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Morphometric analysis of developing rat cerebral cortex following acute prenatal ethanol exposure
EXPERIMENTALNEUROLOGY
106,283-288
(1989)
Morphometric Analysis of Developing Rat Cerebral Cortex following Acute Prenatal Ethanol Exposure ANDSTATANORTON
LOISA.KOTKOSKIE
University ofKansas Medical Center Department ofPharmacology, Toxicology, and Therapeutics, 39th Street and Rainbow Boulevard, Kansas City, Kansas 66103
Morphologic alterations of fetal rat cerebral cortex were quantified by morphometric analysis following acute ethanol exposure on Gestational Day 14, a critical period of development of cerebral cortex. Pregnant rats were intubated with a total dose of 9 g/kg of ethanol on Gestational Day 14. Maternal blood ethanol levels ranged from 51 to 202 mg% during the period of ethanol exposure. Fetal brains were examined on Gestational Day 15, 24 h after the last dose of ethanol. The acute morphologic changes associated with ethanol exposure include enlargement of subventricular zone nuclei, cortical swelling, and dilation of pial blood vessels over the affected cortex. In some fetuses, cortical swelling was accompanied by the protrusion into the lateral ventricles of cytoplasmic blebs of ventricular zone cells. It is concluded that maternal ethanol exposure during a critical period of brain development produces measurable morphologic changes in fetal rat cerebral cortex within 24 h after ethanol exposure. o 1st~ Academic Press,
Inc.
INTRODUCTION Ethanol ingestion during pregnancy has been associated with serious disturbances in fetal development including the fetal alcohol syndrome (10). Heterotopias and disorganization of brain structure resulting from widespread abnormalities in neuronal and glial migration have been reported in human neonates exposed to ethanol in utero (5,18). Recent studies have shown that the neuropathology of human fetuses from alcoholic mothers can be reproduced in rats and primates by acute ethanol exposure during critical periods of gestation (7,14). However, the morphologic changes in the developing brain immediately following acute ethanol exposure have not been well documented. The purpose of this study was to describe and quantify the morphologic changes occurring in fetal rat cerebral cortex 24 h after short-term maternal ethanol exposure. Ethanol was administered on Gestational Day 14, a critical period of development of the cerebral cortex (8,9). The formation of the cortical plate from neuroblasts migrating from the germinal matrix
begins about Gestational Day 14. Rapid proliferation of the germinal matrix begins following closure of the anterior neuropore by Gestational Day 11. Migration to form the cortical plate begins after the germinal matrix has grown from a layer of 1 cell to a layer lo-12 cells thick. This process has been analyzed in detail for the rat (2). MATERIALS
AND METHODS
Breeding and Ethanol Exposure Female, Sprague-Dawley-derived rats (CD Strain, Charles River), 225-325 g, were housed in a temperature-controlled room with a 12-h light/dark cycle and were fed Purina Rat Chow and water ad Zibitum. Females were bred with males of the same strain and the morning that a vaginal smear was positive for sperm was counted as Day 1 of gestation. Ten pregnant females were randomly assigned to control (n = 5) or ethanol (n = 5) groups. On Gestational Day 14, animals were dosed by gavage under light ether anesthesia. Animals in the ethanol-exposed group received 4.5 g/kg ethanol (80%) at 11:00 AM and 3:00 PM on Gestational Day 14 for a total dose of 9 g/kg ethanol. Blood ethanol levels were determined 2 h after each dose of ethanol from 70 111of tail blood (Sigma Kit 322-UV, Sigma Chemical Co., St. Louis, MO). To control for the stress of dosing, two control rats received 5% glucose at 11:00 AM and 3:00 PM on Gestational Day 14 in a volume equal to that received by the ethanolexposed animals. Three control rats were untreated. All control animals were restricted from food between 8:00 AM and 6:00 PM on Gestational Day 14 to control for the period of reduced food consumption associated with ethanol exposure. Histological Preparation of Tissues Pregnant animals were anesthetized with ether and decapitated at 3:00 PM on Gestational Day 1524 h after the last dose of ethanol or glucose. Fetuses were fixed in Karnovsky’s fixative (12). For light microscopy, fetal heads were postfixed in osmic acid and embedded in LR White acrylic resin. Tissue blocks were trimmed to the
283 Copyright 0 1989 A11 rights of reproduction
0014-48&S/89 $3.00 by Academic Press, Inc. in any form reserved.
