GLIA 4:384-392 (1991)
Astroglial Alterations in Rat Hippocampus During Chronic Lead Exposure ASIA SELV~N-TESTA,J.J. LOPEZ-COSTA, A.C. NESSI DE AVIRON, AND J. PECCI SAAVEDRA Znstituto de Biologia Celular, University of Buenos Aires, School of Medicine, Buenos Aires 1121, R. Argentina
KEY WORDS
Astrocyte, Glial fibrillary acidic protein, Immunohistochemistry
ABSTRACT The present study was performed in order to follow the response of astroglial cells in the rat hippocampus to chronic low-level lead exposure. The experiments combined immunohistochemistry using anti-glial fibrillary acidic protein (GFAP) antibody and conventional transmission electron microscopy (EM). Chronic administration with drinking water [l g% wlv (subclinical dose) of lead acetate dissolved in distilled water] was started through the mother’s milk when pups were 7 days old. Following weaning, experimental offspring were treated for 3 months with the same concentration of adulterated water. The group of intoxicated animals and their controls were sacrificed by perfusion-fixation at 30,60, and 90 days of exposure. After 60 days of lead treatment, staining of GFAP-positive cells demonstrated an astroglial transformation from the quiescent to the reactive state, characterized by an increase in GFAP. In control rats no changes in GFAP immunostaining were observed. The intensity of the astroglial response was enhanced after 90 days of lead intoxication, showing an increment of GFAP immunoreactivity. Quantification of these changes was made by computerized image analysis, confirming that the sectional areas of the astroglia in lead-exposed animals were larger than those in controls. These results are consistent with the ultrastructural alterations. Simultaneously with the increment in gliofilaments, intranuclear inclusions were seen in some astrocytes. The mechanisms by which lead affects astrocytes are unknown. Probably the astroglial changes induced by lead intoxication produce microenvironmental modifications that may disturb the neuronal function.
INTRODUCTION It is known that functional and structural abnormalities of the brain accompany acute and chronic lead intoxication. Significant neurological and behavioral sequelae can result after persistent exposure to small doses of lead over long periods of time (Landrigan et al., 1980; Needleman et al., 1990). The ubiquitous nature of lead, its potential hazards to the human being, and, in particular, the great susceptibility of the nervous system of young children to prolonged low-level exposure (Beneton-Marantidou et al., 1988, Chisolm, 1984) confirm the importance of the problem. Experimental studies in which the influence of this toxicant in young animals was investigated (Baraldi et al., 1988; McCauley et al., 1982) provide information for a better comprehension of the action of lead in the central nervous system (CNS). In the rat hippocam: @1991 Wiley-Liss, Inc.
pus the main developmental processes in neuronal differentiation and maturation take place after birth. Since it has been shown that the hippocampus is one of the regions of the CNS more susceptible to lead exposure (Collins et al., 1982; Slomianka et al., 19891, we selected this region to study the effect of chronic lead intoxication in postnatal rats. Trophic influences of glial cells on developing neurons and the exchange of information between neurons and glial cells are well documented (Lauder and McCarthy, 1986). Changes produced in astroglial cells after the lead treatment could modify the neuronal microenvironment, affecting the cells’ functions. Received May 9,1990; accepted November 26,1990. Address reprint requests to Dr. Asia Selvin-Testa, Arenales 2655,1425 Buenos Aires, R. Argentina.
