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Ocular changes in the mouse embryo following acute maternal ethanol intoxication
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OCULAR CHANGES IN THE MOUSE EMBRYO FOLLOWING ACUTE MATERNAL ETHANOL INTOXICATION L. A. Department
of Pharmacology.
KENNEDY
and
M.
J. ELLIOT-I
Teratology Research Laboratory. College of Medicine, Saskatoon. Saskatchewan. Canada S7N OWO (Acwprrd
16 January
University
of Saskatchewan,
1986)
Abstract-The development of the eye was investigated in the mouse embryo following a single administration of ethanol plus [‘Hlthymidine to the dam on day I3 of gestation. After 1 hr there was no difference in the number of labelled cells/lott urn’ in the neural layer of the retina compared to controls. but there was an alcohol-related reduction in labelling density. After 24 hr there was an increase in the numbers of both pyknotic cells and mitotic figures. breaks occurred in the inner surface of the retina and cell debris was being extruded into the posterior chamber. At 48 hr the increase in pyknotic cells persisted, but there was less evidence of cell debris and the borders had been repaired. The estimated cell cycle time in the neural progenitor cells following maternal alcohol administration was increased 7-fold compared to controls. Morphometric analysis revealed that after 48 hr there were significant alcoholrelated reductions in the width and depth of the eye, in the thickness of the neural layer and in the interocular distance. It appears that many of the ophthalmic abnormalities reported in human fetal alcohol syndrome can be produced in the mouse embryo following a single episode of acute maternal intoxication during a critical period of ocular ontogeny, and that they evolve primarily from disturbances in the normal patterns of recruitment and loss of neural progenitor cells in the developing retina. Key Norris: Retinal
precursor
cells. Alcohol
embryopathy
The most obvious feature of the fetal alcohol syndrome (FAS) is a cluster of craniofacial abnormalities which include growth deficits as well as minor structural malformations of the lips, eyes and nose. Human studies have reported microphthalmia, narrow palpebral fissures, epicanthal folds, hypoteliorism, blepharophimosis, esotropia, atrophy of the disc, hypotrophy of the optic nerve and tortuosity of the retinal vessels. 4.h.X.20. Animal studies of FAS have shown that the typical craniofacial and brain malformations can be produced following one brief period of maternal alcohol intoxication during gastrulation. 23 Depending on the severity and the timing of alcohol intoxication relative to critical periods of development, abnormal patterns of neural cell proliferation, growth, differentiation and organization in many regions of the developing brain have been reported. Morphological and biochemical changes have been demonstrated which likely underlie the central nervous system dysfunctions frequently observed in the offspring of alcohol-consuming women. ‘JJ~‘~.‘~-‘~~~’ Although the eye develops as an outgrowth of the central nervous system, the pathogenesis of the ophthalmological abnormalities in FAS has not been studied. Using a combination of autoradiographical, histological and morphometric techniques, we have investigated the development of the eye in the mouse embryo during and following a single episode of maternal alcohol intoxication.
EXPERIMENTAL
PROCEDURES
Primiparous Swiss-Webster mice, age 8-12 weeks, were used in this experiment and were maintained under constant environmental conditions (21°C; 40-50% relative humidity; 12L 12D photoperiod). Day 1 of gestation was the day on which a copulation plug was found. There were six pregnant mice in the control group and six in the ethanol-treated group. On day 13 at T,,, each dam was administered a single injection (i.p.) of 0.5 ml (500 &i) [‘Hlthymidine (obtained from New England Nuclear) in combination with 0.22 ml ethanol (95% v/v) or water. Two dams were killed from each treatment group at time intervals of 1 hr (T,,+ 1 hr), 24 hr (T,,+24 hr) and 48 hr (T,,+48 hr) after the injection. Embryonic heads were placed in Bouin’s fixative for 1 hr. then placed in new fixative on a shaker table overnight. The heads were dissected the next morning as : Author
to whom correspondence
and reprint
requests should be addressed.
311
312
L.. A. Kennedy
and M. J. Elliott
shown in Fig. la, dehydrated in alcohol and embedded in methacrylate. Six heads were randomly selected from each treatment group for each of the three time intervals (a total of 18 heads from each treatment group) and processed as follows. Serial frontal sections (1 pm) beginning at the optic chiasma were placed on ultraclean slides. Because of the lateral orientation of the eye in the embryonic mouse, these frontal sections presented the structures normally seen in anteroposterior sections of adult human eye. On each slide there were four sections from an isotopetreated embryo and one control section from an isotope-untreated embryo to obtain background counts. Six slides/brain were dried on a hotplate (60°C) overnight and processed using routine procedures for liquid emulsion.‘7,‘2 After 2 weeks of exposure in light-tight boxes the slides were developed, dried, stained and mounted. Using an eyepiece micrometer, the eye on the section with the largest ocular dimensions was selected and used for all of the following analyses.
sterior chamber
Fig. I. (a) Embryonic heads were prcparcd for embedding and sectioning by removing the lower portion of the face with a frontal cut (F) and the lower portion of the head with a horizontal cut (H) through the mouth at right angles to F. (h) Frontal sections of the embryonic head provided an ‘antero-posterior‘ aspect of the eye seen in saggittal sections of the human adult eye. The lines demonstrate the distances measured for the morphometric analysis. The lot1 pm’ area of the neural layer used for the autoradiographical and histological analyses of the retina k indictttcd.
