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A contribution to the postnatal enzymatic state of maturity of gyrus hippocampi in the embryofetal alcohol syndrome of the rat
Exp. Path.1S, 170-174 (1980) Institute of Pathology, Medical Academy, Magdeburg (Head: Prof. Dr. sc. med. W. KUHNE)
A contribution to the postnatal enzymatic state of maturity of gyrus hippocampi in the embryofetal alcohol syndrome of the rat By K.-H. ULLRICH and K. DIETZMANN With 3 figures (Received October 8, 1979)
Address for correspondence: Dr. K. DIETZMANN, Institut fUr Pathologie der Medizinischen Akademie Magdeburg, Leipziger StraBe 44, DDR - 3010 Magdeburg Key words: embryofetal alcohol syndrome; gyrus hippocampi, maturation; alcoholism; brain development; dehydrogenases, neurons; alcohol damage, nervous system; myelinisation; formazan formation; RNA
Summary The influence of maternal chronic alcoholism on fetal brain development was investigated in rats. A decrease in the activity of succinate, glycerin-I-phosphate, glucose-6-phosphate dehydrogenases and of tetrazolium reductases was found. The amount of total RNA was reduced in the neonates with alcohol damage. Moreover, a retardation of myelinisation at the time of birth was observed. Observations on the harmfulness of alcohol intake and the consequences of permanent abuse have been made since men began to ingest alcoholic beverages. As early as in 62, PLINIUS described for alcoholics visionary phenomena and loss of memory. Around the year 100, PLUTARCH warned fathers of the intoxicating beverage since offspring produced in drunkenness would become habitual drunkards (HERBER 1971). In the past a multitude of established alcohol-specific parenchyma damage has been compiled. The increasing number of alcoholic mothers has most recently added to our knowledge in terms of another alcohol-induced clinical picture. In 1968 LEMOINE et al. described for the first time severe alcohol-induced anomalies in newborns. The clinical manifestation became generally known as the embryofetal alcohol syndrome not before 1973 (JONES et al. 1973). BIERICH (1978a, b) summarized the clinical picture in four groups of symptoms: 1. Pre- and postnatal hypotrophy. 2. Typical face dysmorphy; blepharophim()sis, epicanthus, microgenia, saddle nose. 3. Malformations on thorax, heart, genitalia, joints, and fingers.
4. Microcephaly, brain malformations, mental deficiency, and psychic retardation. However, not all of those afflicted reveal the full picture of this alcoholic embryofetopathy. Type and severity vary so that also weak forms with barely evident malformations were reported by MAJEWSKI et al. (1976). The prognosis of such children is essentially governed by the cerebral damage. Starting from this aspect we performed experimental investigations into the CNS of animals in order to obtain further findings on the fetal damage following intrauterine exposure, with particular interest being focused on the "outfit" of neurons with some strategic enzymes· at the time of birth.
170
1\1 aterial and 1\1ethods In twenty adult female rats obtained from the FALCKE Breeding Centre, Randau, G.D.R., chronic alcoholism was induced at by daily administration of 30 %sweetened ethanol. Subsequently, the animals were allowed to mate. Those rats that, with simultaneous supply of water, alcohol and varied food ad libitum, ingested exclusively alcohol were qualified as addicted to alcohol. The brains of 27 one-day-old rat neonates born at the calculated date, of addicted dams were examined together with 60 control brains. Brain preparation was followed by weighing and immediate freezing in carbon dioxide ice. The following enzymes were qualitatively and quantitatively studied on cryostat slices of gyrus hippocampi: 1. NADH2 and NADPH 2 tetrazolium reductase according to HESS et al. (1958) (E.C. 1.6.2.1.). 2. SDH according to SCHMIDT et al. (1970) (E.C. 1.3.99.1). 3. NADP dependent Glu-6-pDH according to MEIJER (1973) (E.C. 1.1.49). 4. Glycerin-l-pDH according to MEIJER (1973) (E.C. 1.1.1.8). Quantitative histochemical evaluation was made by a one-line scanning unit. The amount of enzymatically developed MTT formazan was determined by planimetry, elution and calibration by means of standard curves in fl·g/mm3 grey matter/unit time (ALTMAN 1969). Quantitative RNA determination according to BRESNICK (1965) was made on another eight brains of the controls and 16 brains with intrauterine alcohol damage.
