EXPERIMENTAL
NEUROLOGY
107,97-105
(1990)
Effects of Postnatal Ethanol Exposure on Glial Cell Development in Rat Optic Nerve D. E. PHILLIPS
AND S. K. KRUEGER
Department of Biology and WAMZ Medical Education Program, Montana State University, Bozeman, Montana 59717
dence of alcohol-induced abnormalities in neuronal migration (11, 18, 19) which could relate to abnormal glial development since astrocytes are important in normal neuronal migration during development (27). Also, some of the most dramatic reductions in brain size occur in experimental animals when exposures to alcohol take place during a period of development when the most rapid glial proliferation and development occurs (9, 24, 39), implying a potential effect on the proliferation, growth, or maturation of glia. Despite these evidences, experimental studies of the potential effects of alcohol on glial development have generally been limited to studies of cultured astrocytes in which high doses of ethanol impair cellular growth rate and enzyme maturation (7, 13, 14). The brain growth spurt, which peaks during the third trimester in humans and during the first 7-10 postnatal days in rats, is a crucial time in brain development. Most myelination is initiated, myelin and glial cells are rapidly forming and maturing, neurons are completing migration, and synapses are developing during this period (9). Exposures to alcohol during the brain growth spurt have often been provided to rat pups via the milk of the nursing mother but there are difficulties in controlling such exposures since alcohol negatively affects maternal lactation, and because the doses of alcohol ingested by the pup are relatively low and uncontrolled (34, 42). These problems can be minimized by using artificial rearing, in which ethanol-containing and control diets are fed to rat pups via a gastrostomy tube (29, 41), but, except for studies of neurons in the hippocampus and cerebellum (24, 40, 41), this method has not been used for experimental studies of the pathological effects of alcohol on specific cell populations in the developing nervous system. This study was designed to determine what effects a limited postnatal ethanol exposure has on the development and maturation of glial cells in rat optic nerve. The optic nerve of the rat is an excellent tissue for such a study because it is a simplified and well-studied model of the development of CNS glial cells and myelin (31, 35, 37). The exposure can be provided during the most active phases of growth in the optic nerve when oligoden-
This study morphologically evaluated the effects of limited postnatal alcohol exposure on the development of glial cells in the rat optic nerve. Rat pups were artificially reared on Days 5-18 with a supplemented milk diet fed via a chronic gastrostomy tube. Experimental animals received 4% ethanol in their diet on Days 5-9, otherwise the experimental and control animals received identical diets. Optic nerve tissues were prepared for electron microscopy on Days 10, 16,22,29, and 90. There were fewer glial cells per cross section and the cross-sectional areas of optic nerves were smaller on Days 10 and 16 in the ethanol-exposed animals. The alcohol caused a delay in the maturation of oligodendroglial cells at 10 days as evidenced by decreases in the total number of oligodendroglia present and by a delay in the appearance of immature cells within the oligodendroglial lineage. All of these effects were compensated for at later ages. There was no evidence of alcohol-induced degeneration of glial cells or their organelles. Thus, postnatal alcohol exposure causes a delay in oligodendrocyte maturation but appears to have no long-term effects on the glial cell population of rat optic nerve. 0 1990 Academic Press, IIS.
INTRODUCTION
Children with fetal alcohol syndrome (FAS), caused by in hero exposure to alcohol, are characterized by behavioral, neurological, and intellectual manifestations of developmental brain damage (12,33). Numerous studies have examined the effects of alcohol on neuronal development (for review see (42)) but potential effects on glial cells have not been well studied. Abnormalities in glial development are suspected of contributing to alcohol-induced brain effects for several reasons. There is evidence of abnormal glial migration in postmortem studies of FAS victims (3,4) as well as in primate models of FAS (5). Experimental evidence exists for alcohol-induced delays in myelin acquisition (10,15,22,28) which could be related to effects on the development of the oligodendroglial cells which produce myelin. There is evi-
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AND
droglia increase dramatically in number, mature morphologically, and initiate the production of myelin (31, 32,35). MATERIALS
AND
METHODS
The animal rearing and exposure methods used in this study are the same as those reported earlier in more detail (22) and are similar to those reported by Samson and Diaz (29) and West et al. (41). Three to five litters from nulliparous Sprague-Dawley rats were allowed to nurse until Day 4 (96 f 12 h) when the pups were weighed and 20 animals of either sex selected for gastrostomy and an artificial rearing “run.” The animals selected for each run averaged 10.1 to 10.5 g and were divided into weightmatched experimental and control groups. Because of mortalities, several rearing “runs” were required to produce at least four experimental and four control animals at each of five ages studied. All diet was fed through an implanted gastrostomy cannula from syringes on infusion pumps. The supplemented milk diet (8) was infused from Day 5 through Day 18 when the gastrostomy tube was sealed. The animals were then raised in standard cages on standard lab chow. Experimental animals received 4% (v/v) ethanol in their diet on Days 5 through 9, otherwise the experimental and control animals received identical diets in identical volumes. Ether-anesthetized animals were perfused with 4% paraformaldehyde and 2% glutaraldehyde in 0.1 M cacodylate buffer on Postnatal Days 10, 16, 22, 29, and 90 and the optic nerve was prepared for electron microscopic study. Because of the possibility of glial maturation occurring at different rates along the length of the nerve (32), cross sections were always taken from a region extending from 1 mm behind the bulb to approximately one-third the distance to the chiasm. One-micrometer sections for light microscopy were stained with basic toluidine blue. Thin sections for electron microscopy were mounted on uncoated copper grids and stained with methanolic uranyl acetate and aqueous lead citrate. The cross-sectional areas of the optic nerves were measured in l-pm plastic sections at 150 or 300X by tracing the phototube image of the nerve on a computerized bit pad (Zeiss Interactive Digital Analysis System-ZIDAS). Areas of blood vessels were traced and excluded from the gross area. Care was taken to be sure that the nerve sections had a majority of the fibers cut in near circular profile anti thus were not oblique sections of the nerve. Sections from at least two blocks per animal were measured for area, and then an average was determined for each animal. Counts of glidl cells were made in the same light microscopic cross sections that were measured for area.
KRUEGER
The counts were done at 400X by identifying glial cell nuclei and excluding pial, endothelial, and pericytic nuclei. Phototube tracings onto grid paper were used to subdivide the fields and the cells were marked to avoid repetitive counts. Glial cell densities were calculated by dividing glial cells per section by the net area of the optic nerve cross section. Glial cell morphology was studied both directly on the electron microscope and in electron micrographs. Cells were examined for any indication of degeneration or abnormal morphology and cells from experimental and control animals were compared as to organelle content, degree of maturation, and lineage development. Differential counts of developing cell types were done in random sections of optic nerve cross section that did not include the external limiting membrane. All glial cells from one section per animal were identified and characterized as either microglia, glioblasts, immature oligodendroglia, active oligodendroglia, mature oligodendroglia, or astrocytes using characteristics defined in developing optic nerve (31,35,29) and spinal cord white matter (21). Cells were identified directly from electron microscopic sections in more mature animals, while in younger animals, electron micrographs of the cells were used for identi,fication. Care was taken to record individual cell detail and to map sections so that cells were not counted more than once. Electron micrographic identifications were repeated by a second observer blind to the tissue source. For a given animal all cells were totaled and the differential counts were converted to percentages. The total numbers of cells in given sections varied so that the numbers of cells characterized in each animal ranged from 15 to 69 (mean, 30 per animal). The total numbers of cells characterized in experimental and control animals, respectively, were 159 and 107 at 10 days; 96 and 104 at 16 days; 101 and 133 at 22 days; and 123 and 137 at 90 days. Projected numbers of cell types per cross section were calculated by multiplying the total number of glial cells in a cross section (as counted in lpm sections) by the percentage of a given cell type from that same tissue block. Statistical comparisons used an ANOVA to compare the means of at least four experimental and four control animals,at each age evaluated. OBSERVATIONS
The body and brain weights from these animals have been previously reported (22). Generally, the alcohol-exposed animals were slightly, but not significantly, smaller than control animals at all ages studied. Brain weights and brain weight to body weight ratios were always less in ethanol-exposed animals but the differences were statistically significant only at 10 days (by approximately 25%).