A morphometric analysis was performed using an eyepiece micrometer at x 400 magnification. The section was first oriented along a base line (AB) which bissected and was at right angles to the line joining the eyelids (Fig. lb). The distances measured were as follows: AB, the distance along the baseline between the outer limits of surface ectoderm and neural layer, or the horizontal depth of the eye; BC, the distance along AB between the outer and inner limits of the neural layer of the retina, or the thickness of the posterior neural layer; DE, the distance between the two outer limits of the pigment layer along a line bissecting and at right angles to AB, or the vertical depth of the eye; FG, the distance along DE between the two outer limits of the anterior portion of the neural layer; and HI, the distance along DE between the inner limits of the anterior neural layer, or the vertical depth of the posterior chamber. The mean thickness of the anterior portion of the neural layer was then calculated (FG-HI + 2). The interocular distance was the shortest distance between the two eyes on the section.
Ocular changes in the mouse embryo folIowing acute maternal ethanol intoxication
313
Histological and autoradiographical analyses were performed using an eyepiece micrometer at x 1000 magnification under oil in a 100 pm2 area of the neural layer of the retina (Fig. lb). This area was bordering on and at right angles to BC and began at the inner limit of the neural layer. The total numbers of nuclei, mitotic figures, pyknotic cells and labelled nuclei were counted in the area, and the corrected grain count per nucleus (mean number of grains/nucleus on the isotope-treated section minus the background counts as determined on the isotope-untreated section on the same slide) was calculated. The numbers of grains/nucleus was then plotted on a semi-log scale as a function of time after the injection. The period of time in which the mean grain count/nucleus was reduced to half its original (T,,+ 1 hr) value was determined from the regression line. Grain halving time (TgYz) was used as an indirect estimate of mean cell cycle time of the neural progenitor cells in the developing retina and is based on the assumption that the labelled DNA of the parent cell is distributed relatively equaily between the two daughter cells. As a result, the number of grains per nucleus is reduced to approximately half after each complete division and the overall decline in the numbers of grains/nucleus with time is therefore exponential. ” All data were analysed statistically for treatment-related differences using a one-way analysis of variance. RESULTS The administration of 0.22 ml of absolute ethanol mixed with 0.5 ml [“Hfthymidine is, in effect, a single administration of 0.72 ml of 29% (v/v) ethanol to each dam. This treatment resulted in a mean dose of 5.4 ml abs. EtOHlkg body weight on day 13 of gestation, and was associated with severe locomotor ataxia progressing to stupor, the occasional loss of righting reflex, and hypothermia. The period of severe intoxication lasted up to 1 hr and was followed by a quick recovery. In preliminary experiments with non-pregnant animals of the same strain, this dose resulted in blood alcohol levels in the range of 380 mg/dl, 30 min post-injection. The results of the histological and autoradiographical analyses are summarized in Table 1. At 1 hr post-injection (7’,,+ 1 hr) there was no difference between the treatment groups with respect to the total numbers of nuclei, pyknotic cells, mitotic figures or labelled cells in the 100 pm* area of the retina, but there was a significant reduction in the numbers of grains per labelled nucleus. Twenty-four hours post-injection (T,,+24 hr), there was a significant increase in the numbers of mitotic figures and pyknotic cells in the retinas of embryos obtained from ethanol-treated dams compared to controls. histologically, the continuity of the inner surface of the retina was disrupted and cell debris was being extruded into the posterior chamber. This appeared to be due to rupture of the neural cell membranes. There was no difference in the total numbers of nuclei or labelled cells in the area, or in the labelling density (grains/nucleus). Forty-eight hours after the injection (7’,,+48 hr) the alcohol-related increase in pyknotic cells persisted and there was a tendency, which was not statistically significant (0.05
314
L.
A. Kennedy and M. .I. Elliott
Table 1. Changing patterns of neural cell proliferation and death in the retina of the embryonic mouse following acute maternal ethanol administration. Values presented are mean numbers/ 100 pm’with S.E.M. in parentheses Autoradioyraphy
Histology Mitotic figures
Pyknotic cells
Lahelled cells (‘X,)
Grains/ l~~belledcell
34.3 (3.Y)
(I
51.0 (2.X)
IX.‘, (0.3)
141.7
36.7
0
(6.9
(4.Y)
a.7 (6.X)
13.0 (0.5))
Treatment group
Nuelci
T,,tI hr Control (N= l:11=3)’
IJO.
EtOH. 5.8 ml/kg (N= l.tr=h) Fs(f.7)
(X.4)
0.01 NS
0.0X NS
_
0. IY NS
15.5 I”cCO.01
T,;t24hr
I .o
Control
126.X
53.3
43.5
1(1.1!