KOTKOSKIE
AND
NORTON
and dehydrating with ethanol prior to critical point drying with carbon dioxide (Balzer 020 CPD). Dried brains were mounted on aluminum stubs and sputter coated with a thin conductive layer of gold/palladium alloy (Technic’s Hummer I). A JEOL-35 scanning electron microscope was used to examine the lateral ventricular surfaces of control and ethanol brains at accelerating voltages from 15 to 30 kV.
Morphometric
FIG. 1. a Gestational morphometric
Drawing of a transverse section through the forebrain of Day 15 rat showing (A) area of cerebral cortex used for analysis, (B) corpus striatum, and (Cl eye.
level of the telencephalon, sectioned at 2 pm, and stained with toluidine blue. Lateral ventricular surfaces of Gestational Day 15 brains were prepared for scanning electron microscopy by dissecting fixed brains to expose the lateral ventricles
FIG. 2. pial blood
Developing cerebral cortex vessels are dilated (arrowhead)
of Gestational Day and the ventricular
Methods
Morphometric analysis of Gestational Day 15 fetal cerebral cortex, using a-pm-thick sections, toluidine blue stained, was performed using a light microscope equipped with the ZIDAS optical system (Carl Zeiss, Inc.) for image analysis. The cortices of four to seven fetuses per litter were analyzed blind with respect to treatment groups. Zones of the developing cortex were identified following the Boulder conventions (3). In a 15day fetus, the ventricular zone consists of l- to 3-cell layers showing various mitotic figures and intervening stages of mitosis. Above this zone is the subventricular zone which is 8- to lo-cell layers in height and cells are more densely packed than in other zones. An area in the center of this zone, 5- to 6-cell layers in height and width, was selected for measurement of nuclei. Under low
15 rat fetuses. (A) Control. (B) Ethanol. margin has a ruffled appearance (arrow).
The cortical thickness Bar is 50 pm.
is increased,
the
ETHANOL
FIG. 3. fluid-filled
Developing blebs (arrows)
AND
THE
cerebral cortex of Gestational Day 15 rat fetuses along the ventricular margin. Bar is 25 pm.
power of the microscope the same cortical area was selected for measurement of each brain (Fig. 1, arrow A) in the same anterior-posterior location. The total number of nuclei present in 2500 pm2 of the subventricular zone in a section 2 pm thick varies from 25 to 30. About half of these have nucleoli. To measure those nuclei cut through the center of the nucleus, the 10 largest nuclei with one or more nucleoli were selected. The luminal areas in cross section of all pial blood vessels were determined along 1 mm of cortex (linear measurement along the ventricular margin). A sample size of 10 nuclear areas and 20-30 pial blood vessel areas per animal resulted in a standard error of less than 8%. Cortical thickness was measured at the same point on each fetal brain, as shown in Fig. 1A. The linear measurement was made
DEVELOPING
24 h after
285
BRAIN
acute ethanol
exposure
on Gestational
from the pial surface to the ventricular dicular to the cortical margins.
Day 14 demonstrating
surface, perpen-
Statistics Nuclear area was normally distributed and was analyzed by one-way ANOVA. Cortical thickness and pial blood vessel area data were not normally distributed and so were evaluated by the Kolmogorov-Smirnov, twosample, two-sided test (23). Because several variables were measured on each fetus, the CYlevel for statistical significance was set at P G 0.01. The relationship of maternal blood ethanol concentration with morphometric parameters was assessedby Spearman’s rank correlation coefficient.
286
FIG. 4. the surface
KOTKOSKIE
Scanning electron photomicrograph of the lateral ventricles. (B) Ethanol.
of lateral Microvilli
AND
NORTON
ventricular surfaces of Gestational Day 15 rat fetuses. (A) Control. Microvilli have been replaced by large protrusions of ventricular zone cells (X5200).
RESULTS
Blood ethanol levels ranged between 51 and 189 mg% 2 h after the first dose of ethanol and between 95 and 202 mg% 2 h after the last dose of ethanol on Gestational Day 14. Animals exposed to ethanol became ataxic for several hours following each dose of ethanol. No maternal respiratory depression was noted. All animals recovered and appeared healthy after the period of ethanol intoxication. Normal architecture of Gestational Day 15 fetal cerebral cortex is shown in Fig. 2. Ethanol administration on Gestational Day 14 did not cause cell death or alter the
line
laminar organization of immature neurons within developing cerebral cortex. However, 24 h following ethanol exposure on Gestational Day 14, swelling of the cerebral cortex and dilation of pial blood vessels were present in most ethanol-exposed fetuses (Fig. 2). Other areas of the telencephalon, such as the developing corpus striatum, appeared unaffected by acute ethanol exposure on Gestational Day 14. In many ethanol-exposed fetuses, the increase in cortical thickness was so great as to cause protrusion of ventricular zone cells into the lateral ventricles. This caused the ventricular margin of ethanol-exposed fetuses to assume a ruffled appearance (Fig. 2). Additionally, in sev-
’
TABLE
ETHANOL
AND
THE
1
Morphometric Analysis of Gestational Day 15 Fetal Rat Cerebral Cortex Group Control n=25(5)’ Ethanol n = 25 (5)
Nuclear area (pm’) Mean ? SE
Cortical thickness Mean (range)
30.4 + 0.4
125 (110-148)
47.4 + 0.71b
140 (110-187)’
’ Number of fetuses (number b Significantly different from ’ Significantly different from nov.
