Ultrastructural Pathology, 2014; 38(3): 224–236 ! Informa Healthcare USA, Inc. ISSN: 0191-3123 print / 1521-0758 online DOI: 10.3109/01913123.2014.889259
ORIGINAL ARTICLE
Ameliorative Effect of Pimpinella anisum Oil on Immunohistochemical and Ultrastuctural Changes of Cerebellum of Albino Rats Induced by Aspartame
Department of Zoology, Faculty of Science, Beni-Suef University, Beni Suef, Egypt
ABSTRACT The study aims to investigate the protective effect of Pimpinella anisum oil on aspartame (ASP) which resulted in cerebellar changes. The rats were divided into four equal groups: Group 1: (control group): served as control animals. Group 2: control P. anisum oil received .5 mL/kg/d/b wt. once daily. Group 3 (ASP group): received daily 250 mg/kg/b wt. of ASP dissolved in distilled water and given orally to the animals by intra-gastric tube for 2 months. Group 4: received .5 mL/kg/b wt. of prophylactic P. anisum oil once daily, followed by ASP after 2 h for 2 months. The histopathological approach revealed marked changes in the Purkinje cells, myleinated nerve fibers and granular cells of ASP-treated animals. Some of these cells appeared with deeply stained cytoplasm. Ultrastructural examination showed Purkinje cells with dilated rough endoplasmic reticulum and condensed mitochondria. Granular cells appeared with less c nuclei and surrounded by dissolution of most Mossy rosettes structures. Most myelinated nerve fibers showed thickening of myelinated sheath and others showed splitting of their myelin sheath. The histopathological, immunohistochemical and ultrastructural alterations were much less observed in concomitant use of P. anisum oil with ASP. Cerebellar cortex is considered target areas of ASP neurotoxicity, while P. anisum oil, when used in combination with ASP displays a protective action against neurotoxicity. Keywords: Aspartame, cerebellum, histopathology, immunohistochemistry, Pimpinella anisum oil, ultrastructure
INTRODUCTION
non-caloric although its energy value is 4 kcal/g of ASP [4]. ASP can enter the bloodstream and circulate through the entire body, including the brain, because it can penetrate the blood–brain barrier. It might cause excitotoxicity of brain cells [5], which is damage to the brain and nervous system. It can be metabolized to yield phenylalanine, aspartic acid, and methanol and is then later oxidized into formaldehyde and formic acid in many tissues. Formic acid was considered the principal metabolite responsible for the deleterious effects of acute intoxication by methanol in humans and animals [6]. Furthermore, elevated phenylalanine levels are associated with neurologic function, such as phenylketonuria and mental retardation [7]. Some authors reported that ASP could be metabolized
Aspartame (ASP) is the most widely used artificial sweetener. It is a methyl ester of a dipeptide (L-aspartyl-L-phenylalanine methyl ester) [1]. It was found in more than 6000 products, including carbonated soft drinks, hot chocolate, chewing gum, candy, deserts, tabletop sweeteners and some pharmaceutical products, such as vitamins and sugar-free cough drops [2]. It was estimated by the ASP Information Center [3] to be consumed by more 200 million people worldwide, including children between 2 and 5 years of age and pregnant females. This white, crystalline, and odorless powder is 180–200 times sweeter than sucrose. The intense sensation of sweetness allows the use of such small doses so that the product is almost
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Manal Abdul-Hamid, PhD and Sanaa Rida Gallaly, PhD
Received 23 December 2013; Revised 26 January 2014; Accepted 27 January 2014; Published online 28 March 2014 Correspondence: Manal Abdul-Hamid, Ass. Prof. of Histology and Cytology, Department of Zoology, Faculty of Science, Beni-Suef University, Beni Suef 62511, Egypt. Fax: +20 822327986. E-mail: [email protected]
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Pimpinella anisum oil protects cerebellum in cell mitochondria, inducing mitochondrial and nuclear DNA damage and affecting its ability to produce inadequate and incomplete energy metabolism, which gives rise to highly damaging free radicals [8]. Moreover, free radical scavengers including the functional reserve of antioxidant, vitamins, and minerals could be necessary for neural protection and regeneration [9], because improvement from ASPinduced toxicity was gradual and incomplete as reported by Christian et al. [10]. ASP consumption was suggested to be implicated with various symptoms from the central nervous system such as seizures [6], memory loss [11], cholinergic symptoms, headaches [12], and oncogenesis [2]. It had been reported that the astrocyte-specific protein, the glial fibrillary acidic protein (GFAP), this intermediate filaments found in astrocyte, could also serve as a marker for glial cells [13]. Cyclooxygenase (COX) is a membrane-bound enzyme responsible for the oxidation of arachidonic acid to prostaglandin G2 and its subsequent reduction to prostaglandin H2 [14,15]. COX is expressed in at least two different isoforms, a constitutively expressed form COX-1 and an inducible form COX-2 [15,16]. COX-1 is expressed in many normal tissues and is involved in a number of homeostatic body functions, such as hemostasis, vasodilatation in renal vessels and cytoprotection of the gastric mucosa [16]. The expression of COX-2 is induced by various stimuli, such as growth factors and cytokines and is involved in chronic inflammatory pathologies such as inflammatory bowel disease [17]. Minghetti [18] reported that over-expression of COX-2 has been associated with neurotoxicity in acute conditions, such as hypoxia/ ischemia and seizures. However, the beneficial or detrimental role played by COX-2 in inflammatory and neurodegenerative brain pathologies is still controversial. Plants and their extracts offer a unique opportunity in this regard since aromatic plants and their extracts have been used traditionally in the therapy of some diseases for a long time through the world [19]. Aniseed (Pimpinella anisum L.), an aromatic plant, is an annual herb cultivated in many countries but indigenous to Iran, India, and Turkey [20]. Chemical studies have demonstrated that the aniseed contains anethole (85%) as an active ingredient, in addition to eugenol, methylchavicol, anisaldehyde, and estragole [21]. Pimpinella anisum has been used for different purposes as an antioxidant, hepatoprotective, antiinflammatory agent, and toxicity of the pituitary thyroid axis [22]. Pimpinella anisum may have therapeutic effects in diseases such as digestive, gynacologic, neurologic, and respiratory disorders [23]. The purpose of this study was to evaluate the protective effect of P. anisum oil on ASP-induced cerebellum changes, by observing histopathological, immunohistochemical and ultrastructural changes. !
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MATERIALS AND METHODS Chemicals ASP was obtained from Al-Ameriya Pharma Company (Alexandria, Egypt). It is available in the form of tablets, each one containing 20 mg of ASP. The tablets were dissolved in distilled water and administered to the rats. Pimpinella anisum oil was obtained from an aromatics shop in Beni-Suef (Egypt).
Animals and experimental design Twenty male adult albino rats, weighing about 140–180 g, were used in this study. All the animals were maintained under standard laboratory conditions of temperature (25 C) and 12 h light and 12 h dark cycles throughout the experimental period. They were housed in standard cages and had free access to water and standard diet at the research center in Beni-Suef University. Experiments were conducted as per the guidelines of Institutional Animal Ethical Committee, Beni-Suef University. The animals were divided into four groups (five each) as the following: Group 1 (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. Group 2 (P. anisum oil group): These animals received a daily dose of .5 mL/kg/b wt. P. anisum oil (according to Lershin [24]) for 2 months. Group 3 (ASP group): Rats of this group received daily oral dose of 250 mg/kg/b wt. of ASP dissolved in distilled water by intra-gastric tube for 2 months [25]. ASP tablets, each one containing 20 mg, were obtained from Al-Ameriya Pharma Company. This dose used is corresponding to the human dose, which is 40–50 mg/kg/b wt. after species factor correction requiring 5–6 times higher dose than man, as rat metabolizes ASP faster than human [26]. Group 4 (P. anisum oil + ASP group): Animals of this group received .5 mL/kg/d/b wt. of prophylactic P. anisum oil once daily [24], followed by a daily dose of 250 mg/kg/b wt. of ASP after 2 h for 2 months.
Light microscopic examination For light microscopic study, samples from cerebellum were taken; after fixation in 10% neutral buffered formalin, they were dehydrated through alcohols, cleared in xylene and embedded in paraffin wax.
226 M. Abdul-Hamid and S. R. Gallaly Later on, 5-mm-thick sections were stained with hematoxylin and eosin [27].
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Immunohistochemical staining for GFAP of astrocytes For immunohistochemical study, staining was performed for GFAP as an indicator for glial reactivity. Primary antibodies monoclonal mouse anti-human GFAP was purchased from Dako, Carpenteria, CA. Serial paraffin sections of 5 mm thickness were deparaffinized and dehydrated, including positive control sections from the rat cerebellar cortex matter. The endogenous peroxidase activity was blocked with 0.05% hydrogen peroxide in absolute alcohol for 30 min. The slides were washed for 5 min in phosphate buffered saline (PBS) at pH 7.4. To unmask the antigenic sites, sections were placed in .01 mol/L citrate buffer (pH 6) in a microwave for 5 min. The slides were incubated in 1% BSA dissolved in PBS for 30 min at 37 C in order to prevent nonspecific background staining. Slides were incubated with the primary antibody (1:500 monoclonal mouse antiGFAP) at 4 C for 18–20 h and after washing, they were incubated with biotinylated secondary antibodies and then with avidin–biotin complex. Finally, sections were developed with .05% 3,3-diaminobenzidine for 15 min. Slides were counterstained with Mayer’s hematoxylin, dehydration, clearing and mounting were done by DPX [28].
solution (1 drop of DAB chromogen/1 mL of substrate buffer) was removed from 2 to 8 C storage and applied on specimens for 10 min. Slides were rinsed gently in distilled water, immersed in hematoxylin for .5 min and were rinsed in tab water until blue. Slides were dehydrated in ascending grades of alcohol, cleared in xylol, mounted by Canada balsam, and covered with a cover slip. Negative control slides were prepared by the same steps except they were incubated with the antibody diluent instead of primary antibody. 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].
