8.1 The effect of pre- and postnatal exposure to organic solvents onthe development of the cerebellar cortex in the rat
GISELA STOLTENBURG-DIDINGER
Institute of Neuropathology, Klinikum Steglitz, Freie Universitiit, Berlin (FRG)
Introduction
The cerebellum of the neonatal rat presents a unique opportunity for the study of neuronalmaturation. This structure is comparatively immature at birth (ADDISON 1911) and developsrapidly during the first month of extrauterine life (ALTMAN 1972). Moreover, the adult cerebellarcortex possesses well known and characteristic anatomical features which facilitate developmentalcompansons.
The cerebellum has been subjected to a large number of enzyme histochemical studies (FRIEDE1957; BARGETON-FARKAS and PEARSE 1965; KUCKUK 1967; ALTMAN 1972). The enzyme maturation pattern of the cerebellum was studied as correlated with its morphology. This could best be
accomplished by enzyme histochemistry which allows investigation of enzyme activities at thecellular level during development.
The purpose of enzyme histochemistry is the localization of specific enzyme activities to intacttissue components. The characterization and quantitation of the enzyme activities mediating thehistochemical reaction is as integral a function of this discipline as the localization of the activitiesthemselves.
Enzyme histochemical studies on normal rat cerebellum have indicated a high and early activityof enzymes involved in anaerobic metabolism, while enzymes involved in aerobic metabolism
increase rapidly after birth. The aerobic pathway soon becomes quantitatively the most importantone in the postnatal cerebellum (ROBINS and LOWE 1961; BARGETON-FARKAS and PEARSE 1965).The external and internal granular cells exhibit weak oxidative enzyme activity at all ages. The
Purkinje cells show increasing oxidative enzyme activity from birth, reaching adult activity at the
end of the 4th week in normal ratss. The aim of the present study was to investigate whether anydifferences occur in the enzyme maturation pattern between solvent-exposed and normal rat
cerebellum as correlated with changes in its morphology, caused by pre- and postnatal exposure
to hexacarbons.
228 . G. Stoltenburg-Didinger
Although the neurotoxic effects of organic solvents in the adult organism have been well
studied, there is a paucity of information on the impact of in utero exposure to solvents on the
developing brain (ALTENKIRCH et al. 1978; 1982a, b; SPENCER and SCHAUMBURG 1975; STOLTENBURG-DJDINGER and ALTENKIRCH 1988). Animal studies demonstrate that a variety of solvents
readily cross the placenta (PELKONEN 1985) and that maternal inhalation of various solventsresults in neurodevelopmental deficits in the newborn rodent (Bus et al. 1979; MARKS et al. 1980;
DEACON et al. 1981). In utero exposure to organic solvents may also affect the development of the
human brain (TOUTANT and LIPPMANN 1979; HOLMBERG et al. 1980; HERSH et al. 1985, ESKENASI
et al. 1988).The primary fissure of cerebellar vermis was used as an enzymatically and morphologically
defined and homogeneous model system of an early maturing region (LARSELL 1952; KUCKUK
1967; ALTMAN 1972). A unique property of the commonly-used histochemical oxidation-reduc
tion indicators, the tetrazolium salts, is the reversible binding in their oxidized tetrazole form to
cytoplasmic components of almost all cells (ADAMS 1965). The binding of the tetrazolium salt to
tissue components permits electron transport in situ to form the insoluble colored reduced
product, the formazan, near the site of enzymatic activity.
Materials and methods
Animals: Virgin rats of the Wistar strain, 3 months of age, were mated with males varying in age from 4 to8 months. The time of fertilization was determined by vaginal smears taken daily. The mode of inhalation was23 h a day, 7days a week. Controls were kept under the same conditions but without solvent exposure. Thesolvent was pumped on a glass frit through which filtered air was conducted. The hexane concentration wasmonitored with a continously measuring flame ionization detector.
In the first solvent experiment only n-hexane was used, and the animals were exposed to low concentrations (500ppm) during prenatal development (21 days).
In the second solvent experiment we used higher concentrations and exposed one group only prenatally(21 days) and a second group also postnatally (42 days) in order to include the growth spurt of the cerebelluminto the exposure period. n-Hexane and MEK were studied in this experiment.
In the third solvent experiment we used even higher concentrations (1000 ppm, initially 1500 ppm). Therewere likewise two groups: one was exposed prenatally (21 days), the other pre- and postnatally (51 days).
The substances used were n-hexane (Merck, Darmstadt, nr.4367 99%), methyl-ethyl-ketone (Merck,Darmstadt, nr.6014 99%) and a mixture of both (hex/MEK = 1200:300).
The newborn rats were examined for effects on body and brain maturation, weight development, fitness forsurvival and the possible occurrence of deformities.
