 hypothermia,” placental dysfunction’4,‘X,2’ and endocrine disturbances.4.‘h.‘2 Although the pathogenesis of alcoholrelated developmental abnormalities is not clearly understood, it is evident from epidemiological and experimental studies that the risk to embryofetal development increases with increasing alcohol consumption and in the presence of other alcohol-related maternal risk facfors.15,‘8.‘9.27 In previous experiments using the same mouse strain and a chronic model, we found that prolonged maternal alcohol consumption during the early proliferative phase of the mouse brain growth spurt (Ell-E18) resulted in changes in the proliferative cells of the embryonic neocortex which were detectable in vitro and in situ. These changes, however, were seen in association with significant reductions in brain weight and general body growth deficits.‘” The present investigations were conducted to determine whether similar changes in the proliferative neural cells would be seen using a ‘binge’ or acute model of alcohol abuse during the same developmental period. Preliminary trials using a single dose of 0.2 ml of 0, 10, 15 or 20% (v/v) EtOH resulted in blood alcohol levels of 0, 55, 98 and 125 mg/dl in non-pregnant adult mice 30 min later. Higher peak levels would be expected in the present experiments with two doses of 0.3 ml of the ethanol solutions administered 2 h apart. Based on the severity and duration of locomotor impairment the ucute model resulted in higher peak blood alcohol levels than were achieved in the chronic model, but the periods of intoxication were shorter and the daily doses were lower. There were no alcohol-related differences in prenatal mortality or morbidity in either model. In contrast to the chronic model, the acute model did not result in impairment in embryofetal body or brain growth, nor were there any detectable alcohol-related changes in the proliferative neural cells. In this investigation, the ventricular SI was used as an indicator of neural stem cell proliferation, the number and distribution of non-surface MFs were used as indicators of the replication and migration of the early glioblasts, and the thickness of the layers of the dorsal roof was used as a general indicator of cellular growth, differentiation and organization. Although the actual number of cells studied may be relatively small, this area of the developing neocortex has been used extensively to study neural cell proliferation, and the behavior of these cells may be considered representative of other populations of cortical progenitor cells. During the treatment period used in this study, neuroblast replication is at a peak, glial replication is rising to a peak which occurs postnatally and progenitor cells are migrating outward to their final location.h*8726Our results indicate that acute maternal alcohol intoxication during the early proliferative phase of the mouse brain growth spurt (corresponding to the second trimester of human pregnancy) is not associated with any lasting impairment in the recruitment of neural precursor cells in the absence of general body growth impairment. Acknowledgements-We gratefully acknowledge the financial support of the Saskatchewan Health Research Board and the University of Saskatchewan. Mrs. M. Matheson did the typing. Thanks to Dr. P. Flood and M. Diocee for the use of their JB4 microtome. This work has been presented at the Western Pharmacological Society Meetings in Rena, Nevada. January 1984, and the 27th Annual Meeting of Canadian Federation of Biological Societies, Saskatoon, Saskatchewan, June 1984.
315
Alcohol and neural cell proliferation
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