Electron microscopic examination For electron microscopy, rats of all groups were dissected and other parts of the specimens were immersed in 2.5% glutraldehyde in phosphate buffer (pH 7.4) for 4 h and then post fixed in 1% osmium tetra oxide at 4 C, dehydrated and embedded in epoxy resin. Semithin sections were cut and stained with Toluidine blue. Ultra-thin sections were stained with 2% uranyl acetate and 2% lead citrate [31] and examined and photographed by transmission electron microscope (JEOL-Ex1010 TEM) in AinShams University.
RESULTS Immunohistochemical staining for localization of COX-2
Light microscopic evaluation of cerebellar cortex
The expression of COX-2 was detected using rabbit polyclonal anti-human COX-2 specific IgG (H-62, sc-7951, Santa Cruz Biotechnology, Dallas, TX). Sections (of cerebellar cortex) were dewaxed in xylol for 20 min (two changes) and hydrated in descending grades of alcohol down to distilled water. They were immersed into preheated target retrieval solution to 95–99 C (without boiling) in water bath for 40 min, removed from the bath and allowed to cool for 20 min at room temperature. Sections were rinsed three times with PBS. Excess liquid was tapped off the slides. Enough hydrogen peroxide was applied to cover the specimen for 5 min, then slides were rinsed gently with PBS and excess liquid was tapped off. Enough amount of primary antibody (dilution 1:200) was applied on specimens and was incubated for 2 h in a humid chamber at room temperature. Slides were rinsed in PBS. Biotinylated link was applied on specimens for 10 min and sections were rinsed in PBS. Streptavidin HRP reagent was applied on specimens for 10 min and sections were then rinsed in PBS. Freshly prepared DAB substrate chromogen
Examination of sections of the control group (Figure 1a and b) showed the three layers forming the cerebellar cortex, the molecular layer, Purkinje cell layer, and the granular layer. The Purkinje cell layer showed Purkinje cells with pyriform shaped cell bodies and central rounded vesicular nuclei and prominent nucleoli. The cytoplasm was finely granular. The granular layer contained numerous small granule cells with relatively dense nuclei and scanty cytoplasm. Control oil P. anisum group did not show any histopathological changes. Examination of the ASP-treated group revealed Purkinje cells with irregular shrunken outline of their perikarya associated with deeply stained cytoplasm and pyknotic nuclei (Figure 2a). Some shrunken pyknotic cells lost their characteristic pyriform shape and were surrounded by vacuoles due to fallen off cells, leaving empty spaces (Figure 2b). Some vacuolations (spongiosis) appeared between cells of the granular layer and in the molecular layer (Figure 2b). Examination of P. anisum oil + ASP-supplemented group showed that the majority of Purkinje cells Ultrastructural Pathology
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FIGURE 1. Photomicrograph of the cerebellar cortex of control brain showing (a) three layers forming the cerebellar cortex, the molecular layer (M), Purkinje cell layer (arrows) and the granular layer (G). (b) The outer molecular layer contains few small scattered cells. Purkinje cell layer contains Purkinje cells (arrows) appeared with pyriform shaped cell bodies and central rounded vesicular nuclei and prominent nucleoli. The granular layer contains numerous small granule cells with relatively dense nuclei and scanty cytoplasm with cerebellar islands (C) in-between (H&E 400, 1000).
FIGURE 2. Photomicrograph of the cerebellar cortex of ASP-treated group brain showing (a) granular layer (G), molecular layer (M) and Purkinje cells with irregular shrunken outline losing their characteristic pyriform shape associated with deeply stained cytoplasm and pyknotic nuclei (arrows). (b) The shrunken pyknotic Purkinje cells (arrows). Some vacuolations (spongiosis) (V) appeared between cells of the granular layer and in the molecular layer (H&E 400, 1000, respectively).
appeared normal with well-defined nuclei and prominent nucleoli (Figure 3a and b). Few cells were still shrunken with deeply stained cytoplasm (Figure 3b). Semithin section in the cerebellar cortex of control rat showed Purkinje cell with pyriform shaped cell body and defined nucleus with prominent nucleolus (Figure 4a and b). The ASP-treated animal showed Purkinje cells with darkly stained cytoplasm and hardly identified nuclei (Figure 5). Cerebellar cortex of P. anisum oil + ASP-treated group showed some Purkinje cells regaining their pyriform shape but others with darkly stained cytoplasm and hardly identified nucleus (Figure 6). !
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Immunohistochemical evaluation For GFAP Immunohistochemical examination of positive control sections of the rat cerebellum stained with GFAP showed the presence of GFAP-positive represented by elongated cells and their processes in the molecular layer with pale brownish immuno-reaction, while granular layer showed few brownish star-shaped cells (Figure 7a and b). Marked increase in the size and intensity of brownish immuno-reaction of GFAP positive astrocytes was observed in ASP-treated rats in the granular
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FIGURE 3. (a and b) Photomicrographs of the cerebellar cortex of P. anisum oil + ASP group showing granular layer (G) and molecular layer (M) the majority of Purkinje cells (arrows) regain their shape with well-defined nuclei and prominent nucleoli (H&E 400, 1000, respectively).
FIGURE 4. (a and b) Photomicrograph of semithin section in the cerebellar cortex of control rat showing Purkinje cell (arrow) with pyriform shaped cell body and defined nucleus (arrow head) with prominent nucleolus (Toluidine blue, 1000).
layer. In the molecular layer, the elongated cells and their processes also appeared with deeply stained brownish color (Figure 8a and b). Immunohistochemichal stained sections for GFAP in P. anisum oil + ASP group showed few brownish star-shaped cells in the granular layer in-between cells. Elongated cells and their processes appeared with faint brownish immuno-reaction in the molecular layer (Figure 9a and b).
In the ASP group a strong positive reaction was observed in the form of brown color within the molecular layer, Purkinje cells and granular layer (Figure 11a and b). The majority of Purkinje cells showed a negative immune reaction of Cox-2 in P. anisum oil + ASP group (Figure 12).
Electron microscopic evaluation of cerebellar cortex For Cox-2 immuno-stain The immunohistochemical sections of Cox-2 of control groups showed negative immune reaction for Cox-2 in molecular layer, Purkinje cells, and granular layer (Figure 10).
In the ultrastructural examination of the control group granule cells appeared small with large and slightly darker nuclei with thin rim of cytoplasm. These cells surrounded the Mossy rosettes containing many Ultrastructural Pathology
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FIGURE 5. A photomicrograph of a semithin section of the cerebellar cortex of ASP-treated animals showing Purkinje cells (arrow) with darkly stained cytoplasm and hardly identified nuclei (Toluidine blue, 1000).
FIGURE 6. Photomicrograph of semithin section in the cerebellar cortex of P. anisum oil + ASP group showing some Purkinje cells regaining their pyriform shape (arrow) but other Purkinje cells with darkly stained cytoplasm and hardly identified nucleus (arrow head) (Toluidine blue, 1000).
mitochondria and synaptic vesicles (Figure 13a). In the granular layer of ASP group, granule cells nuclei showed some indentation and surrounded by damaged mitochondria (Figure 13b). Other parts showed granular cells with condensed nuclei and surrounded by dissolution of most Mossy rosettes structures (Figure 13c). In the cerebellar cortex of P. anisum oil + ASP group the granular layer showed normal granule cells with relatively darker nuclei. !
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FIGURE 7. (a and b) Photomicrographs of a section of control group showing elongated cells and their processes appear in the molecular layer with faint brownish immuno-reaction and few brownish star-shaped cells in the granular layer (GFAP 400 and 1000, respectively).
Normal Mossy rosettes with numerous spherical mitochondria and normal myelin sheath were observed (Figure 13d). Purkinje cells of the control group appeared with large nucleus and apparent nucleoli (Figure 14a). Cisternea of rER, ribosomes, mitochondria, appeared in the cytoplasm (Figure 14b). Purkinje cells of the
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FIGURE 8. (a and b) Photomicrographs of a section of ASP group showing marked increase in size and intensity of brownish immuno-reaction of star-shaped cells (short arrow) in the granular layer. Elongated cells and their processes appear in the molecular layer (long arrow) with deeply stained brownish color (GFAP 400 and 1000, respectively).
FIGURE 9. (a and b) Photomicrograph of a section of P. anisum oil + ASP group showing less brownish intensity and number of starshaped cells in the granular layer and elongated cells in molecular layer compared to the ASP group (GFAP 400 and 1000, respectively).