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High doses of lead induce encephalopathy in postnatal rats with morphological alterations in glial cells. Swelling of astrocytes and the presence of cytoplasmic electron-dense bodies and intranuclear inclusions are some of the cell responses to the toxic action of this type of lead intoxication (Holtzman et al., 1984; Lefauconnier et al., 1983).Astroglial reactivity and proliferation have also been reported with higher levels of lead exposure in postnatal rats (Goyer and Rhyne, 1973). These conditions appear to be distinct from those observed by one of us in a previous report (Selvin-Testa and Palacios-Prii, 1988) in which we described certain changes in the hippocampus that appeared after a few months of chronic low-level lead exposure when the treatment was started in the first postnatal week. Some of these morphological modifications occurred in glial cells, in the absence of edema and local alterations or destructive lesions in surrounding areas. These ultrastructural findings induced us to study astroglial reactivity after chronic low-level lead treatment, using immunocytochemistry with an antibody specific to glial fibrillary acidic protein (GFAP) (Dahl and Bignami, 1985). The purpose of the present report was to follow the response of hippocampal astrocytes during chronic low-level lead intoxication. This research combined immunohistochemical study with conventional transmission electron microscopy. MATERIALS AND METHODS Drugs were purchased from Merck Co. and 3,3'diaminobenzidine, polyclonal rabbit antiserum against GFAP, sheep serum, and secondary antibodies used for immunohistochemistry studies were obtained from Sigma Chemical Co. Pregnant Wistar rats were kept in individual cages and fed with laboratory chow and water ad libitum. After birth each untreated dam fed eight male pups but the treated dams fed only six pups to avoid decrements in body weight (Alfano and Petit, 1982). Once the pups were 7 days old, dams with six pups were exposed to subclinical doses of lead (Averill and Needleman, 1980) by giving them a lead acetate solution as drinking water [lg% (w/v)l.In order to prevent the precipitation of lead, solutions were prepared in freshly boiled distilled water. Control mothers with eight pups received distilled water for drinking. Experimental and control pups were weaned at 25 days. Experimental pups were subsequently treated with 1g% (w/v)lead acetate aqueous solution, as drinking water, during 3 months of lead administration. Control animals received distilled water and were maintained in the same experimental conditions as treated rats. Experimental and control animals were weighed twice a week, in order to detect alterations in the somatic development. The intoxicated and control animals were killed by perfusion-fixation after 30, 60, and 90 days of lead administration. A minimum of six rats from each time
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point was used in each of the microscope study groups. Animals were anesthetized with 0.1 mVlOO g of body weight (i.p.1 of 28% (w/v) chloral hydrate, and perfused through the abdominal aorta (Gonzalez Aguilar and De Robertis, 1963) with a mixture of aldehydes. Before perfusion, blood samples were collected in heparinized glass vials for lead analysis. Immunohistochemical Labeling Rats were perfused with 4% paraformaldehyde, 0.25% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. Before fixation a brief rinse of the circulatory system with 30 ml of solution containing 0.8% sodium chloride, 0.4%sucrose, and 0.4%glucose (w/v)(Gonzalez Aguilar and De Robertis, 1963)was done. Following the perfusion-fixation, the brain was removed and postfixed for an additional 30 min in the same fixative. Three washings in phosphate buffer plus 5% (w/v) sucrose were performed and then the brain was left in this washing solution for 18 h at 4°C. Coronal sections (40 pm thick) from the brain containing the hippocampus were cut in an Oxford vibratome. Representative sections were collected in phosphate-buffered saline (PBS) at 4°C. To obtain specific labeling of the astroglial cells with a better preservation of the tissue, we introduced a modification to the method of Streefkerk (1971) to inhibit endogenous peroxidase (Selvin-Testaet al., 1988). Sections from controls and treated animals were always processed simultaneously, using the same amount of time for each step of the immunohistochemical procedure. Sections were immunohistochemically stained for GFAP according to the peroxidase-antiperoxidase (PAP) method of Sternberger (1986) in the following sequence: 1) incubation in 3% normal sheep serum (NSS) for 30 min; 2) incubation overnight at 4°C in the primary antibody, anti-GFAP, diluted 1:1,000 in 1% NSS; 3) incubation in the secondary antibody, sheepanti-rabbit diluted 1 5 0 in 1%NSS, for 45 min; 4) incubation in PAP complex (developed in rabbit in our laboratory), diluted 1:lOO in 1%NSS, for 45 min; and 5 ) development in a solution containing 0.5% 3,3'diaminobenzidine + 0.01% H202in PBS for 15-30 min. Sections were mounted on glass slides, dehydrated, and examined in a Zeiss MC63 photomicroscope. Electron Microscopy