Preparation: All rats were decapitated. The skull was opened immidiately with scissors. Cerebellum andcerebrum were lifted out. Each cerebellar hemisphere was cut sagittally with a razor blade and each opposinghalf hemisphere was mounted onto a cork, then immidiately quenched in liquid nitrogen.
All cryostat blocks were stored at a temperature of at least -20°C and sectioned at the same temperature in7!!m thick sections.
Sections were immidiately incubated for enzyme histochemistry of succinic dehydrogenase (SDH) andNADH tetrazolium reductase (NADH-Tr). All enzyme histochemical reactions were performed according tothe methods described by NOVIKOFF (1960). Nitro Blue Tetrazolium (NBT) was used as hydrogen acceptor.All incubation solutions were freshly prepared. The chemicals were obtained from Sigma Chemical Companyand from E. Merck AG, Darmstadt. All incubations were made for 30 min at a temperature of 37°C (SDH) or20°C for NADH-Tr respectively. Paired substrate-free sections were used as negative controls in order toexclude nonspecific reduction of NBT.
The enzyme histochemical activity as revealed by its formazan deposition was studied in the primaryfissure of the cerebellar vermis at postnatal days 1, 9 and 21.
Organic solvents and cerebellar cortex . 229
Results
Differences in formazan deposition between SDH and NADH-Tr activity could not be detected. Because of the finer granules of the reaction product figures were selected from NADHTr.
Normal development. Activity of the oxidative enzymes SDH and NADH-Tr as visualized byformazan deposition was present already at birth in Purkinje cells. All activity was located in thecytoplasm. The activity at birth appeared weak, then gradually increased, by the third day strongSDH and NADH-Tr activity was present in the bodies of Purkinje cells. The layer of Purkinjecells remained the only one with marked activity until the ninth day (Fig. 2), when dendrites withmarked oxidative activity grow into the molecular layer (Figs. 2a, 3a).
The external granular cells showed a low activity, which appeared as a thin rim of perikaryalactivity, equal in the proliferative and premigratory zones. As the molecular layer expanded andregression of the external granular layer continued, NADH-Tr was seen closer to the subpialsurface, reflecting the outgrowth of Purkinje cell dendrites, thereby providing a clear picture ofthe Purkinje cell dendritic tree (Fig. 3a). At day 9 the first faint trace of oxidative activity was seenin the internal granular cells. This activity remained low to moderate at all ages into adult life.
Development of solvent-exposed rats. The development of SDH and NADH-Tr activity paralleled that of normal rats with a delay. After prenatal exposure only the activity of the Purkinjecell apical cones was higher at day 9 compared to normal rats, reflecting the delayed outgrowth ofthe Purkinje cell apical dendritic tree (Fig. 3b). After day 21, both groups showed equal formazandeposition in the Purkinje cells. No differences in SDH and NADH-Tr activity between prenatally exposed and normal rats could be seen either in the external and internal granular cells.
After pre- and postnatal exposure, the Purkinje cells of exposed rats showed a higher SDH andNADH-Tr activity at day 9 than those of normal rats.
Pre- and postnatal exposure to n-hexane resulted in a persisting apical cone and delayedformation of the apical dendritic tree of the Purkinje cells in the cerebellum from a 9-day-old rat(Fig. 3c).
After pre- and postnatal exposure to the mixture of n-hexane and methyl-ethyl-ketone (MEK),the retardation of cell maturation was even more pronounced (Fig. 3d). At day 9, the Purkinjecells showed persisting maximal perikaryal formazan coloration, indicating a high, concentratedNADH-Tr activity. Furthermore, the difference in width of the molecular layer between pre- andpostnatally exposed and normal rats was greater as a result of the retarded apical dendrite formation (Fig. 3a-d). This could be demonstrated by enzyme histochemistry much more impressingthan by conventional hematoxylin-eosin-staining (Figs. 1 and 2).
All solvents studied delayed the histogenesis of the cerebellar cortex in the experimental animals at all concentrations examined (Figs. 1,2, 3a-d). Even at the lowest concentration, appliedonly prenatally as in the first solvent experiment, the histological preparations of frozen sectionsof the fissura prima of the vermis cerebelli showed a delay in migration of the outer granular cellsand a persistence of Purkinje cells at a lower stage of development (Figs. 1b, 2 b, 3b). Again it wasproven that postnatal exposure aggravated the developmental delay (Figure 1c, 2c, 3c). Methylethyl-ketone (MEK) potentiated n-hexarie-neurotoxicity (Figs. 1d, 2d, 3d).
230 . G. Stoltenburg-Didinger
2dFig. 1. Frozen sections of rat cerebellum, sagittal view of fissura prima of vermis (early developing region),third solvent experiment, postnatal day 9, HE. - x 300. a: Control; b: MEK prenatal exposure; c: n-Heyane,prenatal exposure; d: n-Hexane/MEK, pre- and postnatal exposureNote differences in thicknes of outer granular layer and molecular layer as well as differences in cell density ininner and outer granular layer.
