Ethanol is the main addictive and neurotoxic constituent of alcohol. Ethanol exposure during embryonic development causes dysfunction of the central nervous system (CNS) and leads to fetal alcohol spectrum disorders. The cerebellum is one of the CNS regions that are particularly vulnerable to the toxic effect of ethanol .
Consumption of alcohol during pregnancy affects brain growth and induces significant alterations in the histological architecture of the cerebellum of growing rats in the form of a decrease in the diameter of Purkinje cells and in the width of molecular and granular layers .
Ethanol crosses the placental membrane freely and accumulates in the fetus. In the fetus, it is distributed to different organs including the brain .
Acute and chronic ethanol exposure produces profound impairments in motor functioning, as well as impairments that are responsible for millions of injuries and deaths worldwide .
Alcohol can exert teratogenic effects on the CNS through multiple mechanisms, which may be influenced by maternal and fetal characteristics and by the pattern of exposure .
Cerebellum is a key target of alcohol toxicity in the CNS .
The mechanisms of ethanol-induced cerebellar degeneration include thiamine deficiency and defects in energy production, deficiency in growth factors, and apoptosis . Alcohol interacts with neurotrophins, the roles of which in the developing and mature cerebellum are critical .
Alcohol exposure during brain development induces neuronal cell death in the brain . Chronic ethanol exposure produces an apparent decrease in the density of cerebellar Purkinje cells in the developing guinea pig  and in deep cerebellar nuclear cells .
Ethanol exposure during brain growth spurt (postnatal days 4–9) causes a differential loss of Purkinje cells; the loss of cerebellar granule cells has been shown to be parallel to the loss of Purkinje cells .
Little information is known about the long-lasting effect of ethanol exposure on brain growth; hence, the aim of this work was to study the effect of alcohol ingestion on the postnatal development of the cerebellar cortex of offspring from birth until adult stage by using light and electron microscopes.
Materials and methods
Twenty pregnant rats were used in this study. They were purchased from Assiut University Animal Breeding Unit. All animals were kept in clean, properly ventilated, separate cages under similar environmental conditions and fed the same laboratory chow and tap water.
They were divided into two groups. Each group consisted of 10 pregnant rats.
Group I: The pregnant animals of the first group were considered as controls and were kept after delivery with their offspring for 3 weeks for lactation. They were given distilled water orally daily by means of a gastric tube
Group II: The pregnant animals of the second group were given 10% ethyl alcohol (El-Nasr Company for intermediate chemicals, Egypt) using an oral tube at a dose of 2.5 gm/kg from day 11 of gestation, which was continued for 3 weeks after delivery [13,14].
Determination of the date of pregnancy
Vaginal smears were taken by inserting a glass rod smoothly into the vagina; the smears were spread on a slide and stained by Shori stain. The estrus period is characterized by the presence of cornified nonnucleated epithelial cells without leukocytes. After appearance of the mucous plug, the vaginal smear of pregnant rats was found to contain cornified nonnucleated epithelial cells, leukocytes, and a large quantity of mucous .
The offspring from the two groups were sacrificed at the following ages: at 1, 2, and 3 weeks and at 3 months (five animals at each age). Specimens from the cerebella were rapidly excised and fixed in Bouins solution, dehydrated in ascending series of ethanol, cleared in methyl benzoate, and embedded in paraffin wax. Paraffin sections of 5 microns were prepared and stained with Einerson's gallocyanine for examination of Nissl's granules .
Cerebella from two mice aged 3 weeks and from two adult mice from both groups were fixed in Golgi–Cox solution for 2 months to stain the neurons and their processes. Thereafter, specimens were processed using the usual paraffin technique. Ten-micron paraffin sections were cut and placed in 10% ammonia solution for 1 h. Sections were dehydrated, cleared, and mounted by DPX (A mixture of Distyrene, a Plasticizer, and Xylene) .
