Neuro-amelioration of cinnamaldehyde in aluminum-induced Alzheimer’s disease rat model

Journal of Histotechnology

ISSN: 0147-8885 (Print) 2046-0236 (Online) Journal homepage: https://www.tandfonline.com/loi/yhis20

Neuro-amelioration of cinnamaldehyde in aluminum-induced Alzheimer’s disease rat model Hesham N. Mustafa To cite this article: Hesham N. Mustafa (2019): Neuro-amelioration of cinnamaldehyde in aluminum-induced Alzheimer’s disease rat model, Journal of Histotechnology To link to this article: https://doi.org/10.1080/01478885.2019.1652994

Published online: 28 Aug 2019.

Submit your article to this journal

View related articles

View Crossmark data

Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=yhis20

JOURNAL OF HISTOTECHNOLOGY https://doi.org/10.1080/01478885.2019.1652994

Neuro-amelioration of cinnamaldehyde in aluminum-induced Alzheimer’s disease rat model Hesham N. Mustafa Anatomy Department, Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia ABSTRACT

KEYWORDS

Aluminum (Al) is a neurotoxic substance which has played an important role in the etiology, pathogenesis, and development of amyloid-β (Aβ) plaques. This study was carried out to evaluate the neuroprotective effect of aqueous cinnamon extract against aluminum chloride (AlCl3)induced Alzheimer’s disease. Forty adult male albino rats, randomly divided into four equal groups. Control group; ACE200 group administered aqueous cinnamon extract (ACE) orally; AlCl3 group received daily intraperitoneal (i.p.) injection of AlCl3 for 60 days to induce neurotoxicity and AlCl3 + ACE200 group received a combination of AlCl3 and ACE in the same dose and route as previous groups. Aluminum administration significantly enhanced the memory impairment and the Aβ formation in the rat model. The cerebellum exhibited a significant reduced number of Purkinje cells, marked decrease in the density of dendritic arborization and prominent perineuronal spaces in the molecular layer. There was loss of dendritic spines, neurofibrillary degeneration, and appearance of neuritic plaques. Concomitant administration of AlCl3 and ACE displayed an observable protection against these changes with progressive improvement in memory and intellectual performance. In conclusion, ACE may play a protective role against formation of amyloid-β plaques in cerebellum.

Alzheimer; aluminum chloride; cinnamon; memory; amyloid beta; apoptosis

Introduction Aluminum (Al) is a neurotoxin that leads to development of anxiety disorders, depression, memory deficits, and symptoms similar to those for Alzheimer′s disease (AD) [1]. Aluminum accumulates in the body through medical interventions such as renal dialysis, vaccines, antiperspirants, and allergy desensitization injections [2]. Bondy reported that Al can induce programmed cell death, vacuolar spaces, distortion in the architecture and loss of Purkinje cell layer of cerebellar cortex [3].

In addition, amyloid-β (Aβ) oxidative stress has a critical role in Aβ-mediated neuronal cytotoxicity by triggering neurodegeneration in AD [4]. Oxidative damage and excessive reactive oxygen species (ROS) production are initiated during earlier stages of AD and induce mild cognitive impairment [5].

Additionally, oxidative damage has been associated with mitochondrial membrane damage, dysfunction, and lipid peroxidation elevation that plays a significant role in the pathogenesis of brain disorders induced by Al [6].

Astrocytes are vital for the optimal physiological functions and existence of neurons as these are the CONTACT Hesham N. Mustafa Saudi Arabia

hesham977@hotmail.com

© 2019 National Society for Histotechnology

most plentiful glial cell type in the nervous system [7–9]. Glial fibrillary acidic protein (GFAP) is the chief intermediate filament protein of mature astrocytes and is known as the astrocyte-specific marker responsible for controlling astrocyte movement and shape; thus, GFAP has the crucial role in modulating synaptic efficiency [9,10].

Aqueous (aq) cinnamon extract (ACE) has been associated with a variety of beneficial effects. The antioxidant properties are attributed to cinnamaldehyde and polymeric polyphenol molecules known as proanthocyanidins [11]. These molecules have inhibited amyloid fibril formation by interacting with the polyphenols and Aβ [12,13] and through a high binding affinity of proanthocyanidins to unstructured proteins rich in proline [14]. The aq. cinnamon extract effectively inhibited aggregation of tau related to AD, and this inhibitory activity was attributed to both a proanthocyanidin trimer and cinnamaldehyde [13]. Studies showed that the potentially toxic compounds in cinnamon bark were found in lipid-soluble fractions, while low levels of these compounds were in a water-soluble extract [15] that is considered safer for uptake.