ETHANOL-INDUCED
EFFECTS
TABLE Glial Cell Nuclei per Cross-Section;
99
GLIOGENESIS
1
Optic Nerve Cross-Sectional
Area”
16 days
22 days
156 (15.7)** 195 (12.8) 80 0.009
229 (37.9) 289 (35.7) 79 0.06
276 (7.5)* 300 (16.7) 92 0.04
212 256
2.56 (0.51)* 3.79 (0.80) 68 0.04
6.63 (1.26)* 8.55 (0.74) 78 0.04
10.9 (0.4) 11.4 (1.4) 96 0.49
10.9 (2.1) 12.6 (1.0) a7 0.18
10 days Number of glial nuclei Experimental Control E/C as % P Optic nerve area minus mm2 X lo-’ Experimental Control E/C as % P
ON
29 days
(20.2) (36.6) 83 0.08
90 days
254 (20.2) 271 (29.6) 94 0.39
vessel areas,
a Means and (standard deviations) on sections from at least two tissue * P < 0.05, **P < 0.01, ANOVA.
of four experimental blocks for each animal
and four control animals at each age. Glial cells and areas were counted and then averaged for the animal. E/C = Experimental/Control ratio.
The gross numbers of glial cells per cross section of optic nerve (Table l), as determined by light microscopic counts, were reduced by approximately 20% in ethanolexposed animals at 10 and 16 days and to a lesser extent at 22 days (-8% reduction) and 29 days (-17% reduction) but were not dramatically or significantly different than control animals at 90 days. This reduction in number paralleled a considerable reduction in the net cross sectional area of the same sections of nerve at 10 and 16 days (Table 1). When glial number was divided by area to calculate the approximate glial cell density, there were no dramatic or statistically significant differences in alcohol-exposed animals. The ultrastructural morphology of glial cell bodies and their processes was similar in both experimental and control animals and was similar to the descriptions in the literature of glial cells in developing rat optic nerve (31, 35, 37). There was no evidence in any of the glial cell types of alcohol-induced pathology or degeneration. Proportions of glial cell types (Table 2) were calculated from electron microscopic counts made on the basis of the morphological specializations described. Microglia. Microglial cells had a dense, angular, and often elongated nucleus. The cytoplasm was often dense and contained rough endoplasmic reticulum which was elongated and stringy in appearance. The cytoplasm often contained lipid droplets or dense bodies. Microglia never accounted for more than 5% of the glial cells in either experimental or control animals and generally were present in the O&2% range (Table 2). Although the average numbers of microglia were higher in the experimental animals, the absolute number present was so low (0 in some animals in both experimental and control groups) as to preclude comparisons. Glioblusts. Glioblasts were characterized by a relatively large nucleus with a thin rim of cytoplasm which
19.4 (3.3) 20.0 (1.6) 97 0.74 and measured
contained only scattered mitochondria and ribosomes. Microtubules, glial filaments, glycogen, or elements of endoplasmic reticulum were not normally present. Glioblasts were only encountered at 10 and 16 days in experimental animals and at 10 days in control animals. The glioblasts were too few in number to allow statistical comparisons, but there were more glioblasts encountered in experimental animals than in control animals, particularly at 10 days. Immature oligodendroglia. Immature oligodendroglia commonly occurred either as elongate or rounded cells with processes at one or both ends. The cells had a fairly electron lucid cytoplasm and a nucleus with evenly scattered chromatin. Microtubules were common throughout the cytoplasm as were obvious ribosomes. The short cisternae of rough endoplasmic reticulum contained material of about the same density as the cytoplasm. There were numerous small, well-organized areas of Golgi apparatus. Immature oligodendroglia were relatively common at 10 days in both experimental and control animals (Table 2); they were fewer but still present at 16 days, but were not encountered at 22 days and after. There were fewer immature oligodendroglia at 10 days (-35% fewer, P = 0.09) but not at 16 days in ethanol-exposed animals. Active oligodendroglia. Active oligodendroglia (Fig. 1) are generally considered to be the cells actively involved in the initiation and formation of myelin sheaths (21, 35, 37). Their morphology was representative of a transition between immature and mature oligodendroglia. The cells were fairly large and usually had clearly identifiable processes that could sometimes be traced to continuity with developing myelin. The nucleus was rounded with some chromatin clumping and the cytoplasm was commonly, but not always, somewhat electron dense. Scattered microtubules were common, espe-
100
PHILLIPS
AND KRUEGER
TABLE
2
Glial Cellsby Type, as Percentages” 10 days
16 days
22 days
90 days
0.9 (1.8) 0.5 (1.0)
4.1 (3.7) 2.9 (3.8)
55.9 (18.0) 57.6 (10.6) 97 0.87
44.2 (17.2) 38.4 (11.9) 115 0.60
Microglia Experimental Control
1.3 (0.9) 0.0 (-)
2.0 (4.0) 1.8 (2.0)
Glioblasts Experimental Control
6.9 (5.1) 1.3 (2.6)
1.3 (2.5) 0.0 (-)
Astrocytes Experimental Control E/C as % P
54.2 (13.5) 42.5 (5.4) 128 0.15
48.9 (8.7) 43.2 (16.2) 113 0.55
Immature oligodendroglia Experimental Control E/C as % P
25.4 (8.9) 40.2 (13.2) 63 0.09
17.7 (13.1) 18.0 (13.3) 98 0.97
Mature and active Experimental Control E/C as % P
11.3 (7.0) 16.0 (12.6) 71 0.50
30.2 (7.0) 37.1 (19.4) 81 0.53
41.2 (19.9) 39.4 (6.0) 105 0.86
53.9 (15.1) 58.7 (9.8) 92 0.61
36.7 (9.1)** 56.2 (4.9) 65 0.006
47.9 (10.7) 55.1 (16.0) 87 0.48
41.2 (19.9) 4O.lt6.9) 103 0.92
53.9 (15.1) 58.7 (9.8) 92 0.61
Oligodendroglia, Experimental Control E/C as % P n Means and (standard ** P c 0.01, ANOVA.
oligodendroglia
total
of all types
deviations)
of four experimental
and four
control
cially in the processes. Numerous ribosomes contributed to the density of the cytoplasm. The characteristic rough endoplasmic reticulum was composed of compact cisternae with an electron lucid content and were usually arranged in stacks. Mitochondria were small, elongate, electron dense, and often located in a submembrane position. Active oligodendroglia were most common in the younger animals studied (10 and 16 days) and were unusual after 22 days. Transitional cells between active and mature oligodendroglia were common, making absolute assignment of specific cell type difficult, so active oligodendroglia were grouped with mature oligodendroglia for differential counts (Table 2). Mature oligodendrogliu. Relative to active oligodendroglia, mature oligodendroglia (Fig. 2) were smaller and more rounded, and their processes were less obvious. There were both light and dark oligodendroglia. In the dark cells the cytoplasm was electron dense and the chromatin was clumped beneath the nuclear envelope. Cell processes could be found occasionally but were difficult to trace. Stacks of rough endoplasmic reticulum were encountered less often than in active oligodendrog-
animals.