Electron microscopic study
Small pieces about 1 mm3 from the cerebella of adult mice from both groups were fixed in 2.5% glutaraldehyde for 24 h. Specimens from both groups were fixed in 0.1 mol/l phosphate buffer at 4°C and then postfixed in 1% osmium tetroxide at room temperature. Specimens were dehydrated in ascending grades of ethyl alcohol and then embedded in Epon resin. Semithin sections (1 μm) were stained with toluidine blue in borax and examined using a light microscope. Ultrathin sections (50 nm) were cut, mounted on copper grids, and stained with uranyl acetate and lead citrate 11. Specimens were examined using a transmission electron microscope Jeol-1010 (Tokyo, Japan) and photographed at 100 kV (Sohag University) to show the ultrastructure of the neurons .
For histological techniques, the following were assessed in the cerebellum:
- (1) Estimation of the thickness of the external granular layer and the molecular and internal granular layers of control and ethyl alcohol-treated rats (at magnification × 200).
- (2) Linear density of Purkinje cells by counting Purkinje cells per millimeter line length throughout the section . Measurements were taken on 5-μm-thick gallocyanine-stained sections, from 10 fields from three sections belonging to three animals (at magnification × 200) from each age group. Neurons were counted by nuclei.
- (3) Estimation of the density of internal granule cells and deep cerebellar nuclei (dentate) per unit area (0.7 ml) 2 of control and ethyl alcohol-treated rats, from 10 fields from three sections belonging to three animals (at magnification × 400) from each group.
- (4) Estimation of the nuclear diameter of Purkinje cells: The longest axis of the nucleus (average of the greatest and least dimensions for each nucleus) was measured as the major diameter, according to the method described in Maricich et al. .
All previous parameters were measured using an image analysis system (Digimizer; version 3.7.0, MedCalc Software, Broekstraat 52, 9030 Mariakerke, Belgium).
Variables are represented as mean ± SD. The unpaired Student t-test was used when comparing the means of these variables between different groups, according to the method described in Saunders and Trapp .
The external granular layer
In group I, at 1 and 2 weeks of age, the external granular layer was formed consisting of small closely packed cells with deeply stained rounded nuclei. The thickness started to decrease at 1 and 3 weeks of age and appeared as a thin layer formed of one or two rows comprising a few widely separated cells. Complete disappearance of this layer was noticed at 12 weeks of age (Figs 1–3).
In group II, at 1 week of age, there was an apparent decrease in the thickness of the external granular layer, compared with group I. However, at 2 weeks of age, the thickness showed an apparent increase, compared with group I. In the third week it appeared to be formed of a few widely separated cells (Figs 5–7).
The molecular layer
In group I, the molecular layer appeared as a clear zone containing a few widely separated cells midway between the external granular and Purkinje cell layer. It showed a gradual increase in thickness with age to reach its maximum size at adult stage (Figs 1–4).
In group II, there was an apparent reduction in the thickness of the molecular layer at all age groups compared with group I (Figs 5-8).
The Purkinje cell layer
In group I, the Purkinje cell layer at 1 week of age appeared to be formed of several layers of well-defined large cells arranged parallel to the pial surface. At 2 weeks of age it appeared as a single layer (Figs 1 and 2). At 3 weeks, these cells appeared rounded with large nuclei and pale basophilic cytoplasm and were arranged in a single row (Figs 3 and 9).
In the adult cerebellum, the Purkinje cell layer consisted of one row of flask-shaped cells with large, rounded, vesicular nuclei and pale basophilic cytoplasm. These cells appeared along the upper margin of the granular layer. In between the Purkinje cells, large Golgi cells and small darkly stained granule cells could be detected (Figs 4,11).
With the Golgi–Cox stain, the Purkinje cells appeared flask shaped with two or three primary dendrites arising from the apical pole of the cell. From these primary dendrites appeared numerous extended secondary dendrites, which reached up to the pial surface. These branches increased in number from 3 weeks to adult age (Figs 13,15).
In group II, contrary to group I, the Purkinje cells were arranged in more than one layer until the age of 3 weeks. The cells appeared smaller, with a pale cytoplasm (Figs 5-7 and 10).
Mature Purkinje cells lost their flask shape and appeared smaller with irregular, less vesicular nuclei (Figs 8 and 12).
