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 Table of Contents  
ORIGINAL ARTICLE
Year : 2016  |  Volume : 29  |  Issue : 3  |  Page : 728-735

Immunohistological study of the effect of extravirgin olive oil on aspartame-treated cerebellum of male albino rat


Department of Anatomy, Faculty of Medicine, Minia University, El Minia, Egypt

Date of Submission27-Sep-2015
Date of Acceptance29-Nov-2015
Date of Web Publication23-Jan-2017

Correspondence Address:
Fatma Alzhraa F Abdel Baky
Lecturer, Department of Anatomy, Faculty of Medicine, Minia University, El Minia, 61519
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-2098.198791

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  Abstract 

Objective
The aim of this study was to determine the possible protective effects of extravirgin olive oil on aspartame (ASP)-treated rats, which causes cerebellar changes.
Background
ASP is found in many products and is used all over the world. It has been reported that the consumption of ASP could cause neurological and behavioral changes such as headache, insomnia, and seizures. This study discussed the possible protective effects of extravirgin olive oil on the cerebellum of rats previously treated with ASP.
Materials and methods
Thirty adult male albino rats were used in the present study. The animals were divided into three equal groups (10 rats in each). The first group served as the control group. In the second group, the rats were administered ASP at a dose of 75 mg/kg body weight daily for 3 months by using an intragastric tube. In the third group, the rats were administered olive oil in a dose of 0.5 mg/kg body weight, followed by ASP in the same dose as the rats in group 2 through the same route. In addition, cyclooxygenase-2 immunohistological and hematoxylin and eosin light microscopic, and morphometric analyses were carried out.
Results
In the ASP-treated group, disorganization of the three layers of the cerebellar cortex had occurred. ASP caused marked changes in the histological picture of the normal cerebellum, indicated by an increase in the number of vacuolated spaces, necrosis, and apoptosis in the three layers. Furthermore, there was a decrease in the mean value (±SD) of Purkinje cell count and the area of Purkinje cell layer in the ASP-treated group. The administration of extravirgin olive oil in conjunction with ASP resulted in significant improvement in the organization of cellular layers of the cerebellar cortex and ameliorated the effects of ASP on the cerebellum. There was a significant improvement in the morphometric results with the use of the oil.
Conclusion
Administration of extravirgin olive oil ameliorates the neuropathological changes caused by aspartame on cerebellum of albino rats.

Keywords: aspartame, cerebellum, extravirgin olive oil


How to cite this article:
Abdel Baky FF. Immunohistological study of the effect of extravirgin olive oil on aspartame-treated cerebellum of male albino rat. Menoufia Med J 2016;29:728-35

How to cite this URL:
Abdel Baky FF. Immunohistological study of the effect of extravirgin olive oil on aspartame-treated cerebellum of male albino rat. Menoufia Med J [serial online] 2016 [cited 2020 Jun 1];29:728-35. Available from: http://www.mmj.eg.net/text.asp?2016/29/3/728/198791


  Introduction Top


Aspartame (ASP) is a methyl ester of a dipeptide. It is composed of phenylalanine (50%), aspartic acid (40%), and methanol (10%) [1] . It is synthetic, white, odorless, and a crystalline powder sweetener. It is 180-200 times sweeter than sucrose. It is used as a substitute for sugar in some foods and beverages [2] . Many people use ASP in diet soft drinks, though it can be found in many other products such as fruit drinks and chewing gum [3] . It is also present in hot chocolate, candy, tabletop sweeteners, and some pharmaceutical products such as vitamins and sugar-free cough drops [4] .

Consumption of ASP in a single large dose may have an effect on the plasma amino acid levels and brain neurotransmitter levels. ASP can be metabolized into three components - phenylalanine, aspartic acid, and methanol [5] . Phenylalanine plays an important role in neurotransmitter regulation, and aspartic acid is believed to play a role as an excitatory neurotransmitter in the central nervous system (CNS) [6] . It was reported that, elevated levels of phenylalanine and aspartic acid in plasma can be associated with many neurologic disorders such as phenylketonuria and mental retardation [7] .

If ASP enters the bloodstream, it can penetrate the blood-brain barrier and thus it might causes neurotoxicity of the brain cells and damage of the brain [8] , resulting in many neurological disorders such as memory loss and seizures [9] .

It was reported that ASP could be metabolized in the mitochondria of cells causing mitochondrial, nuclear DNA damage, and inadequate energy metabolism, resulting in highly damaging free radicals [10] . These free radical include the functional reserve of antioxidant, vitamins, and minerals that is necessary for neural protection and regeneration [11] .