lia. Microtubules were present, although they were less obvious than in active oligodendrocytes. Mitochondria were generally of small caliber and often were in a characteristic submembrane location; their matrix was dense and their cisternae were electron lucid. In dense oligodendroglia there were numerous ribosomes packing the cytoplasm and contributing to its electron density. Light oligodendroglia were generally similar in details of organelle structure and arrangement except that the cytoplasm was less electron dense, there were fewer ribosomes, and the nucleus had less obvious chromatin clumping. Mature oligodendroglia were hardly ever encountered at 10 days, and only occasionally at 16 days. At 22 days they were common and by 90 days they comprised virtually all of the oligodendroglial population. As a combined group, active and mature oligodendroglia in control animals increased from 16% at Day 10 to 59% at Day 90 (Table 2). The proportion of this combined category of cells was never dramatically or statistically different in ethanol-exposed animals. The counts of specific cell types (Table 2) revealed that alcohol caused a marked delay in oligodendroglial
ETHANOL-INDUCED
EFFECTS
ON
GLIOGENESIS
101
FIG. 1. Active oligodendroglia (0) from a 16-day ethanol-exposed animal, 11,900X. Active oligodendroglia are relatively large with a rounded nucleus with some marginal chromatin clumping. Processes (arrows) are easily identified and can sometimes be traced to continuity with developing myelin. The cytoplasm is commonly, but not always, electron dense. Scattered microtubules (T) are common, especially in processes, and can contribute to the density of the cytoplasm. The rough endoplasmic reticulum is composed of compact cisternae often arranged in stacks (arrowheads). Mitochondria are small, elongate, often electron dense, and often in a submembrane location. Such cells showed no evidence of ethanol-induced alterations in structure.
development, which was particularly obvious at 10 days. When all cells of the oligodendrocyte lineage (immature, active, and mature oligodendroglia) were grouped together, there was a dramatic and statistically significant decrease in the proportion of cells in that lineage at 10 days in the ethanol-exposed animals (21 to 55% fewer, mean of 35% fewer, P = 0.006). The projected number of all oligodendroglia per cross section was dramatically less in experimental animals on Day 10 (16 to 62% fewer, mean of 48% fewer, P = 0.005) and although they were still apparently fewer in number on Day 16, the mean difference was not statistically significant (as much as 40% fewer, mean of 27% fewer, P = 0.10). When all immature cells contributing to the oligodendroglia lineage (glioblasts and immature oligodendroglia) were considered it was apparent that, in younger animals, there was a delay in the progression of maturation of oligodendroglia in ethanol-exposed animals. Astrocytes. Mature astrocytes (Fig. 2) had more electron lucid cytoplasm than other glial cells. The nuclear
chromatin was generally evenly distributed with some slight submembrane clumping. Cells had numerous processes with fine branches filling spaces between other glial elements and axons. Groups of filaments were easy to identify but microtubules were only occasionally present. Glycogen granules could be identified in scattered locations. The characteristic rough endoplasmic reticulum consisted of cisternae that were slightly expanded and contained a flocculent, slightly electron dense material. Golgi were scattered and consisted of four or five stacks of relatively short cisternae. Mitochondria, located in random locations in the cytoplasm, were elongated, plump, and with a matrix slightly more dense than the cytoplasm. Dense bodies were common. Immature astrocytes were similar in most ways to mature astrocytes except that microtubules were more common and glial filaments were less common. Filaments in immature astrocytes were often scattered in the cytoplasm rather than in bundles. There was relatively less cytoplasm and it was slightly more electron dense. Im-
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AND
KRUEGER
FIG. 2. Mature astrocytes (A) and a mature oligodendroglia (0) from a 22-day ethanol-exposed animal, 16,000X. Astrocytes are characterized by a relatively electron lucid cytoplasm and nuclear chromatin that is evenly distributed with some slight membrane clumping. The cells have numerous processes with fine branches filling excess space (arrows). Groups of filaments (F) are obvious. The cistemae of rough cytoplasmic reticulum (arrowheads) are slightly expanded and contain a flocculent, slightly electron-dense material. Golgi are scattered and consist of four or five stacks of relatively short cisternae. Mitochondria (M) are elongated, plump, and with a matrix only slightly more dense than the cytoplasm. Dense bodies (D) are common. Mature oligodendroglia are smaller and more rounded, and the processes are less obvious, relative to active oligodendroglia. The cytoplasm is usually quite dense and the stacks of RER are less conspicuous. The dense mitochondria (M) are often in a submembrane location. The mature glial cells had no structural evidence of changes due to the developmental ethanol exposure.
mature astrocytes were only present in the youngest animals studied and it was difficult to always separate them from mature cells, so the two types were combined in the differential counts (Table 2). Astrocytes in control animals accounted for 42 to 58% of the glial cells present from Days 10-90. They were present in relatively greater proportions at 10 and 16 days in ethanol-exposed animals (although the differences were not statistically significant) but the proportion of astrocytes in experimental and control animals was not markedly different at 22 or 90 days. The total projected number of astrocytes per cross section was essentially the same in experimental and control animals at each age studied, including at 10 and 16 days when oligodendroglia were less common and the total number of cells was less. In general, the progression of development of astrocytes was not delayed by the ethanol exposure as was the development of oligodendroglia.
