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Effect of aluminum on the histological structure of rats' cerebellar cortex and possible protection by melatonin

EL–Shafei, M. Deiaa El-Din M.a; Kamel, Ashraf M.F.a; Mostafa, Mohamed E.A.b

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The Egyptian Journal of Histology: June 2011 - Volume 34 - Issue 2 - p 239-250
doi: 10.1097/01.EHX.0000396640.10505.da
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Abstract

Introduction

Aluminum (AL) is a light metal that makes up 7% of the earth's crust, and is the third most abundant element after oxygen and silicon [1]. AL enters the atmosphere through industrial and occupational sources of exposure, which contribute significantly toward increased levels of AL in environment [2].

AL can enter the body through drinking water, food intake, inhalation, and skin contact. In addition, specific medical interventions, such as dialysis or certain AL-containing drugs, may lead to AL accumulation in the tissues [3]. Antiperspirants, vaccines, allergy desensitization injections, and AL-based oral antiacids can also contribute significantly to total human AL exposure in some people [4].

The primary dietary sources of AL in the United States are food and beverages, including tea. Average daily AL intake is typically 5–10 mg [5]. Tea typically contains 2–4 mg AL/L [6]. Herbal tea contains less AL [7]. Drinking water provides approximately 0.1 mg of AL (1.5% of total daily dietary AL intake), whereas in countries where AL from other sources is relatively small and tea consumption is relatively large, as in the UK, tea may contribute up to 50% of total daily AL intake [8].

Oral bioavailability (fractional absorption) is the amount absorbed compared with the amount administered. It is aprroximately 0.37% from tea, 0.3% from water, 0.1–0.3% from basic sodium aluminum phosphate in cheese, and only 0.1% from biscuits [9].

AL is known as a potent neurotoxin in animal cells, and its relevance to Alzheimer's disease is hotly debated [10]. AL toxicity induces programmed cell death in yeast [11], plants [12], and animals [13].

AL can produce toxicity to the central nervous, skeletal, and hematopoietic systems. It can produce an encephalopathy in renal-impaired humans (dialysis encephalopathy), cognitive deficits in young children, low-turnover bone disease, and microcytic hypochromic anemia. It has been shown to contribute to some neurodegenerative diseases, including Alzheimer's disease [4,14].

Exposure to AL may also contribute to inflammatory events and/or breakdown of the blood–brain barrier [15]. Disruption of the blood–brain barrier is associated with the development and/or progression of stroke, ischemia/reperfusion, and vascular dysfunction [16].

Increased oxidative stress plays a significant role in the pathogenesis of brain disorders induced by AL. This is based on the fact that AL is a redox inactive metal but is known to possess prooxidant activity [17]. Different mechanisms have been proposed to account for the oxidant activity of AL. These mechanisms include influence on the membrane lipids [18]. AL was proposed to elevate lipid peroxidation and induce changes in the antioxidant defense system of the brain [19].

Melatonin (N-acetyl-5 metoksitriptamin) is produced mainly by the pineal gland. It is also produced in small amounts in the retina, gastrointestinal system, and by leucocytes [20]. Melatonin (MEL) is found abundantly in all parts of the cell as it is minute and highly lipophilic [21]. It protects the DNA, lipids, and proteins against oxidative damage [22]. The free radical scavenging and antioxidant effects of MEL have been demonstrated in several studies. Many studies have shown that MEL reduced lipid peroxidation caused by various conditions both by its free radical scavenging effect, and also by directly increasing natural antioxidant activity [23,24]. Similar studies have indicated that MEL reduced lipid peroxidation in the thyroid [25], oxidative damage in the liver [26], and gastric damage caused by indomethacin [27].

Although the brain is the primary organ affected by the AL toxicity, only few studies have described its effect on the structure of the cerebellum. Furthermore, the use of MEL as a protective measure against AL toxicity needs to be investigated. Therefore, the aim of this study was to clarify the effect of AL administration on the histological structure of the cerebellum and to assess any possible protection by concomitant administration of MEL.

Materials and methods

Drugs and chemicals

Aluminum chloride and MEL were purchased from Sigma (St Louis, Missouri, USA). Other reagents were of analytical grade and were obtained from commercial sources.