(N=7:u=h)
(13.3)
(2.0)
(0.3)
(2.4)
(0.7)
EtOH. 5.3 ml/kg (N=Z:rz=h)
119.7 (5.6)
70.3 (4.3)
1Y.2 (3.1)
-II.’ (1.7)
Y“ (0 7)
0.5 NS
1h NS
Fs(l,
If))
T,,-I-~X hr Control (N-Z:,t=h) &OH. 5.5 ml/kg (N= I.r?=h) Fs (I. 10)
(I.2 NS
10.7 1’<0.01
7X.2 /‘<0.00I
151.7 (15.2)
102.7 (Y.2)
0.5 (0.-1,
3.6 (1.6)
8.3 (0.3)
110.5 (12.5)
X5.X (3.7)
15.0 (5.X)
24.5 (3.3)
X.4 (0.3)
3.6 NS
2.0 NS
5.1 1’<0.05
,’ N = number of dams from which the embryonic number of embryonic heads included in the analysis.
heads analysed
0.06 NS were taken;
O.l)l NS II = total
the developing brain, little attention has been given to the nature or the pathogenesis of the ophthalmologic abnormalities. During ontogeny, all organ systems pass through periods of very rapid development which are considered to be once-only opportunities for growth and during which development is exquisitely vulnerable to disturbance. Derangements in the processes of cell replication or cell death during the critical period of ocular ontogeny could irreversibly influence the ultimate size, organization and function of the eye. Using a combination of autoradiographical, histological and morphometric techniques, we have studied the patterns of neural cell proliferation and death in the eye of the mouse embryo during and following a single episode of maternal alcohol intoxication. The animal model we used in this experiment was designed to mimic a ‘binge’ abuse of alcohol. Because of the rapid rate of development in the mouse embryo, the exposure period used would actually span a longer period of human development. The equivalent developmental age in humans would occur during the sixth week of pregnancy, or between 44 and 48 days postconception.‘” During this developmental period (stage 21) the lens vesicle has lost its lumen and is a solid sphere, and the vitreous body is extremely small. In the retina. neural progenitor cells are proliferating rapidly and differentiation is beginning. A layer of nerve fibres is seen over the retina but the nuclear layers of the retina are largely indistinguishable.~~ Disturbances of devetopmental processes during this critical period of organogenesis frequently result in congenital malformation of the eye.” At 1 hr post-injection, the dams were recovering from the intoxication but, because of the kinetics of amniotic fluid circulation, the embryo would still be exposed to higher levels than those seen in the maternal blood.’ As might be expected, there was no difference in the cell density (nuclei/lO~) Fm’) of the neural layer so soon after treatment. There was no treatmentrelated difference in the numbers of labelled cells, pyknotic cells or mitotic figures, and no detectable histological change. Since hypoxia-related changes in the fetal brain can be detected hisfetal hypoxia was a major tologically as early as 15 min, ’ it is unlikely that acute alcohol-related
Ocular Table
changes in the mouse embryo
2. Morphometric administration.
following
acute maternal
ethanol
315
intoxication
changes in the eye of the mouse embryo following acute maternal Values presented are mean distances (urn) with S.E.M. in parentheses
ethanol
Intraoculardimensions Total horizontal depth
Total vertical depth
Depth of posterior chamber
Depth of posterior neural layer
Depth of anterior neural layer
Interocular distance
461.7 (20.3)
593.3 (5.4)
320.11
143.7 (11.9)
116.7 (3.6)
2426.7 (87.6)
44.5 .o (16.1)
s15.0 (33.6)
280.X (54.4)
141.7
102.3 (12.5)
2111.7 (42.2)
0.3 NS
2. I NS
0.2 NS
Control (N=2,11=6)
s71.7 (17.6)
635.8 (27.2)
326.7 (47. I)
154.2 (13.3)
EtOH. 5.3 ml/kg (N=2:,1=6)
547.5 (34.0)
646.7 (44.7)
36X.0 (37.6) 0.3 NS
l.Y NS
2.3 NS
Treatment group T,,+ 1hr Control (IV= l:ri=3)” EtOH. S.gml/kg (N= l:,1=h) Fs(1.7)
(8.1)
(9.6) 0.01 NS
0,s NS
10.4 P
T,,+24 hr
Fs(1.
0.04 NS
0.3 NS
IO)
T<,,+-tX hr Control (N=2;,1=6) EtOH. 5.5 ml/kg (N= I:rt=6) Fs(I.10)
2393.3 (96.7)
128.0
Il6.0
(8.7)
(X.6)
2425.0 (84.8) 0.05 NS
769.2 (43.8)
x1x.3 (47.9)
448.3 (62.9)
IXO.0 (15.1)
167.5 (7.3)
273X.3 (X2.1)
536.7 (33.9)
685.0 (40. 1)
348.3 (23.6)
12x.3 (11.4)
125,s (13.3)
2310.0 (59.2)
14.6 Pto.ot)s
3.x NS
I.9 NS
:’ N= number of dams from which the embryonic embryonic heads included in the analysis.
%
141.7 (11.5)
6.0 P
heads analyzed
64 PiO.05
were taken;
14.Y P
,I= total number
of
5-
%
I I I
/ I
0 1
2
3
4 5676910
20
30 4050
100
200 300
Hours after treatment Fig. 2. The decline over time in the mean number of grains/nu~ieus in the neural layer of the retina in control (--O--) and ethanol-treated (-A-) mouse embryos. Grain halving time (T&h) is the point on the regression line at which the mean number of grains/nucleus is reduced to half and indicates the time theoretically required for this halving to occur in each treatment group.
to our subsequent results. There was, however, an ethanol-related reduction (31%) in labelling density (grains/nucleus). Since the numbers of mitotic figures and labelled cells (and thus the number of dividing cells) were not reduced and since in preliminary experiments we found that by halving the amount of isotope injected, we halved the grain count/nucleus, it is possible that this observation reflects less isotope being available to the dividing cells. This could result from stasis in the peripheral and utero-placental circulation or placental dysfunction, both of which have been shown to occur during ethanol exposure,7,‘2.‘y or less likely, from changes in isotope absorption due to the presence of aicohol. At 24 hr after treatment, there was no difference in cell density in the retina, but there was a dramatic (19-fold) increase in the numbers of contributor
316
L.