Anatomy Department, Faculty of Medicine, King Abdulaziz University, JEDDAH 21589,

2

H. N. MUSTAFA

Therefore, the aim of this study was to clarify the protective effect of aq. cinnamon extract on aluminum neurotoxicity model for AD on the behavior changes and cerebellar pathology in rats.

Material and methods Ethical approval This study was conducted after approval by the Medical Research Ethics Committee of the Faculty of Medicine, King Abdulaziz University [Reference No 220–19].

Animals Forty male adult Wistar rats (6 wk of age) weighing 200 ± 20 g were obtained from the university Animal House and were distributed randomly into four groups of animals (n = 10). The rats were individually housed in stainless steel cages at controlled temperature (22 ± 2°C) and humidity (55 ± 10%) for a 12/12 h cycle of light/dark with access to food and drinking water ad libitum. The experimental procedures were carried out in accordance with the international guidelines for the care and use of animals in the laboratory.

according to a previously reported dose that caused neurotoxicity [17–19]. AlCl3 exposure was chosen according to European Food Safety Authority that recommended the aluminum mean occupational exposure of adult humans (0.2–1.5 mg/kg-week) [20]. The combination AlCl3 and ACE200 group received the same doses and routes as the separate AlCl3 group and ACE200 group.

Rewarded T-maze test The neurocognitive function was evaluated by the rewarded T-maze test for rats as described by Deacon and Rawlins [21]. Before the experiment, the rat is trained where it is allowed to explore the whole maze and rewarded with food at completion of the test. The rats were denied food for 24 h but allowed to have water. At the start of the test, the rat is placed in the start location, and the time in second which the rat spends to reach the end of each arm was recorded using an auto stopwatch. The test was done within 4 days of training rats to perform. All groups were subjected to the rewarded T-maze test which was done: Trial 1 was at zero time before starting, Trial 2 was 24 h after AlCl3 and Trial 3 was 24 h after the last dose of the drugs for all groups [22].

Measurement of aluminum level in cerebella

Chemicals Aluminum chloride (AlCl3) (Cat No 8010810500; MilliporeSigma, St Louis, MO, USA).

Preparation of cinnamon extract Cinnamomum cassia obtained from local spice market at Jeddah, Saudi Arabia and ground into a powder then 50 g of cinnamon powder was dissolved in 500 ml of distilled water (dH2O) and boiled for 3 h. This mixture was concentrated to make an oily extract using a rotary evaporator (EYELA, Rotary Vacuum Evaporator, N-1000 series, Tokyo Rikakikai Co., Ltd., Chuo-ku, Tokyo, Japan) and lyophilized to obtain 12.48 g of cinnamon powder [16].

Experimental design Control group received dH2O through oral gavage.

ACE200 group were administered 200 mg/kg b. w./ day aq cinnamon extract (ACE) orally for 60 days [11,14] and the selected dose was based on toxicity studies carried out in our laboratory. AlCl3 group was given 100 mg/kg b.w. intraperitoneally (i.p.) for 60 days

The whole cerebella of randomly chosen rats (n = 4) from each group were carefully separated, removed and washed by ice-cold (4ºC) normal saline, weighed and put into a solution containing 0.05 ml nitric acid (1004551000, MilliporeSigma, St. Louis, MO, USA) and 0.2 ml hydrogen peroxide (H2O2) (386790, MilliporeSigma), then whole tissue mixture was incubated at 120°C for 2 h. The aluminum level in cerebella measured by µg/g was determined by an atomic absorption spectrophotometer (PinAAcle500, Perkin Elmer, Waltham, MA, USA) [23,24].

Cerebellum histology At the end of the experiment, the remaining animals were euthanized. The cerebellum surgically removed, weighed, and fixed in 10% neutral buffered formalin (NBF) for 24 h. Tissues were processed through an ascending ethyl alcohol gradient (50%, 70%, 90%, and 95%) 30 min each for, 100% ethyl alcohol for 1 h (two changes), cleared in xylene for 1 h (two changes), infiltrated with paraffin at 60°C for 2 h and embedded in paraffin. Sections 5 µm thick were cut using a rotatory microtome (Shandon,

JOURNAL OF HISTOTECHNOLOGY

Finesse 325, ThermoFisher Scientific, Luton, England), and mounted on slides precoated with an egg albumin–glycerol adhesive. Sections were deparaffinized in xylene (three changes, 15 min each) and rehydrated through a descending alcohol gradient (100%, 90%, 70%) 5–10 min each change to diH20.

Sections were stained 10 min in Harris hematoxylin (HHS16, MilliporeSigma,), washed in tap H2O to ‘blue’ the nuclei, counterstained in Alcoholic Eosin Y (515, 3,801,615; Leica Biosystems Inc., Buffalo Grove, IL, USA) for 5 min., dehydrated through an ascending alcohol gradient, cleared in xylene, and coverslipped using Canada balsam (C1795, MilliporeSigma) [10].