Animals and treatment

This study was carried out on 50 adult male albino rats, weighing 190–220 g. The rats were obtained from the Animal House of the Faculty of Medicine, Cairo University. Animals were housed in stainless steel cages under normal hygienic conditions and allowed water and food (laboratory chow) ad libitum throughout the study in accordance with the international guidelines for the care and use of laboratory animals. They were divided into five groups, 10 rats each: Group I (control group) received no treatment. Group II (saline/ethanol-treated group) received daily intraperitoneal (i.p.) injection of ½ ml 0.9% saline containing 2% ethanol, for 2 months. Group III (MEL-treated group) received daily i.p. injection of MEL in a dose of 10 mg/kg bw dissolved in ½ ml 0.9% saline plus 2% ethanol, for 2 months [28]. Group IV (AL-treated group) received daily i.p. injection of aluminum chloride in a dose of 10 mg/kg bw dissolved in ½ ml saline for 2 months [29]. Group V (AL-treated group and MEL-treated group) received daily i.p. injection of MEL and aluminum chloride in the same doses as in groups III and IV for 2 months.

One day after the last dose, the animals of all groups were weighed and anesthetized with i.p. injection of thiopental sodium (50 mg/kg) [30]. The animals of each group were randomly divided into two equal subgroups (five rats each); the first was processed for light and the second for electron microscopic study. Fixation of the cerebellum was performed by perfusion rather than by immersion, as perfusion provides much more constant preservation of cytological details [31].

Light microscopic study

After the chest wall was opened, animals were perfused transcardially through the left ventricle with 10% formol saline [28]. Before perfusion, the descending aorta was ligated and the right atrium was opened once perfusion has started [32]. The perfusion was stopped when the venous return from the right atrium became clear. The skull was opened and the brain was removed and weighed. The cerebellum was excised and a midsagittal section of the cerebellar vermis was made in order to obtain the 10 cerebellar lobules intact in one section. The tissue was fixed in 10% formol saline, processed into 5μm-thick paraffin sections, and then stained with hematoxylin and eosin [33].

Semithin and transmission electron microscopic studies

The animals were perfused, as described before, transcardially using 4% glutaraldehyde in 0.9% saline [34]. The cerebellum was obtained as above and fixed in fresh 3% glutaraldehyde at 4°C for 4 h. Then, 1×1 mm specimens were cut and washed in 0.15 mol/l phosphate buffer, pH 7.4, for 2 h (two changes), then postfixed in 1% osmium tetroxide for 1 h at 4°C. The specimens were dehydrated and embedded in epoxy resin. Semithin sections were cut at 0.5 μm thickness by an ultramicrotome, stained with 1% toluidine blue, and examined by a light microscope. For electron microscopy, ultrathin sections (50–80 nm thick) were cut using the same ultramicrotome and stained with uranyl acetate and lead citrate [35]. The sections were examined by a JEM-1400A transmission electron microscope (JEOL, Tokyo, Japan) operated at 80 kv at the Faculty of Agriculture Research Park, Cairo University.

Quantitative morphometric study

Images were analysed using computer-based image analysis software (Leica Qwin 500; Imaging Systems, Cambridge, UK). All Purkinje cells were counted in each of the 10 cerebellar lobules, using light microscopy at 200× magnification. For each rat, the number of Purkinje cells was determined for each of the 10 cerebellar lobules of each section, and then the average value for the 10 lobules was calculated for each section. The total length of the cerebellar folia in the 10 lobules was estimated in μm then converted to mm. Purkinje cell number was expressed as a mean value of the cell number per millimeter length of the cerebellar folia [36].

Statistical study

The quantitative variables of body and brain weights and also the number of Purkinje cells/mm length were presented as mean±standard deviation. The statistical analysis was conducted using analysis of variance followed by post-hoc Tukey Honestly Significantly Different test to compare variables among the different groups. A P value of less than 0.05 was considered to be significant [37].

Results

Body and brain weights

Statistical analysis of the mean body and brain weights revealed no significant change in groups II and III versus group I. However, there was a significant decrease (P<0.01) in the body and brain weights after AL administration (group III) compared with the control group. Treatment with MEL alone did not cause any significant change in the body and brain weights of the animals when compared with control animals. In contrast, there was a significant increase (P<0.01) in both the body and brain weights in rats treated with both MEL and AL compared with rats treated with AL only (Table 1 and Histogram 1).