A.
Kennedy and
M.
J.
Elliott
pyknotic cells as well as histological evidence of tissue damage, with rupture of ceil membranes and cell debris being extruded into the posterior chamber. These observations indicate an ethanol-related increase in neural cell death and cell loss. It is unknown whether these changes result from the direct effects of alcohol or are secondary to narcosis, hypothermia, impaired utero-placental circulation, and/or embryofetal hypoxia and acidosis, ail of which have been “.‘2.iy There was again no differshown to occur foliowing acute maternal alcohol intoxication. ence in the numbers of labeiled cells and the reduction in the labeliing density seen at 1 hr was virtually eliminated by 24 hr, possibly the result of grain dilution with progressive cell replications. Forty-eight hours after treatment, the increase in cell pyknosis remained, but histologically it appeared that repair processes were already contributing to recovery. There was a strong tendency, which was not statistically signi~cant (0.05 < P
Ocular
changes in the mouse embryo
following
acute maternal
ethanol
intoxication
317
REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. IS. 16. 17. lg. 19. 20. 21. 22. 23. 24.
Bauer-Moffett C. and Altman .I. (1977) The effect of ethanol chronically administered to pre-weanling rats on cerebellar development. A morphological study. Bruin Res. 119, 249-268. Brien J. F.. Loomis C. W.. Tranmer J. and MacGrath M. (1983) Disposition of ethanol in human maternal venous blood and amniotic fluid. Am. J. Obstet. Gynecol. 146, 181-186. 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 Alcohol Depend. 2, 421429. Gonzalez E. R. (1981) New ophthalmic findings in fetal alcohol syndrome. J.A.M.A. 245, 108. Hammer R. P. and Schiebel A. B. (1981) Morphologic evidence for a delay in neuronal maturation in fetal alcohol exposure. Exp. Neurobiol. 74, S87-S96. Hanson J. W., Jones K. L. and Smith D. W. (1976) Fetal alcohol syndrome: experience with 41 patients. J.A.M.A. 235, 145%1460. Henderson G. I.. Patwardhan R. V.. McLeroy S. and Schenker S. (1982) Inhibition of placental amino acid uptake in rats following acute and chronic ethanol exposure. Alcoholism 6, 495-505. Jones K. L., Smith D. W. and Hanson J. W. (1976) The fetal alcohol syndrome: clinical delineation. Ann. N.Y. Amd. SC;. 273, 130-137. Kennedy L. A. and Persaud T. V. N. (1977) Pathogenesis of developmental defects induced in the rat by amniotic sac puncture. Acta Anat. 97, 23-35. Kennedy L. A. (1983) Changes in the proliferative cells in the fetal mouse neopallium associated with maternal alcohol consumption. /tn. J. devl Neurosci. 1, 234 (Abstract). Kennedy L. A. (1984) The pathogenesis of brain malformations in the fetal alcohol syndrome. An integrating hypothesis. Teratology 29, 36>368. Kennedy L. A. (1984) Changes in the term mouse placenta associated with alcohol consumption and fetal growth deficits. Am. J. Obstet. Gynecol. 149, 5112-522. 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 consumptions. fttt. J. devl Neurosci. 2, 437446. Kennedy L. A. and Elliott M. J. (1985) Ceil proliferation in the embryonic mouse neocortex following acute maternal alcohol intoxication. htt. J. devl Neurosci. 3, 31 I-315. Kennedy L. A. and Mukerji S. (1986) Ethanol neurotoxicity. 1. Direct effects on replicating astrocytes. Neurobehav. Toxicol. Teratol. 8. Kennedy L. A. and Mukerji S. (1986) Ethanol neurotoxicity. 2. Direct effects on differentiating astrocytes. Neurobehav. Toxicol. Teratol. 8. Korr H., Schultze B. and Maurer W. (197.5) Autoradiographic investigations of glial proliferation in the brain of adult mice. J. camp. Neural. 160, 477-490. Moore K. L. (1982) The Developing Human: Clinically Oriented Embryology. W. B. Saunders Co.. Toronto, p. 152. Mukerjee A. B. and Hodgen G. D. (1982) Maternal ethanol exposure induces transient impairment of umbilical circulation and fetal hypoxia in monkeys. Science 218, 700-702. Rabinowicz I. M. (1980) Ophthalmologic findings in fetal alcohol syndrome. Ophrhalmologica 87 (Suppl.), 93. Rawatt A. (1976) Ribosomal protein synthesis in the fetal and neonatal rat brain as influenced by maternal alcohol consumption. Res. Commun. them. Pathol. Pharmac. 12, 723-732. Sidman R. L. (1961) Histogenesis of mouse retina studied with [‘HI-thymidine. In The Sfructure of the Eye (ed. Smelser G. K.). pp. 487-505. Academic Press. New York. Sulik K. K., Lauder J. M. and Dehart D. (1984) Brain malformations in prenatal mice following acute maternal ethanol administration. Int. J. devl Neurosci. 2, 203-214. Theiler K. (1972) The House Mouse, pp. 94-99. Springer-Verlag, New York.