Olive oil is used commonly in cooking, cosmetics, and pharmaceuticals. Extravirgin olive oil becomes more important in daily diets, especially in Mediterranean diet, because of its beneficial effects on the human health [12] . It is derived from the first pressing of olive, and has many antioxidant benefits [13] . It is rich in omega-3 and omega-6 fatty acids that help to improve memory [14] . It was reported that, extravirgin olive oil has a beneficial effect on the CNS by aiding nerves to do their function, and by increasing the serotonin level in the brain [15] . It contains variable amounts of phenolic antioxidants [16] . Hydroxytyrosol (3,4-DHPEA) and its derivatives are among these phenols; they are responsible for the antioxidant activity of olive oil; it is absorbed in rats and humans only if administered orally [17],[18] . Its antioxidant activity has been shown both in vitro [19],[20] and in vivo [21] . Cyclooxygenase (COX) is a membrane-bound enzyme that is responsible for the oxidation of arachidonic acid to prostaglandin (G2) and its subsequent reduction to prostaglandin (H2) [22] . It is expressed in at least two forms, the expressed form, COX-1, and the inducible form, COX-2 [23] . COX-1 can be expressed in many normal tissues and is involved in a number of homeostatic body functions such as hemostasis [24] . The expression of COX-2 can be induced by different stimuli such as growth factors and cytokines [25] . A strong expression of COX-2 has been associated with chronic inflammatory diseases and neurotoxic conditions such as seizures and hypoxia [26] .


  Materials and methods Top


Animals

This study was conducted in the Anatomy Department of the Faculty of Medicine, El-Minia University. Thirty adult male albino rats weighing about125-150 g were included in the study. Animals were housed in standard clean plastic cages and were given regular diet and water ad libitum under controlled conditions. The experiment was approved by the Ethical Committee for animal handling for research work in El-Minia University.

The experimental design

The animals were divided into three equal groups (10 rats each).

Group 1 served as the control group. Animals of this group received a daily dose of distilled water comparable to the dose given to the other groups throughout the experiment.

In group 2, the animals were administered ASP at a dose of 75 mg/kg body weight daily [1] for 3 months using an intragastric tube. The ASP tablets used, each one containing 20 mg, were obtained from the Sigma (Saint Louis, Missouri, USA).

In group 3, the animals were administered extravirgin olive oil in a dose of 0.5 mg/kg body weight followed by ASP in the same dose as the rats in group 2.

After the end of the experiment, the rats were sacrificed by decapitation under halothane anesthesia. The brain was removed and the cerebellum was taken out and divided into two halves.

Light microscopic examination

For light microscopic study, samples from the cerebellum of rats were taken and fixed in 10% neutral buffered formalin. Samples were dehydrated by using alcohols, cleared in xylene, and then embedded in paraffin wax. Then, 5-mm-thick sections were stained with hematoxylin and eosin.

Immunohistochemical staining for localization of cyclooxygenase-2

The expression of COX-2 was detected  by using the primary antibody anti-rabbit, anti-mouse polyclonal COX-2-specific IgG (SAB4200576; Sigma).

The sections of the cerebellar cortex were dewaxed in xylol for 20 min (two changes) and hydrated in descending grades of alcohol down to distilled water.  The primary antibody sections were incubated at an appropriate degree of dilution in immunohistochemical (IHC)-Tek, antibody diluent (Cat. #IW-1000 or #IW-1001) for 1 h at room temperature or overnight. Peroxidase blocking was carried out after incubation for 10 min at room temperature. For acetone-fixed frozen sections, this peroxidase blocking step was performed using 0.3% hydrogen peroxide in methanol before primary antibody  incubation to avoid tissue destruction.

Sections were rinsed three times with PBS between 20 for 3 × 2 min, and excess liquid was tapped off the slides. Enough hydrogen peroxide was applied to cover the specimen for 5 min, and then the slides were rinsed gently with PBS and excess liquid was tapped off.

Enough amount of primary antibody (dilution 1 : 200) was applied on the specimens, which were then incubated for 2 h in a humid chamber at room temperature. Then the slides were rinsed in PBS.

Biotinylated link was applied on the specimens for 10 min and the sections were rinsed in PBS.

Streptavidin horseradish peroxidase reagent was applied on the specimens for 10 min and then the sections were rinsed in PBS. Freshly prepared 3,3'-diaminobenzidine substrate chromogen solution (one drop of 3,3'- diaminobenzidine chromogen/1 ml of substrate buffer) was removed from 2 to 8°C storage and applied on the specimens for 10 min.