Table 1
Table 1:
The body and brain weights (mean±standard deviation) of the studied groups
Histogram 1
Histogram 1:
Histogram 1. (a) The mean body weights and (b) mean brain weights of the studied groups. •P<0.01when compared with the control group. *P<0.01 when compared with the aluminium-treated group.

Light microscopic study

Control adult rats showed the usual architecture of the cerebellum with folia separated by narrow sulci. Each folium consisted of a mantle of cerebellar cortex and a core of white matter (Fig. 1). The cerebellar cortex was made up of molecular, Purkinje cell and granular layers (Figs 2–4). The molecular layer was formed of few small stellate and basket cells together with numerous fibers. Stellate cells were located superficially in this layer, whereas basket cells were found in the deeper parts near Purkinje cell bodies (Fig. 3). The Purkinje cell layer was seen arranged in one row along the outer margin of the granular layer. It consisted of a single row of large pyriform somata of Purkinje neurons (Figs 3 and 4) with clear vesicular nuclei and prominent nucleoli (Fig. 4). The granular layer was composed of tightly packed small rounded cells with deeply stained nuclei (Fig. 3).

Figure 1
Figure 1:
A photomicrograph of a section in the cerebellum of a control rat (group I) showing folia, separated by narrow sulci. Each folium consists of a mantle of cerebellar cortex with a core of white matter (W). The covering pia mater can be observed (arrow).H&E×100.
Figure 2
Figure 2:
A photomicrograph of a section in the cerebellar cortex of a control rat (group I) exhibiting that it is made up of molecular layer (M), Purkinje cell layer (P), and granular layer (G). H&E×200.
Figure 3
Figure 3:
A photomicrograph of a section in the cerebellar cortex of a control rat (group I) displaying molecular layer (M) formed of few small stellate (SC) and basket cells (BC) together with numerous fibers. Purkinje cell layer (P) consists of large pyriform cells (arrow), whereas the granular layer (G) is composed of tightly packed small rounded cells with deeply stained nuclei. H&E×400.
Figure 4
Figure 4

Light microscopic examination of groups II and III revealed no observable differences from the control group.

The cerebellar cortex of the AL-treated group (group IV) exhibited marked reduction in the number of Purkinje cells that were hardly detected whereas the granular layer appeared unaffected (Figs 5–7). The few Purkinje cells seen showed darkly stained nuclei and a darkly stained cytoplasm (Figs 6 and 7). Prominent perineuronal spaces were observed in the molecular layer around both basket and stellate cells (Fig. 6).

Figure 5
Figure 5:
A photomicrograph of a section in the cerebellar cortex of an aluminium-treated rat (group IV) exhibiting apparently normal molecular layer (M). Purkinje cells (arrow) are hardly found in the Purkinje cell layer (P), whereas the granular layer (G) appears unaffected. Note the presence of a congested meningeal blood vessel (BV).H&E×200.
Figure 6
Figure 6:
A photomicrograph of a section in the cerebellar cortex of an aluminium-treated rat (group IV) showing a Purkinje cell with a darkly stained nucleus (arrow) and darkly stained cytoplasm in the Purkinje cell layer (P). Prominent perineuronal spaces (arrow head) are seen around basket (BC) and stellate cells (SC) in the molecular layer (M), whereas the granular layer (G) appears unaffected.H&E×400.
Figure 7
Figure 7:
A photomicrograph of a section in the cerebellar cortex of an aluminium-treated rat (group IV) displaying one Purkinje cell with a pyknotic nucleus (arrow) and darkly stained cytoplasm in the Purkinje cell layer (P). Perineuronal space (arrow head) is seen around a basket cell in the molecular layer (M), whereas the granular layer (G) appears unaffected.Toluidine blue×1000.

Light microscopic examination of the cerebellar cortex of group V (treated with both AL and MEL) displayed nearly normal appearance of the molecular, granular, and Purkinje cell layers (Figs 8–10). Compared with the control group, the Purkinje cells were slightly reduced in number (Figs 8 and 9) and seemed smaller in size (Fig. 10). They exhibited darker nuclei and cytoplasm compared with those of the control group (Fig. 10).