OCULAR CHANGES IN THE MOUSE EMBRYO FOLLOWING ACUTE MATERNAL ETHANOL INTOXICATION L. A. Department
of Pharmacology.
KENNEDY
and
M.
J. ELLIOT-I
Teratology Research Laboratory. College of Medicine, Saskatoon. Saskatchewan. Canada S7N OWO (Acwprrd
16 January
University
of Saskatchewan,
1986)
Abstract-The development of the eye was investigated in the mouse embryo following a single administration of ethanol plus [‘Hlthymidine to the dam on day I3 of gestation. After 1 hr there was no difference in the number of labelled cells/lott urn’ in the neural layer of the retina compared to controls. but there was an alcohol-related reduction in labelling density. After 24 hr there was an increase in the numbers of both pyknotic cells and mitotic figures. breaks occurred in the inner surface of the retina and cell debris was being extruded into the posterior chamber. At 48 hr the increase in pyknotic cells persisted, but there was less evidence of cell debris and the borders had been repaired. The estimated cell cycle time in the neural progenitor cells following maternal alcohol administration was increased 7-fold compared to controls. Morphometric analysis revealed that after 48 hr there were significant alcoholrelated reductions in the width and depth of the eye, in the thickness of the neural layer and in the interocular distance. It appears that many of the ophthalmic abnormalities reported in human fetal alcohol syndrome can be produced in the mouse embryo following a single episode of acute maternal intoxication during a critical period of ocular ontogeny, and that they evolve primarily from disturbances in the normal patterns of recruitment and loss of neural progenitor cells in the developing retina. Key Norris: Retinal
precursor
cells. Alcohol
embryopathy
The most obvious feature of the fetal alcohol syndrome (FAS) is a cluster of craniofacial abnormalities which include growth deficits as well as minor structural malformations of the lips, eyes and nose. Human studies have reported microphthalmia, narrow palpebral fissures, epicanthal folds, hypoteliorism, blepharophimosis, esotropia, atrophy of the disc, hypotrophy of the optic nerve and tortuosity of the retinal vessels. 4.h.X.20. Animal studies of FAS have shown that the typical craniofacial and brain malformations can be produced following one brief period of maternal alcohol intoxication during gastrulation. 23 Depending on the severity and the timing of alcohol intoxication relative to critical periods of development, abnormal patterns of neural cell proliferation, growth, differentiation and organization in many regions of the developing brain have been reported. Morphological and biochemical changes have been demonstrated which likely underlie the central nervous system dysfunctions frequently observed in the offspring of alcohol-consuming women. ‘JJ~‘~.‘~-‘~~~’ Although the eye develops as an outgrowth of the central nervous system, the pathogenesis of the ophthalmological abnormalities in FAS has not been studied. Using a combination of autoradiographical, histological and morphometric techniques, we have investigated the development of the eye in the mouse embryo during and following a single episode of maternal alcohol intoxication.
EXPERIMENTAL
PROCEDURES
Primiparous Swiss-Webster mice, age 8-12 weeks, were used in this experiment and were maintained under constant environmental conditions (21°C; 40-50% relative humidity; 12L 12D photoperiod). Day 1 of gestation was the day on which a copulation plug was found. There were six pregnant mice in the control group and six in the ethanol-treated group. On day 13 at T,,, each dam was administered a single injection (i.p.) of 0.5 ml (500 &i) [‘Hlthymidine (obtained from New England Nuclear) in combination with 0.22 ml ethanol (95% v/v) or water. Two dams were killed from each treatment group at time intervals of 1 hr (T,,+ 1 hr), 24 hr (T,,+24 hr) and 48 hr (T,,+48 hr) after the injection. Embryonic heads were placed in Bouin’s fixative for 1 hr. then placed in new fixative on a shaker table overnight. The heads were dissected the next morning as : Author
to whom correspondence
and reprint
requests should be addressed.
311
312
L.. A. Kennedy
and M. J. Elliott
shown in Fig. la, dehydrated in alcohol and embedded in methacrylate. Six heads were randomly selected from each treatment group for each of the three time intervals (a total of 18 heads from each treatment group) and processed as follows. Serial frontal sections (1 pm) beginning at the optic chiasma were placed on ultraclean slides. Because of the lateral orientation of the eye in the embryonic mouse, these frontal sections presented the structures normally seen in anteroposterior sections of adult human eye. On each slide there were four sections from an isotopetreated embryo and one control section from an isotope-untreated embryo to obtain background counts. Six slides/brain were dried on a hotplate (60°C) overnight and processed using routine procedures for liquid emulsion.‘7,‘2 After 2 weeks of exposure in light-tight boxes the slides were developed, dried, stained and mounted. Using an eyepiece micrometer, the eye on the section with the largest ocular dimensions was selected and used for all of the following analyses.
sterior chamber
Fig. I. (a) Embryonic heads were prcparcd for embedding and sectioning by removing the lower portion of the face with a frontal cut (F) and the lower portion of the head with a horizontal cut (H) through the mouth at right angles to F. (h) Frontal sections of the embryonic head provided an ‘antero-posterior‘ aspect of the eye seen in saggittal sections of the human adult eye. The lines demonstrate the distances measured for the morphometric analysis. The lot1 pm’ area of the neural layer used for the autoradiographical and histological analyses of the retina k indictttcd.