Congo red stain for the amyloid β Sections were deparaffinized and rehydrated as previously described, and stained in Congo red solution (1% Congo red in dH2O) (C-6277, MilliporeSigma) for 30–60 min, then rinse in distilled water. Differentiated (5–10 dips) in alkaline alcohol solution (1% sodium hydroxide + 50% alcohol). Then counterstained in hematoxylin (23–750016, MilliporeSigma) for 30 sec, blued in ammonia water for 30 sec, rinsed in tap H2O for 5 min, dehydrated through 95% and 100% alcohols, cleared in xylene and cover glass mounted with resinous mounting medium [6,7].

Bielschowsky silver stain for amyloid plaques Bielshowsky silver stain can be used for diagnosis of AD. Sections were deparaffinized and rehydrated as described above. Slides are placed in pre-warmed (40ºC) silver nitrate (AgNO3) solution (0.1 mol Titrisol, 109990, MilliporeSigma) for 15 min until sections become a brown color then washed in diH2O 3 times. The ammoniacal silver stain solution (AgNO3/NH4OH) was prepared as follows: conc. ammonium hydroxide (NH4OH, 221228, MilliporeSigma), is added to the AgNO3 solution drop by drop until the precipitate formed just turns clear.

Slides were returned to the AgNO3/NH4OH solution for 30 min in 40ºC oven followed by direct immersion into the developer solution for about 1 min. The developer is made with 20 ml of 40% formaldehyde (818708, MilliporeSigma), 100 ml dH2O, 20 µl conc. nitric acid (1004551000, MilliporeSigma), and 0.5 g citric acid (sodium citrate, S4641, MilliporeSigma). Slides were dipped for 1 min in 1% NH4OH to stop the silver reaction, washed in dH2O 3 times, and placed in 5% aq. sodium thiosulfate for 5 min. Sections were dehydrated, cleared, and coverslipped [7].

3

Immunohistochemical (IHC) study Immunostaining for glial fibrillary acidic protein (GFAP) in the astrocytes was done on deparaffinized sections after antigen retrieval and removal of endogenous peroxidase as done by Saleh et al. [7]. The Histostain-Plus IHC Kit, HRP, broad spectrum (859,043, Invitrogen, Carlsbad CA, USA) with diaminobenzidene (DAB) chromogen was used according to kit instructions. The primary antibody was Anti-GFAP (Anti-glial fibrillary acidic protein, mouse monoclonal, IgG, clone GA5, MAB3402, RRID: AB_94844 MilliporeSigma) diluted 1:1000 and incubated overnight at 4°C. The negative control was sectioned from dH2O control group with PBS replacing the anti-GFAP antibody. GFAP-positive (GFAP+) astrocytes will display brown cellular membranes and cytoplasm with blue nuclei [7,9].

Quantitative morphometric study Sections from all groups were examined using an Olympus BX53 microscope fitted with a DP73 camera (Olympus, Tokyo, Japan). Ten slides of nonoverlapping fields from each group with one slide from each animal were analyzed with Image-Pro Plus v6 (Media Cybernetics Inc., Bethesda, MD, USA). For each rat in all groups, the number of Purkinje cells were counted from 10 lobules in each cerebellar section at 200x magnification. The average value of Purkinje cells was calculated for these 10 lobules per section. The total length of the cerebellar folia in the 10 lobules was estimated in µm then converted into millimeter (mm). Purkinje cells = mean value of cell number ÷ length (mm) of the cerebellar folia according to McGoey et. al [25].

Also, the area percent for GFAP expressed in astrocytes and in their processes in cerebellar cortices were measured.

Statistical analysis Quantitative data were expressed as the mean and standard deviations of different parameters (transit time spent in the T-maze test, linear density of Purkinje cells/mm length of the folia and area percent of GFAP+ astrocytes) between the treated groups. Data were analyzed using a one-way analysis of variance (ANOVA) followed by a least significant difference (LSD) post hoc test. All statistical analyses were implemented using the Statistical Package for the Social Sciences (SPSS), version 23. The values were considered significant when p < 0.05.

4

H. N. MUSTAFA

Table 2. Therapeutic effects of ACE on AL level in different groups.

Results T-maze test The results demonstrated a significant increase in time (seconds) taken by rats in the AlCl3 group to reach the food in the T-maze indicating a deteriorated neurocognitive function. Whereas the AlCl3 + ACE200 group showed a significant decrease in time taken by rats to reach food in the T-Maze indicating improved cognitive abilities as compared to the AlCl3 group. Additionally, the ACE200 group showed a significant decrease in time to achieve the task, as compared to the control group (Table 1).