of litters). controls, control,
P < 0.01, P < 0.01,
(pm)
one-way ANOVA. Kolmogorov-Smir-
era1 ethanol-exposed fetuses ventricular zone cells bulged into the lateral ventricles and appeared as blebs along the ventricular margin in the light microscope (Fig. 3). The blebbing was so severe that microvilli lining the ventricular surface were seen in the scanning electron microscope as large round processes of the ventricular zone cells (Fig. 4). Morphometric measurements of Gestational Day 15 fetal cerebral cortex are given in Table 1 and Fig. 5. There were no statistical differences between untreated controls and glucose controls in any of the morphometric parameters measured. Therefore, both groups of control fetuses were combined into one control group. Cortical thickness and subventricular zone nuclear area were increased in ethanol-exposed fetuses compared with controls 24 h after ethanol exposure on Gestational Day 14. There were no significant correlations between maternal blood ethanol levels and nuclear area or cortical thickness. A histogram of pial blood vessel area (Fig. 5) shows that Gestational Day 15 control fetuses had many small vessels and few large vessels, while Gestational Day 15 ethanol-exposed fetuses had few small vessels and many large vessels in the same region above the developing cerebral cortex. Therefore, quantitative analysis of Gestational Day 15 cerebral cortex reveals that ethanol-exposed fetuses had markedly dilated pial blood vessels and swelling of developing cerebral cortex 24 h after acute ethanol exposure.
DEVELOPING
287
BRAIN
since it has been shown that ethanol freely crosses the placental barrier of the rat (13). Swelling of the cerebral cortex and dilation of pial blood vessels were observed in Gestational Day 15 fetuses following ethanol exposure on Gestational Day 14. These acute morphologic changes were present even though pregnant rats did not attain very high blood ethanol levels. It is possible that ethanol acted directly and/ or indirectly on the fetus to evoke morphologic changes. Ethanol has been proposed to act directly on cells to increase membrane fluidity and therefore affect the function of membrane-bound enzymes (21). Rudeen and Guerri (22) reported that chronic prenatal and postnatal ethanol exposure decreased the activities of Na/KATPase and Ca-ATPase, two membrane-bound enzymes necessary to maintain ionic gradients across neuronal cell membranes. A brief decrease in the activity of these and other membrane-bound enzymes following acute ethanol exposure could account for the cell swelling observed in this study. Several investigators have hypothesized that ethanol exerts some of its actions indirectly by causing fetal hypoxia (11,15). Experimentally produced fetal hypoxia in primates results in the same morphologic changes found in this study, specifically, swelling of the brain and dilation of pial blood vessels (16,20). Therefore, some of the morphologic changes observed in this study may be due to direct effects of ethanol and some to indirect effects of fetal hypoxia following ethanol exposure. In several ethanol-exposed fetuses, blebbing of ventricular zone cells into the lateral ventricle was found. It appeared that the cells within the cerebral cortex became so enlarged that the tissue reached its limits of expansion, and the only cells able to expand were those lining the ventricle. Blebbing of ventricular zone cells has also been described in fetal rats 24 h after exposure to 6-aminonicotinamide on Gestational Day 15 by Chamberlain (4). He hypothesized that the blebs secreted fluid from the cortex into the lateral ventricles to cause congenital hydrocephalus. In this study, the blebs
h
s
3Otl” 0 I
CONTROL ETHANOL’
DISCUSSION The administration of 9 g/kg of ethanol to pregnant rats on Gestational Day 14 resulted in slight maternal ethanol intoxication. Comparable human blood ethanol levels can be obtained after the ingestion of 3 oz of pure alcohol (1). The variability of maternal blood ethanol levels may have been due to differences in pharmacokinetic parameters such as the rate of absorption and biotransformation of ethanol. Fetal blood ethanol levels were probably the same as maternal blood ethanol levels,
PIAL BLOOD VESSEL
AREA (pm2)
FIG. 5. Pial blood vessel area distribution of Gestational Day 15 control and ethanol-exposed fetuses (n = 20 per group). Eight percent of ethanol values are greater than 800 Brn’. Control (494) and ethanol (446) distributions are significantly different, *P < 0.001, Kolmogorov-Smirnov.