A morphometric analysis was performed using an eyepiece micrometer at x 400 magnification. The section was first oriented along a base line (AB) which bissected and was at right angles to the line joining the eyelids (Fig. lb). The distances measured were as follows: AB, the distance along the baseline between the outer limits of surface ectoderm and neural layer, or the horizontal depth of the eye; BC, the distance along AB between the outer and inner limits of the neural layer of the retina, or the thickness of the posterior neural layer; DE, the distance between the two outer limits of the pigment layer along a line bissecting and at right angles to AB, or the vertical depth of the eye; FG, the distance along DE between the two outer limits of the anterior portion of the neural layer; and HI, the distance along DE between the inner limits of the anterior neural layer, or the vertical depth of the posterior chamber. The mean thickness of the anterior portion of the neural layer was then calculated (FG-HI + 2). The interocular distance was the shortest distance between the two eyes on the section.
Ocular changes in the mouse embryo folIowing acute maternal ethanol intoxication
313
Histological and autoradiographical analyses were performed using an eyepiece micrometer at x 1000 magnification under oil in a 100 pm2 area of the neural layer of the retina (Fig. lb). This area was bordering on and at right angles to BC and began at the inner limit of the neural layer. The total numbers of nuclei, mitotic figures, pyknotic cells and labelled nuclei were counted in the area, and the corrected grain count per nucleus (mean number of grains/nucleus on the isotope-treated section minus the background counts as determined on the isotope-untreated section on the same slide) was calculated. The numbers of grains/nucleus was then plotted on a semi-log scale as a function of time after the injection. The period of time in which the mean grain count/nucleus was reduced to half its original (T,,+ 1 hr) value was determined from the regression line. Grain halving time (TgYz) was used as an indirect estimate of mean cell cycle time of the neural progenitor cells in the developing retina and is based on the assumption that the labelled DNA of the parent cell is distributed relatively equaily between the two daughter cells. As a result, the number of grains per nucleus is reduced to approximately half after each complete division and the overall decline in the numbers of grains/nucleus with time is therefore exponential. ” All data were analysed statistically for treatment-related differences using a one-way analysis of variance. RESULTS The administration of 0.22 ml of absolute ethanol mixed with 0.5 ml [“Hfthymidine is, in effect, a single administration of 0.72 ml of 29% (v/v) ethanol to each dam. This treatment resulted in a mean dose of 5.4 ml abs. EtOHlkg body weight on day 13 of gestation, and was associated with severe locomotor ataxia progressing to stupor, the occasional loss of righting reflex, and hypothermia. The period of severe intoxication lasted up to 1 hr and was followed by a quick recovery. In preliminary experiments with non-pregnant animals of the same strain, this dose resulted in blood alcohol levels in the range of 380 mg/dl, 30 min post-injection. The results of the histological and autoradiographical analyses are summarized in Table 1. At 1 hr post-injection (7’,,+ 1 hr) there was no difference between the treatment groups with respect to the total numbers of nuclei, pyknotic cells, mitotic figures or labelled cells in the 100 pm* area of the retina, but there was a significant reduction in the numbers of grains per labelled nucleus. Twenty-four hours post-injection (T,,+24 hr), there was a significant increase in the numbers of mitotic figures and pyknotic cells in the retinas of embryos obtained from ethanol-treated dams compared to controls. histologically, the continuity of the inner surface of the retina was disrupted and cell debris was being extruded into the posterior chamber. This appeared to be due to rupture of the neural cell membranes. There was no difference in the total numbers of nuclei or labelled cells in the area, or in the labelling density (grains/nucleus). Forty-eight hours after the injection (7’,,+48 hr) the alcohol-related increase in pyknotic cells persisted and there was a tendency, which was not statistically significant (0.05
314
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A. Kennedy and M. .I. Elliott
Table 1. Changing patterns of neural cell proliferation and death in the retina of the embryonic mouse following acute maternal ethanol administration. Values presented are mean numbers/ 100 pm’with S.E.M. in parentheses Autoradioyraphy
Histology Mitotic figures
Pyknotic cells
Lahelled cells (‘X,)
Grains/ l~~belledcell
34.3 (3.Y)
(I
51.0 (2.X)
IX.‘, (0.3)
141.7
36.7
0
(6.9
(4.Y)
a.7 (6.X)
13.0 (0.5))
Treatment group
Nuelci
T,,tI hr Control (N= l:11=3)’
IJO.
EtOH. 5.8 ml/kg (N= l.tr=h) Fs(f.7)
(X.4)
0.01 NS
0.0X NS
_
0. IY NS
15.5 I”cCO.01
T,;t24hr
I .o
Control
126.X
53.3
43.5
1(1.1!