Results Comparison of body weights already revealed an average difference about 2 g between controls and alcohol-damaged newborns. The means (± S.D.) for the former were found to be 6.99 ± 0.96 while 4.96 ± 1.09 calculated for rat neonates with intrauterine alcohol damage. This difference also referred to the brain weights (see fig. 1) indicating a prenatal growth retardation. Macroscopically, no malformations were found on the rat newborns after intrauterine exposure to alcohol. Topochemical preparation of some oxido-reductases in gyrus hippocampi revealed pronounced deviations from normal. Enzymatically formed stain precipitates were found in stratum pyramidale, granulosum and oriens only. They were not present or hardly detectable in stratum radiatum, lacunosum, moleculare cornus ammonis and fasciae dentatae. In the controls the succinate dehydrogenase, glucose-6-phosphate dehydrogenase, glycerin-1phosphate dehydrogenase and tetrazolium reductases under investigation were found in all the above-mentioned cell layers. Verification of the topochemical enzyme findings was substantiated by elution of mono-
formazan. The results are shown in figs. 2 and 3.
0.5 0,4
0,3 1=301
.r2 =o,()()()3
11= 12
.....~
0.1
~I.. 265 293:xJ() max. (;m,Y ofc(assps In mg
Fig. 1. Brain weights of newborn rats with experimental embryofetal alcohol syndrome (A) and an untreated control group (K).
171
73 12
11 10 9 ~
8 .S: E 7
~ 3
6 ......
.~
It)
It)
""
'<
....Cb
~
~
"l
E
5 2
~
....~ ~
<.)
"l
3 ......~ t-.. 2 ....
E ......
.... t-..
~
1
~
~
~
Fig. 2. Enzymatic forma zan formation in the cortex of the brain of newborn rats with intrauterine ethanol exposure. Shaded: Animals with alcohol exposure. White bar: Controls. I: G-l-P-DH; II: NADH 2 -Diaph.; III: NADPH 2-Diaph.; IV: SDH; V: G-6-P-DH. frequency SDH /.3.9S./
0.35 0.30 0.25 0.20 0.75 0.70 0.05
~
~~~~~~~~~~~~~~~------~ 0.15 0.25 0.35 0,,5 0.55 0.65 0.75 0.85 095 7.05 7.75 725 735 7A5 7.55 7.65 775 795 extinction
Fig. 3. Frequency distributions of measured succinate dehydrogenase extinctions in controls (1111111) and animals with alcohol exposure (rig!!;!). In addition, the RNA in the brain at the time of birth was assayed in both groups using a colorimetric orcein procedure. By use of the selective U-test according to MANN and WHITNEY (cited in WEBER 1957) the results were significantly different (IX = 0.025 and 0.050) between the animals with intrauterine alcohol damage and the untreated rat neonates for both unidirectional and bidirectional procedures, respectively. The results obtained (mean ± S.D.) were 0.307 ± 0.024 flg RNA/mg brain for normal animals and 0.259 ± 0.066flg RNA/mg brain tissue for newborns with experimental embryofetal alcohol syndrome.