288
KOTKOSKIE
may function in the same manner to reduce cortical swelling after ethanol exposure. It is not known if hydrocephalus could be a long-term consequence of acute ethanol exposure on Gestational Day 14. Acute ethanol exposure on Gestational Day 14 was also associated with enlargement of subventricular zone nuclei 24 h after exposure. Nuclear enlargement may have occurred as a response involving increased protein synthesis for repair of ethanol-induced damage within the cell. For example, it has been shown that acute ethanol exposure increases synthesis of heat shock protein in rats (17). Other investigators have reported that the peak blood ethanol concentration threshold for producing brain damage in the rat (19) and the primate (6) was approximately 150 mg%. The results of this study support this concept because most of the maternal animals achieved blood ethanol levels near or above 150 mg%. However, we were unable to show a significant correlation between maternal blood ethanol levels and fetal outcome, probably because we could not use peak blood ethanol concentrations in our statistical analysis. The results of this study provide evidence that acute ethanol exposure in pregnant rats with blood ethanol levels at 200 mg% or less during a critical period of brain development induced measurable morphologic changes within the developing cerebral cortex. Cortical swelling and pial blood vessel dilation were present in ethanolexposed fetuses. Since cell death was not observed, these morphologic changes may be temporary in nature and may subside once cellular damage caused by ethanol has been repaired. However, the long-term morphologic and functional consequences of an acute alcoholic exposure at these blood levels on Gestational Day 14 are not known. ACKNOWLEDGMENTS This research was supported in part by Grants NS16694 and ES07079. L.A.K. was supported by a Procter & Gamble Fellowship.
REFERENCES 1. ANDREWS, L. S., AND R. SNYDER. 1986. Toxic effects of solvents and vapors. Pages 636-668 in C. D. Klaassen, M. 0. Amdur, and J. Doull, Eds., Casarett and DouZl’s Toxicology, 3rd ed. Macmillan, New York. 2. BERRY, M. 1974. Development of the cerebral neocortex of the rat. Pages 7-67 in G. Gottlieb, Ed., Aspects of Neurogenesis. Academic Press, New York. 3. Boulder Committee. 1970. Embryonic vertebrate central nervous system: Revised terminology. Anat. Rec. 166: 257-262. 4. CHAMBERLAIN, J. G. 1970. Early neurovascular abnormalities underlying 6-aminonicotinamide (6-AN)-induced congenital hydrocephalus in rats. Teratokgy 3: 377-388.
AND NORTON 5. CLARREN, S. K., E. C. ALVORD, JR., S. M. SUMI, A. P. STREISSGUTH, AND D. W. SMITH. 1978. Brain malformations related to prenatal exposure to ethanol. J. Pediatr. 92: 64-67. 6. CLARREN, S. K., S. J. ASTLEY, AND D. M. BOWDEN. 1988. Physical anomalies and developmental delays in nonhuman primate infants exposed to weekly doses of ethanol during gestation. Teratology 37: 561-569. 7. CLARREN, S. K., AND D. M. BOWDEN. 1982. Fetal alcohol syndrome: A new primate model for binge drinking and its relevance to human ethanol teratogenesis. J. Pediatr. 101: 819-824. 8. HICKS, S. P. 1954. The effects of ionizing radiation, certain hormones and radiomimetic drugs on the developing nervous system. J. Cell Comp. Physiol. 43(Suppl. 1): 151-178. 9. HICKS, S. P., C. J. D’AMATO, AND M. J. LOWE. 1959. The development of the mammalian nervous system. J. Comp. Neurol. 113: 435-470. 10. JONES, K. L., D. W. SMITH, C. N. ULLELAND, AND A. P. STREISS. GUTH. 1973. Pattern of malformation in offspring of chronic alcoholic mothers. Lancet 1: 1267-1271. 