Slides were rinsed gently in distilled water, immersed in hematoxylin for 5 min, and were rinsed in tap water until the color turned blue; then the slides were dehydrated in ascending grades of alcohol, cleared in xylol, mounted by Canada balsam, and covered with a cover slip.

At the time of assessment, tissues were independently assessed by two observers for positive or negative cases. Positive immunostaining gave nuclear and/or cytoplasmic dark brown granules. Then the number of positive cells that gave brown cytoplasmic staining system under light microscope were counted. The extent of the IHC signal was determined in 10 fields (×100 magnification). In each field the total number of cells was counted and the extent of cytoplasmic staining cells was determined as a percent.

The total staining score was divided by the number of whole cells per field in 10 fields, and the percentage of positively stained cells in the 10 fields was calculated for each case by taking the mean of the percentage of the positively stained cell in the 10 fields. COX-2 immunoreactivity was assessed as being positive [27] . Negative control slides were prepared by using the same steps except they were incubated with the antibody diluents instead of primary antibody [28] .

Sections prepared from the kidney tissue of control animals were used as positive control slides for COX-2 [29] .

Positive reaction appeared brown in color [30] .

Immunohistochemistry assessment

There are no established cutoff points available for quantitative COX-2 expression as there are relatively few articles reporting a wide variations in the incidence of COX-2 overexpression ranging from 32 to 67.7%. COX-2 expression was categorized by using an H score [31] , which combines the intensity of staining in each cell and percentage of stained cells. In brief, staining intensity score ranges from 0 to 3 (I0, I1-I3), and the percent of stained cells was recorded in 5% increments from a range of 0 to 100 (P0, P1-P3). For each spot analyzed, a score was generated form the product of intensity and percent of stained cells. A final H score (range 0-300) was obtained by adding the sum of individual scores obtained for each tissue microarray spot (H score Ό= I1 × P1 + I2 × P2 + I3 × P3). X-tile plots are constructed for assessment of biomarker and optimization of cut off points based on outcome. The X-Tile plots allow determination of an optimal cut point while correcting for the use of minimum P statistics. Using the X-Tile program, an optimal cut point for COX-2 expression was determined at 160, with a Miller-Seigmund P value of 0.5950 as determined by X-Tile. Cells with H score <160 were classified as low expressers (n = 58; 39.7%), and those with H score >160 were classified as high expressers (n = 88; 60.3%).

Golgi-Cox method [32]

For each group, the specimens were fixed in a Golgi-Cox solution for 2 months and embedded in cellodin. It was prepared as follows: 5% potassium dichromate (10 volumes), 5% mercuric chloride (10 volumes), 5% potassium chromate (eight volumes), and distilled water (15-20 volumes). The fluid was put in a dark bottle containing a pad of cotton at the bottom to ensure complete bathing, after which the specimens were left in this fluid for 6 weeks in the dark.

After 6 weeks, the specimens were washed in 80° ethyl alcohol for 0.5-2 h, and then in a mixture of equal volumes of absolute alcohol and acetone (three changes) for 24 h. The specimens were transferred into equal parts of absolute alcohol and ether for 4 h, and then passed through celloidin of ascending grades; 2-8% embedding was carried out in thick celloidin 12% concentration, which was hardened in chloroform vapor. The blocks were stored in 70% ethyl alcohol, and sections were cut on a sliding microtome at a thickness of 90 μm in a horizontal plane, and developed on 5% potassium sulfate, to which few drops of 5% oxalic acid had been previously added, till the sections became greenish gray in color.

The sections were then rinsed in distilled water for 15 min; then dehydration was carried out in ascending grades of alcohol, and lastly in a mixture of equal volumes of absolute alcohol and ether. They were arranged on slides and finally mounted in thick Canada balsam, covered and lifted to dry in the incubator. Sections were examined by using a Leitz Orthopan light microscope (Leitz, Wetzlar, Germany): the nerve cells and their processes appeared black.

Morphometric analysis

The data were obtained using a computerized image analyzer (Lecia Imaging System Ltd, Cambridge, UK). Cerebellar sections were randomly selected for morphometric measurements. The image analyzer consisted of a colored video camera (Panasonic Color CCTV Camera; Matsushita Communication Industrial Co. Ltd, Kτhoku-ku, Yokohama, Japan), colored monitor, and hard disc of IBM personal computer connected to a light microscope (Olympus BX-40; Olympus Optical Co. Ltd, Yoyohata-chτ, Hatagaya, Japan) and controlled by a Leica Qwin 500 software (Leica). The image analyzer was first calibrated automatically to convert the measurement units (pixels) produced by the image analyzer program into actual micrometer units. Ten readings were obtained for each specimen and also the mean values were obtained. Using the interactive measure, the number of Purkinje cells were measured using a magnification of ×400 with a measure frame of 7381.11 μm [33] .