DISCUSSION
The primary effect of postnatal ethanol exposure on developing glial cells in the rat optic nerve was a delay in the maturation of oligodendroglia. This was evidenced by decreases in the total number of oligodendroglia present, especially at 10 days, and by a delay in the appearance of immature cells within the oligodendroglial lineage. The proportion of cells of different types was similar in older animals, and by 90 days there was no evidence of differences in cell structure or in the relative proportions of the various cell types. The numbers of astrocytes and the progression of their maturation did not appear to be affected by the postnatal alcohol exposure. There was no evidence of abnormal glial cell placements, such as the abnormal loci of cells reported in postmortem examination of human and primate brains after developmental exposure to alcohol (3-5). This is
ETHANOL-INDUCED
EFFECTS
despite the fact that this ethanol exposure occurred at a time of rapid glial maturation and migration in the optic nerve (31, 32, 37) when such ectopic placements might be expected to originate from such an insult. Perhaps the dosage did not occur early enough to cause such ectopia or perhaps the oligodendroglia, which are so actively maturing and migrating at this time, are simply not involved. It is not surprising that this particular ethanol exposure exerts its primary effect on oligodendroglia and not astrocytes. This is because most astrocytes in the rat optic nerve (particularly radial or Type I astrocytes) form in late gestation and have undergone much of their maturation before Days 5-9 (2531) when the exposure occurred. Conversely, the oligodendroglia are proliferating and maturing at their greatest rate during Days 5 to 10 (25, 31, 32, 35), at the time of this ethanol exposure. Thus if alcohol has a significant effect on the processes of cell acquisition and maturation in glial cells, as it does in neurons (2,19,20,23,38), one would expect the timing of this exposure to favor damage to maturing oligodendroglia rather than astrocytes. Although alcohol-induced delays in glial proliferation and maturation have been reported in in vitro studies, those studies have all focused on cultured astrocytes. Acute exposure to ethanol does not affect the cellular morphology or the concentration of filament proteins, but does increase the amounts of certain nuclear proteins of cultured l-day rat astrocytes (16). Longer exposures cause a generalized delay in the proliferation and maturation of developing astrocytes cultured from chick and mouse (7,13,14). It is quite possible that the effects on oligodendroglia, which are rapidly proliferating and maturing in optic nerve at the time of the exposure (31, 35), are a reflection of cellular changes similar to those reported in the in vitro studies of proliferating and maturing astrocytes. Small et al. (32) have reported that the progenitor cells for oligodendroglia and some astrocytes (Type II) migrate into the rat optic nerve from its chiasmal end, starting at birth. Perhaps the delay in the maturation of oligodendroglia could simply be an alcohol-induced delay in the migration of the progenitor cells. Because the radial glia (Type I astrocytes) in the optic nerve generally form earlier (at Embryonic Day 16) (25, 31) and do not migrate to their position, they would not be as markedly affected by an ethanol-induced delay of postnatal migration. Another potential cause of delayed oligodendroglial maturation could be related to a delay in the maturation rate of optic nerve axons, thus effectively delaying the “trigger” mechanisms that initiate myelin formation and thus influencing oligodendroglial maturation. Such a delay in axonal growth (reported in some optic axons from these same animals, along with a delay in myelin
ON
GLIOGENESIS
103
acquisition (22)), could alter the feedback mechanisms which regulate oligodendroglial formation and maturation. Although few details have been specifically defined, such feedback seems implicit in the current understanding of myelination (26,43). There was no morphological evidence in this study of ethanol-induced pathological alterations of individual glial cells or organelles. This is in contrast to the suggestion that morphological alterations of mitochondria may be an important feature of developmental alcohol exposure in neurons (1). Even though ethanol is reported to inhibit ATPase activity in synaptosomal preparations from adult CNS (17), there was no consistent evidence in this study of the cellular swelling expected if such an inhibition of ATPase activity occurred in developing glial cells (36). Perhaps glial cells are not as susceptible to such cellular alterations as neurons or perhaps the ethanol exposure provided in this study was not provided early enough, long enough, or in high enough doses to produce such damage. Although the overall average effects of ethanol are subtle, some individual ethanol-treated animals were severely affected compared to other ethanol-exposed or control animals. One ethanol exposed animal at 10 days had a 55% smaller proportion of oligodendroglia and 62% fewer projected total number of oligodendroglia than the average of control animals. The most extreme examples of ethanol-induced pathology are partially masked by evaluation of means and consideration of such results needs to include the concept of the possibility of potential for more extreme pathology in individual instances. Despite this extreme effect in those animals most affected, there was no consistent evidence of cellular degeneration. The fact that there was no apparent evidence of cellular degeneration or permanent glial cell loss due to the ethanol exposure is not surprising since some developmentally exposed nerve cell populations do not show dramatic evidences of ethanol-induced cell death, but rather, there is evidence of similar delays in their maturation or migration (18,20,38,42). However, other neuronal populations do undergo obvious cellular degeneration during or after developmental exposures to ethanol (2, 6, 23) and in some instances all evidence of such activity is gone within 1 or 2 days (6,23). There is a possibility that glial cell degeneration could have occurred in this study during the 5- to g-day exposure period, but that the evidence of such degeneration was gone by 10 days when the tissue was first examined. It is difficult to project the long-term implications of this reported delay in oligodendroglial maturation, particularly since the developmental exposure to alcohol has apparently not affected the number, distribution, or morphology of glial cells in mature animals. Obviously the delay in oligodendroglial maturation could be related
PHILLIPS
AND KRUEGER
to the delay in myelin acquisition reported in these same animals (22). Such delays in myelin acquisition could, in turn, by modifying spontaneous patterns of action potentials and axon conduction, affect the maturation of other elements in the neuronal pathway (30). Such changes in neuronal pathways could help explain subtle changes in nervous system function caused by ethanol exposures. ACKNOWLEDGMENTS This work was supported by Research Grants AA07042 from NIAAA, MBRS-SO608218 from NIH, and ISPS011449 from NSF-EPSCoR. Acknowledgments are due J. Pembroke, L. Fritzler, and J. Rydquist for technical assistance.
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14. KENNEDY, L. A., AND S. MUKERJI. 1986b. Ethanol neurotoxicity.
2. Direct effects on differentiating astrocytes. Neurobehau. Tori8: 17-21. LANCASTER, F. C., S. M. PHILLIPS, P. P. PATSALOS, AND R. C. WIGGINS. 1984. Brain myelination in the offspring of ethanoltreated rats: In utero versus lactational exposure by crossfostering offspring of control, pair fed and ethanol treated dams. Brain Res. 309: 209-216. LIPSKY, R. H., M. C. VEMURI, AND S. J. SILVERMAN. 1988. Changes in nuclear proteins of astrocytes as a result of acute ammonia or ethanol exposure. Neurochem. Znt. 12: 519-524. MARQUES, A., AND C. GUERRI. 1988. Effects of ethanol on rat brain (Na+, K+)ATPase from native and delipidized synaptic membranes. Biochem. Phurmacol. 37: 601-606. MILLER, M. W. 1986. Effects of alcohol on the generation and migration of cerebral cortical neurons. Science 233: 1308-1311. MILLER, M. W. 1988. Effect of prenatal exposure to ethanol on the development of cerebral cortex. I. Neuronal generation. Alcohol: Clin. Exp. Res. 12: 440-449. MOHAMED, S. A., E. J. NATHANIEL, D. R. NATHANIEL, AND L. SNELL. 1987. Altered Purkinje cell maturation in rats exposed prenatally to ethanol. I. Cytology. Exp. Neurol. 97: 35-52. PHILLIPS, D. E. 1973. An electron microscopic study of macroglial and microglial development in the lateral funiculus of the spinal cord in the fetal monkey. 2. Zellforsch. Microsk. Anut. 140:145-167. PHILLIPS, D. E. 1989. Effects of limited postnatal ethanol exposure on the development of myelin and nerve fibers in rat optic nerve. Exp. Neurol. 103: 90-100. PHILLIPS, S. C., AND B. G. CRAGG. 1982. A change in susceptibility of rat cerebellar Purkinje cells to damage by alcohol during fetal, neonatal and adult life. Neuropathol. Appl. Neurobiol. 8: 441-454. PIERCE, D. R., AND J. R. WEST. 1987. Differential deficits in regional brain growth induced by postnatal alcohol. Neurotoxicol. Teratol. 9: 129-141. RAFF, M. C., E. R. ABNEY, AND R. H. MILLER. 1984. Two glial cell lineages diverge prenatally in rat optic nerve. Deu. Biol. 106: 53-60. RAINE, C. S. 1984. Morphology of myelin and myelination. Pages l-50 in P. Morell, Ed., Myelin. Plenum, New York. RAKIC, P. 1985. Mechanisms of neuronal migration in developing cerebellar cortex. Pages 139-160 in G. M. Edelman, W. E. Gall, and W. M. Cowan, Eds., Molecular Basis of Neural Development. Wiley, New York. SAMORAJSKI, T., F. LANCASTER, AND R. C. WIGGINS. 1986. Fetal ethanol exposure: A morphometric analysis of myelination in the optic nerve. Int. J. Dev. Neurosci. 4: 369-374. SAMSON, H. H., AND J. DIAZ. 1982. Effects of neonatal ethanol exposure on brain development in rodents. Pages 131-151 in E. L. Abel, Ed., Fetal Alcohol Syndrome, Vol. III. Animal Studies. CRC Press, Boca Raton, FL. SHATZ, C. J., AND M. P. STRYKER. 1988. Prenatal tetrodoxin infusion blocks segregation of retinogeniculate afferents. Science. 242: 87-89. SKOFF, R. P., D. L. PRICE, AND A. STOCKS. 1976. Electron microscopic autoradiographic studies of gliogenesis in rat optic nerve. II. Time of origin. J. Comp. Neurol. 169: 313-334. SMALL, R. K., P. RIDDLE, AND M. NOBLE. 1987. Evidence for migration of oligodendrocyte-type-2 astrocyte progenitor cells into the developing rat optic nerve. Nature (London) 328: 155157. col. Teratol.