For electron microscopic studies, animals were perfused with an aldehyde mixture (4%paraformaldehyde, 5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2) similar to that used by Reese and Karnovsky (1967). Before fixation a brief rinse of the circulatory system with 30 ml of a solution containing 0.8% NaC1, 0.4% glucose, 0.4% sucrose (w/v), was done. Two hours after the perfusion, brains were removed and then both hippocampi were dissected. Blocks of areas CA1 (Lorente de NO, 1934) and CA4, the hilus of the fascia dentata
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(Blackstad, 1956), were taken. Samples were kept in cold fixative overnight and then rinsed in 0.1 M cacodylate buffer, pH 7.2, and postfixed in 1%osmium tetroxide dissolved in the same buffer. Afterwards, tissues were dehydrated in graded ethanols and propylene oxide and embedded in LX-112 resin. Sections 1p.m thick, stained were toluidine blue, were made and used to identify histological structures at low magnification and t o select the areas to be studied by electron microscopy. Ultrathin sections from selected areas were stained with uranyl acetate and lead citrate, and examined in a Hitachi H 500 and a Siemens Elmiskop I electron microscope.
TABLE 1 . Body weight and blood lead level in exDerimenta1 and control rats Experimental group
30 days control n=8 30 days lead n=6 60 days control n=8 60 days lead n=6 90 days control n=8 90 days lead n=6
Body weight (9)
Blood lead concentration(pg/dl)
105.5 f 5.0
6.00 f 1.0
100.9 f 5.8 P < 0.2 237.7 14.5
*
112.1 f 4.88 P < 0.001 8.25 f 0.75
213.2 f 39.1 P < 0.2 346.8 f 48.6
97.33 f 3.77 P < 0.001 6.00 f 1.0
291.8 f 4.4 P < 0.05
102.25
*
5.76 P < 0.001
Computerized Image Analysis
Statistical Analysis
The distribution of labeled cells was measured quantitatively using computer digital analysis in sections of six rats from both groups. The selected hippocampal fields were located in the CA1 strata oriens and radiatum, in the molecular layer of the suprapyramidal leaf of the dentate gyrus and the dentate hilus. Sections with adequate labeling and penetration of the antibody were chosen. For each animal, mapping was made from representative sections 40 p,m thick. Data from 40 selected hippocampal and dentate gyrus fields were averaged for each animal. Photographs (1,250x ) were taken of randomly selected fields with a Zeiss MC63 photomicroscope and mapped with a computerized morphometric digitizing system. Photographs of individual cells were used in order to avoid interference from overlapping images. Photomicrographs were scanned onto 5 bitplane-high resolution and interlaced screens. The images captured by a black and white video camera (Crest) were then transformed into digits with the help of an analog/digital conversor (Digi-View) connected to an Amiga 1000 personal computer as described elsewhere (Nessi de Avinon, 1989a,b). The scanned images were then analyzed by ad hoc image processor software using a scale composed of 16 gray levels with the darkest values belonging to the higher numbers and the lightest to the lower. The digitized astroglial cell bodies were evaluated separately from their processes. The scanned images were placed permanently on the computer screen, and each level of gray was made to blink into a different color. In this way it was possible to recognize the patterns of gray levels in different cell regions. Whereas the levels corresponding to the cell body depicted on the screen matched from 10 to 13, cell processes, since they exhibited lighter optic densities than the soma, varied from 6 to 9. Areas of soma and processes were later calculated from the area data of different gray levels provided by the computer (Nessi de Avinon, 1990). Images were measured in pixel counts and transformed into square micrometers, in order to get a more accurate evaluation of astrocyte size (in current measurement units) from both treated and control animals.