Statistical analysis

The mean and SD were determined for each parameter in each group, and the significance of differences observed were pooled and assessed using P values. All the analyses were performed using the SPSS (version 13; SPSS Inc., Chicago, Illinois, USA).


  Results Top


Light microscopic evaluation of the cerebellar cortex

In the present study, the examination of sections of the control group ([Figure 1]) showed the three layers of the cerebellar cortex: the molecular layer, which was formed of scattered nerve cells and fibers, Purkinje cell layer, and the granular layer. The Purkinje cell layer appeared with large cell bodies pyriform in shape and contained rounded pale-stained nuclei with prominent nucleoli. The granular layer contained small granule cells with dense nuclei and scanty cytoplasm. Examination of group 2 (ASP-treated group) showed the Purkinje cells with irregular shrunken outline with deeply stained cytoplasm and pyknotic nuclei ([Figure 2]). Many shrunken cells lost their characteristic pyriform shape and become surrounded by many vacuoles due to fallen off cells, leaving empty spaces ([Figure 2]). Some vacuolations appeared between the cells of the granular layer and in the molecular layer ([Figure 2]). Examination of group 3 (extravirgin olive oil + ASP-supplemented group) showed that many Purkinje cells appeared normal with well-defined nuclei ([Figure 3]); some cells were still shrunken with deeply stained cytoplasm, and also few areas of vacuolations were still present ([Figure 3]).
Figure 1: Photomicrograph of the cerebellar cortex of the control group showing three layers forming the cerebellar cortex - molecular layer (M), Purkinje cell layer (P), and the granular layer (G). The outer molecular layer contains fewer small scattered cells. The Purkinje cell layer contains Purkinje cells, which have a pyriform-shaped cell body with their dendrites toward the molecular layer (arrow). Hematoxylin and eosin, ×400.

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Figure 2: Photomicrograph of the cerebellar cortex of aspartame-treated group 2 showing the granular layer (G), molecular layer (M), and Purkinje cells (P) with irregular shrunken outline losing their characteristic pyriform shape (thin arrow). Some vacuolations appeared between the cells of Purkinje cell layer (dark arrows). Hematoxylin and eosin, ×400.

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Figure 3: Photomicrograph of the cerebellar cortex of extravirgin olive oil + aspartame group 3 showing the granular (G) and molecular layer (M). The majority of the Purkinje cells (arrow) regain their shape. Hematoxylin and eosin, ×400.

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Golgi-Cox evaluation

In Golgi-Cox-stained sections, the examination of Purkinje cells of group 1 (control) showed that the primary dendrites arise from the tapering end of the Purkinje cell body; they divide into a tree-like secondary dendrites ([Figure 4]). Purkinje cells of group 2 (the ASP-treated group) showed less dendritic branching compared with that of the control group ([Figure 5]). Examination of group 3 (ASP+extravirgn olive oil) showed that the Purkinje cells' dendritic branching was obvious but still less than that of the control group ([Figure 6])
Figure 4: Photomicrograph of a Purkinje cell (P) of the control group 1; they appeared with extensively tree-like dendritic branches; primary dendrites (PD), and secondry dendrites (SD). Golgi-Cox, ×400.

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Figure 5: Photomicrograph of a Purkinje cell (P) of the control group 1; they appeared with extensively tree-like dendritic branches; primary dendrites (PD), and secondry dendrites (SD). Golgi-Cox, ×400.
Figure 5
Photomicrograph of a Purkinje cell (P) of the aspartame-treated group 2; there was less branching of the primary dendrites (PD) and the secondary dendrites (SD). Golgi-Cox, ×400.


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Figure 6: Photomicrograph of a Purkinje cell (P) of the extravirgin olive oil+aspartame group 3 showing the Purkinje cell with branching close to that of the control, with primary dendrites (PD) and secondary dendrites (SD). Golgi-Cox, ×400.