Figure 8
Figure 8:
A photomicrograph of a section in the cerebellar cortex of a rat treated by both aluminium and melatonin (group V) showing that the molecular (M), the granular (G), and the Purkinje cell layers (P) have an appearance near to that of the control group. The Purkinje cells (arrows) appear slightly reduced in number compared with the control group.H&E×200.
Figure 9
Figure 9
Figure 10
Figure 10:
A photomicrograph of a section in the cerebellar cortex of a rat treated by both aluminium and melatonin (group V) displaying nearly normal appearance of the molecular (M), granular, (G) and Purkinje cell layers (P). The Purkinje cells (arrows) seem smaller in size, compared with the control group, and exhibit darker nuclei and cytoplasm.Toluidine blue×1000.

Transmission electron microscopic study

Ultrathin sections of the cerebellar cortex of group I revealed that Purkinje cells were distinguished by their position, large size, euchromatic nuclei, and well-defined nucleoli. The nuclear envelope showed shallow dimples (Fig. 11). The cytoplasm was rich in mitochondria with intact membranes and regular cristae, and with cisternae of rough endoplasmic reticulum as well (Fig. 12). Bergmann astrocytes were observed between the Purkinje cells with their euchromatic nuclei and processes (Fig. 13). Granule cells were seen with their rounded heterochromatic nuclei and a shell of cytoplasm that exhibited strands of rough endoplasmic reticulum and free ribosomes (Fig. 14).

Figure 11
Figure 11:
An electron micrograph of a section in the cerebellar cortex of a control rat (group I) showing a Purkinje cell (PC) that is large in size with a euchromatic nucleus (N) and well-defined nucleolus (arrow head). The nuclear envelope shows shallow dimples (arrows). The cytoplasm is rich in organelles. Granule cells (GC) can be seen on one side of the Purkinje cell.×2000.
Figure 12
Figure 12:
An electron micrograph of a section in the cerebellar cortex of a control rat (group I) displaying the cytoplasm of a Purkinje cell containing numerous mitochondria (M) with intact membranes and regular cristae, and also cisternae of rough endoplasmic reticulum (rER).×15 000.
Figure 13
Figure 13:
An electron micrograph of a section in the cerebellar cortex of a control rat (group I) exhibiting a Bergmann astrocyte (arrow) with a euchromatic nucleus (N). One of its processes (arrow head) can be observed.×4000.
Figure 14
Figure 14

The cerebellar cortex of groups II and III showed no ultrastructural deviation from group I.

After AL administration (group IV), features of neuronal insult were prominent. Some of the few encountered Purkinje cells were shrunken with an electron-dense cytoplasm and ill-distinct nuclei (Fig. 15), whereas in others the nuclei were rarefied and hardly identified exhibiting only faint outlines of a nucleus or a nuclear ghost (Fig. 16). Some of the Purkinje cell mitochondria appeared swollen with ruptured membranes and disrupted cristae (Fig. 17). Bergmann astrocytes revealed a euchromatic nucleus and prominent cytoplasmic organelles (Fig. 16). Granule cells displayed increased condensation of their nuclear chromatin (Fig. 18).

Figure 15
Figure 15:
An electron micrograph of a section of the cerebellar cortex of an aluminium-treated rat (group IV) showing a shrunken Purkinje cell (PC) with an electron-dense cytoplasm and an ill-distinct nucleus (N). Granule cells (GC) with increased condensation of their nuclear chromatin are observed on one side of the Purkinje cell.×2000.
Figure 16
Figure 16:
An electron micrograph of a section in the cerebellar cortex of an aluminium-treated rat (group IV) showing Bergmann astrocyte (thick arrow) besides a degenerating Purkinje cell (PC) with a rarefied nucleus (N1) showing only faint outlines (nuclear ghost). Bergmann astrocyte exhibits a euchromatic nucleus (N2) and prominent cytoplasmic organelles (arrow).×4000.
Figure 17
Figure 17:
An electron micrograph of a section in the cerebellar cortex of an aluminium-treated rat (group IV) showing the cytoplasm of a Purkinje cell containing swollen mitochondria (M), some of which exhibit ruptured membranes (arrow) and cristae.×15000.
Figure 18
Figure 18:
An electron micrograph of a section in the cerebellar cortex of an aluminium-treated rat (group IV) showing increased condensation of nuclear chromatin inside granule cells nuclei (N).×4000.