ASP group showed nuclei with more condensed chromatin (Figure 14c). Vacuolations and dilated rough endoplasmic reticulum were seen. Some mitochondria appeared with more condensed matrix (Figure 14d). Pimpinella anisum oil + ASP group revealed normal Purkinje cells with euochromatic nucleus (Figure 14e). Figure 14(f) revealed normal profiles of rER cisternae, free ribosomes, and many normal mitochondria. Myelinated fibers of control group could be detected between cells (Figure 15a). Some myelinated nerve fibers of ASP showed dissolution of their myelin sheath and others contained damaged mitochondria (Figure 15b). Most myelinated nerve fibers showed thickening of myelinated sheath and other
showed splitting of their myelin sheath (Figure 15c). Pimpinella anisum oil + ASP group showed normal myelinated nerve fibers with regular compact myelin sheath (Figure 15d).
DISCUSSION Numerous reports and various issues, concerning the toxic effects of ASP have continued to be raised (more than 20 years after its approval by FDA) [10]. So, the specific aim of this study was to determine if long-term ASP administration (for 2 months) would lead to cytopathological effect on cerebellar cortex. Ultrastructural Pathology
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In this work, cerebellar cortex of ASP-treated animals showed prominent structural lesions in the Purkinje cell layer. Irregular, shrunken outline of their perikarya with deeply stained cytoplasm and nuclei were frequently observed and loss of the pyriform shape of the Purkinje cell body. Histopathological changes observed in cerebellar tissue of the present study are supported by Abd El-Samad [32]. Similar alterations in cisplatin-treated cerebellum were reported, where histological examination
revealed degenerative changes in Purkinje cells ranging from pyknosis to cell death [33]. These changes in Purkinje cells revealed affection of the main cells of the cerebellar cortex as they are the only cells of the cerebellum to send information to the outside [34]. Recent in vitro experiments have shown that ASP induces tubule formation, reactive oxygen species (ROS) formation and cytotoxicity [35]. The present ultrastructure result of ASP group showed damaged mitochondria and dissolution of
FIGURE 10. Photomicrographs of the of control cerebellum group showing faint cytoplasmic reaction to COX-2 in molecular layer, Purkinje cells (arrow) and granular layer (immunohistochemical stain, 400).
FIGURE 12. Photomicrographs of the cerebellar cortex of P. anisum oil + ASP group showing moderate reaction to COX-2 in molecular layer, Purkinje cells (arrow) and granular layer (immunohistochemical stain, 400).
FIGURE 11. (a and b) Photomicrographs of cerebellum of ASP group showing a strong positive reaction to COX-2 in molecular layer, Purkinje cells (arrows) and granular layer (immunohistochemical stain, 400). !
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FIGURE 13. (a) Electron micrograph of control group showing small granule cells (G) appear with slightly darker nuclei. Mossy rosettes (R) are seen containing many mitochondria and synaptic vesicles. Scale bar = 2 mm. Electron micrograph of ASP group showing (b) granule cells (G) nuclei showed some indentation and surrounded by damaged mitochondria (arrow). Scale bar = 2 mm. (c) Other part showing granular cells (G) with condensed nuclei and surrounded by dissolution of most Mossy rosettes structures (d). Scale bar = 2 mm. (d) Electron micrograph of P. anisum oil + ASP group showing granular layer normal granule cells (G) with relatively darker nuclei. Normal Mossy rosettes with numerous spherical mitochondria (arrow head) and normal myelin sheath (arrow) were observed. Scale bar = 2 mm.
most Mossy rosettes structures of granular cells. Condensed nucleus, dilated rough endoplasmic reticulum of Purkinje cells, and dissolution of some myelinated nerve fibers were also seen. Similar results were observed by Abd El-Samad [32]. Previous investigators reported that excessive ASP stimulation could trigger the generation of large numbers of free radical species, both as nitrogen and oxygen species. These free radicals had been shown to damage cellular proteins and DNA. The most immediate DNA damage was to the mitochondrial DNA [9]. Because the mitochondria are important for energy production within a neuron, a change in their function
may damage the cell. In addition, chronic mitochondrial alterations in cerebellum could be involved in occurrence of depression in rats [36]. The production and maintenance of myelin is essential to normal CNS function. Even small changes in myelin indices could lead to changes in conduction speed and signal timing which is crucial for the proper function of integrated neuronal circuits [37]. In the current study, P. anisum oil + ASP group was performed to evaluate the effects of P. anisum oil on the structure of the cerebellar cortex. This group showed regaining of normal shape and nuclei of the Purkinje cells. The ultrastructure of these cells Ultrastructural Pathology
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Pimpinella anisum oil protects cerebellum
FIGURE 14. Electron micrograph of control group showing (a) Purkinje cell with large nucleus (N) and apparent nucleolus (arrow). (b) Cisternea of rER (curved arrow), mitochondria (arrow heads) free, ribosomes (r) appear in the cytoplasm and lipid droplet (arrow). Scale bar = 2 mm and 500 nm, respectively. Electron micrograph of ASP group showing (c) Purkinje cells nucleus (N) with more condensed chromatin and dilated rough endoplasmic reticulum (arrow). Scale bar = 2 mm. (d) Vacuolations and dilated rough endoplasmic reticulum (arrow) were seen. Some mitochondria (curved arrow) appeared with more condensed matrix. Scale bar = 500 nm. Electron micrograph of P. anisum oil + ASP group showing (e) normal Purkinje cells of with normal euochromatic nucleus (N). Scale bar = 2 mm. (f) Normal profiles of rER cisternae (curved arrow), free ribosomes and many normal mitochondria. Scale bar = 500 nm.
revealed euchromatic nucleus, nearly normal mitochondria and normal profiles of rER cisternae. These findings denoted restoration of Purkinje cells to their normal structure after treatment with oil. This improvement might be secondary to the !
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antioxidant ability of anise, which attacks ROS and thus neutralizes their harmful effects on the tissues. It was found that anise could have a radical scavenging effect, inhibiting H2O2-chelating, and Fe2+-chelating activity by more than 70% [38].
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FIGURE 15. (a) Electron micrograph of control group of myelin sheath (arrow) and part of granular cells (G). Scale bar = 500 nm. (b) Electron micrograph of ASP group of myelinated fibers showing dissolution of their myelin sheath (arrow) and damaged mitochondria. Scale bar = 2 mm. (c) Most myelinated nerve fibers of ASP group showing thickening of myelin sheath (arrow head) and other showed splitting of their myelin sheath (arrow). Scale bar = 2 mm. (d) Electron micrograph of P. anisum oil + ASP group showing normal myelinated nerve fibers (arrows) with regular compact myelin sheath and normal granular cell (G) were observed. Scale bar = 2 mm.
Similar results were observed by El Haliem [22] who reported that the prolonged consumption of ASPinduced disturbance in the pituitary thyroid axis, where the use of P. anisum decreased the toxic effect of ASP. In this study, marked increase in the size and intensity of brownish immuno-reaction of GFAP positive astrocytes was observed in ASP-treated rats in the granular layer. In the molecular layer, the elongated cells and their processes also appeared with deeply stained brownish color. Aspartate also resembled in structure to the glutamate, which is a transmitter at excitatory synapses in the cerebellum,
as cerebellar molecular and granular cell layer, the parallel fiber and mossy fiber synapses [10]. Glutamine was the precursor for releasable aspartate via glutaminase in the parallel and mossy fibers. After release of glutamate and aspartate, they were taken up by the processes of the surrounding glial cells. GABA, the inhibitory neurotransmitter of the cerebellum, was also synthesized from glutamate in the Golgi type II cell terminals and taken up by surrounding glial cells [39]. These findings could explain why the GFAP immuno-stain in this study which showed increased number, size, and immunoreaction of the glial cells in the three layers of the Ultrastructural Pathology
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Pimpinella anisum oil protects cerebellum cerebellar cortex. In the immunohistochemical examination, many star-shaped cells appeared in-between cells of the granular layer. These findings of this study might be in correlation with many authors who reported the high incidence of brain tumors mainly the gliomas in human [5] and in rats [2]. The GFAP-stained glial cells decreased in P. anisum oil + ASP group compared to ASP group. This finding coincided with an author who reported that microglial cells might support the neuronal recovery through release of some cytokines and growth factors [32,40]. GFAP may have CNS-damaging effect as well; astrocytes react rapidly to any CNS insult by producing various neurotoxic substances and more GFAP, which was considered a marker protein for astrogliosis [41]. In the present immunohistochemical result ASP group showed strong positive reaction in the form of brown color within molecular layer, Purkinje cells, and granular layer. Previous studies reported lowlevel staining of COX-2 antibodies in the cortex and hippocampus is seen in the control mice. Intensive accumulation of COX-2 immunoreactivity in the cortex layer and throughout the hippocampus is unique to the Tg mouse [42]. The pathogenic effect of COX-2 relates to its role in the injury caused by inflammation [43]. In the brain as in other organs, COX-2 is markedly upregulated in pathologies associated with inflammation and contributes to cytotoxicity both in vitro and in vivo [44]. Increased neuronal COX-2 expression is associated with neuropathology, including traumatic brain injury [45] and chronic neurodegenerative conditions such as Alzheimer’s disease [46]. Upregulation of neuronal COX-2 is associated with ischemia and excitotoxicity, suggesting that COX-2 is involved in neurotoxic mechanisms. Increased susceptibility to excitotoxicity in COX-2 overexpressing neurons and neuroprotection by COX-2 inhibition has been shown in several experimental models [47]. Nonetheless, increased COX-2 expression could be an adaptive reaction to pathological events, such as cerebrovascular dysfunction, early inflammatory processes, or oxidative stress, in the attempt to restore lost physiological functions [18]. In conclusion, the present results indicated that the cerebellar cortex is particularly susceptible to ASP-induced histopathological, immunohistochemical, and ultrastructural changes which may lead to the development of neurodegenerative diseases, as well as the protective effect of P. anisum against the changes induced by ASP.