Data are presented as mean ± SE (n = 10). Mean with different superscripts (a, b, c, d, e) are significant at p ≤ 0.05. Trial 1 was at zero time before starting, Trial 2 was 24 h after AlCl3 and Trial 3 was 24 h after the last dose for all groups. T-maze transit time is in sec.

Group Control (n = 4) ACE200 (n = 4) AlCl3 (n = 4)

Al level (µg/g) wet tissue.

3.14 ± 0.12

AlCl3 + ACE200 (n = 4)

3.92 ± 0.81 10.34 ± 0.49 P1 < 0.001 P2 < 0.001 6.27 ± 1.90 P1 < 0.01 P2 < 0.05 P3 < 0.001

Each value represents the mean ± S.D.; P1: compared to Control, P2: compared to ACE200, P3: compared to AlCl3. n= number of rats

Each value represents the mean ± S.D.; P1: compared to control, P2: compared to ACE200, P3: compared to AlCl3.

Cerebellar histology

Aluminum level in cerebella AlCl3 levels in cerebella were detected by atomic absorption spectrophotometry. Results showed that AlCl3 treatment had a significantly elevated Al level as compared to control group. Otherwise, ACE200 administration inhibited the increase of Al level (Figure 1, Table 2).

Table 1. Therapeutic effects of ACE on the transit time spent in the T-maze by experimental groups.

Groups Control ACE200 AlCl3 AlCl3 + ACE200

Trial 1 (sec.)

13.12 ± 2.12 a 12.73 a ± 0.94 18.92 d ± 3.91 15.27 a ± 2.09

Trial 2 (sec.)

16.57 c ± 4.70 14.45 c ± 0.98 25.39 e ± 3.12 20.00 b ± 1.57

Trial 3 (sec.)

14.88 a ± 1.14 15.00 a ± 2.77 23.36 e ± 4.66 18.43 c ± 3.82

Cell alteration and disintegration were compared to control. Neurons were morphologically damaged and showed shrunken pyknotic hyperchromatic nuclei in AlCl3 group.

In AlCl3 + ACE200 group, the extent of neuronal damage was declined significantly. Also, Cellular morphology was improved and no sign of degeneration was observed as compared to controls (Figure 2).

In AlCl3 group, Congo red staining results demonstrated that noticeable amyloid β plaques were distributed in the molecular layer and rare amyloid plaques were seen in the granular layer. Amyloid plaques exhibited a light red mass without distinct borders. In AlCl3 + ACE200 group, the positively stained areas of amyloid plaques were markedly reduced with a normal, restored appearance and numbers of Purkinje cell layer (Figure 3).

Immunohistochemical results AlCl3 group showed many GFAP+ hypertrophic astrocytes with extensive branching of processes extending into the molecular cell layer. In the AlCl3 + ACE200 group, there was a decrease in the number of GFAP+ astrocytes (Figure 4), and these findings are supported statistically (Figure 4).

Morphometric and statistical results

Figure 1. Therapeutic effects of ACE on aluminum level in different groups.

As compared with the control group, the Purkinje cells in the AlCl3 group were significantly reduced in number although Purkinje cells in the AlCl3 + ACE200 group were significantly increased in number. There was also a significant increase in the mean number of astrocytes in AlCl3 group as compared to astrocyte numbers in the other three groups. There was a significant decrease in the

JOURNAL OF HISTOTECHNOLOGY

5

Figure 2. Photomicrographs of cerebellar cortex sections from experimental groups. (a) Control group molecular layer (M) has small stellate cells (SC), and basket cells (BC). The Purkinje cell layer (P) has large pyriform somata with prominent nucleoli, and the granular layer (G) shows tightly packed small rounded cells with deeply stained nuclei. (b) ACE200 group exhibited normal morphology. (c) AlCl3 group exhibited a normal molecular layer (M). Few Purkinje cells (arrow) are found in the Purkinje cell layer (P) and have irregular size, shape, darkly stained nuclei and cytoplasm (arrow). Prominent perineuronal spaces (stars) are seen around basket (BC) and stellate cells (SC) in the molecular layer (M). The granular layer (G) appears unaffected but obvious amyloid plaques (Aβ) were detected. (d) AlCl3 + ACE200 group molecular (M), the granular (G), and the Purkinje cell layers (P) have restored appearance and numbers. The Purkinje cells (arrows) are slightly reduced in number. (H&E, Scale bar = 20 µm).