With the Golgi–Cox stain, the extent of Purkinje cells’ dendritic tree and their branches were reduced compared with group I at both 3 weeks and adult stage (Figs 14 and 16).
The internal granular layer
In group I, this layer increased in thickness with age and appeared to comprise small, deeply stained internal granular cells (Figs 1-4).
In group II, the internal granular layer at 1 week of age was delayed in its differentiation from the white matter, compared with group I (Figs 5-8).
Deep cerebellar nuclei
In group I, these nuclei were well defined in their groups: from lateral to medial, the dentate, the interpositus (anterior and posterior), and the fastigi. A fine white matter capsule separated each nucleus from the other (Fig. 21). The cells of the deep cerebellar nuclei were well defined into two types of cells: small and large cells (Fig. 21).
In group II, there was loss of differentiation of deep cerebellar nuclei compared with group I (Fig. 23). Most of the large cells showed an irregular outline and deeply stained nuclei (Fig. 24).
In group I, Golgi cells appeared larger than granule cells, with large nuclei and a thin rim of cytoplasm with a few organelles in it (Fig. 17). Granule cells appeared more or less equal in size with large nuclei containing clumps of heterochromatin and a thin layer of cytoplasm having a few organelles (Fig. 19).
In group II, granule cells showed karyolytic changes with the presence of coarse clumps of chromatin. The cytoplasm was nearly devoid of organelles (Fig. 20). Golgi cells appeared smaller in size than the cells of the control group, with irregular, damaged nucleus (Fig. 18).
Morphometric and statistical results
The external granular layer. In the control group, the thickness of the external granular layer appeared at maximum size at 1 week (154.12 ± 1). The thickness then started to reduce and became approximately 69.28 ± 1 at 2 weeks, and at 3 weeks it appeared as a single layer of cells (32.86 ± 1).
In the treated group, the external granular layer reduced in thickness in the treated group compared with the control group at 1 week of age (111.3 ± 2) and the difference was statistically significant (P-value < 0.001).
The thickness increased in the treated group compared with the control group at 2 weeks (149.93 ± 1), and the difference was statistically significant (P-value < 0.001). In the third week, the thickness reduced to 72.60 ± 1 and the difference was highly significant (P-value < 0.001) (Table 1 and Histogram 1).
The molecular layer. In the control group, the molecular layer showed an increase in thickness with age. It was about 208.6 μm at 1 week, 521.9 μm at 2 weeks, 538.4 μm at 3 weeks, and 708.1 μm at adult stage.
In the treated group, the molecular layer of the treated group was reduced in thickness compared with that of the age-matched controls at all ages examined. It was approximately 130 μm at 1 week, 182.5 μm at 2 weeks, 337 μm at 3 weeks, and 505.3 μm at adult stage. The difference was very highly significant (P-value < 0.001) (Table 2 and Histogram 2).
The internal granular layer. In the control group, the thickness of this layer increased with age. It was approximately 365.2 ± 4 at 1 week, 579.12 ± 6 at 2 weeks, 619.2 ± 7 μm at 3 weeks, and 645.11 ± 6μm at adult stage.
In the treated group, this layer was reduced in thickness compared with the control group at all ages examined. It reached approximately 286.4 ± 2 at 1 week, 347.3 ± 5 at 2 weeks, 494.4 ± 8μm at 3 weeks, and 500 ± 5 μm at adult stage. The difference was very highly significant (P-value < 0.001) at all ages (Table 3 and Histogram 3).
The neuronal density of Purkinje cells in the treated group was lower than that of the control group at all ages. At 1 week it was approximately 14 ± 2 compared with 17.38 ± 1 for controls. At 2 weeks it was approximately 10 ± 0 compared with 13.15 ± 1 for controls. At 3 weeks it was approximately 8.69 ± 1 compared with 10.76 ± 1 for controls. At adult age it was approximately 9.15 ± 1 compared with 11.38 ± 1 for controls. The difference was very highly significant (***P≤0.001) at 1 and 2 weeks and highly significant (**P≤0.01) at 3 weeks and at adult stage (Table 4 and Histogram 4).