Discussion et al. (1975), LOSER (1977), YOLK (1977), CLARREN et al. (1978), and coworkers (1978) the pathogenesis of the embryo fetal alcohol syndrome is still unclear Ethanol directly overcomes the placental barrier and could, either by itself or through its metabolites, exert a teratogenetic effect on the fetus, As reported by
HANSON
172
(1978),
MANN
MAJEWSKI
especially since PIKKARAINEN and RATHA (1968) could not demonstrate an activity of alcohol dehydrogenase in the embryonal liver and hardly found such activity in the fetus. The liver damage of the dam must be included as another causal factor. TEWARI and NOBLE (1975) found a decrease in cerebral m- and r-RNA in the polysomal fractions after alcohol administration. These findings are supported by the present results. The orcein method used was not suited for differentiation into various kinds of RNA so that no meaningful information can be provided as to a consecutively altered protein synthesis. However, RAW AT (1975) found a reduced protein synthesis under the influence of alcohol on the brains of neonates. The similar decrease in enzymes with such a dissimilar importance and status in the intermediary metabolism such as tetrazolium reductases, succinate, glucose-6-phosphate and glycerin-1-phosphate dehydrogenase might, however, suggest a diminished de novo-synthesis in the brain material under investigation. There exist common dependencies between lipid and phosphatide syntheses and glycerin1-phosphate-oxido-reductase as well as glucose-6-phosphate dehydrogenase. The activity decreases found for the above-mentioned enzymes correlate with a retarded myelinisation of the nerve fibres. At the date of birth, the animals with intrauterine alcohol damage did not yet show myelinisation in the total brain. In another hypotrophy model (DIETZ MANN and LESSEL 1976) rat babies of the same age exhibited a timely state of myelinisation. The observations made by Ho et al. (1972) who found an alcohol accumulation in the grey matter with peak values in regions including the gyrus hippocampi, are of interest. As the latter brain region is particularly involved in mnestic performance, further investigations into this area are believed to be desirable in animals with embryo fetal alcohol syndrome.
Literature ALTMAN, F. P., The quantitative elution of nitro-blue formazan from tissue sections. Histochemie 17, 319-326 (1969). BIERICH, J. R, Pranatale Schadigung durch Alkohol. Internist 19, 131-139 (1978a). - Pranatale Schadigung durch Alkohol. Gynakologe 11, 142-150 (1978b). BRESNICK, E., Early changes in pyrimidine biosynthesis after partial hepatectomy. J. BioI. Chern. 240, 2550 (1965), cited in HOPPE-SEYLER-THIERFELDER, Hb. physiol. path. Analyse, Bd. IV/2, p. 1308. Springer, Berlin-Gottingen-Heidelberg 1960. CLARREN, S. K., E. C. ALVORD Jr., S. M. SUMI, A. P. STREISSGUTH and D. W. SMITH, Brain malformations related to prenatal exposure to ethanol. J. of Pediatrics 92, 64-67 (1978). DIETZMANN, K., and W. LESSEL, An experimental model for inducing fetal hypotrophy in the rabbit_ Exp. Path. 12, 309-314 (1976). HANSON, J. W., A. PYTKOWICZ-STREISSGUTH and D. W. SMITH, The effects of moderate alcohol consumption during pregnancy on fetal growth and morphogenesis. J. Pediatrics 92, 457 -460 (1978). HERBER, F., Alkohol -- Prozente, Promille, Probleme. VEB Verlag Volk und Gesundheit, Berlin 1971. HESS, R, D. G. SCARPELLI and A. G. E. PEARSE, The cytologic demonstration of oxidative enzymes. II. Pyridine nucleotide, linked dehydrogenase. J. Biophys. biochem. Cytol. 4, 753 -760 (1958). Ho, B. T., G. E. FRITCHIE, J. E. IDANPAAN-HEIKKILA and W. M. McISAAC, Placental transfer and tissue distribution of ethanol-p4C. Quart. J. Stud. Alcohol. 