DECLARATION OF INTEREST The authors declare that there are no conflicts of interest. !
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REFERENCES 1. Trocho C, Pardo R, Rafecas I, et al. Formaldehyde derived from dietary aspartame binds to tissue components in vivo. Life Sci 1998;63: 337–49. 2. Soffritti M, Belpoggi F, Degli Esposti D, et al. First experimental demonstration of the multipotential carcinogenic effects of aspartame administered in the feed to Sprague-Dawley rats. Environ Health Perspect 2006;114: 379–85. 3. Aspartame Information Center. Aspartame Information Center Homepage. Available from: http://www.aspar tame.org/ [last accessed 27 Oct 2005]. 4. Rowe RC, Sheskey PJ, Quinn ME. Hand book of pharmaceutical excipients, 6th ed. London: Pharmaceutical Press and American Pharmacists Association; 2009. 5. Olney JW, Farber NB, Spitznagel E, et al. Increasing brain tumor rates: Is there a link to aspartame? J Neuropathol Exp Neurol 1996;55: 1115–23. 6. Camfield PR, Camfield CS, Dooley JM, et al. Aspartame exacerbates EEG spike wave discharge in children with generalized absence epilepsy: A double-blind controlled study. Neurology 1992;42: 1000–3. 7. Mackey SA, Berlin Jr CM. Effect of dietary aspartame on plasma concentrations of phenylalanine and tyrosine in normal and homozygous phenylketonuric patients. Clin Pediatr (Phila) 1992;31: 394–9. 8. El-Haliem NGA, Mohamed DS. The effect of aspartame on the histological structure of the liver and renal cortex of adult male albino rat and the possible protective effect of Pimpinella anisum oil. Egypt J Histol 2011;34: 715–26. 9. Beal MF, Hyman BT, Koroshetz W. Do defects in mitochondrial energy metabolism underlie the pathology of neurodegenerative diseases? Trends Neurosci 1993;16: 125–31. 10. Christian B, McConnaughey K, Bethea E, et al. Chronic aspartame affects T-maze performance, brain cholinergic receptors and Na+, K+-ATPase in rats. Pharmacol Biochem Behav 2004;78: 121–7. 11. Moser RH. Aspartame and memory loss. JAMA 1994;272: 1543. 12. Van den Eeden SK, Koepsell TD, et al. Aspartame ingestion and headaches: A randomized crossover trial. Neurology 1994;44: 1787–93. 13. Brock TO, O’Callaghan JP. Quantitative changes in the synaptic vesicle proteins synapsin I and p38 and the astrocyte specific protein glial fibrillary acidic protein are associated with chemical-induced injury to the rat central nervous system. J Neurosci 1987;7: 931–42. 14. Marnett LJ. Cyclooxygenase mechanisms. Curr Opin Chem Biol 2000;4: 545–52. 15. Smith WL, DeWitt DL, Garavito RM. Cyclooxygenases: Structural, cellular and molecular biology. Annu Rev Biochem 2000;69: 145–82. 16. Smith WL, Garavito RM, DeWitt DL. Prostaglandin endoperoxide H synthases (cyclooxygenases)-1 and -2. J Biol Chem 1996;271: 33157–60. 17. Singer II, Kawka DW, Schloemann S, et al. Cyclooxygenase 2 is induced in colonic epithelial cells in inflammatory bowel disease. Gastroenterology 1998;115: 297–306. 18. Minghetti L. Cyclooxygenase-2 (COX-2) in inflammatory and degenerative brain diseases. J Neuropathol Exp Neurol 2004;63: 901–10. 19. Benchaar C, Calsamiglia S, Chaves AV, et al. A review of plant-derived essential oils in ruminant nutrition and production. Anim Feed Sci Technol 2008;145: 209–28. 20. Kucukkurt G, Avci A, Eryavuz I, et al. Uyarlar effects of supplementation of aniseed (Pimpinella anisum L.) at various amounts to diets on lipid peroxidation,
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21.
22.
23.
Ultrastruct Pathol Downloaded from informahealthcare.com by Radboud Universiteit Nijmegen on 11/25/14 For personal use only.
24. 25.
26.
27. 28.
29.
30.
31.
32.
33.
antioxidant activity and some biochemical parameters in laying quails (Coturnix coturnix japonica) Kocatepe. Vet J 2009;2: 1–5. Gulcin U, Oktay M, Kirecci E, Kufrevioglu U. Screening of antioxidant and antimicrobial activities of anise (Pimpinella anisum) seed extracts. Food Chem 2003;83: 371–82. El-Haliem NGA. The effect of aspartame on the pituitary thyroid axis of adult male albino rat and the possible protective effect of Pimpinella anisum oil: Histological and immunohistochemical study. Egypt J Histol 2013;36: 195–205. Aboabrahim Z. Zakhirah kharazmshahi, vol. 2. Tehran: National Works Publications, 1970. Lershin BI. Experimental drug therapy of toxic hepatitis. Patol Fiziol Eksp Ter 1972;16: 66–7. Fernstrom JD. Oral aspartame and plasma phenylalanine: Pharmacokinetic difference between rodents and man and relevance to CNS effects of phenylalanine. Short note. J Neural Transm 1989;75: 159–64. Hjelle JJ, Dudley RE, Marietta, MP, et al. Plasma concentrations and pharmacokinetics of phenylalanine in rats and mice administered aspartame. Pharmacology 1992;44: 48–60. Bancroft JD, Stevens A. Theory and practice of histological techniques, 4th ed. Edinburgh: Churchill Livingstone; 1996. Kiernan JA. Histological and histochemical methods: Theory and practice. 3rd ed. Oxford: Butterworth–Heinemann; 1999. Therland KL, Stubbe J, Thiesson HC, et al. Cycloxygenase2 is expressed in vasculature of normal and ischemic adult human kidney and is colocalized with vascular prostaglandin E2 EP4 receptors. J Am Soc Nephrol 2004;15: 1189–98. Giorno R. A comparison of two immunoperoxidase staining methods based on the avidin–biotin interaction. Diagn Immunol 1984;2: 161–6. Hayat MA. Principles and techniques of electron microscopy. Biological applications, 3rd ed. Hampshire and London: CRC Press; 1989: 24–74. Abd El-Samad AA. Light and electron microscopic study on the effect of aspartame on the cerebellar cortex of male albino rat. Egypt J Histol 2010;33: 419–30. Magnuson BA, Burdock GA, Doull J, et al. Aspartame: A safety evaluation based on current use levels,
34. 35.
36.
37.
38.
39.
40. 41. 42.
43.
44.
45.
46.
47.
regulations and toxicological and epidemiological studies. Crit Rev Toxicol 2007;37: 629–727. Gartner LP, Hiatt JL. Color textbook of histology, 2nd ed. Baltimore: Lippincott Williams & Wilkins; 2001. Alleva R, Borghi B, Santarelli L, et al. In vitro effect of aspartame in angiogenesis induction. Toxicol In Vitro 2011; 25: 286–93. Rezin GT, Cardoso MR, Gonc¸alves CL, et al. Inhibition of mitochondrial respiratory chain in brain of rats subjected to an experimental model of depression. Neurochem Int 2008;53: 395–400. Felts PA, Baker TA, Smith, KJ. Conduction in segmentally demyelinated mammalian central axons. J Neurosci 1997;17: 7267–77. Elsn KS. Radical scavenging and iron-chelating activities of some greens used as traditional dishes in Mediterranean diet. Int J Food Sci Nutr 2004;55: 67–74. Barbour B, Keller BU, Llano I, Marty A. Prolonged presence of glutamate during excitatory synaptic transmission to cerebellar Purkinje cells. Neuron 1994;12: 1331–43. Bruce Keller AJ. Microglial-neuronal interactions in synaptic damage and recovery. J Neurosci Res 1999;58: 191–201. Eng LF, Ghirnikar RS. GFAP and astrogliosis. Brain Pathol 1994;4: 229–37. Hwang DY, ChaEe KR, Kang TS, et al. Alterations in behavior, amyloid-42, caspase-3, and Cox-2 in mutant PS2 transgenic mouse model of Alzheimer’s disease. FASEB J 2002;16: 805–13. Koki A, Khan NK, Woerner BM, et al. Cyclooxygenase-2 in human pathological disease. Adv Exp Med Biol 2002;507: 177–84. Boje KM, Jaworowicz Jr D, Raybon JJ. Neuroinflammatory role of prostaglandins during experimental meningitis: Evidence suggestive of an in vivo relationship between nitric oxide and prostaglandins. J Pharmacol Exp Ther 2003; 304: 319–25. Strauss KI, Barbe MF, Marshall R, et al. Prolonged cyclooxygenase-2 induction in neurons and glia following traumatic brain injury in the rat. J Neurotrauma 2000;17: 695–711. Pasinetti GM, Aisen PS. Cyclooxygenase-2 expression is increased in frontal cortex of Alzheimer’s disease brain. Neuroscience 1998;87: 319–24. Aisen PS. The potential of anti-inflammatory drugs for the treatment of Alzheimer’s disease. Lancet Neurol 2002;1: 279–84.