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Immunohistochemical evaluation for cyclooxygenase-2

IHC sections of COX-2 of the control group 1 showed less intensity of immune reaction for COX-2, with an H score of less than 160 (n1/8 =58; 39.7%) in the molecular layer, Purkinje cells, and the granular layer ([Figure 7]). In the ASP-treated group 2, a strong positive high-intensity COX-2 reaction was observed in the form of brown color, with an H score of greater than 160 (nΌ =88; 60.3%) in the molecular layer, Purkinje cells, and the granular layer ([Figure 8]). The majority of the Purkinje cells showed moderate-intensity immune reaction of COX-2 in group 3 (extravirgin olive oil + ASP group 3) ([Figure 9] and [Table 1]).
Figure 7: Low-intensity cytoplasmic reaction to cyclooxygenase-2 in the molecular layer, Purkinje cells, and the granular layer in the control group 1. Immunohistochemical stain, ×400.

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Figure 8: Strong-intensity positive reaction to cyclooxygenase-2 in the molecular layer, Purkinje cells, and the granular layer in aspartame-treated group 2. Immunohistochemical stain, ×400.

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Figure 9: Moderate-intensity reaction to cyclooxygenase-2 in the molecular layer, Purkinje cells, and the granular layer in the aspartame+extravirgin olive oil group 3. Immunohistochemical stain, ×400.

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Table 1 Comparison between the three groups as regards immunohistochemical expression


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Morphometric results

[Table 2] shows that, the difference in Purkinje cell count, area of Purkinje cell, and the distance between Purkinje cells between groups 1, 2, and 3 was statistically significant (P = 0.000).
Table 2 Comparison between the three studied groups as regards mean±SD


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  Discussion Top


The aim of this study was to determine whether chronic ASP administration (for 3 months) will lead to histopathological changes of the cerebellar cortex of albino rats and to evaluate the effect of extravirgin olive oil on these changes. In the present study, the cerebellar cortex of ASP-treated animals showed obvious structural lesions in the Purkinje cell layer, which are in line with the results obtained by Abd El-Samad [34] . These changes in Purkinje cells revealed pathological affection of the chief cells of the cerebellar cortex that are the only cells of the cerebellum that send information to other parts of the brain [35] . In addition, these pathologic changes may be due to the fact that ASP can penetrate the blood-brain barrier and cause neurotoxicity of brain cells and damage of the brain [8] , resulting in many neurological disorders as such as memory loss and seizures [9] .

In the present study, extravirgin olive oil was administered with ASP in group 3 to evaluate its effects on the structure of the cerebellar cortex affected by ASP. This group showed improvement in the cells of the cerebellar cortex; this improvement might be attributed to the fact that extravirgin olive oil is rich in phenolic antioxidant activity [13],[16] . These antioxidants are amphipathic molecules and can easily pass through the blood-brain barrier. This is an evidence of the neuroprotective activity of olive oil phenols when administered orally in mice [36] . Moreover, this improvement might be due to the fact that, extravirgin olive oil has beneficial effects on the CNS by aiding nerves to do their function, and by increasing the serotonin level in the brain [15] .

In the present study, IHC result of the ASP group showed strong positive reaction; this pathogenic effect of COX-2 is due to its role in inflammation-induced injury [37] . In many organs including the brain, COX-2 is markedly upregulated in many pathological conditions associated with inflammation and cytotoxicity both in vitro and in vivo [38] . Strong positive COX-2 expression is associated with multiple neuropathologies such as traumatic brain injury [39] , and chronic neurodegenerative conditions such as Alzheimer's disease [40] . High intensity of COX-2 expression might be an adaptive reaction to many pathological conditions, such as early inflammatory processes and oxidative stress [26] . Dendrites and dendritic spine formation are important for proper brain function, and thus, in the present study, we studied the effect of ASP and evaluated the effect of extravirgin olive oil on them. Using the Golgi-Cox technique, the Purkinje cells dendritic arborization in ASP-treated group showed a marked impairment, which might be related to an impairment of mechanisms underlying neuroplasticity [41] . In the extravirgin olive oil+ASP group, a clear improvement in the arborization of Purkinje cells was seen. Morphometric results in our study revealed a signficant decrease in the Purkinje cell count and the area of Purkinje cell layer in the ASP-treated group 2 compared with the ASP+extravirgin olive oil group 3; furthermore, the distance between Purkinje cells was significantly increased in both groups 2 and 3, which revealed an increase in the areas of vacuolations caused by apoptosis, necrosis, and death of Purkinje cells, and also the use of extravirgin olive oil moderately affected these changes caused by chronic use of ASP.


  Conclusion Top


Administration of extravirgin olive oil ameliorates the neuropathological changes caused by aspartame on cerebellum of albino rats.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9]
 
 
    Tables

  [Table 1], [Table 2]



 

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