33, 485-494 (1972). JONES, M. D., D. W. SMITH, C. N. LILLELAND and A. P. STREISSGUTH, Pattern of malformation in offsprings of chronic alcoholic mothers. Lancet 1, 1267 -1271 (1973). LEMOINE, P., H. HAROUSEAU, J. BORTEGRY and J. G. MENuET, Les enfants des parents alcoholiques: anomalies observe's, apropos de 127 cas. Arch_ Franc. Pediat. 25, 831-838 (1968). LOSER, H., Alkoholembryopathie - ein hiiufiges Fehlbildungssyndrom. Hippokrates 48, 272-273 (1977). MAJEWSKI, F., J. R BIERICH, H. LOSER, R MICHAELIS, B. LEIBER und F. BETTECKEN, Zur Klinik und Pathogenese der Alkohol-Embryopathie. Munch. med. Wschr. 118, 1635-1642 (1976). MANN, 1. J., A. BHAKTHAVATHSALAN, M. Lm and PH. MAKOWSKI, Effect of alcohol on fetal cerebri function and metabolism. Amer. J. Obstet. Gynec. 122, 845-851 (1975). MEIJER, A. E. F. H., Semipermeable membranes for improving the histochemical demonstration of enzyme activites in tissue sections. Histochemie 36, 165 -172 (1973). PIKKARAINEN, P., and N. C. R RATHA, Development of alcohol dehydrogenase activity in the human liver. Pediat. Res. 1, 165 -172 (1968). 12 Exp. Path. 18, H. 3
173
RAWAT, A. K., Ribosomal protein synthesis in the fetal and neonatal rat brain as influenced by maternal ethanol consumption. Res. Commun. Chern. Pathol. Pharmacol. 12, 723-732 (1975). SCHMIDT, W., W. GABLER und D. WENDLER, Zur quantitativen Auswertbarkeit des histochemischen Succinat-Dehydrogenase-Nachweises und dessen Abhiingigkeit von unterschiedlichen Reaktionsbedingungen. Acta histochem. 37, 250-258 (1970). TEWARI, S., and E. P. NOBLE, Alteration in cerebral polynucleotide metabolism following chronic ethanol ingestion. Adv. expo Med. Biol., vol. 59. Plenum Press, New York 1975. VOLK, B., Verziigerte Kleinhirnentwicklung im Rahmen des "embryofetalen Alkoholsyndroms". Acta neuropath. (Berl.) 39, 157 -163 (1977). WEBER, E., GrundriB der biologischen Statistik, 3. Auflage. VEB Gustav Fischer Verlag, Jena 1957.
174
A contribution to the postnatal enzymatic state of maturity of gyrus hippocampi in the embryofetal alcohol syndrome of the rat By K.-H. ULLRICH and K. DIETZMANN With 3 figures (Received October 8, 1979)
Address for correspondence: Dr. K. DIETZMANN, Institut fUr Pathologie der Medizinischen Akademie Magdeburg, Leipziger StraBe 44, DDR - 3010 Magdeburg Key words: embryofetal alcohol syndrome; gyrus hippocampi, maturation; alcoholism; brain development; dehydrogenases, neurons; alcohol damage, nervous system; myelinisation; formazan formation; RNA
Summary The influence of maternal chronic alcoholism on fetal brain development was investigated in rats. A decrease in the activity of succinate, glycerin-I-phosphate, glucose-6-phosphate dehydrogenases and of tetrazolium reductases was found. The amount of total RNA was reduced in the neonates with alcohol damage. Moreover, a retardation of myelinisation at the time of birth was observed. Observations on the harmfulness of alcohol intake and the consequences of permanent abuse have been made since men began to ingest alcoholic beverages. As early as in 62, PLINIUS described for alcoholics visionary phenomena and loss of memory. Around the year 100, PLUTARCH warned fathers of the intoxicating beverage since offspring produced in drunkenness would become habitual drunkards (HERBER 1971). In the past a multitude of established alcohol-specific parenchyma damage has been compiled. The increasing number of alcoholic mothers has most recently added to our knowledge in terms of another alcohol-induced clinical picture. In 1968 LEMOINE et al. described for the first time severe alcohol-induced anomalies in newborns. The clinical manifestation became generally known as the embryofetal alcohol syndrome not before 1973 (JONES et al. 1973). BIERICH (1978a, b) summarized the clinical picture in four groups of symptoms: 1. Pre- and postnatal hypotrophy. 2. Typical face dysmorphy; blepharophim()sis, epicanthus, microgenia, saddle nose. 3. Malformations on thorax, heart, genitalia, joints, and fingers.