Ultrastructural Pathology
ORIGINAL ARTICLE
Ameliorative Effect of Pimpinella anisum Oil on Immunohistochemical and Ultrastuctural Changes of Cerebellum of Albino Rats Induced by Aspartame
Department of Zoology, Faculty of Science, Beni-Suef University, Beni Suef, Egypt
ABSTRACT The study aims to investigate the protective effect of Pimpinella anisum oil on aspartame (ASP) which resulted in cerebellar changes. The rats were divided into four equal groups: Group 1: (control group): served as control animals. Group 2: control P. anisum oil received .5 mL/kg/d/b wt. once daily. Group 3 (ASP group): received daily 250 mg/kg/b wt. of ASP dissolved in distilled water and given orally to the animals by intra-gastric tube for 2 months. Group 4: received .5 mL/kg/b wt. of prophylactic P. anisum oil once daily, followed by ASP after 2 h for 2 months. The histopathological approach revealed marked changes in the Purkinje cells, myleinated nerve fibers and granular cells of ASP-treated animals. Some of these cells appeared with deeply stained cytoplasm. Ultrastructural examination showed Purkinje cells with dilated rough endoplasmic reticulum and condensed mitochondria. Granular cells appeared with less c nuclei and surrounded by dissolution of most Mossy rosettes structures. Most myelinated nerve fibers showed thickening of myelinated sheath and others showed splitting of their myelin sheath. The histopathological, immunohistochemical and ultrastructural alterations were much less observed in concomitant use of P. anisum oil with ASP. Cerebellar cortex is considered target areas of ASP neurotoxicity, while P. anisum oil, when used in combination with ASP displays a protective action against neurotoxicity. Keywords: Aspartame, cerebellum, histopathology, immunohistochemistry, Pimpinella anisum oil, ultrastructure
INTRODUCTION
non-caloric although its energy value is 4 kcal/g of ASP [4]. ASP can enter the bloodstream and circulate through the entire body, including the brain, because it can penetrate the blood–brain barrier. It might cause excitotoxicity of brain cells [5], which is damage to the brain and nervous system. It can be metabolized to yield phenylalanine, aspartic acid, and methanol and is then later oxidized into formaldehyde and formic acid in many tissues. Formic acid was considered the principal metabolite responsible for the deleterious effects of acute intoxication by methanol in humans and animals [6]. Furthermore, elevated phenylalanine levels are associated with neurologic function, such as phenylketonuria and mental retardation [7]. Some authors reported that ASP could be metabolized
Aspartame (ASP) is the most widely used artificial sweetener. It is a methyl ester of a dipeptide (L-aspartyl-L-phenylalanine methyl ester) [1]. It was found in more than 6000 products, including carbonated soft drinks, hot chocolate, chewing gum, candy, deserts, tabletop sweeteners and some pharmaceutical products, such as vitamins and sugar-free cough drops [2]. It was estimated by the ASP Information Center [3] to be consumed by more 200 million people worldwide, including children between 2 and 5 years of age and pregnant females. This white, crystalline, and odorless powder is 180–200 times sweeter than sucrose. The intense sensation of sweetness allows the use of such small doses so that the product is almost
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Manal Abdul-Hamid, PhD and Sanaa Rida Gallaly, PhD
Received 23 December 2013; Revised 26 January 2014; Accepted 27 January 2014; Published online 28 March 2014 Correspondence: Manal Abdul-Hamid, Ass. Prof. of Histology and Cytology, Department of Zoology, Faculty of Science, Beni-Suef University, Beni Suef 62511, Egypt. Fax: +20 822327986. E-mail: [email protected]
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Pimpinella anisum oil protects cerebellum in cell mitochondria, inducing mitochondrial and nuclear DNA damage and affecting its ability to produce inadequate and incomplete energy metabolism, which gives rise to highly damaging free radicals [8]. Moreover, free radical scavengers including the functional reserve of antioxidant, vitamins, and minerals could be necessary for neural protection and regeneration [9], because improvement from ASPinduced toxicity was gradual and incomplete as reported by Christian et al. [10]. ASP consumption was suggested to be implicated with various symptoms from the central nervous system such as seizures [6], memory loss [11], cholinergic symptoms, headaches [12], and oncogenesis [2]. It had been reported that the astrocyte-specific protein, the glial fibrillary acidic protein (GFAP), this intermediate filaments found in astrocyte, could also serve as a marker for glial cells [13]. Cyclooxygenase (COX) is a membrane-bound enzyme responsible for the oxidation of arachidonic acid to prostaglandin G2 and its subsequent reduction to prostaglandin H2 [14,15]. COX is expressed in at least two different isoforms, a constitutively expressed form COX-1 and an inducible form COX-2 [15,16]. COX-1 is expressed in many normal tissues and is involved in a number of homeostatic body functions, such as hemostasis, vasodilatation in renal vessels and cytoprotection of the gastric mucosa [16]. The expression of COX-2 is induced by various stimuli, such as growth factors and cytokines and is involved in chronic inflammatory pathologies such as inflammatory bowel disease [17]. Minghetti [18] reported that over-expression of COX-2 has been associated with neurotoxicity in acute conditions, such as hypoxia/ ischemia and seizures. However, the beneficial or detrimental role played by COX-2 in inflammatory and neurodegenerative brain pathologies is still controversial. Plants and their extracts offer a unique opportunity in this regard since aromatic plants and their extracts have been used traditionally in the therapy of some diseases for a long time through the world [19]. Aniseed (Pimpinella anisum L.), an aromatic plant, is an annual herb cultivated in many countries but indigenous to Iran, India, and Turkey [20]. Chemical studies have demonstrated that the aniseed contains anethole (85%) as an active ingredient, in addition to eugenol, methylchavicol, anisaldehyde, and estragole [21]. Pimpinella anisum has been used for different purposes as an antioxidant, hepatoprotective, antiinflammatory agent, and toxicity of the pituitary thyroid axis [22]. Pimpinella anisum may have therapeutic effects in diseases such as digestive, gynacologic, neurologic, and respiratory disorders [23]. The purpose of this study was to evaluate the protective effect of P. anisum oil on ASP-induced cerebellum changes, by observing histopathological, immunohistochemical and ultrastructural changes. !
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MATERIALS AND METHODS Chemicals ASP was obtained from Al-Ameriya Pharma Company (Alexandria, Egypt). It is available in the form of tablets, each one containing 20 mg of ASP. The tablets were dissolved in distilled water and administered to the rats. Pimpinella anisum oil was obtained from an aromatics shop in Beni-Suef (Egypt).
Animals and experimental design Twenty male adult albino rats, weighing about 140–180 g, were used in this study. All the animals were maintained under standard laboratory conditions of temperature (25 C) and 12 h light and 12 h dark cycles throughout the experimental period. They were housed in standard cages and had free access to water and standard diet at the research center in Beni-Suef University. Experiments were conducted as per the guidelines of Institutional Animal Ethical Committee, Beni-Suef University. The animals were divided into four groups (five each) as the following: Group 1 (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. Group 2 (P. anisum oil group): These animals received a daily dose of .5 mL/kg/b wt. P. anisum oil (according to Lershin [24]) for 2 months. Group 3 (ASP group): Rats of this group received daily oral dose of 250 mg/kg/b wt. of ASP dissolved in distilled water by intra-gastric tube for 2 months [25]. ASP tablets, each one containing 20 mg, were obtained from Al-Ameriya Pharma Company. This dose used is corresponding to the human dose, which is 40–50 mg/kg/b wt. after species factor correction requiring 5–6 times higher dose than man, as rat metabolizes ASP faster than human [26]. Group 4 (P. anisum oil + ASP group): Animals of this group received .5 mL/kg/d/b wt. of prophylactic P. anisum oil once daily [24], followed by a daily dose of 250 mg/kg/b wt. of ASP after 2 h for 2 months.
Light microscopic examination For light microscopic study, samples from cerebellum were taken; after fixation in 10% neutral buffered formalin, they were dehydrated through alcohols, cleared in xylene and embedded in paraffin wax.
226 M. Abdul-Hamid and S. R. Gallaly Later on, 5-mm-thick sections were stained with hematoxylin and eosin [27].
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Immunohistochemical staining for GFAP of astrocytes For immunohistochemical study, staining was performed for GFAP as an indicator for glial reactivity. Primary antibodies monoclonal mouse anti-human GFAP was purchased from Dako, Carpenteria, CA. Serial paraffin sections of 5 mm thickness were deparaffinized and dehydrated, including positive control sections from the rat cerebellar cortex matter. The endogenous peroxidase activity was blocked with 0.05% hydrogen peroxide in absolute alcohol for 30 min. The slides were washed for 5 min in phosphate buffered saline (PBS) at pH 7.4. To unmask the antigenic sites, sections were placed in .01 mol/L citrate buffer (pH 6) in a microwave for 5 min. The slides were incubated in 1% BSA dissolved in PBS for 30 min at 37 C in order to prevent nonspecific background staining. Slides were incubated with the primary antibody (1:500 monoclonal mouse antiGFAP) at 4 C for 18–20 h and after washing, they were incubated with biotinylated secondary antibodies and then with avidin–biotin complex. Finally, sections were developed with .05% 3,3-diaminobenzidine for 15 min. Slides were counterstained with Mayer’s hematoxylin, dehydration, clearing and mounting were done by DPX [28].
solution (1 drop of DAB chromogen/1 mL of substrate buffer) was removed from 2 to 8 C storage and applied on specimens for 10 min. Slides were rinsed gently in distilled water, immersed in hematoxylin for .5 min and were rinsed in tab water until blue. Slides were dehydrated in ascending grades of alcohol, cleared in xylol, mounted by Canada balsam, and covered with a cover slip. Negative control slides were prepared by the same steps except they were incubated with the antibody diluent instead of primary antibody. 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].