15’
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18. 19.
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ETHANOL-INDUCED
EFFECTS
33. STREISSGUTH, A. P. 1987. The behavioral teratology of alcohol: Performance, behavioral, and intellectual deficits in prenatally exposed children. Pages 3-44 in J. R. West, Ed., Alcohol and Brain Development. Oxford Univ. Press, New York. 34. SWIATEK, K. R., G. J. DOMBROWSKI, JR., AND K. CHAO. 1986. The inefficient transfer of maternally fed alcohol to nursing rats. Alcohol 3: 169-174. 35. TENNEKOON, G. I., Y. KISHIMOTO, I. SINGH, G. NONAKA, AND J. BOURRE. 1980. The differentiation of oligodendrocytes in rat optic nerve. Dev. Biol. 79: 149-158. 36. TRUMP, B. F., B. P. CROKER, AND W. J. MERGNER. 1971. The role of energy metabolism, ion, and water shifts in the pathogenesis of cell injury. Pages 84-128 in G. W. Richter and D. G. Scarpelli, Eds., Cell Membranes: Biological and Pathological Aspects. Williams & Wilkins, Baltimore. 37. VAUGHN, J. E. 1969. An electron microscopic analysis of gliogenesis in rat optic newes. 2. Zellforsch. 94: 293-324.
ON GLIOGENESIS
105
38. VOLK, B., J. MALETZ, M. TIEDEMANN, G. MALL, C. KLEIN, AND H. H. BERLET. 1981. Impaired maturation of Purkinje cells in the fetal alcohol syndrome of the rat. Acta Neuropathol. (Berlin) 64: 19-29. WEST, J. R. 1987. Fetal alcohol-induced brain damage and the 3g. problem of determining temporal vulnerability: A review. Alcohol Drug Res. 7: 423441. 40. WEST, J. R., AND K. M. HAMRE. 1985. Effects of alcohol exposure during different periods of development: Changes in hippocampal mossy fibers. Dev. Brain Res. 17: 280-284. 41. WEST, J. R., K. M. HAMRE, AND D. R. PIERCE. 1984. Delay in brain development induced by alcohol in artificially reared rat pups. Alcohol 1: 213-222. 42. WEST, J. R., AND D. R. PIERCE. 1984. Perinatal alcohol exposure and neuronal damage. Pages 120-157 in J. R. West, Ed., Alcohol and Brain Development. Oxford Univ. Press, New York. 43. WIGGINS, R. C. 1986. Myelination: A critical stage in development. Neurotoxicology 7: 103-120.
NEUROLOGY
107,97-105
(1990)
Effects of Postnatal Ethanol Exposure on Glial Cell Development in Rat Optic Nerve D. E. PHILLIPS
AND S. K. KRUEGER
Department of Biology and WAMZ Medical Education Program, Montana State University, Bozeman, Montana 59717
dence of alcohol-induced abnormalities in neuronal migration (11, 18, 19) which could relate to abnormal glial development since astrocytes are important in normal neuronal migration during development (27). Also, some of the most dramatic reductions in brain size occur in experimental animals when exposures to alcohol take place during a period of development when the most rapid glial proliferation and development occurs (9, 24, 39), implying a potential effect on the proliferation, growth, or maturation of glia. Despite these evidences, experimental studies of the potential effects of alcohol on glial development have generally been limited to studies of cultured astrocytes in which high doses of ethanol impair cellular growth rate and enzyme maturation (7, 13, 14). The brain growth spurt, which peaks during the third trimester in humans and during the first 7-10 postnatal days in rats, is a crucial time in brain development. Most myelination is initiated, myelin and glial cells are rapidly forming and maturing, neurons are completing migration, and synapses are developing during this period (9). Exposures to alcohol during the brain growth spurt have often been provided to rat pups via the milk of the nursing mother but there are difficulties in controlling such exposures since alcohol negatively affects maternal lactation, and because the doses of alcohol ingested by the pup are relatively low and uncontrolled (34, 42). These problems can be minimized by using artificial rearing, in which ethanol-containing and control diets are fed to rat pups via a gastrostomy tube (29, 41), but, except for studies of neurons in the hippocampus and cerebellum (24, 40, 41), this method has not been used for experimental studies of the pathological effects of alcohol on specific cell populations in the developing nervous system. This study was designed to determine what effects a limited postnatal ethanol exposure has on the development and maturation of glial cells in rat optic nerve. The optic nerve of the rat is an excellent tissue for such a study because it is a simplified and well-studied model of the development of CNS glial cells and myelin (31, 35, 37). The exposure can be provided during the most active phases of growth in the optic nerve when oligoden-
This study morphologically evaluated the effects of limited postnatal alcohol exposure on the development of glial cells in the rat optic nerve. Rat pups were artificially reared on Days 5-18 with a supplemented milk diet fed via a chronic gastrostomy tube. Experimental animals received 4% ethanol in their diet on Days 5-9, otherwise the experimental and control animals received identical diets. Optic nerve tissues were prepared for electron microscopy on Days 10, 16,22,29, and 90. There were fewer glial cells per cross section and the cross-sectional areas of optic nerves were smaller on Days 10 and 16 in the ethanol-exposed animals. The alcohol caused a delay in the maturation of oligodendroglial cells at 10 days as evidenced by decreases in the total number of oligodendroglia present and by a delay in the appearance of immature cells within the oligodendroglial lineage. All of these effects were compensated for at later ages. There was no evidence of alcohol-induced degeneration of glial cells or their organelles. Thus, postnatal alcohol exposure causes a delay in oligodendrocyte maturation but appears to have no long-term effects on the glial cell population of rat optic nerve. 0 1990 Academic Press, IIS.
INTRODUCTION
Children with fetal alcohol syndrome (FAS), caused by in hero exposure to alcohol, are characterized by behavioral, neurological, and intellectual manifestations of developmental brain damage (12,33). Numerous studies have examined the effects of alcohol on neuronal development (for review see (42)) but potential effects on glial cells have not been well studied. Abnormalities in glial development are suspected of contributing to alcohol-induced brain effects for several reasons. There is evidence of abnormal glial migration in postmortem studies of FAS victims (3,4) as well as in primate models of FAS (5). Experimental evidence exists for alcohol-induced delays in myelin acquisition (10,15,22,28) which could be related to effects on the development of the oligodendroglial cells which produce myelin. There is evi-
97 Copyright 0 1990 All rights of reproduction
0014-4666/90 $3.00 by Academic Press, Inc. in any form reserved.