4. Microcephaly, brain malformations, mental deficiency, and psychic retardation. However, not all of those afflicted reveal the full picture of this alcoholic embryofetopathy. Type and severity vary so that also weak forms with barely evident malformations were reported by MAJEWSKI et al. (1976). The prognosis of such children is essentially governed by the cerebral damage. Starting from this aspect we performed experimental investigations into the CNS of animals in order to obtain further findings on the fetal damage following intrauterine exposure, with particular interest being focused on the "outfit" of neurons with some strategic enzymes· at the time of birth.
170
1\1 aterial and 1\1ethods In twenty adult female rats obtained from the FALCKE Breeding Centre, Randau, G.D.R., chronic alcoholism was induced at by daily administration of 30 %sweetened ethanol. Subsequently, the animals were allowed to mate. Those rats that, with simultaneous supply of water, alcohol and varied food ad libitum, ingested exclusively alcohol were qualified as addicted to alcohol. The brains of 27 one-day-old rat neonates born at the calculated date, of addicted dams were examined together with 60 control brains. Brain preparation was followed by weighing and immediate freezing in carbon dioxide ice. The following enzymes were qualitatively and quantitatively studied on cryostat slices of gyrus hippocampi: 1. NADH2 and NADPH 2 tetrazolium reductase according to HESS et al. (1958) (E.C. 1.6.2.1.). 2. SDH according to SCHMIDT et al. (1970) (E.C. 1.3.99.1). 3. NADP dependent Glu-6-pDH according to MEIJER (1973) (E.C. 1.1.49). 4. Glycerin-l-pDH according to MEIJER (1973) (E.C. 1.1.1.8). Quantitative histochemical evaluation was made by a one-line scanning unit. The amount of enzymatically developed MTT formazan was determined by planimetry, elution and calibration by means of standard curves in fl·g/mm3 grey matter/unit time (ALTMAN 1969). Quantitative RNA determination according to BRESNICK (1965) was made on another eight brains of the controls and 16 brains with intrauterine alcohol damage.
Results Comparison of body weights already revealed an average difference about 2 g between controls and alcohol-damaged newborns. The means (± S.D.) for the former were found to be 6.99 ± 0.96 while 4.96 ± 1.09 calculated for rat neonates with intrauterine alcohol damage. This difference also referred to the brain weights (see fig. 1) indicating a prenatal growth retardation. Macroscopically, no malformations were found on the rat newborns after intrauterine exposure to alcohol. Topochemical preparation of some oxido-reductases in gyrus hippocampi revealed pronounced deviations from normal. Enzymatically formed stain precipitates were found in stratum pyramidale, granulosum and oriens only. They were not present or hardly detectable in stratum radiatum, lacunosum, moleculare cornus ammonis and fasciae dentatae. In the controls the succinate dehydrogenase, glucose-6-phosphate dehydrogenase, glycerin-1phosphate dehydrogenase and tetrazolium reductases under investigation were found in all the above-mentioned cell layers. Verification of the topochemical enzyme findings was substantiated by elution of mono-
formazan. The results are shown in figs. 2 and 3.
0.5 0,4
0,3 1=301
.r2 =o,()()()3
11= 12
.....~
0.1
~I.. 265 293:xJ() max. (;m,Y ofc(assps In mg
Fig. 1. Brain weights of newborn rats with experimental embryofetal alcohol syndrome (A) and an untreated control group (K).
171
73 12
11 10 9 ~
8 .S: E 7
~ 3
6 ......
.~
It)
It)
""
'<
....Cb
~
~
"l
E
5 2
~
....~ ~
<.)
"l
3 ......~ t-.. 2 ....
E ......
.... t-..