Figure 3. Photomicrographs of cerebellar cortex sections from experimental groups. (a) Control with the three cerebellar layers, Purkinje (P), molecular (M) and granular (G). (b) ACE200 group exhibits normal morphology. (c) AlCl3 group shows a reduced number of Purkinje layer cells (arrow) with irregular darkly stained cytoplasm (arrow), and amyloid plaques (Aβ). (d) AlCl3 + ACE200 group shows the Purkinje layer (P) has a restored appearance and number of cells. (Congo red, Scale bar = 20 µm).

6

H. N. MUSTAFA

Figure 4. Photomicrographs of cerebellar cortex sections from experimental groups. (a) Control group shows GFAP+ astrocytes with long and thin processes (star) and granular layer protoplasmic astrocytes with thick processes (arrowhead). (b) ACE200 group exhibited GFAP+ astrocytes with small oligodendrocytes (arrowhead) and spindle-shaped microglia (star). (c) AlCl3 group exhibited an increase in the number of GFAP+ astrocytes with relatively longer processes (arrowheads). (d) AlCl3 + ACE200 group showed relatively fewer numbers of astrocytes with thin processes (arrowheads) (GFAP, scale bar = 20 µm).

a

b

25

6

P1 < 0.001 P2 < 0.001 P3 < 0.001

20

15

10 P1 < 0.001 P2 < 0.001

5

5

Area percent of GFAP+ astrocytes

Linear density of Purkinje cells/mm length of the folia

P1 < 0.001 P2 < 0.001

4 P1 < 0.001 P2 < 0.001 P3 < 0.001

3 2 1 0

0

Control

ACE200

AlCl3

AlCl3 + ACE200

Control

ACE200

AlCl3

AlCl3 + ACE200

Figure 5. (a) The linear density of Purkinje cells/mm length of the folia. (b) Area percent of GFAP+ astrocytes.

mean number of astrocytes in AlCl3 + ACE200 group (Figures 5 a,b, Table 3).

Data are represented as mean ± SD. P1: as compared to control, P2: as compared to ACE200, P3: as compared to AlCl3.

Data are represented as mean ± SD. P1: as compared to control, P2: as compared to ACE200, P3: as compared to AlCl3.

Bielschowsky results In AlCl3 group, modified Bielschowsky results demonstrated an obvious heavily stained brown amyloid plaques with irregular border in the molecular layer. In AlCl3 + ACE200 group, the amyloid plaques were markedly reduced with improvements in the morphology of the cerebellum (Figure 6).

JOURNAL OF HISTOTECHNOLOGY

Table 3. The linear density of Purkinje cells/mm length of the folia and area percent of GFAP+ astrocytes.

Groups Control ACE200 AlCl3 AlCl3+ ACE200

Purkinje cells 21.74 ± 0.98 21.57 ± 0.96 3.35 ± 0.71 P1 < 0.001 P2 < 0.001 15.73 ± 1.02 P1 < 0.001 P2 < 0.001 P3 < 0.001

GFAP+ (n = 200) 1.37 ± 0.43 1.54 ± 0.23 4.63 ± 0.35 P1 < 0.001 P2 < 0.001 2.28 ± 0.83 P1 < 0.001 P2 < 0.001 P3 < 0.001

Data are represented as mean ± SD. P1: compared to control, P2: compared to ACE200, P3: compared to AlCl3. n = 200 is the number of cells counted.

Discussion The findings of the current study are in accordance with other studies for cerebellum in Alzheimer’s disease, with deterioration of the cerebellar volume due to damaged Purkinje neurons and smalled cell bodies. Dendrites disintegration, decline of dendritic fields density, dendritic spines were lost and a marked increase of focal lipid storage within the dendritic arborization [4,12,26].

The duration and dose of AlCl3 administration was selected to induce AD symptoms based on previous findings [27,28]. Even though the dose of AlCl3 may be higher than routine human exposure (0.4–1.7 mg/kg b.w./day), humans are sometimes exposed to higher levels of AlCl3 during occupational toxicity and dialysis encephalopathy [29–31]. Moreover, humans are exposed to aluminum through various ways such as cooking utensils and drinking water [32].

7

Aluminum exposure caused a significant decrease in body and brain weights in rats in a study by Mohamed and Abd El-Moneium [33] and this could be attributed to the interference by the aluminum on the hormonal status and/or protein synthesis [34]. Furthermore, the decrease in brain weight might be due to increased lipid peroxidation as a consequence of oxidative stress [31].

The AlCl3 group showed a significant decrease in the behavior scores as compared with the control and AlCl3+ ACE200 group in T-maze test. This study coincided with Wu, Li et al. that proved the deposition of Aβ plaques in AD brains impairs learning and memory [35].

Cinnamaldehyde is effective in preventing the tau knots by prohibiting oxidative stress, as cinnamaldehyde binds to two residues of the amino cysteine on the tau protein. The cysteine residues are vulnerable to these modifications, which have contributed to the development of Alzheimer’s disease. This could explain why ACE reduced the cerebellar Al level in the current study, may be the other mechanism related to neuroprotective effects by ACE [36].