Compared with the control group, the cerebellar cortex of rats of group V (treated with both AL and MEL) showed nearly normal ultrastructure of Purkinje cells with euchromatic nuclei and a slightly electron-dense cytoplasm (Fig. 19) and the mitochondria seemed like those of the control group (Fig. 20). Both Bergmann astrocytes (Fig. 21) and granule cells (Fig. 22) displayed a very similar ultrastructure to that of the control.

Figure 19
Figure 19
Figure 20
Figure 20:
An electron micrograph of a section in the cerebellar cortex of a rat treated by both aluminium and melatonin (group V) showing the cytoplasm of a Purkinje cell with mitochondria (M) exhibiting a similar appearance to that of the control group. Note the presence of intact cisternae of rough endoplasmic reticulum (rER).×15 000.
Figure 21
Figure 21:
An electron micrograph of a section in the cerebellar cortex of a rat treated by both aluminium and melatonin (group V) showing Bergmann astrocyte (arrow) exhibiting a similar appearance to that of the control group with a euchromatic nucleus (N2) and cytoplasmic process (arrow head). Note the presence of a part of an apparently intact Purkinje cell (PC) having a euchromatic nucleus (N1).×4000.
Figure 22
Figure 22:
An electron micrograph of a section in the cerebellar cortex of a rat treated by both aluminium and melatonin (group V) showing granule cells with nuclei (N) similar to that of the control group.×4000.

Quantitative morphometric study

Semithin sections in the control cerebellar cortex presented a mean number of Purkinje cells/mm length of 25.6±1.82 (Table 2 and Histogram 2). Statistical analysis of the mean number of Purkinje cells/mm length revealed no significant change in groups II and III versus group I. In AL-treated group, the mean number decreased significantly (P<0.01) compared with the control group to become 3.3±0.18. Treatment with MEL alone did not cause any significant change in the mean number of Purkinje cells/mm length of cerebellar cortex compared with the control group. In contrast, this mean number increased significantly (P<0.01) in rats treated with both AL and MEL compared with the AL-treated group to become 23.8±1.15.

Table 2
Table 2:
The mean and standard deviation of the linear density of Purkinje cells/mm length of the folia in different groups
Histogram 2
Histogram 2:
Histogram 2. The mean and standard deviation (SD) of the number of Purkinje cells/mm length of the folia in different groups. •P<0.01 when compared with the control group. *P<0.01 when compared with the aluminium-treated group.

Discussion

The human population is constantly exposed to AL through various sources such as AL cooking utensils, certain beverages, and drinking water [38].

In this study, AL exposure resulted in a significant decrease in body and brain weights. Similar results were also observed by earlier researchers [39]. The decrease in the body weight could be attributed to the interference of AL with the hormonal status and/or protein synthesis [40]. Moreover, the decrease in the brain weight might be due to increased peroxidation of lipids as a consequence of oxidative stress [41].

In this study, there was a significant reduction in the number of Purkinje cells that were hardly detected. Disorganization of the Purkinje cell layer with loss of the Purkinje cells was previously reported with AL exposure [41]. The few observed Purkinje cells in this study showed a darkly stained cytoplasm and dark (pyknotic) nuclei. Pyknosis was described as irreversible condensation of chromatin in the nucleus of the cell undergoing programmed cell death or apoptosis [28]. Ultrastructurally, some Purkinje cells were shrunken with an electron-dense cytoplasm and ill-distinct nuclei, whereas others showed rarefied nuclei with faint outlines like a nuclear ghost. Their mitochondria appeared swollen with ruptured membranes and disrupted cristae. Changes in the mitochondria observed in this study could be explained by the effect of reactive oxygen species (ROS) generated by AL exposure. This exposure resulted in high mitochondrial membrane potential and elevated levels of oxidized proteins and lipids [10].