~
1
~
~
~
Fig. 2. Enzymatic forma zan formation in the cortex of the brain of newborn rats with intrauterine ethanol exposure. Shaded: Animals with alcohol exposure. White bar: Controls. I: G-l-P-DH; II: NADH 2 -Diaph.; III: NADPH 2-Diaph.; IV: SDH; V: G-6-P-DH. frequency SDH /.3.9S./
0.35 0.30 0.25 0.20 0.75 0.70 0.05
~
~~~~~~~~~~~~~~~------~ 0.15 0.25 0.35 0,,5 0.55 0.65 0.75 0.85 095 7.05 7.75 725 735 7A5 7.55 7.65 775 795 extinction
Fig. 3. Frequency distributions of measured succinate dehydrogenase extinctions in controls (1111111) and animals with alcohol exposure (rig!!;!). In addition, the RNA in the brain at the time of birth was assayed in both groups using a colorimetric orcein procedure. By use of the selective U-test according to MANN and WHITNEY (cited in WEBER 1957) the results were significantly different (IX = 0.025 and 0.050) between the animals with intrauterine alcohol damage and the untreated rat neonates for both unidirectional and bidirectional procedures, respectively. The results obtained (mean ± S.D.) were 0.307 ± 0.024 flg RNA/mg brain for normal animals and 0.259 ± 0.066flg RNA/mg brain tissue for newborns with experimental embryofetal alcohol syndrome.
Discussion et al. (1975), LOSER (1977), YOLK (1977), CLARREN et al. (1978), and coworkers (1978) the pathogenesis of the embryo fetal alcohol syndrome is still unclear Ethanol directly overcomes the placental barrier and could, either by itself or through its metabolites, exert a teratogenetic effect on the fetus, As reported by
HANSON
172
(1978),
MANN
MAJEWSKI
especially since PIKKARAINEN and RATHA (1968) could not demonstrate an activity of alcohol dehydrogenase in the embryonal liver and hardly found such activity in the fetus. The liver damage of the dam must be included as another causal factor. TEWARI and NOBLE (1975) found a decrease in cerebral m- and r-RNA in the polysomal fractions after alcohol administration. These findings are supported by the present results. The orcein method used was not suited for differentiation into various kinds of RNA so that no meaningful information can be provided as to a consecutively altered protein synthesis. However, RAW AT (1975) found a reduced protein synthesis under the influence of alcohol on the brains of neonates. The similar decrease in enzymes with such a dissimilar importance and status in the intermediary metabolism such as tetrazolium reductases, succinate, glucose-6-phosphate and glycerin-1-phosphate dehydrogenase might, however, suggest a diminished de novo-synthesis in the brain material under investigation. There exist common dependencies between lipid and phosphatide syntheses and glycerin1-phosphate-oxido-reductase as well as glucose-6-phosphate dehydrogenase. The activity decreases found for the above-mentioned enzymes correlate with a retarded myelinisation of the nerve fibres. At the date of birth, the animals with intrauterine alcohol damage did not yet show myelinisation in the total brain. In another hypotrophy model (DIETZ MANN and LESSEL 1976) rat babies of the same age exhibited a timely state of myelinisation. The observations made by Ho et al. (1972) who found an alcohol accumulation in the grey matter with peak values in regions including the gyrus hippocampi, are of interest. As the latter brain region is particularly involved in mnestic performance, further investigations into this area are believed to be desirable in animals with embryo fetal alcohol syndrome.