Fig. 2. Specimen identical to Fig. 1, NADH-Tr. - x 300.Note differences in cell size and staining intensity of Purkinje cells.
Organic solvents and cerebellar cortex . 231
Discussion
The biological oxidation mechanisms, whereby electrons and frequently hydrogen are transferred through one or more intermediates to hydrogen, afford the major energy source of the livingcell. Within the mitochondrion the electron carrier components are bound to one another (ADAMS1965). The activity of oxidative enzymes in a given region may be considered an approximateparameter of the intensity of the oxidative metabolism (WOODWART et al. 1969). A comparison ofthese patterns provides a quite reliable picture of the functional activity of a given region (FRIEDEand PAX 1961).
In rats prenatally exposed to n-hexane or methyl-ethyl-ketone (MEK), a delay in the development of the high oxidative enzyme activity of the Purkinje cell apical cone was noticed. Thisretarded increase in enzyme activity was followed by a delayed decrease in activity of the Purkinjecell apical cone parallel to the delayed formation and differentiation of the Purkinje cell apicaldendritic tree (SIMA and PERSSON 1975). It seems reasonable to suppose that the delayed increaseof enzyme activity in the apical cone prior to the dendritic formation and the delayed decrease ofactivity in the Purkinje cell apical cone concomitant to the retarded development of the dendritictree may be related to inhibition of glycolysis by n-hexane and its main metabolite 2,5-hexanedione (SABRI et al. 1979; SPENCER et al. 1979; SPENCER et al. 1980). This molecular mechanism isyet not the only mode of action (DE CAPRIO 1985). Toxicity of n-hexane is mediated by 2,5hexane-dione, its main metabolite, which plays a major role in the neurotoxicity of this compound. Neurotoxicity of n-hexane depends on the toxicogenic transformation in the organism,enzyme induction having an additive effect. In the immature liver of fetuses and newborn animals,the biotransformation systems are, at any rate, developed only incompletely or not at all. Thus,the newborn animals receive a loading with diketones via the placenta or the mother's milk. Sinceactive metabolization of hexacarbons to diketones is not possible in young animals, they do notdevelop any clinically detectable peripheral paralyses (STOLTENBURG-DIDINGER et al. 1990). Thusthe peripheral nervous system of adult rats seems to be more vulnerable to solvents than theimmature one, possibly because of its paucity in neurofilaments as well.
It is concluded that prenatal solvent exposure causes a retarded enzymatic development of themolecular and internal granular layers as well as of the Purkinje cells in rat cerebellum, followedby catch-up growth and maturation. This is consistent with other investigations where mild ormissing developmental deficit was found after solvent exposure during pregnancy (Bus et al.1979; MARKS et al. 1980; DEACON et al. 1981; BHATT et al. 1988).
The persistent delay in enzymatic development after pre- and postnatal exposure to organicsolvents parallels a morphological retarded development of cerebellar structures and can becompared to the effect of malnutrition (STOLTENBURG-DIDINGER et al. 1990). A retarded differentiation of neurons in early undernutrition has been observed in cerebellum. PERSSON and SIMA(1975) demonstrated a delay in the formation and enzymatic maturation of the Purkinje cell apicaldendritic tree.
After postnatal exposure to hexacarbons there is no catch-up growth because the period of thegrowth spurt of the cerebellum is the most vulnerable (DOBBING and SANDS 1971). It mighttherefore be assumed that the delayed differentiation and migration of the external granular cellsas well as the delayed outgrowth of the Purkinje cell dendritic tree results in a delayed synaptogenesis and, due to permanent loss of cerebellar cortical neurons, probably also a permanentloss of synapses. To prove this by other than enzyme histochemical methods remains a considerable challenge.
232 . G. Stoltenburg-Didinger
3c
Organic solvents and cerebellar cortex . 233
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Fig. 3. Frozen sections of rat cerebellum, sagittal view of fissura prima of vermis, third solvent experiment(1500-1000 ppm), postnatal day 9, NADH-Tr. - x500.a: Control. Purkinje cells of normal rat cerebellum with clear resolution of the apical cones and advancingapical dendrite formation. The dendrites divide into tertiary and quartenary branchlets. The perikaryalformazan deposition is submaximal.b: n-Hexane., prenatal exposure. Note the persisting apical cone with maximal activity. Retarded development of apical dendrites is seen, resulting in a delayed decrease of the apical cone NADH-Tr activityc: n-Hexane, pre- and postnatal exposure. Intense NADH-Tr activity confined to the perikarya of thePurkinje cells. Note the absence of apical cone formation and persistence of numerous somatic dendrites ofthe Purkinje cells, contrary to the finding in normal ratsd: n-Hexane/MEK, pre- and postnatal exposure. Purkinje cells demonstrating high to moderate activitymostly confined to the apical cone and perikaryon. Badly developed dendritic tree, very thin molecular layer
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