11. JONES, P. J. H., J. LEICHTER, AND M. LEE. 1981. Placental blood flow in rate fed alcohol before and during gestation. Life Sci. 29: 1153-1159. 12. KARNOVSKY, M. J. 1965. A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J. Cell Biol. 27: I37A-138A. 13. KESXNIEMI, Y. A., AND H. W. SIPPEL. 1975. Placental and foetal metabolism of acetaldehyde in rat. I. Contents of ethanol and acetaldehyde in placenta and foetus of the pregnant rat during ethanol oxidation. Acta Pharmacol. Toxicol. 37: 43-48. 14. KOTKOSKIE, L. A., AND S. NORTON. 1988. Prenatal brain malformations following acute ethanol exposure in the rat. Alcoholism: Clin. Exp. Res. 12: 831-836. 15. MUKHERJEE, A. B., AND G. D. HODGEN. 1982. Maternal ethanol exposure induces transient impairment of umbilical circulation and fetal hypoxia in monkeys. Science 2 18: 700-702. 16. MYERS, R. E., R. BEARD, AND K. ADAMSONS. 1969. Brain swelling in the newborn rhesus monkey following prolonged partial asphyxia. Neurology 19: 1012-1018. 17. NOVER, L., D. HELLMUND, D. NEUMANN, K. D. SCHARF, AND E. SERFLING. 1984. The heat shock response of eukaryotic cells. Biol. Zentralbl. 103: 357-435. 18. PEIFFER, J., F. MAJEWSKI, H. FISCHBACH, J. R. BIERICH, AND B. VOLK. 1979. Alcohol embryo- and fetopathy: Neuropathology of 3 children and 3 fetuses. J. Neurol. Sci. 41: 125-137. 19. PIERCE, D. R., AND J. R. WEST. 1988. Alcohol-induced microencephaly during the third trimester equivalent: Relationship to dose and blood alcohol concentration. Alcohol 3: 185-191. 20. REIVICH, M., A. W. BRANN, JR., AND R. E. MYERS. 1972. Regional cerebral blood flow during prolonged partial asphyxia. Pages 216-227 in J. S. Meyer, M. Reivich, H. Lechner, and 0. Eichorn, Eds., Research on the Cerebral Circulation, Fifth Znternational Salzburg Conference. Thomas, Springfield, IL. 21. RITCHIE, J. M. 1985. The aliphatic alcohols. Pages 372-386 in A. G. Gilman, L. S. Goodman, T. W. Rall and F. Murad, Ede., Goodman and Gilman’s the Pharmacological Basis of Therapeutics, 7th ed. Macmillan, New York. 22. RUDEEN, K. L., AND C. GUERRI. 1985. The effects of alcohol exposure in utero on acetylcholinesterase, Na/K-ATPase and CaATPase activities in six regions of rat brain. Alcohol Alcohol. 20: 417-425. 23. SIEGEL, S. 1956. Nonparametric Statistics for the Behavioral Sciences. McGraw-Hill, New York.
106,283-288
(1989)
Morphometric Analysis of Developing Rat Cerebral Cortex following Acute Prenatal Ethanol Exposure ANDSTATANORTON
LOISA.KOTKOSKIE
University ofKansas Medical Center Department ofPharmacology, Toxicology, and Therapeutics, 39th Street and Rainbow Boulevard, Kansas City, Kansas 66103
Morphologic alterations of fetal rat cerebral cortex were quantified by morphometric analysis following acute ethanol exposure on Gestational Day 14, a critical period of development of cerebral cortex. Pregnant rats were intubated with a total dose of 9 g/kg of ethanol on Gestational Day 14. Maternal blood ethanol levels ranged from 51 to 202 mg% during the period of ethanol exposure. Fetal brains were examined on Gestational Day 15, 24 h after the last dose of ethanol. The acute morphologic changes associated with ethanol exposure include enlargement of subventricular zone nuclei, cortical swelling, and dilation of pial blood vessels over the affected cortex. In some fetuses, cortical swelling was accompanied by the protrusion into the lateral ventricles of cytoplasmic blebs of ventricular zone cells. It is concluded that maternal ethanol exposure during a critical period of brain development produces measurable morphologic changes in fetal rat cerebral cortex within 24 h after ethanol exposure. o 1st~ Academic Press,
Inc.