Data were used to derive the mean and standard deviation. Control and experimental groups were compared by Student’s t-test; a P value < 0.05 was considered significant. The concentration of lead in blood samples was measured in treated groups and their controls using atomic absorption spectrophotometry.
RESULTS Lead-intoxicated animals showed no significant differences from controls in development, appearance of body hair, or time of eye opening. The treated rats gained weight almost at the same rate as controls. In the first month, they weighed only -4.4% (P < 0.2) less than their controls, although the blood lead level was 121.1 k 4.88 p.g/dl. During the second month of exposure, a difference of -10.3% (P < 0.2) in body weight was detected. In the third month of treatment the weight differences increased to -15.8% (P < 0.051, although lead values were almost stable during the 3 months of lead administration (Table 1). In control animals, immunolabeled hippocampal cells resembled fibrous astrocytes (Fig. la,b), and they showed different branching patterns of processes, depending on their location in hippocampal areas. Some of the processes were seen to extend both around neurons and immediately outside the endothelial cells, outlining perivascular regions. After 30 days of lead treatment, immunohistochemical studies in hippocampal sections revealed that the reaction product associated with gliofilaments followed the pattern of normal astrocytes, in spite of the high blood lead values; the differences from control animals were negligible. After 60 days of lead exposure, immunostaining revealed the presence of a great number of GFAP-immunolabeled cells (Fig. 2a). Individual astrocytes (Fig. 2b) presented a pattern of staining (Fig. 2a,b) different from that observed in the respective controls (Fig. la,b). Processes seemed thicker and less highly branched than
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Fig. 1. Astrocytes in the CA1 region of the hippocampus. GFAP-immunoreactivecells show a uniform staining of astroglial cells with thin, wavering rocesses. Many processes can be seen associated with the blood vessels. Control rat, 2 months old. a: [ow magnification of a histological section. ~ 4 0 0 Scale . bar = 25 pm. b: High magnification from another histological section of other control rat. x 1,000.Scale bar = 10 pm.
in controls (Fig. 2a,b). After 90 days of lead treatment, an increment of the diaminobenzidine reaction product was seen. GFAP immunoreactivity was concentrated in the cytoplasm surrounding the nucleus, and in proximal parts of processes (Fig. 3a,b). Preliminary image analysis data in six treated animals (2 months of exposure) showed that the individual astrocytes presented a larger sectional area: 6,416 pm2 to 11,628 pm2 with a significant (P < 0.002) increment of GFAP-positivecell areas (average: 8,766 ? 565 pm2). The soma area of GFAP-positivecells occupied 6.54 f 1.91%and the processes 93.44 ? 1.91% (P < 0.005). In six control animals, the area of GFAP-immunoreactive cells (1,458 pm2 to 3,000 pm2) showed a marked difference (P < 0.002; average: 2,487 k 145 pm2) from those of lead-exposured rats. The partial control areas for soma and processes were 15.87 2 4.24% and 84.13 5 4.24%, respectively. Hypertrophic astrocytes were found distributed in the neuropil, and around endothelial cells and neurons. The increased density of GFAP immunoreactivity was seen to be evenly distributed in every astrocyte in the tissue sections. Mitotic figures, local modifications, andor
degenerative lesions were not observed in semithin sections stained with toluidine blue. The neighboring brain areas of the hypertrophic astrocytes had a normal aspect at all times of lead exposure. These results were consistent with our previous observations (Selvin-Testa and Palacios Pru, 1988) and the present results at the ultrastructural level, where an increment in gliofilaments was shown. Thick bundles of filaments surrounded by numerous mitochondria, most of them with a normal aspect, was a constant finding in the soma and astroglial cell processes of treated animals (Figs. 4a and 5; compare with a control astrocyte, Fig. 4b). After 2 months of lead administration, intranuclear inclusion bodies, without limiting membrane, were detected in some astrocytes (Fig. 5).They were of irregular shape with an electron-dense core and amorphous matrix. A denser fibrillar interwoven mesh of fibrils radiating out and giving a spicular border (Fig. 5) could be distinguished in the matrix. The cytoplasm of astroglial cells with intranuclear inclusions always showed an abnormally large accumulation of filaments (Fig. 5). During the period of lead administration no changes in endothelial cells were detected and no reactive micro-
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Fig. 2. After 2 months of lead intoxication astrocytes exhibit an increase in filaments immunolabeled with GFAF'antisera. The soma and rincipal astroglial processes in the CA1 region ofthe hi pocampus are enlar ed and more intensely stainei than in controls.a: Low magnificationof a histologicafsection.X400. Scaletar = 25 pm.b: Hi h magnificationfrom another histologicalsection of area CA1 of another 2 month treated rat. ~ 1 , 0 0 0Scaf? . bar = 10 pm. Compare with Fig. l a and b.