Literature ALTMAN, F. P., The quantitative elution of nitro-blue formazan from tissue sections. Histochemie 17, 319-326 (1969). BIERICH, J. R, Pranatale Schadigung durch Alkohol. Internist 19, 131-139 (1978a). - Pranatale Schadigung durch Alkohol. Gynakologe 11, 142-150 (1978b). BRESNICK, E., Early changes in pyrimidine biosynthesis after partial hepatectomy. J. BioI. Chern. 240, 2550 (1965), cited in HOPPE-SEYLER-THIERFELDER, Hb. physiol. path. Analyse, Bd. IV/2, p. 1308. Springer, Berlin-Gottingen-Heidelberg 1960. CLARREN, S. K., E. C. ALVORD Jr., S. M. SUMI, A. P. STREISSGUTH and D. W. SMITH, Brain malformations related to prenatal exposure to ethanol. J. of Pediatrics 92, 64-67 (1978). DIETZMANN, K., and W. LESSEL, An experimental model for inducing fetal hypotrophy in the rabbit_ Exp. Path. 12, 309-314 (1976). HANSON, J. W., A. PYTKOWICZ-STREISSGUTH and D. W. SMITH, The effects of moderate alcohol consumption during pregnancy on fetal growth and morphogenesis. J. Pediatrics 92, 457 -460 (1978). HERBER, F., Alkohol -- Prozente, Promille, Probleme. VEB Verlag Volk und Gesundheit, Berlin 1971. HESS, R, D. G. SCARPELLI and A. G. E. PEARSE, The cytologic demonstration of oxidative enzymes. II. Pyridine nucleotide, linked dehydrogenase. J. Biophys. biochem. Cytol. 4, 753 -760 (1958). Ho, B. T., G. E. FRITCHIE, J. E. IDANPAAN-HEIKKILA and W. M. McISAAC, Placental transfer and tissue distribution of ethanol-p4C. Quart. J. Stud. Alcohol. 33, 485-494 (1972). JONES, M. D., D. W. SMITH, C. N. LILLELAND and A. P. STREISSGUTH, Pattern of malformation in offsprings of chronic alcoholic mothers. Lancet 1, 1267 -1271 (1973). LEMOINE, P., H. HAROUSEAU, J. BORTEGRY and J. G. MENuET, Les enfants des parents alcoholiques: anomalies observe's, apropos de 127 cas. Arch_ Franc. Pediat. 25, 831-838 (1968). LOSER, H., Alkoholembryopathie - ein hiiufiges Fehlbildungssyndrom. Hippokrates 48, 272-273 (1977). MAJEWSKI, F., J. R BIERICH, H. LOSER, R MICHAELIS, B. LEIBER und F. BETTECKEN, Zur Klinik und Pathogenese der Alkohol-Embryopathie. Munch. med. Wschr. 118, 1635-1642 (1976). MANN, 1. J., A. BHAKTHAVATHSALAN, M. Lm and PH. MAKOWSKI, Effect of alcohol on fetal cerebri function and metabolism. Amer. J. Obstet. Gynec. 122, 845-851 (1975). MEIJER, A. E. F. H., Semipermeable membranes for improving the histochemical demonstration of enzyme activites in tissue sections. Histochemie 36, 165 -172 (1973). PIKKARAINEN, P., and N. C. R RATHA, Development of alcohol dehydrogenase activity in the human liver. Pediat. Res. 1, 165 -172 (1968). 12 Exp. Path. 18, H. 3
173
RAWAT, A. K., Ribosomal protein synthesis in the fetal and neonatal rat brain as influenced by maternal ethanol consumption. Res. Commun. Chern. Pathol. Pharmacol. 12, 723-732 (1975). SCHMIDT, W., W. GABLER und D. WENDLER, Zur quantitativen Auswertbarkeit des histochemischen Succinat-Dehydrogenase-Nachweises und dessen Abhiingigkeit von unterschiedlichen Reaktionsbedingungen. Acta histochem. 37, 250-258 (1970). TEWARI, S., and E. P. NOBLE, Alteration in cerebral polynucleotide metabolism following chronic ethanol ingestion. Adv. expo Med. Biol., vol. 59. Plenum Press, New York 1975. VOLK, B., Verziigerte Kleinhirnentwicklung im Rahmen des "embryofetalen Alkoholsyndroms". Acta neuropath. (Berl.) 39, 157 -163 (1977). WEBER, E., GrundriB der biologischen Statistik, 3. Auflage. VEB Gustav Fischer Verlag, Jena 1957.
174