(N=7:u=h)
(13.3)
(2.0)
(0.3)
(2.4)
(0.7)
EtOH. 5.3 ml/kg (N=Z:rz=h)
119.7 (5.6)
70.3 (4.3)
1Y.2 (3.1)
-II.’ (1.7)
Y“ (0 7)
0.5 NS
1h NS
Fs(l,
If))
T,,-I-~X hr Control (N-Z:,t=h) &OH. 5.5 ml/kg (N= I.r?=h) Fs (I. 10)
(I.2 NS
10.7 1’<0.01
7X.2 /‘<0.00I
151.7 (15.2)
102.7 (Y.2)
0.5 (0.-1,
3.6 (1.6)
8.3 (0.3)
110.5 (12.5)
X5.X (3.7)
15.0 (5.X)
24.5 (3.3)
X.4 (0.3)
3.6 NS
2.0 NS
5.1 1’<0.05
,’ N = number of dams from which the embryonic number of embryonic heads included in the analysis.
heads analysed
0.06 NS were taken;
O.l)l NS II = total
the developing brain, little attention has been given to the nature or the pathogenesis of the ophthalmologic abnormalities. During ontogeny, all organ systems pass through periods of very rapid development which are considered to be once-only opportunities for growth and during which development is exquisitely vulnerable to disturbance. Derangements in the processes of cell replication or cell death during the critical period of ocular ontogeny could irreversibly influence the ultimate size, organization and function of the eye. Using a combination of autoradiographical, histological and morphometric techniques, we have studied the patterns of neural cell proliferation and death in the eye of the mouse embryo during and following a single episode of maternal alcohol intoxication. The animal model we used in this experiment was designed to mimic a ‘binge’ abuse of alcohol. Because of the rapid rate of development in the mouse embryo, the exposure period used would actually span a longer period of human development. The equivalent developmental age in humans would occur during the sixth week of pregnancy, or between 44 and 48 days postconception.‘” During this developmental period (stage 21) the lens vesicle has lost its lumen and is a solid sphere, and the vitreous body is extremely small. In the retina. neural progenitor cells are proliferating rapidly and differentiation is beginning. A layer of nerve fibres is seen over the retina but the nuclear layers of the retina are largely indistinguishable.~~ Disturbances of devetopmental processes during this critical period of organogenesis frequently result in congenital malformation of the eye.” At 1 hr post-injection, the dams were recovering from the intoxication but, because of the kinetics of amniotic fluid circulation, the embryo would still be exposed to higher levels than those seen in the maternal blood.’ As might be expected, there was no difference in the cell density (nuclei/lO~) Fm’) of the neural layer so soon after treatment. There was no treatmentrelated difference in the numbers of labelled cells, pyknotic cells or mitotic figures, and no detectable histological change. Since hypoxia-related changes in the fetal brain can be detected hisfetal hypoxia was a major tologically as early as 15 min, ’ it is unlikely that acute alcohol-related
Ocular Table
changes in the mouse embryo
2. Morphometric administration.
following
acute maternal
ethanol
315
intoxication
changes in the eye of the mouse embryo following acute maternal Values presented are mean distances (urn) with S.E.M. in parentheses
ethanol
Intraoculardimensions Total horizontal depth
Total vertical depth
Depth of posterior chamber
Depth of posterior neural layer
Depth of anterior neural layer
Interocular distance
461.7 (20.3)
593.3 (5.4)
320.11
143.7 (11.9)
116.7 (3.6)
2426.7 (87.6)
44.5 .o (16.1)
s15.0 (33.6)
280.X (54.4)
141.7
102.3 (12.5)
2111.7 (42.2)
0.3 NS
2. I NS
0.2 NS
Control (N=2,11=6)
s71.7 (17.6)
635.8 (27.2)
326.7 (47. I)
154.2 (13.3)
EtOH. 5.3 ml/kg (N=2:,1=6)
547.5 (34.0)
646.7 (44.7)
36X.0 (37.6) 0.3 NS
l.Y NS
2.3 NS
Treatment group T,,+ 1hr Control (IV= l:ri=3)” EtOH. S.gml/kg (N= l:,1=h) Fs(1.7)
(8.1)
(9.6) 0.01 NS
0,s NS
10.4 P
T,,+24 hr
Fs(1.
0.04 NS
0.3 NS
IO)
T<,,+-tX hr Control (N=2;,1=6) EtOH. 5.5 ml/kg (N= I:rt=6) Fs(I.10)
2393.3 (96.7)
128.0
Il6.0
(8.7)
(X.6)
2425.0 (84.8) 0.05 NS
769.2 (43.8)
x1x.3 (47.9)
448.3 (62.9)
IXO.0 (15.1)
167.5 (7.3)
273X.3 (X2.1)
536.7 (33.9)
685.0 (40. 1)
348.3 (23.6)
12x.3 (11.4)
125,s (13.3)
2310.0 (59.2)
14.6 Pto.ot)s
3.x NS
I.9 NS
:’ N= number of dams from which the embryonic embryonic heads included in the analysis.
%
141.7 (11.5)
6.0 P
heads analyzed
64 PiO.05
were taken;
14.Y P
,I= total number
of
5-
%
I I I
/ I
0 1
2
3
4 5676910
20
30 4050
100
200 300
Hours after treatment Fig. 2. The decline over time in the mean number of grains/nu~ieus in the neural layer of the retina in control (--O--) and ethanol-treated (-A-) mouse embryos. Grain halving time (T&h) is the point on the regression line at which the mean number of grains/nucleus is reduced to half and indicates the time theoretically required for this halving to occur in each treatment group.
to our subsequent results. There was, however, an ethanol-related reduction (31%) in labelling density (grains/nucleus). Since the numbers of mitotic figures and labelled cells (and thus the number of dividing cells) were not reduced and since in preliminary experiments we found that by halving the amount of isotope injected, we halved the grain count/nucleus, it is possible that this observation reflects less isotope being available to the dividing cells. This could result from stasis in the peripheral and utero-placental circulation or placental dysfunction, both of which have been shown to occur during ethanol exposure,7,‘2.‘y or less likely, from changes in isotope absorption due to the presence of aicohol. At 24 hr after treatment, there was no difference in cell density in the retina, but there was a dramatic (19-fold) increase in the numbers of contributor
316
L.