Electron microscopic examination For electron microscopy, rats of all groups were dissected and other parts of the specimens were immersed in 2.5% glutraldehyde in phosphate buffer (pH 7.4) for 4 h and then post fixed in 1% osmium tetra oxide at 4 C, dehydrated and embedded in epoxy resin. Semithin sections were cut and stained with Toluidine blue. Ultra-thin sections were stained with 2% uranyl acetate and 2% lead citrate [31] and examined and photographed by transmission electron microscope (JEOL-Ex1010 TEM) in AinShams University.
RESULTS Immunohistochemical staining for localization of COX-2
Light microscopic evaluation of cerebellar cortex
The expression of COX-2 was detected using rabbit polyclonal anti-human COX-2 specific IgG (H-62, sc-7951, Santa Cruz Biotechnology, Dallas, TX). Sections (of cerebellar cortex) were dewaxed in xylol for 20 min (two changes) and hydrated in descending grades of alcohol down to distilled water. They were immersed into preheated target retrieval solution to 95–99 C (without boiling) in water bath for 40 min, removed from the bath and allowed to cool for 20 min at room temperature. Sections were rinsed three times with PBS. Excess liquid was tapped off the slides. Enough hydrogen peroxide was applied to cover the specimen for 5 min, then slides were rinsed gently with PBS and excess liquid was tapped off. Enough amount of primary antibody (dilution 1:200) was applied on specimens and was incubated for 2 h in a humid chamber at room temperature. Slides were rinsed in PBS. Biotinylated link was applied on specimens for 10 min and sections were rinsed in PBS. Streptavidin HRP reagent was applied on specimens for 10 min and sections were then rinsed in PBS. Freshly prepared DAB substrate chromogen
Examination of sections of the control group (Figure 1a and b) showed the three layers forming the cerebellar cortex, the molecular layer, Purkinje cell layer, and the granular layer. The Purkinje cell layer showed Purkinje cells with pyriform shaped cell bodies and central rounded vesicular nuclei and prominent nucleoli. The cytoplasm was finely granular. The granular layer contained numerous small granule cells with relatively dense nuclei and scanty cytoplasm. Control oil P. anisum group did not show any histopathological changes. Examination of the ASP-treated group revealed Purkinje cells with irregular shrunken outline of their perikarya associated with deeply stained cytoplasm and pyknotic nuclei (Figure 2a). Some shrunken pyknotic cells lost their characteristic pyriform shape and were surrounded by vacuoles due to fallen off cells, leaving empty spaces (Figure 2b). Some vacuolations (spongiosis) appeared between cells of the granular layer and in the molecular layer (Figure 2b). Examination of P. anisum oil + ASP-supplemented group showed that the majority of Purkinje cells Ultrastructural Pathology
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FIGURE 1. Photomicrograph of the cerebellar cortex of control brain showing (a) three layers forming the cerebellar cortex, the molecular layer (M), Purkinje cell layer (arrows) and the granular layer (G). (b) The outer molecular layer contains few small scattered cells. Purkinje cell layer contains Purkinje cells (arrows) appeared with pyriform shaped cell bodies and central rounded vesicular nuclei and prominent nucleoli. The granular layer contains numerous small granule cells with relatively dense nuclei and scanty cytoplasm with cerebellar islands (C) in-between (H&E 400, 1000).
FIGURE 2. Photomicrograph of the cerebellar cortex of ASP-treated group brain showing (a) granular layer (G), molecular layer (M) and Purkinje cells with irregular shrunken outline losing their characteristic pyriform shape associated with deeply stained cytoplasm and pyknotic nuclei (arrows). (b) The shrunken pyknotic Purkinje cells (arrows). Some vacuolations (spongiosis) (V) appeared between cells of the granular layer and in the molecular layer (H&E 400, 1000, respectively).
appeared normal with well-defined nuclei and prominent nucleoli (Figure 3a and b). Few cells were still shrunken with deeply stained cytoplasm (Figure 3b). Semithin section in the cerebellar cortex of control rat showed Purkinje cell with pyriform shaped cell body and defined nucleus with prominent nucleolus (Figure 4a and b). The ASP-treated animal showed Purkinje cells with darkly stained cytoplasm and hardly identified nuclei (Figure 5). Cerebellar cortex of P. anisum oil + ASP-treated group showed some Purkinje cells regaining their pyriform shape but others with darkly stained cytoplasm and hardly identified nucleus (Figure 6). !
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Immunohistochemical evaluation For GFAP Immunohistochemical examination of positive control sections of the rat cerebellum stained with GFAP showed the presence of GFAP-positive represented by elongated cells and their processes in the molecular layer with pale brownish immuno-reaction, while granular layer showed few brownish star-shaped cells (Figure 7a and b). Marked increase in the size and intensity of brownish immuno-reaction of GFAP positive astrocytes was observed in ASP-treated rats in the granular
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FIGURE 3. (a and b) Photomicrographs of the cerebellar cortex of P. anisum oil + ASP group showing granular layer (G) and molecular layer (M) the majority of Purkinje cells (arrows) regain their shape with well-defined nuclei and prominent nucleoli (H&E 400, 1000, respectively).
FIGURE 4. (a and b) Photomicrograph of semithin section in the cerebellar cortex of control rat showing Purkinje cell (arrow) with pyriform shaped cell body and defined nucleus (arrow head) with prominent nucleolus (Toluidine blue, 1000).
layer. In the molecular layer, the elongated cells and their processes also appeared with deeply stained brownish color (Figure 8a and b). Immunohistochemichal stained sections for GFAP in P. anisum oil + ASP group showed few brownish star-shaped cells in the granular layer in-between cells. Elongated cells and their processes appeared with faint brownish immuno-reaction in the molecular layer (Figure 9a and b).
In the ASP group a strong positive reaction was observed in the form of brown color within the molecular layer, Purkinje cells and granular layer (Figure 11a and b). The majority of Purkinje cells showed a negative immune reaction of Cox-2 in P. anisum oil + ASP group (Figure 12).
Electron microscopic evaluation of cerebellar cortex For Cox-2 immuno-stain The immunohistochemical sections of Cox-2 of control groups showed negative immune reaction for Cox-2 in molecular layer, Purkinje cells, and granular layer (Figure 10).
In the ultrastructural examination of the control group granule cells appeared small with large and slightly darker nuclei with thin rim of cytoplasm. These cells surrounded the Mossy rosettes containing many Ultrastructural Pathology
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FIGURE 5. A photomicrograph of a semithin section of the cerebellar cortex of ASP-treated animals showing Purkinje cells (arrow) with darkly stained cytoplasm and hardly identified nuclei (Toluidine blue, 1000).
FIGURE 6. Photomicrograph of semithin section in the cerebellar cortex of P. anisum oil + ASP group showing some Purkinje cells regaining their pyriform shape (arrow) but other Purkinje cells with darkly stained cytoplasm and hardly identified nucleus (arrow head) (Toluidine blue, 1000).
mitochondria and synaptic vesicles (Figure 13a). In the granular layer of ASP group, granule cells nuclei showed some indentation and surrounded by damaged mitochondria (Figure 13b). Other parts showed granular cells with condensed nuclei and surrounded by dissolution of most Mossy rosettes structures (Figure 13c). In the cerebellar cortex of P. anisum oil + ASP group the granular layer showed normal granule cells with relatively darker nuclei. !
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FIGURE 7. (a and b) Photomicrographs of a section of control group showing elongated cells and their processes appear in the molecular layer with faint brownish immuno-reaction and few brownish star-shaped cells in the granular layer (GFAP 400 and 1000, respectively).
Normal Mossy rosettes with numerous spherical mitochondria and normal myelin sheath were observed (Figure 13d). Purkinje cells of the control group appeared with large nucleus and apparent nucleoli (Figure 14a). Cisternea of rER, ribosomes, mitochondria, appeared in the cytoplasm (Figure 14b). Purkinje cells of the
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FIGURE 8. (a and b) Photomicrographs of a section of ASP group showing marked increase in size and intensity of brownish immuno-reaction of star-shaped cells (short arrow) in the granular layer. Elongated cells and their processes appear in the molecular layer (long arrow) with deeply stained brownish color (GFAP 400 and 1000, respectively).
FIGURE 9. (a and b) Photomicrograph of a section of P. anisum oil + ASP group showing less brownish intensity and number of starshaped cells in the granular layer and elongated cells in molecular layer compared to the ASP group (GFAP 400 and 1000, respectively).
ASP group showed nuclei with more condensed chromatin (Figure 14c). Vacuolations and dilated rough endoplasmic reticulum were seen. Some mitochondria appeared with more condensed matrix (Figure 14d). Pimpinella anisum oil + ASP group revealed normal Purkinje cells with euochromatic nucleus (Figure 14e). Figure 14(f) revealed normal profiles of rER cisternae, free ribosomes, and many normal mitochondria. Myelinated fibers of control group could be detected between cells (Figure 15a). Some myelinated nerve fibers of ASP showed dissolution of their myelin sheath and others contained damaged mitochondria (Figure 15b). Most myelinated nerve fibers showed thickening of myelinated sheath and other
showed splitting of their myelin sheath (Figure 15c). Pimpinella anisum oil + ASP group showed normal myelinated nerve fibers with regular compact myelin sheath (Figure 15d).