INTRODUCTION Ethanol ingestion during pregnancy has been associated with serious disturbances in fetal development including the fetal alcohol syndrome (10). Heterotopias and disorganization of brain structure resulting from widespread abnormalities in neuronal and glial migration have been reported in human neonates exposed to ethanol in utero (5,18). Recent studies have shown that the neuropathology of human fetuses from alcoholic mothers can be reproduced in rats and primates by acute ethanol exposure during critical periods of gestation (7,14). However, the morphologic changes in the developing brain immediately following acute ethanol exposure have not been well documented. The purpose of this study was to describe and quantify the morphologic changes occurring in fetal rat cerebral cortex 24 h after short-term maternal ethanol exposure. Ethanol was administered on Gestational Day 14, a critical period of development of the cerebral cortex (8,9). The formation of the cortical plate from neuroblasts migrating from the germinal matrix
begins about Gestational Day 14. Rapid proliferation of the germinal matrix begins following closure of the anterior neuropore by Gestational Day 11. Migration to form the cortical plate begins after the germinal matrix has grown from a layer of 1 cell to a layer lo-12 cells thick. This process has been analyzed in detail for the rat (2). MATERIALS
AND METHODS
Breeding and Ethanol Exposure Female, Sprague-Dawley-derived rats (CD Strain, Charles River), 225-325 g, were housed in a temperature-controlled room with a 12-h light/dark cycle and were fed Purina Rat Chow and water ad Zibitum. Females were bred with males of the same strain and the morning that a vaginal smear was positive for sperm was counted as Day 1 of gestation. Ten pregnant females were randomly assigned to control (n = 5) or ethanol (n = 5) groups. On Gestational Day 14, animals were dosed by gavage under light ether anesthesia. Animals in the ethanol-exposed group received 4.5 g/kg ethanol (80%) at 11:00 AM and 3:00 PM on Gestational Day 14 for a total dose of 9 g/kg ethanol. Blood ethanol levels were determined 2 h after each dose of ethanol from 70 111of tail blood (Sigma Kit 322-UV, Sigma Chemical Co., St. Louis, MO). To control for the stress of dosing, two control rats received 5% glucose at 11:00 AM and 3:00 PM on Gestational Day 14 in a volume equal to that received by the ethanolexposed animals. Three control rats were untreated. All control animals were restricted from food between 8:00 AM and 6:00 PM on Gestational Day 14 to control for the period of reduced food consumption associated with ethanol exposure. Histological Preparation of Tissues Pregnant animals were anesthetized with ether and decapitated at 3:00 PM on Gestational Day 1524 h after the last dose of ethanol or glucose. Fetuses were fixed in Karnovsky’s fixative (12). For light microscopy, fetal heads were postfixed in osmic acid and embedded in LR White acrylic resin. Tissue blocks were trimmed to the
283 Copyright 0 1989 A11 rights of reproduction
0014-48&S/89 $3.00 by Academic Press, Inc. in any form reserved.
KOTKOSKIE
AND
NORTON
and dehydrating with ethanol prior to critical point drying with carbon dioxide (Balzer 020 CPD). Dried brains were mounted on aluminum stubs and sputter coated with a thin conductive layer of gold/palladium alloy (Technic’s Hummer I). A JEOL-35 scanning electron microscope was used to examine the lateral ventricular surfaces of control and ethanol brains at accelerating voltages from 15 to 30 kV.
Morphometric
FIG. 1. a Gestational morphometric
Drawing of a transverse section through the forebrain of Day 15 rat showing (A) area of cerebral cortex used for analysis, (B) corpus striatum, and (Cl eye.
level of the telencephalon, sectioned at 2 pm, and stained with toluidine blue. Lateral ventricular surfaces of Gestational Day 15 brains were prepared for scanning electron microscopy by dissecting fixed brains to expose the lateral ventricles
FIG. 2. pial blood
Developing cerebral cortex vessels are dilated (arrowhead)
of Gestational Day and the ventricular
Methods
Morphometric analysis of Gestational Day 15 fetal cerebral cortex, using a-pm-thick sections, toluidine blue stained, was performed using a light microscope equipped with the ZIDAS optical system (Carl Zeiss, Inc.) for image analysis. The cortices of four to seven fetuses per litter were analyzed blind with respect to treatment groups. Zones of the developing cortex were identified following the Boulder conventions (3). In a 15day fetus, the ventricular zone consists of l- to 3-cell layers showing various mitotic figures and intervening stages of mitosis. Above this zone is the subventricular zone which is 8- to lo-cell layers in height and cells are more densely packed than in other zones. An area in the center of this zone, 5- to 6-cell layers in height and width, was selected for measurement of nuclei. Under low
15 rat fetuses. (A) Control. (B) Ethanol. margin has a ruffled appearance (arrow).