98
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AND
droglia increase dramatically in number, mature morphologically, and initiate the production of myelin (31, 32,35). MATERIALS
AND
METHODS
The animal rearing and exposure methods used in this study are the same as those reported earlier in more detail (22) and are similar to those reported by Samson and Diaz (29) and West et al. (41). Three to five litters from nulliparous Sprague-Dawley rats were allowed to nurse until Day 4 (96 f 12 h) when the pups were weighed and 20 animals of either sex selected for gastrostomy and an artificial rearing “run.” The animals selected for each run averaged 10.1 to 10.5 g and were divided into weightmatched experimental and control groups. Because of mortalities, several rearing “runs” were required to produce at least four experimental and four control animals at each of five ages studied. All diet was fed through an implanted gastrostomy cannula from syringes on infusion pumps. The supplemented milk diet (8) was infused from Day 5 through Day 18 when the gastrostomy tube was sealed. The animals were then raised in standard cages on standard lab chow. Experimental animals received 4% (v/v) ethanol in their diet on Days 5 through 9, otherwise the experimental and control animals received identical diets in identical volumes. Ether-anesthetized animals were perfused with 4% paraformaldehyde and 2% glutaraldehyde in 0.1 M cacodylate buffer on Postnatal Days 10, 16, 22, 29, and 90 and the optic nerve was prepared for electron microscopic study. Because of the possibility of glial maturation occurring at different rates along the length of the nerve (32), cross sections were always taken from a region extending from 1 mm behind the bulb to approximately one-third the distance to the chiasm. One-micrometer sections for light microscopy were stained with basic toluidine blue. Thin sections for electron microscopy were mounted on uncoated copper grids and stained with methanolic uranyl acetate and aqueous lead citrate. The cross-sectional areas of the optic nerves were measured in l-pm plastic sections at 150 or 300X by tracing the phototube image of the nerve on a computerized bit pad (Zeiss Interactive Digital Analysis System-ZIDAS). Areas of blood vessels were traced and excluded from the gross area. Care was taken to be sure that the nerve sections had a majority of the fibers cut in near circular profile anti thus were not oblique sections of the nerve. Sections from at least two blocks per animal were measured for area, and then an average was determined for each animal. Counts of glidl cells were made in the same light microscopic cross sections that were measured for area.
KRUEGER
The counts were done at 400X by identifying glial cell nuclei and excluding pial, endothelial, and pericytic nuclei. Phototube tracings onto grid paper were used to subdivide the fields and the cells were marked to avoid repetitive counts. Glial cell densities were calculated by dividing glial cells per section by the net area of the optic nerve cross section. Glial cell morphology was studied both directly on the electron microscope and in electron micrographs. Cells were examined for any indication of degeneration or abnormal morphology and cells from experimental and control animals were compared as to organelle content, degree of maturation, and lineage development. Differential counts of developing cell types were done in random sections of optic nerve cross section that did not include the external limiting membrane. All glial cells from one section per animal were identified and characterized as either microglia, glioblasts, immature oligodendroglia, active oligodendroglia, mature oligodendroglia, or astrocytes using characteristics defined in developing optic nerve (31,35,29) and spinal cord white matter (21). Cells were identified directly from electron microscopic sections in more mature animals, while in younger animals, electron micrographs of the cells were used for identi,fication. Care was taken to record individual cell detail and to map sections so that cells were not counted more than once. Electron micrographic identifications were repeated by a second observer blind to the tissue source. For a given animal all cells were totaled and the differential counts were converted to percentages. The total numbers of cells in given sections varied so that the numbers of cells characterized in each animal ranged from 15 to 69 (mean, 30 per animal). The total numbers of cells characterized in experimental and control animals, respectively, were 159 and 107 at 10 days; 96 and 104 at 16 days; 101 and 133 at 22 days; and 123 and 137 at 90 days. Projected numbers of cell types per cross section were calculated by multiplying the total number of glial cells in a cross section (as counted in lpm sections) by the percentage of a given cell type from that same tissue block. Statistical comparisons used an ANOVA to compare the means of at least four experimental and four control animals,at each age evaluated. OBSERVATIONS
The body and brain weights from these animals have been previously reported (22). Generally, the alcohol-exposed animals were slightly, but not significantly, smaller than control animals at all ages studied. Brain weights and brain weight to body weight ratios were always less in ethanol-exposed animals but the differences were statistically significant only at 10 days (by approximately 25%).
ETHANOL-INDUCED
EFFECTS
TABLE Glial Cell Nuclei per Cross-Section;
99
GLIOGENESIS
1
Optic Nerve Cross-Sectional
Area”
16 days
22 days
156 (15.7)** 195 (12.8) 80 0.009
229 (37.9) 289 (35.7) 79 0.06
276 (7.5)* 300 (16.7) 92 0.04
212 256
2.56 (0.51)* 3.79 (0.80) 68 0.04
6.63 (1.26)* 8.55 (0.74) 78 0.04
10.9 (0.4) 11.4 (1.4) 96 0.49
10.9 (2.1) 12.6 (1.0) a7 0.18
10 days Number of glial nuclei Experimental Control E/C as % P Optic nerve area minus mm2 X lo-’ Experimental Control E/C as % P
ON
29 days
(20.2) (36.6) 83 0.08
90 days
254 (20.2) 271 (29.6) 94 0.39
vessel areas,
a Means and (standard deviations) on sections from at least two tissue * P < 0.05, **P < 0.01, ANOVA.
of four experimental blocks for each animal
and four control animals at each age. Glial cells and areas were counted and then averaged for the animal. E/C = Experimental/Control ratio.