glial cells appeared in the vicinity of modified astro- thy, with a high level of lead exposure, induced in cytes. postnatal rats during the lactation period (Goyer and Rhyne, 1973).These findings showed some similarities with our results but they differed in: 1) the higher level of lead administration; 2) the short duration of the DISCUSSION intoxication period (up to weaning); and 3) the beginThe immunohistochemical reaction of GFAF', a glio- ning of lead exposure, the first postnatal day. In the typic protein, has been used to characterize the tempo- present immunohistochemicaldata, 30 day treated rats ral and regional patterns of astrocytic reactivity in did not show an increase in GFAP. These results are lead-treated animals, as was proposed by OCallaghan consistent with our previous ultrastructural observa(1988) after brain insults induced by other neurotoxi- tions (Selvin-Testaand Palacios-Pru, 1988)detected in chronic low-level lead intoxication with short exposure cants. GFAP staining demonstrated clearly the astroglial periods (postnatal days 7-30). However, a transient reaction after 60 days of lead exposure. Passage from astroglial reaction was reported by Sundstrom and the quiescent to the reactive state was characterized by Kalino (1987) in 15 day old rats. This observation is an increment in GFAP immunolabeling in astrocytes difficult to compare with our results because of the during the 3 months of lead administration. These methodological differences (different age and exposure findings were confirmed by computerized image analy- conditions). When the low-level lead exposure was maintained at sis. The electron microscopic appearance of numerous gliofilaments in the perinuclear cytoplasm and in bun- least for 2 months, astrocytic response appeared; GFAP dles converging on large cell processes was consistent immunoreaction was greatly enhanced within individual cells, if lead administration lasted for 3 months. A with the GFAP immunostaining. Glial hypertrophy was described in an encephalopa- comparable glial response was described by us after 8
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Fi 3. a: Astrocytes after 3 months of lead exposure show a strong immunocytochemical reaction to G F h , with evident enlargement of cells. ~ 4 0 0 .Scale bar = 25 pm. b: Note the increased area of GFAP-positive cells with intense immunoreactivit in the soma and processes after 3 months of lead . bar = 10 pm. treatment. A high magnification of photomicrograpg in a. ~ 1 , 0 0 0Scale
months of exposure, using a very low dose (40 mg% of lead acetate in drinking water) (Selvin-Testaand Palacios-Prii,1988). Modification of astroglial cells is a common response after a chemically induced injury (Bjorklund et al., 1986) and other forms of brain lesions (Eng, 1987; Lascano and Berria, 1983). However, in our leadtreated rats, an evident astroglial hypertrophy in the hippocampus was observed in the absence of local destructive lesions or edema responsiblefor gliosis (Klatzo et al., 1980).This glial response presented some similarity to that observed in old rats (Topp et al., 1989). The advantages of differential litter size in the reduction of the body weight differences (Alfano and Petit, 1982),and of initiation time of lead exposure (Holtzman et al., 1982) to avoid possibility of malnutrition, were confirmed in our results. Intranuclear inclusions, an occasional finding, were always seen in astrocytes containing a large number of gliofilaments, and more than 2 months of exposure were necessary for detection. Our results differ from those reported by Powell et al. (1982) in which intranuclear inclusions without degenerative changes appeared in