In the current study, Al exposure resulted in a significant reduction in the number of Purkinje cells.

This agreed with studies that reported disorganization of the Purkinje cell layer with a loss of Purkinje cells with Al exposure [1]. A darkly stained cytoplasm and pyknotic nuclei were observed in the Purkinje cells. Pyknosis was described as irreversible condensation of nuclear chromatin in cells undergoing programmed cell death or apoptosis [37]. These results are in agreement with the

Figure 6. Photomicrographs of cerebellum sections from experimental groups. (a) Control exhibited the three cerebellar layers.

Purkinje (P), molecular (M) and granular (G). (b) ACE200 group displayed the same normal morphological findings as indicated in Figures 1 and 2. (c) ALCl3 group revealed shrunken pyknotic Purkinje cells and with obvious large, dark irregular amyloid plaques (Aβ). (d) AlCl3 + ACE200 group showed notable improvement of any signs of degeneration (Bielschowsky, scale bar = 20 µm).

8

H. N. MUSTAFA

histological findings in the cerebellar cortex after Al treatment investigated by El–Shafei and colleagues [38].

This study showed that the molecular layer was characterized by the presence of diffuse plaques and absence of typical neuritic plaques which was in accordance with work by Mavroudis et. al [4]. The main difference between these two types of plaques was the amyloid-β protein nature that is present. Diffuse plaques in the cerebellum are known to be positive for the end specific monoclonal antibodies Aβ 1–42 but not Aβ 1–40 [12,26]. This agreed with Du et al. who noticed that metabolites from cerebellar neurons encouraged the expression of Aβ degrading enzymes and advance the clearance of Aβ [4].

Astrocytes play active roles in neuronal regulation and modulation [39]. It has also been suggested that the loss of astrocyte functions may precede neurodegeneration and aluminum could be a contributing factor for this loss [40]. Astrocytes are the principal target of the action of aluminum [39] that can cause astrocyte death through apoptosis [41].

The current findings showed a significant increase in GFAP immunoreactivity of astrocytes in AlCl3 group, which is in accordance with previous findings and may be related to a generic response of the central nervous system to neural injury [42]. Injury to the parenchyma of the brain induced many plump reactive astrocytes.

These researchers added that as a response to injury, they also observed the production of a dense network of processes and increased synthesis of GFAP. The role of astrocytes in central nervous system (CNS) disorders remains of interest. The present study showed a significant increase in the number of GFAP+ astrocytes in AlCl3 group and this finding indicated that AlCl3 altered the production and degradation of GFAP, the marker of reactive astrocytosis. Thus, GFAP expression has been a relevant marker for studying neurodegenerative changes. In contrast, other researchers have found decreased GFAP expression in the cerebellar cortex [43,44].

Gliosis might be mediated indirectly through the free radical formation and herbal antioxidants may help in preventing this reactive gliosis possibly by reducing the damaging effects of ROS. Based on this postulation, the use of ACE in the present study significantly reduced GFAP expression in cerebellar cortex thus protecting the memory and learned ability as reported by other authors [9,45].

Conclusion Aqueous Cinnamon Extract (ACE) may be considered an efficacious therapeutic strategy to alleviate amyloid-

β plaques. It is recommended to avoid using of aluminum cooking utensils, water tubing and to control occupational exposure.

Disclosure statement No potential conflict of interest was reported by the author.

ORCID Hesham N. Mustafa 2187

http://orcid.org/0000-0003-1188-

References [1] Bhalla P, Dhawan DK. Protective role of lithium in ameliorating the aluminium-induced oxidative stress and histological changes in rat brain. Cell Mol Neurobiol. 2009;29(4):513–521.

[2] Krewski D, Yokel RA, Nieboer E, et al. Human health risk assessment for aluminium, aluminium oxide, and aluminium hydroxide. J Toxicol Environ Health B Crit Rev. 2007;10(Suppl 1):1–269.

[3] Bondy SC. Low levels of aluminum can lead to behavioral and morphological changes associated with Alzheimer’s disease and age-related neurodegeneration. Neurotoxicol. 2016;52:222–229.

[4] Mavroudis IA, Manani MG, Petrides F, et al. Dendritic and spinal pathology of the Purkinje cells from the human cerebellar vermis in Alzheimer’s disease.

Psychiatr Danub. 2013;25(3):221–226.

[5] Abu-Taweel GM, Ajarem JS, Ahmad M. Neurobehavioral toxic effects of perinatal oral exposure to aluminum on the developmental motor reflexes, learning, memory and brain neurotransmitters of mice offspring. Pharmacol Biochem Behav. 2012;101(1):49–56.