The neuronal density of granule cells in the treated group was lower than that of the control group at all ages. At 1 week it was approximately 317.14 ± 5 compared with 588.39 ± 1 for controls. At 2 weeks it was approximately 531 ± 3 compared with 687.14 ± 2 for controls. At 3 weeks it was approximately 946.4 ± 4 compared with 1143.5 ± 7 for controls. In adults it was approximately 1064.6 ± 5 compared with 1305.7 ± 5 for controls. The difference was very highly significant (P-value < 0.001) at all ages (Table 5 and Histogram 5).
The neuronal density of dentate cells in the treated group was lower than that of the control group at all ages. At 1 week it was approximately 159.74 ± 11.3 compared with 204.40 ± 16 for controls. At 2 weeks it was approximately 145.74 ± 9 compared with 168.8 ± 1 for controls. At 3 weeks it was approximately 133.44 ± 7 compared with 154.74 ± 9 for controls. In adults it was approximately 115.51 ± 8 compared with 148.37 ± 7 for controls. The difference was very highly significant (P-value < 0.001) at all ages (Table 6 and Histogram 6).
The nuclear diameter of Purkinje cells in the treated group was lower than that of the control group at all ages. At 1 week it was approximately 63.7 ± 1 compared with 74.35 ± 2 for controls. At 2 weeks it was approximately 82.8 ± 1 compared with 105.33 ± 3 for controls. At 3 weeks it was approximately 104.6 ± 2 compared with 145.39 ± 1 for controls. In adults it was approximately 117.6 ± 1 compared with 170.6 ± 2 for controls.
The difference was significant (P ≤ 0.05) at 1 week and very highly significant (P-value < 0.001) at 2 weeks, 3 weeks, and at adult stage (Table 7 and Histogram 7).
The cerebellum was chosen in this study as it is considered the most sensitive morphological indicator of fetal alcohol syndrome .
The developing cerebellum is one of the most vulnerable brain structures to prenatal ethanol exposure, and the Purkinje cell is the most susceptible cerebellar cell type .
The results of the present study revealed that the external granular layer in the treated group increased in thickness compared with that of the control group in the second and third weeks postnatally.
The present results are in agreement with previous reports by Shetty and colleagues [24–26].
They reported that the increase in thickness of this layer in treated rats may be due to failure of its cells to achieve their final form: The molecular Purkinje and granular layers. This failure in migration occurs because of an alternation in the function of Bergmann glial cells  as the granule cells migrate in the molecular layer toward the Purkinje cell layer along the processes of Bergmann glial cells. This leads to an increase in the thickness of this layer and decreases the number of cells derived from it .
In contrast, the present results disagreed with previous reports by Hwang and colleagues [9,27,28]. They mentioned a significant decrease in the number of external granular cells as a result of early postnatal ethanol exposure compared with the control group. They reported that this occurred because of an increase in the incidence of cellular death among these cells, which resulted in a decrease in the thickness of this layer.
The present results show that the molecular layer of the treated group is reduced in thickness compared with that of the control group at all ages.
These results are in agreement with previous reports by Rintala and colleagues [29–31]. They mentioned that ethanol treatment resulted in degeneration and regression of Purkinje neuron dendritic arborization, which transferred the molecular layer up toward the surface of the cerebellum and led to a decrease in the thickness of the molecular layer .
The present results showed a reduction in the thickness of the internal granular layer in treated animals at all ages of postnatal life compared with that of the corresponding age-matched controls.
These results are consistent with the previous results of Li and colleagues [32,33]. They mentioned that the decrease in the thickness of the internal granular layer occurred as ethanol interfered with the cyclin-dependent kinase system, which regulates cell division, apoptosis, transcription, differentiation, and nervous system function  and disrupts the cell cycle kinetics in cerebellar granule progenitors found in the external granular layer.
Ethanol affects the expression and activation of retinoic acid receptors in the cerebellum and in cerebellar granule cells, as retinoic acid is required for neuronal development. Therefore, ethanol exposure impairs signaling events and induces harmful effects on the survival and differentiation of cerebellar granule cells .