Schwann, endothelial, and renal tubular cells in the early stages of high-dose lead neuropathy. At variance with our observations made in young adult rats, Holtzman et al. (1984, 1987) observed that intranuclear bodies, without an increase in gliofilaments, were frequently seen in astrocytes of immature lead-exposed animals. The way lead affects astroglial cells is unknown; even the sequence of events occurring in the CNS is not yet established. The cause of astrocytic reaction is probably direct exposure to the neurotoxicant, which induces metabolic changes. These changes can spread rapidly from cell to cell through gap junctions and provoke cytoskeletal alterations in the astroglial cell bodies and processes. Another possibility could be an indirect glial effect (Eng, 1987) as a consequence of subtle leadinduced neuronal changes (Selvin-Testa and PalaciosPrii, 1988). In this case, neurons, primarily affected, may modify the astroglial cytoskeleton. The initiation and regulation of astroglial reactivity are not understood. Lead, perhaps like other neurotoxicants, facilitates the release of some unknown activator that produces morphogenic alterations in a time-depen-
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Fig. 4. a: A portion of the astroglial process located in the neuropil contains packed bundles of gliofilaments surrounded by mitochondria and glycogen anules. Neighboring structures have a normal aspect. After 2 montg of lead ex osure. X30,OOO. Scale bar = 0.5 pm. b: Astrocyte in the neuropila!o control animal 75 days old. Bundles of gliofilaments are thinner than in treated rats. Compare with Figs. 4a and 5. x 18,000.Scale bar = 0.5 pm.
Fig. 5 . Astrocyte with intranuclear inclusions,after 2 months oflead treatment. The cytoplasm appears filled with com act bundles of gliofilaments. Neuropil elements surrounding the aftered astrocyte show well-preservedstructures. ~24,000.Scale bar = 0.5 pm.
LEAD EFFECT ON HIPPOCAMPAL ASTROCYTES
dent fashion (Miller et al., 1987). However, modifications in the cytoskeletal protein (GFAP) polymerized into gliofilaments (Eng and DeArmond, 1981) could be an unspecific reaction. Astrocytes perform homeostatic regulatory functions, exerting a fine control of the CNS extracellular environment (Pearce and Murphy, 1988), which produces a complex interdependency between neurons and glia (Svaetichin et al., 1965).Astroglial cells, when they lose their normal structural organization, probably change a variety of the active mechanisms that maintain a normal neuronal microenvironment, inducing disturbances in neuronal functions. The way lead enters into the cells is unknown, although Goldstein et al. (1977) proposed that it uses the same carrier as Ca2+.Lead may also directly substitute for, or antagonize, processes that are normally activated by calcium (Audesirk, 1985). It can be assumed that interference with Ca2+metabolism in lead intoxication also mediates morphological changes in the astroglial cytoskeleton. As lead is a common environmental contaminant, it may be considered that its potential neurotoxicant effect provokes alterations in astroglial cells of the human brain-particularly in children-evidenced as functional changes in the nervous system.