[6] Nobakht M, Hoseini SM, Mortazavi P, et al.

Neuropathological changes in brain cortex and hippocampus in a rat model of Alzheimer’s disease. Iran Biomed J. 2011;15(1–2):51–58.

[7] Suvarna KS, Layton C, Bancroft JD. Bancroft’s theory and practice of histological techniques. UK: Elsevier Health Sciences; 2019.

[8] Saleh HA, Abd El-Aziz GS, Mustafa HN, et al.

Thymoquinone ameliorates oxidative damage and histopathological changes of developing brain neurotoxicity. J Histotechnol. 2019;1–12.

[9] Frydman-Marom A, Levin A, Farfara D, et al. Orally administrated cinnamon extract reduces β-amyloid oligomerization and corrects cognitive impairment in Alzheimer’s disease animal models. PloS One. 2011;6 (1):e16564.

[10] Mustafa HN, Hussein AM. Does allicin combined with vitamin B-complex have superior potentials than alpha-tocopherol alone in ameliorating lead acetate-induced Purkinje cell alterations in rats? An immunohistochemical and ultrastructural study. Folia Morphol (Warsz). 2016;75(1):76–86.

JOURNAL OF HISTOTECHNOLOGY

[11] Azab K, Mostafa AH, Ali EM, et al. Cinnamon extract ameliorates ionizing radiation-induced cellular injury in rats. Ecotoxicol Environ Saf. 2011;74 (8):2324–2329.

[12] Mavroudis IA, Fotiou DF, Adipepe LF, et al.

Morphological changes of the human purkinje cells and deposition of neuritic plaques and neurofibrillary tangles on the cerebellar cortex of Alzheimer’s disease. Am J Alzheimers Dis Other Demen. 2010;25(7):585–591.

[13] Peterson DW, George RC, Scaramozzino F, et al.

Cinnamon extract inhibits tau aggregation associated with Alzheimer’s disease in vitro. J Alzheimers Dis. 2009;17(3):585–597.

[14] Morgan AM, El-Ballal SS, El-Bialy BE, et al. Studies on the potential protective effect of cinnamon against bisphenol A- and octylphenol-induced oxidative stress in male albino rats. Toxicol Rep. 2014;1:92–101.

[15] Otto AD. Cinnamon as a supplemental treatment for impaired glucose tolerance and type 2 diabetes. Curr Diab Rep. 2010;10(3):170–172.

[16] Parvazi S, Sadeghi S, Azadi M, et al. The effect of aqueous extract of cinnamon on the metabolome of plasmodium falciparum using (1)HNMR spectroscopy.

J Trop Med. 2016;2016:3174841.

[17] Justin Thenmozhi A, Raja TR, Janakiraman U, et al.

Neuroprotective effect of hesperidin on aluminium chloride induced Alzheimer’s disease in Wistar rats.

Neurochem Res. 2015;40(4):767–776.

[18] Cao Z, Wang F, Xiu C, et al. Hypericum perforatum extract attenuates behavioral, biochemical, and neurochemical abnormalities in Aluminum chlorideinduced Alzheimer’s disease rats.

Biomed Pharmacother. 2017;91:931–937.

[19] Cao Z, Yang X, Zhang H, et al. Aluminum chloride induces neuroinflammation, loss of neuronal dendritic spine and cognition impairment in developing rat.

Chemosphere. 2016;151:289–295.

[20] European Food Safety Authority E. Dietary exposure to aluminium-containing food additives. EFSA Supporting Publ. 2013;10(4):411E. [21] Deacon RM, Rawlins JN. T-maze alternation in the rodent. Nat Protoc. 2006;1(1):7–12.

[22] Farr SA, Ripley JL, Sultana R, et al. Antisense oligonucleotide against GSK-3beta in brain of SAMP8 mice improves learning and memory and decreases oxidative stress: Involvement of transcription factor Nrf2 and implications for Alzheimer disease. Free Radic Biol Med. 2014;67:387–395.

[23] Wei Y, Liu D, Zheng Y, et al. Protective effects of kinetin against aluminum chloride and D-galactose induced cognitive impairment and oxidative damage in mouse. Brain Res Bull. 2017;134:262–272.

[24] Justin Thenmozhi A, Dhivyabharathi M, William Raja TR, et al. Tannoid principles of Emblica officinalis renovate cognitive deficits and attenuate amyloid pathologies against aluminum chloride induced rat model of Alzheimer’s disease. Nutr Neurosci. 2016;19 (6):269–278.

[25] McGoey TN, Reynolds JN, Brien JF. Chronic prenatal ethanol exposure-induced decrease of guinea pig

[26]

[27]

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

9

hippocampal CA1 pyramidal cell and cerebellar Purkinje cell density. Can J Physiol Pharmacol.