A.
Kennedy and
M.
J.
Elliott
pyknotic cells as well as histological evidence of tissue damage, with rupture of ceil membranes and cell debris being extruded into the posterior chamber. These observations indicate an ethanol-related increase in neural cell death and cell loss. It is unknown whether these changes result from the direct effects of alcohol or are secondary to narcosis, hypothermia, impaired utero-placental circulation, and/or embryofetal hypoxia and acidosis, ail of which have been “.‘2.iy There was again no differshown to occur foliowing acute maternal alcohol intoxication. ence in the numbers of labeiled cells and the reduction in the labeliing density seen at 1 hr was virtually eliminated by 24 hr, possibly the result of grain dilution with progressive cell replications. Forty-eight hours after treatment, the increase in cell pyknosis remained, but histologically it appeared that repair processes were already contributing to recovery. There was a strong tendency, which was not statistically signi~cant (0.05 < P
Ocular
changes in the mouse embryo
following
acute maternal
ethanol
intoxication
317
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Bauer-Moffett C. and Altman .I. (1977) The effect of ethanol chronically administered to pre-weanling rats on cerebellar development. A morphological study. Bruin Res. 119, 249-268. Brien J. F.. Loomis C. W.. Tranmer J. and MacGrath M. (1983) Disposition of ethanol in human maternal venous blood and amniotic fluid. Am. J. Obstet. Gynecol. 146, 181-186. 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 Alcohol Depend. 2, 421429. Gonzalez E. R. (1981) New ophthalmic findings in fetal alcohol syndrome. J.A.M.A. 245, 108. Hammer R. P. and Schiebel A. B. (1981) Morphologic evidence for a delay in neuronal maturation in fetal alcohol exposure. Exp. Neurobiol. 74, S87-S96. Hanson J. W., Jones K. L. and Smith D. W. (1976) Fetal alcohol syndrome: experience with 41 patients. J.A.M.A. 235, 145%1460. Henderson G. I.. Patwardhan R. V.. McLeroy S. and Schenker S. (1982) Inhibition of placental amino acid uptake in rats following acute and chronic ethanol exposure. Alcoholism 6, 495-505. Jones K. L., Smith D. W. and Hanson J. W. (1976) The fetal alcohol syndrome: clinical delineation. Ann. N.Y. Amd. SC;. 273, 130-137. Kennedy L. A. and Persaud T. V. N. (1977) Pathogenesis of developmental defects induced in the rat by amniotic sac puncture. Acta Anat. 97, 23-35. Kennedy L. A. (1983) Changes in the proliferative cells in the fetal mouse neopallium associated with maternal alcohol consumption. /tn. J. devl Neurosci. 1, 234 (Abstract). Kennedy L. A. (1984) The pathogenesis of brain malformations in the fetal alcohol syndrome. An integrating hypothesis. Teratology 29, 36>368. Kennedy L. A. (1984) Changes in the term mouse placenta associated with alcohol consumption and fetal growth deficits. Am. J. Obstet. Gynecol. 149, 5112-522. 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 consumptions. fttt. J. devl Neurosci. 2, 437446. Kennedy L. A. and Elliott M. J. (1985) Ceil proliferation in the embryonic mouse neocortex following acute maternal alcohol intoxication. htt. J. devl Neurosci. 3, 31 I-315. Kennedy L. A. and Mukerji S. (1986) Ethanol neurotoxicity. 1. Direct effects on replicating astrocytes. Neurobehav. Toxicol. Teratol. 8. Kennedy L. A. and Mukerji S. (1986) Ethanol neurotoxicity. 2. Direct effects on differentiating astrocytes. Neurobehav. Toxicol. Teratol. 8. Korr H., Schultze B. and Maurer W. (197.5) Autoradiographic investigations of glial proliferation in the brain of adult mice. J. camp. Neural. 160, 477-490. Moore K. L. (1982) The Developing Human: Clinically Oriented Embryology. W. B. Saunders Co.. Toronto, p. 152. Mukerjee A. B. and Hodgen G. D. (1982) Maternal ethanol exposure induces transient impairment of umbilical circulation and fetal hypoxia in monkeys. Science 218, 700-702. Rabinowicz I. M. (1980) Ophthalmologic findings in fetal alcohol syndrome. Ophrhalmologica 87 (Suppl.), 93. Rawatt A. (1976) Ribosomal protein synthesis in the fetal and neonatal rat brain as influenced by maternal alcohol consumption. Res. Commun. them. Pathol. Pharmac. 12, 723-732. Sidman R. L. (1961) Histogenesis of mouse retina studied with [‘HI-thymidine. In The Sfructure of the Eye (ed. Smelser G. K.). pp. 487-505. Academic Press. New York. Sulik K. K., Lauder J. M. and Dehart D. (1984) Brain malformations in prenatal mice following acute maternal ethanol administration. Int. J. devl Neurosci. 2, 203-214. Theiler K. (1972) The House Mouse, pp. 94-99. Springer-Verlag, New York.