Alteration in the Purkinje cell layer after AL exposure may cause changes in motor coordination and changes/loss of motor behavioral activities [42]. It is established that Purkinje cells send inhibitory projections to the deep cerebellar nuclei, and they constitute the sole output of motor coordination in the cerebellar cortex [43].

The granular layer appeared unaffected by light microscopy in this study. However, electron microscopic examination revealed increased condensation of nuclear chromatin in granule cells. This might be a feature of apoptosis. This finding was in agreement with some previous studies [44]. These studies found that the granule cells were a specific target of AL neurotoxicity. They attributed the AL toxicity to impairment of the glutamate-nitric oxide-cyclic GMP pathway in neurons. The changes in the granular layer were thought to be secondary to changes in the Purkinje cells. As the degenerated Purkinje cells failed to establish contact with the granule cells, this led to lack of normal synchronism between them that might minimize their regulatory role. This idea was supported by earlier postulations that assumed that several factors might be able to affect cerebellar interneurones, glial cell appearance, and proliferation in young rats [45].

In this study, prominent perineuronal spaces were observed in the molecular layer around both basket and stellate cells. To our knowledge, no previous study reported the presence of such spaces. However, vacuoles were described around pyramidal cells in the cerebral cortex but not in the cerebellum after AL exposure [46,28].

In this study, prominent cytoplasmic organelles were observed in Bergmann astrocytes after AL exposure. This might reflect a compensatory mechanism in neurodegeneration. This finding indicated that AL enhanced the process of protein synthesis with an increase in the turnover of cytoplasmic organelles with the formation of autophagosomes [47].

Histological alterations in the cerebellum found in this study after AL administration could be explained by increased oxidative stress. This is based on the fact that AL is known to possess prooxidant activity [17]. Different mechanisms have been proposed to account for the oxidant activity of AL, which include its influence on target substances such as membrane lipids [18]. AL has also been shown to elevate lipid peroxidation and cause changes in the antioxidant defense system in the brain [19]. Meanwhile, the brain is highly sensitive to attacks by ROS due to high oxygen uptake [48], neuronal membrane lipids [49], modest antioxidant defenses, and several autooxidizable neurotransmitters [50]. Therefore, in situations in which the generation of free radicals exceeds the capacity of antioxidant defense, oxidative stress may lead to membrane degradation, cellular dysfunction, and also apoptosis [51]. Furthermore, AL treatment caused increased fragmentation of DNA, which is a typical characteristic and one of the markers of apoptosis [52].

In this study, MEL coadministration with AL was able to prevent the body and brain weight losses. MEL administration has been shown to keep the body weight similar to the control group. In the same respect, it was previously reported that MEL therapy decreased lipid peroxidation that resulted in an improvement in the body weight gain [53].

In this study, concomitant administration of MEL decreased the toxic effects of AL on the cerebellum. There was a nearly normal appearance of the molecular, Purkinje, and granular cell layers. However, the Purkinje cells were slightly deceased in number and size compared with the control group, and exhibited darker nuclei and cytoplasm. This was in agreement with a previous study, which examined the oxidant effect of AL on rats and the protective role of MEL on biochemical parameters [54]. They attributed the protective effect of MEL to prevention of lipid peroxidation. The observed improvement in the histological changes in the cerebellum might be ascribed to the neuroprotective role of MEL. It was reported that this protection might be related to decreased levels of ROS and regulation of the antioxidant enzymes, which might play a role in preventing the damage to lipids and other macromolecules [55]. Furthermore, MEL was found to reduce the polymorphonuclear leucocytes and lipid peroxidation products in oxidative damage in the liver [56]. Moreover, MEL was able to protect the membranes of the cell and organelles against free radical damage [57].

Accordingly, it was concluded that AL adversely affected the cerebellum by a triad of gross morphological, histological, and histomorphometric changes. These alterations are dangerous due to heavy human exposure to AL in the environment. Such hazardous effects were protectable with concomitant administration of MEL. It is recommended to abstain the use of AL in cooking utensils, beverages, and drugs. However, this study and its results should be extended to human volunteers in future studies.

Table
Table:
No title available.

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Keywords:

aluminum; cerebellum; melatonin; ultrastructure

© 2011 The Egyptian Journal of Histology