DISCUSSION Numerous reports and various issues, concerning the toxic effects of ASP have continued to be raised (more than 20 years after its approval by FDA) [10]. So, the specific aim of this study was to determine if long-term ASP administration (for 2 months) would lead to cytopathological effect on cerebellar cortex. Ultrastructural Pathology
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In this work, cerebellar cortex of ASP-treated animals showed prominent structural lesions in the Purkinje cell layer. Irregular, shrunken outline of their perikarya with deeply stained cytoplasm and nuclei were frequently observed and loss of the pyriform shape of the Purkinje cell body. Histopathological changes observed in cerebellar tissue of the present study are supported by Abd El-Samad [32]. Similar alterations in cisplatin-treated cerebellum were reported, where histological examination
revealed degenerative changes in Purkinje cells ranging from pyknosis to cell death [33]. These changes in Purkinje cells revealed affection of the main cells of the cerebellar cortex as they are the only cells of the cerebellum to send information to the outside [34]. Recent in vitro experiments have shown that ASP induces tubule formation, reactive oxygen species (ROS) formation and cytotoxicity [35]. The present ultrastructure result of ASP group showed damaged mitochondria and dissolution of
FIGURE 10. Photomicrographs of the of control cerebellum group showing faint cytoplasmic reaction to COX-2 in molecular layer, Purkinje cells (arrow) and granular layer (immunohistochemical stain, 400).
FIGURE 12. Photomicrographs of the cerebellar cortex of P. anisum oil + ASP group showing moderate reaction to COX-2 in molecular layer, Purkinje cells (arrow) and granular layer (immunohistochemical stain, 400).
FIGURE 11. (a and b) Photomicrographs of cerebellum of ASP group showing a strong positive reaction to COX-2 in molecular layer, Purkinje cells (arrows) and granular layer (immunohistochemical stain, 400). !
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FIGURE 13. (a) Electron micrograph of control group showing small granule cells (G) appear with slightly darker nuclei. Mossy rosettes (R) are seen containing many mitochondria and synaptic vesicles. Scale bar = 2 mm. Electron micrograph of ASP group showing (b) granule cells (G) nuclei showed some indentation and surrounded by damaged mitochondria (arrow). Scale bar = 2 mm. (c) Other part showing granular cells (G) with condensed nuclei and surrounded by dissolution of most Mossy rosettes structures (d). Scale bar = 2 mm. (d) Electron micrograph of P. anisum oil + ASP group showing granular layer normal granule cells (G) with relatively darker nuclei. Normal Mossy rosettes with numerous spherical mitochondria (arrow head) and normal myelin sheath (arrow) were observed. Scale bar = 2 mm.
most Mossy rosettes structures of granular cells. Condensed nucleus, dilated rough endoplasmic reticulum of Purkinje cells, and dissolution of some myelinated nerve fibers were also seen. Similar results were observed by Abd El-Samad [32]. Previous investigators reported that excessive ASP stimulation could trigger the generation of large numbers of free radical species, both as nitrogen and oxygen species. These free radicals had been shown to damage cellular proteins and DNA. The most immediate DNA damage was to the mitochondrial DNA [9]. Because the mitochondria are important for energy production within a neuron, a change in their function
may damage the cell. In addition, chronic mitochondrial alterations in cerebellum could be involved in occurrence of depression in rats [36]. The production and maintenance of myelin is essential to normal CNS function. Even small changes in myelin indices could lead to changes in conduction speed and signal timing which is crucial for the proper function of integrated neuronal circuits [37]. In the current study, P. anisum oil + ASP group was performed to evaluate the effects of P. anisum oil on the structure of the cerebellar cortex. This group showed regaining of normal shape and nuclei of the Purkinje cells. The ultrastructure of these cells Ultrastructural Pathology
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Pimpinella anisum oil protects cerebellum
FIGURE 14. Electron micrograph of control group showing (a) Purkinje cell with large nucleus (N) and apparent nucleolus (arrow). (b) Cisternea of rER (curved arrow), mitochondria (arrow heads) free, ribosomes (r) appear in the cytoplasm and lipid droplet (arrow). Scale bar = 2 mm and 500 nm, respectively. Electron micrograph of ASP group showing (c) Purkinje cells nucleus (N) with more condensed chromatin and dilated rough endoplasmic reticulum (arrow). Scale bar = 2 mm. (d) Vacuolations and dilated rough endoplasmic reticulum (arrow) were seen. Some mitochondria (curved arrow) appeared with more condensed matrix. Scale bar = 500 nm. Electron micrograph of P. anisum oil + ASP group showing (e) normal Purkinje cells of with normal euochromatic nucleus (N). Scale bar = 2 mm. (f) Normal profiles of rER cisternae (curved arrow), free ribosomes and many normal mitochondria. Scale bar = 500 nm.
revealed euchromatic nucleus, nearly normal mitochondria and normal profiles of rER cisternae. These findings denoted restoration of Purkinje cells to their normal structure after treatment with oil. This improvement might be secondary to the !
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antioxidant ability of anise, which attacks ROS and thus neutralizes their harmful effects on the tissues. It was found that anise could have a radical scavenging effect, inhibiting H2O2-chelating, and Fe2+-chelating activity by more than 70% [38].
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234 M. Abdul-Hamid and S. R. Gallaly
FIGURE 15. (a) Electron micrograph of control group of myelin sheath (arrow) and part of granular cells (G). Scale bar = 500 nm. (b) Electron micrograph of ASP group of myelinated fibers showing dissolution of their myelin sheath (arrow) and damaged mitochondria. Scale bar = 2 mm. (c) Most myelinated nerve fibers of ASP group showing thickening of myelin sheath (arrow head) and other showed splitting of their myelin sheath (arrow). Scale bar = 2 mm. (d) Electron micrograph of P. anisum oil + ASP group showing normal myelinated nerve fibers (arrows) with regular compact myelin sheath and normal granular cell (G) were observed. Scale bar = 2 mm.
Similar results were observed by El Haliem [22] who reported that the prolonged consumption of ASPinduced disturbance in the pituitary thyroid axis, where the use of P. anisum decreased the toxic effect of ASP. In this study, marked increase in the size and intensity of brownish immuno-reaction of GFAP positive astrocytes was observed in ASP-treated rats in the granular layer. In the molecular layer, the elongated cells and their processes also appeared with deeply stained brownish color. Aspartate also resembled in structure to the glutamate, which is a transmitter at excitatory synapses in the cerebellum,
as cerebellar molecular and granular cell layer, the parallel fiber and mossy fiber synapses [10]. Glutamine was the precursor for releasable aspartate via glutaminase in the parallel and mossy fibers. After release of glutamate and aspartate, they were taken up by the processes of the surrounding glial cells. GABA, the inhibitory neurotransmitter of the cerebellum, was also synthesized from glutamate in the Golgi type II cell terminals and taken up by surrounding glial cells [39]. These findings could explain why the GFAP immuno-stain in this study which showed increased number, size, and immunoreaction of the glial cells in the three layers of the Ultrastructural Pathology
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Pimpinella anisum oil protects cerebellum cerebellar cortex. In the immunohistochemical examination, many star-shaped cells appeared in-between cells of the granular layer. These findings of this study might be in correlation with many authors who reported the high incidence of brain tumors mainly the gliomas in human [5] and in rats [2]. The GFAP-stained glial cells decreased in P. anisum oil + ASP group compared to ASP group. This finding coincided with an author who reported that microglial cells might support the neuronal recovery through release of some cytokines and growth factors [32,40]. GFAP may have CNS-damaging effect as well; astrocytes react rapidly to any CNS insult by producing various neurotoxic substances and more GFAP, which was considered a marker protein for astrogliosis [41]. In the present immunohistochemical result ASP group showed strong positive reaction in the form of brown color within molecular layer, Purkinje cells, and granular layer. Previous studies reported lowlevel staining of COX-2 antibodies in the cortex and hippocampus is seen in the control mice. Intensive accumulation of COX-2 immunoreactivity in the cortex layer and throughout the hippocampus is unique to the Tg mouse [42]. The pathogenic effect of COX-2 relates to its role in the injury caused by inflammation [43]. In the brain as in other organs, COX-2 is markedly upregulated in pathologies associated with inflammation and contributes to cytotoxicity both in vitro and in vivo [44]. Increased neuronal COX-2 expression is associated with neuropathology, including traumatic brain injury [45] and chronic neurodegenerative conditions such as Alzheimer’s disease [46]. Upregulation of neuronal COX-2 is associated with ischemia and excitotoxicity, suggesting that COX-2 is involved in neurotoxic mechanisms. Increased susceptibility to excitotoxicity in COX-2 overexpressing neurons and neuroprotection by COX-2 inhibition has been shown in several experimental models [47]. Nonetheless, increased COX-2 expression could be an adaptive reaction to pathological events, such as cerebrovascular dysfunction, early inflammatory processes, or oxidative stress, in the attempt to restore lost physiological functions [18]. In conclusion, the present results indicated that the cerebellar cortex is particularly susceptible to ASP-induced histopathological, immunohistochemical, and ultrastructural changes which may lead to the development of neurodegenerative diseases, as well as the protective effect of P. anisum against the changes induced by ASP.