The cortical thickness Bar is 50 pm.
is increased,
the
ETHANOL
FIG. 3. fluid-filled
Developing blebs (arrows)
AND
THE
cerebral cortex of Gestational Day 15 rat fetuses along the ventricular margin. Bar is 25 pm.
power of the microscope the same cortical area was selected for measurement of each brain (Fig. 1, arrow A) in the same anterior-posterior location. The total number of nuclei present in 2500 pm2 of the subventricular zone in a section 2 pm thick varies from 25 to 30. About half of these have nucleoli. To measure those nuclei cut through the center of the nucleus, the 10 largest nuclei with one or more nucleoli were selected. The luminal areas in cross section of all pial blood vessels were determined along 1 mm of cortex (linear measurement along the ventricular margin). A sample size of 10 nuclear areas and 20-30 pial blood vessel areas per animal resulted in a standard error of less than 8%. Cortical thickness was measured at the same point on each fetal brain, as shown in Fig. 1A. The linear measurement was made
DEVELOPING
24 h after
285
BRAIN
acute ethanol
exposure
on Gestational
from the pial surface to the ventricular dicular to the cortical margins.
Day 14 demonstrating
surface, perpen-
Statistics Nuclear area was normally distributed and was analyzed by one-way ANOVA. Cortical thickness and pial blood vessel area data were not normally distributed and so were evaluated by the Kolmogorov-Smirnov, twosample, two-sided test (23). Because several variables were measured on each fetus, the CYlevel for statistical significance was set at P G 0.01. The relationship of maternal blood ethanol concentration with morphometric parameters was assessedby Spearman’s rank correlation coefficient.
286
FIG. 4. the surface
KOTKOSKIE
Scanning electron photomicrograph of the lateral ventricles. (B) Ethanol.
of lateral Microvilli
AND
NORTON
ventricular surfaces of Gestational Day 15 rat fetuses. (A) Control. Microvilli have been replaced by large protrusions of ventricular zone cells (X5200).
RESULTS
Blood ethanol levels ranged between 51 and 189 mg% 2 h after the first dose of ethanol and between 95 and 202 mg% 2 h after the last dose of ethanol on Gestational Day 14. Animals exposed to ethanol became ataxic for several hours following each dose of ethanol. No maternal respiratory depression was noted. All animals recovered and appeared healthy after the period of ethanol intoxication. Normal architecture of Gestational Day 15 fetal cerebral cortex is shown in Fig. 2. Ethanol administration on Gestational Day 14 did not cause cell death or alter the
line
laminar organization of immature neurons within developing cerebral cortex. However, 24 h following ethanol exposure on Gestational Day 14, swelling of the cerebral cortex and dilation of pial blood vessels were present in most ethanol-exposed fetuses (Fig. 2). Other areas of the telencephalon, such as the developing corpus striatum, appeared unaffected by acute ethanol exposure on Gestational Day 14. In many ethanol-exposed fetuses, the increase in cortical thickness was so great as to cause protrusion of ventricular zone cells into the lateral ventricles. This caused the ventricular margin of ethanol-exposed fetuses to assume a ruffled appearance (Fig. 2). Additionally, in sev-
’
TABLE
ETHANOL
AND
THE
1
Morphometric Analysis of Gestational Day 15 Fetal Rat Cerebral Cortex Group Control n=25(5)’ Ethanol n = 25 (5)
Nuclear area (pm’) Mean ? SE
Cortical thickness Mean (range)
30.4 + 0.4
125 (110-148)
47.4 + 0.71b
140 (110-187)’
’ Number of fetuses (number b Significantly different from ’ Significantly different from nov.
of litters). controls, control,
P < 0.01, P < 0.01,
(pm)
one-way ANOVA. Kolmogorov-Smir-
era1 ethanol-exposed fetuses ventricular zone cells bulged into the lateral ventricles and appeared as blebs along the ventricular margin in the light microscope (Fig. 3). The blebbing was so severe that microvilli lining the ventricular surface were seen in the scanning electron microscope as large round processes of the ventricular zone cells (Fig. 4). Morphometric measurements of Gestational Day 15 fetal cerebral cortex are given in Table 1 and Fig. 5. There were no statistical differences between untreated controls and glucose controls in any of the morphometric parameters measured. Therefore, both groups of control fetuses were combined into one control group. Cortical thickness and subventricular zone nuclear area were increased in ethanol-exposed fetuses compared with controls 24 h after ethanol exposure on Gestational Day 14. There were no significant correlations between maternal blood ethanol levels and nuclear area or cortical thickness. A histogram of pial blood vessel area (Fig. 5) shows that Gestational Day 15 control fetuses had many small vessels and few large vessels, while Gestational Day 15 ethanol-exposed fetuses had few small vessels and many large vessels in the same region above the developing cerebral cortex. Therefore, quantitative analysis of Gestational Day 15 cerebral cortex reveals that ethanol-exposed fetuses had markedly dilated pial blood vessels and swelling of developing cerebral cortex 24 h after acute ethanol exposure.