In the present study, the neuronal density of Purkinje cells in the treated group decreased at all ages compared with age-matched controls. This is in agreement with previous reports by Napper and colleagues [35–38]. They found that alcohol exposure caused depletion of cerebellar Purkinje cells by inducing oxidative types of alterations that resulted in neurodegeneration of these cells.
Chronic prenatal alcohol intake causes a reduction in the number of Purkinje cells by increasing the apoptotic Purkinje cell nuclei, as Purkinje cells are the most sensitive cells to ethanol [10,39].
In the present study, the neuronal density of the internal granular cells of the treated group decreased at all ages compared with that of age-matched controls, which is in agreement with previous reports by Pantazis and colleagues [40–44]. They reported that alcohol-induced killing of granule cells was the most likely mechanism for the depletion of granule cells in vitro, and it occurred by inhibiting the antiapoptotic action of insulin-like growth factor-mediated cell survival  and by impairing granule cell migration from the external granular layer, thus decreasing the number of granule cells postnatally .
In contrast, the present results disagreed with previous reports by Pentney et al. . They found that chronic ethanol consumption had no effect on the total number of cerebellar granule cells in aged Fischer 344 rats.
Exposure to ethanol during postnatal days 20–25 produced no significant changes in the total number of granule cells in the dentate gyrus .
In the present study, the neuronal density of the dentate nuclei of the treated group decreased at all ages compared with that of age-matched controls. These results are in agreement with previous reports by Green and colleagues [14,47,48].
They mentioned that exposure to ethanol in rats produced a proportional reduction in cell populations of deep cerebellar neurons, which could be either primary degeneration because of a direct toxic action of ethanol or secondary degeneration because of loss of Purkinje cells.
The present work showed that the nuclear diameter of Purkinje cells in the treated group decreased at all ages compared with that of age-matched controls. These results matched with the results of Ismail and colleagues [22,49,50]. They reported that chronic alcohol consumption resulted in a decrease in the mean volume of the Purkinje cell perikaryon by hampering its growth. The decrease in the growth of these cells occurs by inhibition of protein and DNA synthesis and by reduction in the uptake of critical nutrients such as glucose and amino acids , which reflects the morbidity of these cells caused by ethanol toxicity .
The dendritic arborization of Purkinje cells of experimental animals as seen by the Golgi–Cox technique showed manifestation of degeneration in the form of less branching dendrites. This appeared at both 3 weeks and adult stage.
These results were in accordance with previous reports by Tabbaa and colleagues [51–53]. They mentioned that dendritic loss in ethanol treatment may be due to regression in the growth of dendrites resulting from the loss of afferent supply to dendrites. This occurred because of a reduction in the total number of synapses per Purkinje neuron arbor under the effect of ethanol .
In the present study, the ultrastructure of the granule cells and Golgi cells of the treated group reflects degenerative changes in these cells in the form of reduced nuclear size with ill-defined outlines. These results matched the results of Koksal and colleagues [13,54].
They found that, in culture cells, ethanol changed the nuclear envelope (NE) structure by decreasing its permeability. Thus, cells became less permeable for diffusible ions and macromolecules, which finally led to destruction of the cells .
They also found that in Purkinje cells treated with ethanol the continuity of the nuclear membrane was lost and the contents were mixed with the cytoplasm, which also showed changes in the form of degeneration of mitochondria in the form of cristolysis, dilatation of rough endoplasmic reticulum tubuli, and appearance of multivesicular bodies .
Ethanol is a teratogen and its consumption during pregnancy induces harmful effects on the developing fetus, which lead to fetal alcohol syndrome. CNS dysfunctions are the most severe and permanent consequence of maternal alcohol intake and can occur in the absence of gross morphological defects associated with fetal alcohol syndrome .
Alcohol exposure is neurotoxic and must be avoided by pregnant and lactating mothers. Further studies are needed to define the most critical period of ethanol toxicity.
Conflicts of interest
There is no conflict of interest to declare.
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