DECLARATION OF INTEREST The authors declare that there are no conflicts of interest. !
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REFERENCES 1. Trocho C, Pardo R, Rafecas I, et al. Formaldehyde derived from dietary aspartame binds to tissue components in vivo. Life Sci 1998;63: 337–49. 2. Soffritti M, Belpoggi F, Degli Esposti D, et al. First experimental demonstration of the multipotential carcinogenic effects of aspartame administered in the feed to Sprague-Dawley rats. Environ Health Perspect 2006;114: 379–85. 3. Aspartame Information Center. Aspartame Information Center Homepage. Available from: http://www.aspar tame.org/ [last accessed 27 Oct 2005]. 4. Rowe RC, Sheskey PJ, Quinn ME. Hand book of pharmaceutical excipients, 6th ed. London: Pharmaceutical Press and American Pharmacists Association; 2009. 5. Olney JW, Farber NB, Spitznagel E, et al. Increasing brain tumor rates: Is there a link to aspartame? J Neuropathol Exp Neurol 1996;55: 1115–23. 6. Camfield PR, Camfield CS, Dooley JM, et al. Aspartame exacerbates EEG spike wave discharge in children with generalized absence epilepsy: A double-blind controlled study. Neurology 1992;42: 1000–3. 7. Mackey SA, Berlin Jr CM. Effect of dietary aspartame on plasma concentrations of phenylalanine and tyrosine in normal and homozygous phenylketonuric patients. Clin Pediatr (Phila) 1992;31: 394–9. 8. El-Haliem NGA, Mohamed DS. The effect of aspartame on the histological structure of the liver and renal cortex of adult male albino rat and the possible protective effect of Pimpinella anisum oil. Egypt J Histol 2011;34: 715–26. 9. Beal MF, Hyman BT, Koroshetz W. Do defects in mitochondrial energy metabolism underlie the pathology of neurodegenerative diseases? Trends Neurosci 1993;16: 125–31. 10. Christian B, McConnaughey K, Bethea E, et al. Chronic aspartame affects T-maze performance, brain cholinergic receptors and Na+, K+-ATPase in rats. Pharmacol Biochem Behav 2004;78: 121–7. 11. Moser RH. Aspartame and memory loss. JAMA 1994;272: 1543. 12. Van den Eeden SK, Koepsell TD, et al. Aspartame ingestion and headaches: A randomized crossover trial. Neurology 1994;44: 1787–93. 13. Brock TO, O’Callaghan JP. Quantitative changes in the synaptic vesicle proteins synapsin I and p38 and the astrocyte specific protein glial fibrillary acidic protein are associated with chemical-induced injury to the rat central nervous system. J Neurosci 1987;7: 931–42. 14. Marnett LJ. Cyclooxygenase mechanisms. Curr Opin Chem Biol 2000;4: 545–52. 15. Smith WL, DeWitt DL, Garavito RM. Cyclooxygenases: Structural, cellular and molecular biology. Annu Rev Biochem 2000;69: 145–82. 16. Smith WL, Garavito RM, DeWitt DL. Prostaglandin endoperoxide H synthases (cyclooxygenases)-1 and -2. J Biol Chem 1996;271: 33157–60. 17. Singer II, Kawka DW, Schloemann S, et al. Cyclooxygenase 2 is induced in colonic epithelial cells in inflammatory bowel disease. Gastroenterology 1998;115: 297–306. 18. Minghetti L. Cyclooxygenase-2 (COX-2) in inflammatory and degenerative brain diseases. J Neuropathol Exp Neurol 2004;63: 901–10. 19. Benchaar C, Calsamiglia S, Chaves AV, et al. A review of plant-derived essential oils in ruminant nutrition and production. Anim Feed Sci Technol 2008;145: 209–28. 20. Kucukkurt G, Avci A, Eryavuz I, et al. Uyarlar effects of supplementation of aniseed (Pimpinella anisum L.) at various amounts to diets on lipid peroxidation,
236 M. Abdul-Hamid and S. R. Gallaly
21.
22.
23.
Ultrastruct Pathol Downloaded from informahealthcare.com by Radboud Universiteit Nijmegen on 11/25/14 For personal use only.
24. 25.
26.
27. 28.
29.
30.
31.
32.
33.
antioxidant activity and some biochemical parameters in laying quails (Coturnix coturnix japonica) Kocatepe. Vet J 2009;2: 1–5. Gulcin U, Oktay M, Kirecci E, Kufrevioglu U. Screening of antioxidant and antimicrobial activities of anise (Pimpinella anisum) seed extracts. Food Chem 2003;83: 371–82. El-Haliem NGA. The effect of aspartame on the pituitary thyroid axis of adult male albino rat and the possible protective effect of Pimpinella anisum oil: Histological and immunohistochemical study. Egypt J Histol 2013;36: 195–205. Aboabrahim Z. Zakhirah kharazmshahi, vol. 2. Tehran: National Works Publications, 1970. Lershin BI. Experimental drug therapy of toxic hepatitis. Patol Fiziol Eksp Ter 1972;16: 66–7. Fernstrom JD. Oral aspartame and plasma phenylalanine: Pharmacokinetic difference between rodents and man and relevance to CNS effects of phenylalanine. Short note. J Neural Transm 1989;75: 159–64. Hjelle JJ, Dudley RE, Marietta, MP, et al. Plasma concentrations and pharmacokinetics of phenylalanine in rats and mice administered aspartame. Pharmacology 1992;44: 48–60. Bancroft JD, Stevens A. Theory and practice of histological techniques, 4th ed. Edinburgh: Churchill Livingstone; 1996. Kiernan JA. Histological and histochemical methods: Theory and practice. 3rd ed. Oxford: Butterworth–Heinemann; 1999. Therland KL, Stubbe J, Thiesson HC, et al. Cycloxygenase2 is expressed in vasculature of normal and ischemic adult human kidney and is colocalized with vascular prostaglandin E2 EP4 receptors. J Am Soc Nephrol 2004;15: 1189–98. Giorno R. A comparison of two immunoperoxidase staining methods based on the avidin–biotin interaction. Diagn Immunol 1984;2: 161–6. Hayat MA. Principles and techniques of electron microscopy. Biological applications, 3rd ed. Hampshire and London: CRC Press; 1989: 24–74. Abd El-Samad AA. Light and electron microscopic study on the effect of aspartame on the cerebellar cortex of male albino rat. Egypt J Histol 2010;33: 419–30. Magnuson BA, Burdock GA, Doull J, et al. Aspartame: A safety evaluation based on current use levels,
34. 35.
36.
37.
38.
39.
40. 41. 42.
43.
44.
45.
46.
47.
regulations and toxicological and epidemiological studies. Crit Rev Toxicol 2007;37: 629–727. Gartner LP, Hiatt JL. Color textbook of histology, 2nd ed. Baltimore: Lippincott Williams & Wilkins; 2001. Alleva R, Borghi B, Santarelli L, et al. In vitro effect of aspartame in angiogenesis induction. Toxicol In Vitro 2011; 25: 286–93. Rezin GT, Cardoso MR, Gonc¸alves CL, et al. Inhibition of mitochondrial respiratory chain in brain of rats subjected to an experimental model of depression. Neurochem Int 2008;53: 395–400. Felts PA, Baker TA, Smith, KJ. Conduction in segmentally demyelinated mammalian central axons. J Neurosci 1997;17: 7267–77. Elsn KS. Radical scavenging and iron-chelating activities of some greens used as traditional dishes in Mediterranean diet. Int J Food Sci Nutr 2004;55: 67–74. Barbour B, Keller BU, Llano I, Marty A. Prolonged presence of glutamate during excitatory synaptic transmission to cerebellar Purkinje cells. Neuron 1994;12: 1331–43. Bruce Keller AJ. Microglial-neuronal interactions in synaptic damage and recovery. J Neurosci Res 1999;58: 191–201. Eng LF, Ghirnikar RS. GFAP and astrogliosis. Brain Pathol 1994;4: 229–37. Hwang DY, ChaEe KR, Kang TS, et al. Alterations in behavior, amyloid-42, caspase-3, and Cox-2 in mutant PS2 transgenic mouse model of Alzheimer’s disease. FASEB J 2002;16: 805–13. Koki A, Khan NK, Woerner BM, et al. Cyclooxygenase-2 in human pathological disease. Adv Exp Med Biol 2002;507: 177–84. Boje KM, Jaworowicz Jr D, Raybon JJ. Neuroinflammatory role of prostaglandins during experimental meningitis: Evidence suggestive of an in vivo relationship between nitric oxide and prostaglandins. J Pharmacol Exp Ther 2003; 304: 319–25. Strauss KI, Barbe MF, Marshall R, et al. Prolonged cyclooxygenase-2 induction in neurons and glia following traumatic brain injury in the rat. J Neurotrauma 2000;17: 695–711. Pasinetti GM, Aisen PS. Cyclooxygenase-2 expression is increased in frontal cortex of Alzheimer’s disease brain. Neuroscience 1998;87: 319–24. Aisen PS. The potential of anti-inflammatory drugs for the treatment of Alzheimer’s disease. Lancet Neurol 2002;1: 279–84.
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