The gross numbers of glial cells per cross section of optic nerve (Table l), as determined by light microscopic counts, were reduced by approximately 20% in ethanolexposed animals at 10 and 16 days and to a lesser extent at 22 days (-8% reduction) and 29 days (-17% reduction) but were not dramatically or significantly different than control animals at 90 days. This reduction in number paralleled a considerable reduction in the net cross sectional area of the same sections of nerve at 10 and 16 days (Table 1). When glial number was divided by area to calculate the approximate glial cell density, there were no dramatic or statistically significant differences in alcohol-exposed animals. The ultrastructural morphology of glial cell bodies and their processes was similar in both experimental and control animals and was similar to the descriptions in the literature of glial cells in developing rat optic nerve (31, 35, 37). There was no evidence in any of the glial cell types of alcohol-induced pathology or degeneration. Proportions of glial cell types (Table 2) were calculated from electron microscopic counts made on the basis of the morphological specializations described. Microglia. Microglial cells had a dense, angular, and often elongated nucleus. The cytoplasm was often dense and contained rough endoplasmic reticulum which was elongated and stringy in appearance. The cytoplasm often contained lipid droplets or dense bodies. Microglia never accounted for more than 5% of the glial cells in either experimental or control animals and generally were present in the O&2% range (Table 2). Although the average numbers of microglia were higher in the experimental animals, the absolute number present was so low (0 in some animals in both experimental and control groups) as to preclude comparisons. Glioblusts. Glioblasts were characterized by a relatively large nucleus with a thin rim of cytoplasm which
19.4 (3.3) 20.0 (1.6) 97 0.74 and measured
contained only scattered mitochondria and ribosomes. Microtubules, glial filaments, glycogen, or elements of endoplasmic reticulum were not normally present. Glioblasts were only encountered at 10 and 16 days in experimental animals and at 10 days in control animals. The glioblasts were too few in number to allow statistical comparisons, but there were more glioblasts encountered in experimental animals than in control animals, particularly at 10 days. Immature oligodendroglia. Immature oligodendroglia commonly occurred either as elongate or rounded cells with processes at one or both ends. The cells had a fairly electron lucid cytoplasm and a nucleus with evenly scattered chromatin. Microtubules were common throughout the cytoplasm as were obvious ribosomes. The short cisternae of rough endoplasmic reticulum contained material of about the same density as the cytoplasm. There were numerous small, well-organized areas of Golgi apparatus. Immature oligodendroglia were relatively common at 10 days in both experimental and control animals (Table 2); they were fewer but still present at 16 days, but were not encountered at 22 days and after. There were fewer immature oligodendroglia at 10 days (-35% fewer, P = 0.09) but not at 16 days in ethanol-exposed animals. Active oligodendroglia. Active oligodendroglia (Fig. 1) are generally considered to be the cells actively involved in the initiation and formation of myelin sheaths (21, 35, 37). Their morphology was representative of a transition between immature and mature oligodendroglia. The cells were fairly large and usually had clearly identifiable processes that could sometimes be traced to continuity with developing myelin. The nucleus was rounded with some chromatin clumping and the cytoplasm was commonly, but not always, somewhat electron dense. Scattered microtubules were common, espe-
100
PHILLIPS
AND KRUEGER
TABLE
2
Glial Cellsby Type, as Percentages” 10 days
16 days
22 days
90 days
0.9 (1.8) 0.5 (1.0)
4.1 (3.7) 2.9 (3.8)
55.9 (18.0) 57.6 (10.6) 97 0.87
44.2 (17.2) 38.4 (11.9) 115 0.60
Microglia Experimental Control
1.3 (0.9) 0.0 (-)
2.0 (4.0) 1.8 (2.0)
Glioblasts Experimental Control
6.9 (5.1) 1.3 (2.6)
1.3 (2.5) 0.0 (-)
Astrocytes Experimental Control E/C as % P
54.2 (13.5) 42.5 (5.4) 128 0.15
48.9 (8.7) 43.2 (16.2) 113 0.55
Immature oligodendroglia Experimental Control E/C as % P
25.4 (8.9) 40.2 (13.2) 63 0.09
17.7 (13.1) 18.0 (13.3) 98 0.97
Mature and active Experimental Control E/C as % P
11.3 (7.0) 16.0 (12.6) 71 0.50
30.2 (7.0) 37.1 (19.4) 81 0.53
41.2 (19.9) 39.4 (6.0) 105 0.86
53.9 (15.1) 58.7 (9.8) 92 0.61
36.7 (9.1)** 56.2 (4.9) 65 0.006
47.9 (10.7) 55.1 (16.0) 87 0.48
41.2 (19.9) 4O.lt6.9) 103 0.92
53.9 (15.1) 58.7 (9.8) 92 0.61
Oligodendroglia, Experimental Control E/C as % P n Means and (standard ** P c 0.01, ANOVA.
oligodendroglia
total
of all types
deviations)
of four experimental
and four
control
cially in the processes. Numerous ribosomes contributed to the density of the cytoplasm. The characteristic rough endoplasmic reticulum was composed of compact cisternae with an electron lucid content and were usually arranged in stacks. Mitochondria were small, elongate, electron dense, and often located in a submembrane position. Active oligodendroglia were most common in the younger animals studied (10 and 16 days) and were unusual after 22 days. Transitional cells between active and mature oligodendroglia were common, making absolute assignment of specific cell type difficult, so active oligodendroglia were grouped with mature oligodendroglia for differential counts (Table 2). Mature oligodendrogliu. Relative to active oligodendroglia, mature oligodendroglia (Fig. 2) were smaller and more rounded, and their processes were less obvious. There were both light and dark oligodendroglia. In the dark cells the cytoplasm was electron dense and the chromatin was clumped beneath the nuclear envelope. Cell processes could be found occasionally but were difficult to trace. Stacks of rough endoplasmic reticulum were encountered less often than in active oligodendrog-
animals.
lia. Microtubules were present, although they were less obvious than in active oligodendrocytes. Mitochondria were generally of small caliber and often were in a characteristic submembrane location; their matrix was dense and their cisternae were electron lucid. In dense oligodendroglia there were numerous ribosomes packing the cytoplasm and contributing to its electron density. Light oligodendroglia were generally similar in details of organelle structure and arrangement except that the cytoplasm was less electron dense, there were fewer ribosomes, and the nucleus had less obvious chromatin clumping. Mature oligodendroglia were hardly ever encountered at 10 days, and only occasionally at 16 days. At 22 days they were common and by 90 days they comprised virtually all of the oligodendroglial population. As a combined group, active and mature oligodendroglia in control animals increased from 16% at Day 10 to 59% at Day 90 (Table 2). The proportion of this combined category of cells was never dramatically or statistically different in ethanol-exposed animals. The counts of specific cell types (Table 2) revealed that alcohol caused a marked delay in oligodendroglial
ETHANOL-INDUCED
EFFECTS
ON
GLIOGENESIS
101
FIG. 1. Active oligodendroglia (0) from a 16-day ethanol-exposed animal, 11,900X. Active oligodendroglia are relatively large with a rounded nucleus with some marginal chromatin clumping. Processes (arrows) are easily identified and can sometimes be traced to continuity with developing myelin. The cytoplasm is commonly, but not always, electron dense. Scattered microtubules (T) are common, especially in processes, and can contribute to the density of the cytoplasm. The rough endoplasmic reticulum is composed of compact cisternae often arranged in stacks (arrowheads). Mitochondria are small, elongate, often electron dense, and often in a submembrane location. Such cells showed no evidence of ethanol-induced alterations in structure.
development, which was particularly obvious at 10 days. When all cells of the oligodendrocyte lineage (immature, active, and mature oligodendroglia) were grouped together, there was a dramatic and statistically significant decrease in the proportion of cells in that lineage at 10 days in the ethanol-exposed animals (21 to 55% fewer, mean of 35% fewer, P = 0.006). The projected number of all oligodendroglia per cross section was dramatically less in experimental animals on Day 10 (16 to 62% fewer, mean of 48% fewer, P = 0.005) and although they were still apparently fewer in number on Day 16, the mean difference was not statistically significant (as much as 40% fewer, mean of 27% fewer, P = 0.10). When all immature cells contributing to the oligodendroglia lineage (glioblasts and immature oligodendroglia) were considered it was apparent that, in younger animals, there was a delay in the progression of maturation of oligodendroglia in ethanol-exposed animals. Astrocytes. Mature astrocytes (Fig. 2) had more electron lucid cytoplasm than other glial cells. The nuclear
chromatin was generally evenly distributed with some slight submembrane clumping. Cells had numerous processes with fine branches filling spaces between other glial elements and axons. Groups of filaments were easy to identify but microtubules were only occasionally present. Glycogen granules could be identified in scattered locations. The characteristic rough endoplasmic reticulum consisted of cisternae that were slightly expanded and contained a flocculent, slightly electron dense material. Golgi were scattered and consisted of four or five stacks of relatively short cisternae. Mitochondria, located in random locations in the cytoplasm, were elongated, plump, and with a matrix slightly more dense than the cytoplasm. Dense bodies were common. Immature astrocytes were similar in most ways to mature astrocytes except that microtubules were more common and glial filaments were less common. Filaments in immature astrocytes were often scattered in the cytoplasm rather than in bundles. There was relatively less cytoplasm and it was slightly more electron dense. Im-
102
PHILLIPS
AND
KRUEGER
FIG. 2. Mature astrocytes (A) and a mature oligodendroglia (0) from a 22-day ethanol-exposed animal, 16,000X. Astrocytes are characterized by a relatively electron lucid cytoplasm and nuclear chromatin that is evenly distributed with some slight membrane clumping. The cells have numerous processes with fine branches filling excess space (arrows). Groups of filaments (F) are obvious. The cistemae of rough cytoplasmic reticulum (arrowheads) are slightly expanded and contain a flocculent, slightly electron-dense material. Golgi are scattered and consist of four or five stacks of relatively short cisternae. Mitochondria (M) are elongated, plump, and with a matrix only slightly more dense than the cytoplasm. Dense bodies (D) are common. Mature oligodendroglia are smaller and more rounded, and the processes are less obvious, relative to active oligodendroglia. The cytoplasm is usually quite dense and the stacks of RER are less conspicuous. The dense mitochondria (M) are often in a submembrane location. The mature glial cells had no structural evidence of changes due to the developmental ethanol exposure.