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Blackstad, T.W. (1956) Commissural connections of the hippocampal region in the rat with special references to their mode oftermination. J. Comp. Neurol., 105:417-533. Chisolm, J.J. (1984) The continuing hazard of lead expsoure and its effects in children. Neurotoxicogy ,5:23-42. Collins, M.F., Hrdina, P.D., and ittle, E. (1982) Lead in blood and brain regions of rats chronically exposed to low doses of the metal. Toxicol. Appl. Pharmacol., 65:314-322. Dav, D. and Bi ami, A. (1985) Intermediate filaments in nervous tissue. In: C e l c n d Muscle Motzlzty. J.W. Shay, ed. Plenum Press, New York, Vol. 6, pp. 75-96. Eng, L.F. (1987) Experimental models for astrocyte activation and fibrous gliosis. In: Glial-Neuronal Communication in Development and Re eneration. NATO-AS1 series H, Cell Biology. Springer, New York, $01.2, p 27-40. Eng, L.F. and b e b o n d , S.J. (1981) The glial fibrillar acidic (GFA) rotein immunocytochemistry in development and neuro athology. fn: Glial and Neuronal Cell Biology.V. Acosto Vidrio and JFederoff, eds. Alan R. Liss, New York, Vol. 1, p. 65-79 Goldstein,,G.W., Wolinsky, J.S., and Zsejtey, J: (1977) Isolated brain capillaries: A model for the study of lead encephalopathy. Ann Neurol., 1:235-239. Gonzalez Aguilar, F. and De Robertis, E. (1963) A formalin- erfusion fixation method for histophysiological study of the centrar nervous system with electron microscope. Neurology, 13:758-771. Goyer, R.A. and Rhyne, B.C. (1973) Pathological effects of lead. Int. Rev. Exp. Pathol., 129-79. Holtzman, D., De Vries, C., Nguyen, H., Jameson, N., and Olson, J. (19821 Development of resistance to lead encephalopathy during maturation in rat ups. J. Neuropathol. Ex Neurol., 41:652463. Holtzman, D., De d i e s , C., Nguyen, H., OPsbn, J., and Bensch, K. (1984)Maturation of resistance to lead encephalopathy: Cellular and subcellular mechanisms. Neurotoxicology, 597-124. Holtzman, D., Olson, J., De Vries, C., and Bensch, K. (1987) Lead toxicity in primary cultured cerebral astrocytes and cerebellar granular neurons. Toxicol. Appl. Pharmacol., 89:211-235. Klatzo, I., Chui, F., Fujiwara, K., and Spatz, M. (1980) Resolution of vasogenic brain edema. Adv. Neurol., 28:35&373. Landrigan, P.J., Baker, E.L., Whitworth, R.H., and Feldman, R.K. ACKNOWLEDGMENTS (1980) Neuroe idemiological evaluation of children with chronic increased l e a l absorption. In: Low Level Lead Exposure. H.L. Needleman, ed. Raven Press, New York, pp. 17-33. We would like to express our thanks to Dr. A. PelleE.F. and Berria, M.I. (1983) Immunoperoxidase study of grino de Iraldi and Dr. F. Gonzalez-Aguilar for critical Lascano, astrocytic reaction in Junin virus encephalomyelitis of mice. Acta reading of the manuscript, to Dr. E. Villaamil and D. Neuropathol., 59:183-190. J. and McCarthy, K. (1986) Neuronal-glial interactions. In: Gonzalez (Catedra de Toxicologia y Quimica Legal, Fac. Lauder, Astroc tes Biochemist Physiology and Pharmacology of Astrode Farmacia y Bioquimica. Universidad de Buenos cytes. $. Fedoroff and Ayernadakis, eds. Academic Press, New York, Vol. 2, pp. 295-314. Aires) and to Dr. B. Chandler (Instituto de InvestigaJ.M., Hauw, J.J., and Bernard, G. (1983) Regressive or ciones Toxicologicas, Universidad Central de Venezu- Lefauconnier, lethal lead encephalopathy in the suckling rat. Correlations of lead ela) for determinations of blood lead levels, and to P.G. levels and morphological findings. J. Neuropathol. Exp. Neurol., 42:177-190. Testa for assistance in statistical analysis. This work Lorente de No, R. (1934) Studies on the structures of the cerebral was supported by grants from the Consejo Nacional de cortex. 11. Continuation of the study of the ammonic system. J. Investigaciones Cientificas y Tecnicas (CONICET),RePsychol. Neurol., (Leipzi ),46 113 177 McCauley, P.T., Bull, R.J.,%ontij A,