DEVELOPING
287
BRAIN
since it has been shown that ethanol freely crosses the placental barrier of the rat (13). Swelling of the cerebral cortex and dilation of pial blood vessels were observed in Gestational Day 15 fetuses following ethanol exposure on Gestational Day 14. These acute morphologic changes were present even though pregnant rats did not attain very high blood ethanol levels. It is possible that ethanol acted directly and/ or indirectly on the fetus to evoke morphologic changes. Ethanol has been proposed to act directly on cells to increase membrane fluidity and therefore affect the function of membrane-bound enzymes (21). Rudeen and Guerri (22) reported that chronic prenatal and postnatal ethanol exposure decreased the activities of Na/KATPase and Ca-ATPase, two membrane-bound enzymes necessary to maintain ionic gradients across neuronal cell membranes. A brief decrease in the activity of these and other membrane-bound enzymes following acute ethanol exposure could account for the cell swelling observed in this study. Several investigators have hypothesized that ethanol exerts some of its actions indirectly by causing fetal hypoxia (11,15). Experimentally produced fetal hypoxia in primates results in the same morphologic changes found in this study, specifically, swelling of the brain and dilation of pial blood vessels (16,20). Therefore, some of the morphologic changes observed in this study may be due to direct effects of ethanol and some to indirect effects of fetal hypoxia following ethanol exposure. In several ethanol-exposed fetuses, blebbing of ventricular zone cells into the lateral ventricle was found. It appeared that the cells within the cerebral cortex became so enlarged that the tissue reached its limits of expansion, and the only cells able to expand were those lining the ventricle. Blebbing of ventricular zone cells has also been described in fetal rats 24 h after exposure to 6-aminonicotinamide on Gestational Day 15 by Chamberlain (4). He hypothesized that the blebs secreted fluid from the cortex into the lateral ventricles to cause congenital hydrocephalus. In this study, the blebs
h
s
3Otl” 0 I
CONTROL ETHANOL’
DISCUSSION The administration of 9 g/kg of ethanol to pregnant rats on Gestational Day 14 resulted in slight maternal ethanol intoxication. Comparable human blood ethanol levels can be obtained after the ingestion of 3 oz of pure alcohol (1). The variability of maternal blood ethanol levels may have been due to differences in pharmacokinetic parameters such as the rate of absorption and biotransformation of ethanol. Fetal blood ethanol levels were probably the same as maternal blood ethanol levels,
PIAL BLOOD VESSEL
AREA (pm2)
FIG. 5. Pial blood vessel area distribution of Gestational Day 15 control and ethanol-exposed fetuses (n = 20 per group). Eight percent of ethanol values are greater than 800 Brn’. Control (494) and ethanol (446) distributions are significantly different, *P < 0.001, Kolmogorov-Smirnov.
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may function in the same manner to reduce cortical swelling after ethanol exposure. It is not known if hydrocephalus could be a long-term consequence of acute ethanol exposure on Gestational Day 14. Acute ethanol exposure on Gestational Day 14 was also associated with enlargement of subventricular zone nuclei 24 h after exposure. Nuclear enlargement may have occurred as a response involving increased protein synthesis for repair of ethanol-induced damage within the cell. For example, it has been shown that acute ethanol exposure increases synthesis of heat shock protein in rats (17). Other investigators have reported that the peak blood ethanol concentration threshold for producing brain damage in the rat (19) and the primate (6) was approximately 150 mg%. The results of this study support this concept because most of the maternal animals achieved blood ethanol levels near or above 150 mg%. However, we were unable to show a significant correlation between maternal blood ethanol levels and fetal outcome, probably because we could not use peak blood ethanol concentrations in our statistical analysis. The results of this study provide evidence that acute ethanol exposure in pregnant rats with blood ethanol levels at 200 mg% or less during a critical period of brain development induced measurable morphologic changes within the developing cerebral cortex. Cortical swelling and pial blood vessel dilation were present in ethanolexposed fetuses. Since cell death was not observed, these morphologic changes may be temporary in nature and may subside once cellular damage caused by ethanol has been repaired. However, the long-term morphologic and functional consequences of an acute alcoholic exposure at these blood levels on Gestational Day 14 are not known. ACKNOWLEDGMENTS This research was supported in part by Grants NS16694 and ES07079. L.A.K. was supported by a Procter & Gamble Fellowship.
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