mature astrocytes were only present in the youngest animals studied and it was difficult to always separate them from mature cells, so the two types were combined in the differential counts (Table 2). Astrocytes in control animals accounted for 42 to 58% of the glial cells present from Days 10-90. They were present in relatively greater proportions at 10 and 16 days in ethanol-exposed animals (although the differences were not statistically significant) but the proportion of astrocytes in experimental and control animals was not markedly different at 22 or 90 days. The total projected number of astrocytes per cross section was essentially the same in experimental and control animals at each age studied, including at 10 and 16 days when oligodendroglia were less common and the total number of cells was less. In general, the progression of development of astrocytes was not delayed by the ethanol exposure as was the development of oligodendroglia.
DISCUSSION
The primary effect of postnatal ethanol exposure on developing glial cells in the rat optic nerve was a delay in the maturation of oligodendroglia. This was evidenced by decreases in the total number of oligodendroglia present, especially at 10 days, and by a delay in the appearance of immature cells within the oligodendroglial lineage. The proportion of cells of different types was similar in older animals, and by 90 days there was no evidence of differences in cell structure or in the relative proportions of the various cell types. The numbers of astrocytes and the progression of their maturation did not appear to be affected by the postnatal alcohol exposure. There was no evidence of abnormal glial cell placements, such as the abnormal loci of cells reported in postmortem examination of human and primate brains after developmental exposure to alcohol (3-5). This is
ETHANOL-INDUCED
EFFECTS
despite the fact that this ethanol exposure occurred at a time of rapid glial maturation and migration in the optic nerve (31, 32, 37) when such ectopic placements might be expected to originate from such an insult. Perhaps the dosage did not occur early enough to cause such ectopia or perhaps the oligodendroglia, which are so actively maturing and migrating at this time, are simply not involved. It is not surprising that this particular ethanol exposure exerts its primary effect on oligodendroglia and not astrocytes. This is because most astrocytes in the rat optic nerve (particularly radial or Type I astrocytes) form in late gestation and have undergone much of their maturation before Days 5-9 (2531) when the exposure occurred. Conversely, the oligodendroglia are proliferating and maturing at their greatest rate during Days 5 to 10 (25, 31, 32, 35), at the time of this ethanol exposure. Thus if alcohol has a significant effect on the processes of cell acquisition and maturation in glial cells, as it does in neurons (2,19,20,23,38), one would expect the timing of this exposure to favor damage to maturing oligodendroglia rather than astrocytes. Although alcohol-induced delays in glial proliferation and maturation have been reported in in vitro studies, those studies have all focused on cultured astrocytes. Acute exposure to ethanol does not affect the cellular morphology or the concentration of filament proteins, but does increase the amounts of certain nuclear proteins of cultured l-day rat astrocytes (16). Longer exposures cause a generalized delay in the proliferation and maturation of developing astrocytes cultured from chick and mouse (7,13,14). It is quite possible that the effects on oligodendroglia, which are rapidly proliferating and maturing in optic nerve at the time of the exposure (31, 35), are a reflection of cellular changes similar to those reported in the in vitro studies of proliferating and maturing astrocytes. Small et al. (32) have reported that the progenitor cells for oligodendroglia and some astrocytes (Type II) migrate into the rat optic nerve from its chiasmal end, starting at birth. Perhaps the delay in the maturation of oligodendroglia could simply be an alcohol-induced delay in the migration of the progenitor cells. Because the radial glia (Type I astrocytes) in the optic nerve generally form earlier (at Embryonic Day 16) (25, 31) and do not migrate to their position, they would not be as markedly affected by an ethanol-induced delay of postnatal migration. Another potential cause of delayed oligodendroglial maturation could be related to a delay in the maturation rate of optic nerve axons, thus effectively delaying the “trigger” mechanisms that initiate myelin formation and thus influencing oligodendroglial maturation. Such a delay in axonal growth (reported in some optic axons from these same animals, along with a delay in myelin
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acquisition (22)), could alter the feedback mechanisms which regulate oligodendroglial formation and maturation. Although few details have been specifically defined, such feedback seems implicit in the current understanding of myelination (26,43). There was no morphological evidence in this study of ethanol-induced pathological alterations of individual glial cells or organelles. This is in contrast to the suggestion that morphological alterations of mitochondria may be an important feature of developmental alcohol exposure in neurons (1). Even though ethanol is reported to inhibit ATPase activity in synaptosomal preparations from adult CNS (17), there was no consistent evidence in this study of the cellular swelling expected if such an inhibition of ATPase activity occurred in developing glial cells (36). Perhaps glial cells are not as susceptible to such cellular alterations as neurons or perhaps the ethanol exposure provided in this study was not provided early enough, long enough, or in high enough doses to produce such damage. Although the overall average effects of ethanol are subtle, some individual ethanol-treated animals were severely affected compared to other ethanol-exposed or control animals. One ethanol exposed animal at 10 days had a 55% smaller proportion of oligodendroglia and 62% fewer projected total number of oligodendroglia than the average of control animals. The most extreme examples of ethanol-induced pathology are partially masked by evaluation of means and consideration of such results needs to include the concept of the possibility of potential for more extreme pathology in individual instances. Despite this extreme effect in those animals most affected, there was no consistent evidence of cellular degeneration. The fact that there was no apparent evidence of cellular degeneration or permanent glial cell loss due to the ethanol exposure is not surprising since some developmentally exposed nerve cell populations do not show dramatic evidences of ethanol-induced cell death, but rather, there is evidence of similar delays in their maturation or migration (18,20,38,42). However, other neuronal populations do undergo obvious cellular degeneration during or after developmental exposures to ethanol (2, 6, 23) and in some instances all evidence of such activity is gone within 1 or 2 days (6,23). There is a possibility that glial cell degeneration could have occurred in this study during the 5- to g-day exposure period, but that the evidence of such degeneration was gone by 10 days when the tissue was first examined. It is difficult to project the long-term implications of this reported delay in oligodendroglial maturation, particularly since the developmental exposure to alcohol has apparently not affected the number, distribution, or morphology of glial cells in mature animals. Obviously the delay in oligodendroglial maturation could be related
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to the delay in myelin acquisition reported in these same animals (22). Such delays in myelin acquisition could, in turn, by modifying spontaneous patterns of action potentials and axon conduction, affect the maturation of other elements in the neuronal pathway (30). Such changes in neuronal pathways could help explain subtle changes in nervous system function caused by ethanol exposures. ACKNOWLEDGMENTS This work was supported by Research Grants AA07042 from NIAAA, MBRS-SO608218 from NIH, and ISPS011449 from NSF-EPSCoR. Acknowledgments are due J. Pembroke, L. Fritzler, and J. Rydquist for technical assistance.
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