2003;81(5):476–484.

Mavroudis IA, Fotiou DF, Manani MG, et al.

Dendritic pathology and spinal loss in the visual cortex in Alzheimer’s disease: a Golgi study in pathology.

Int J Neurosci. 2011;121(7):347–354.

Nampoothiri M, Kumar N, Venkata Ramalingayya G, et al. Effect of insulin on spatial memory in aluminum chloride-induced dementia in rats. Neuroreport.

2017;28(9):540–544.

Saba K, Rajnala N, Veeraiah P, et al. Energetics of excitatory and inhibitory neurotransmission in aluminum chloride model of Alzheimer’s disease: reversal of behavioral and metabolic deficits by Rasa Sindoor.

Front Mol Neurosci. 2017;10:323.

Niu Q, Yang Y, Zhang Q, et al. The relationship between Bcl-2 gene expression and learning & memory impairment in chronic aluminum-exposed rats.

Neurotox Res. 2007;12(3):163–169.

Bhattacharjee S, Zhao Y, Hill JM, et al. Aluminum and its potential contribution to Alzheimer’s disease (AD).

Front Aging Neurosci. 2014;6:62.

Mathiyazahan DB, Justin Thenmozhi A, Manivasagam T. Protective effect of black tea extract against aluminium chloride-induced Alzheimer’s disease in rats: A behavioural, biochemical and molecular approach. J Funct Foods.

2015;16:423–435.

Ali HA, Afifi M, Abdelazim AM, et al. Quercetin and omega 3 ameliorate oxidative stress induced by aluminium chloride in the brain. J Mol Neurosci. 2014;53 (4):654–660.

Mohamed NE, Abd El-Moneim AE. Ginkgo biloba extract alleviates oxidative stress and some neurotransmitters changes induced by aluminum chloride in rats.

Nutrition. 2017;35:93–99.

Wenting L, Ping L, Haitao J, et al. Therapeutic effect of taurine against aluminum-induced impairment on learning, memory and brain neurotransmitters in rats. Neurol Sci. 2014;35(10):1579–1584.

Wu QY, Li J, Feng ZT, et al. Bone marrow stromal cells of transgenic mice can improve the cognitive ability of an Alzheimer’s disease rat model. Neurosci Lett. 2007;417(3):281–285.

George RC, Lew J, Graves DJ. Interaction of cinnamaldehyde and epicatechin with tau: implications of beneficial effects in modulating Alzheimer’s disease pathogenesis. J Alzheimers Dis. 2013;36 (1):21–40.

Venkataraman P, Selvakumar K, Krishnamoorthy G, et al. Effect of melatonin on PCB (Aroclor 1254) induced neuronal damage and changes in Cu/Zn superoxide dismutase and glutathione peroxidase-4 mRNA expression in cerebral cortex, cerebellum and hippocampus of adult rats. Neurosci Res. 2010;66 (2):189–197.

El–Shafei M-DE-DM, Kamel AMF, Mostafa MEA. Effect of aluminum on the histological structure of ratsʼ cerebellar cortex and possible protection by melatonin. Egypt J Histol. 2011;34(2):239–250.

10

H. N. MUSTAFA

[39] Araque A, Navarrete M. Glial cells in neuronal network function. Philos Trans R Soc Lond B Biol Sci. 2010;365(1551):2375–2381.

[40] Guo GW, Liang YX. Aluminum-induced apoptosis in cultured astrocytes and its effect on calcium homeostasis. Brain Res. 2001;888(2):221–226.

[41] Suarez-Fernandez MB, Soldado AB, Sanz-Medel A, et al. Aluminum-induced degeneration of astrocytes occurs via apoptosis and results in neuronal death.

Brain Res. 1999;835(2):125–136.

[42] Nedzvetsky VS, Tuzcu M, Yasar A, et al. Effects of vitamin E against aluminum neurotoxicity in rats.

Biochemistry (Mosc). 2006;71(3):239–244.

[43] Exley C. Aluminium and Alzheimer’s disease: the science that describes the link. UK: Elsevier Science; 2001.

[44] Silva AF, Aguiar MS, Carvalho OS, et al. Hippocampal neuronal loss, decreased GFAP immunoreactivity and cognitive impairment following experimental intoxication of rats with aluminum citrate. Brain Res. 2013;1491(Supplement C):23–33.

[45] Modi KK, Roy A, Brahmachari S, et al. Cinnamon and its metabolite sodium benzoate attenuate the activation of p21rac and protect memory and learning in an animal model of Alzheimer’s disease. PloS One. 2015;10(6):e0130398.