Effect of Eugenol On Neurotrace Elements, and Histology of the Cerebral Cortex of Wistar Rats Exposed to Aluminium Chloride.

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Mesole S.B.

Department of Human Anatomy Texila American University Zambia.

Ibegbu A.O

Department of Anatomy Alex Ekwueme Federal University Ndufu-Alike, Nigeria

Mesole S.B, Musa S, Bauchi Z, Agbon A.N, Animoku AA and Kolawole OJ

Department of Anatomy, Faculty of Basic Medical Sciences, College of Medical Sciences, Ahmadu Bello University (A.B.U), Zaria, Nigeria.

Okpanachi A.O

Department of Physiology, Kampala International University, Uganda

Mesole S.B., Musa S,, Bauchi Z, Agbon A.N, Animoku AA and Kolawole OJ

Neuroanatomy and Neuroscience Unit, Department of Human Anatomy, A.B.U, Zaria.

Animoku AA

Department of Anatomy, Kogi State University Ayingba Nigeria.

All correspondence to: Mesole S.B e-mail: ms361450@gmail.com

ABSTRACT

Aluminium contamination can occurs via food, vaccines and water. The present study was Acarried out to study the effects of Eugenol on Brain neurotrace elements (Iron Fe; Manganese Mn: Magnesium Mg), cognition using morris water maze and histology of the cerebrum (Layer III and V) following administration of Aluminium chloride on Wistar rats. Materials and Methods. Thirty (30) adult Wistar rats were divided into six (6) groups with five (5) rats in each group. The rats were sacrificed 24 hours after administration of the last dose by 0.8ml/kg of ketamine as an anesthetic agent. Results: Aluminium chloride treatment of rats resulted in significant (p<0.05) elevation of manganese and Aluminium levels in the brain of rats. This is accompanied by a significant decrease (p<0.05) in brain levels of Iron (Fe) and Magnesium. Morris water maze test result revealed a significant (p<0.05) increase in latency time in the rats treated with aluminium chloride when pretreatment is compared to day-21 of treatment. However treatment with eugenol revealed a significant (p<0.05) reduction in latency time. Histological examination of the cerebral cortex Layer III and V using haematoxylin and Eosin revealed pyknosis perineuronal vacuolations of pyramidal cells of group-administered 100 mg/kg of aluminium chloride. However, treatment with Eugenol revealed an almost normal cytoarchitecture of the pyramidal cells of the cerebrum of the Wistar rats. Conclusions: Eugenol has the ability to protect rat brain from the deleterious effect of aluminium chloride on brain neurotrace elements, improve cognition and

preserve cytoarchitecture of the brain of rats.

Keywords: Prineuronal vacoulations, Pyknosis, Pyramidal cells and Anesthetic agent.

It is of interest to note that humans live in what is referred to as ”the Aluminium Age”.Objects made with the metal aluminium are strong, durable, light and corrosion-resistant (Hirata et al.,2011). Relative to bio-availability, the metal can be found in drinking water due to its property as a flocculant, it is a common additive to various processed foods, cosmetics of various types and pharmaceutical products (Tomljenovic and shaw, 2011).
Aluminium  mimics  physiological  elements  such  as magnesium (Mg), calcium (Ca), and iron (Fe) in the human body  hence  results  to  Physiological  alterations  and dysfunction of the body system (Hirata et al., 2011; Wu Zhihao  et  al.,  2012).  Aluminium  can  also  induce neurodegeneration, by increasing the accumulation of iron and  generation  of  reactive  oxygen  species  (ROS) production (Wu Zhihao et al., 2012; Maya et al., 2016).
The physical and chemical properties of aluminium allow it to effectively mimic the above-mentioned metals (Mg, Ca and Fe) in their respective biological functions and trigger a series of physiological abnormalities. Aluminium has been proven to replace Mg and bind to phosphate groups on the cell membrane (Kawahara and Kato., 2011; Singh et al., 2017). Eugenol (4-allyl-2-methoxyphenol), mainly exists in clove oil, camphorated oil, cinnamon leaf oil, and nutmeg oil. At normal temperatures, eugenol is a pale yellow viscous oily liquid with a strong clove flavor and a special hot taste or brown powder in the dried form (Chaieb et al., 2007). Eugenol, which is an active compound (nutraceuticals) in many spice plants such as clove, Ocimum sanctum and Ocimum gratissimum is a well-established antioxidant (Zoppi et al., 2006; Patra et al., 2018). This study was undertaken to assess the protective effect of eugenol on brain neurotrace elements (Mg, Mn and Fe), neurobehavioural (learning and memory) and the histology of the cerebral cortex (layers III and V) following aluminium induced neurotoxicity in rats.

MATERIALS AND METHODS

Chemicals:- Eugenol, a light brownish powdered substance used for this study was obtained from Wuhan JCJ Logis, China, manufactured by Yueyang Jiazhiyuan Biological Co Ltd china (#58-23-4). While aluminium chloride which was used as a neurotoxic agent was obtained from Guandong Guanghua Sci-Tech Co. Ltd China (#7446-70-0).

Animals: A total of thirty (30) apparently healthy Wistar rats of both sex (140 to 160 g) were obtained from the Animal House of the Department of Human Anatomy, Faculty of Basic Medical Sciences, Ahmadu Bello University, Zaria, Kaduna State Nigeria and housed in wired cages in the same facility to acclimatize for a weeks prior to the commencement of the experiments. Ethical approval was obtained from Department of Anatomy Postgraduate research committee Ahmadu Bello University Zaria Nigeria. All rats were given food purchased from Grand Cereals and Oil Mills Limited (GCOML) Jos, Plateau State, Nigeria and water ad libitum. Treatment groups were administered eugenol/aluminium Chloride in addition to water and rat chow.

Experimental Design: Each groups consisted of 5 rats each and all route of administration was via the oral route. E u g e n o l a n d a l u m i n i u m w e r e a d m i n i s t e r e d simultaneously. Group I rats received 300 mg/kg of eugenol (10% LD50) (LD50 3000mg/kg as provided on the safety data sheet), Group II received 150 mg/kg (Mesole et al; 2020) (5% LD50) of eugenol, Group III rats that received 300 mg/kg of eugenol and 100 mg/kg of aluminium chloride, Group IV rats that received 150 mg/kg of eugenol and 100mg/kg of aluminium, Group V rats that received 100 mg/kg of aluminium chloride (Anil et al; 2009; Mesole et al; 2020), Group VI rats served as control and was administered 2 ml/kg of distilled water as placebo. Duration of the entire treatment was for 21 days. Rats were humanely sacrificed 24 hours after the last administration with 0.8 ml/kg (Mesole et al; 2020) of ketamine as anesthesia (Table 1).

Table 1: Animal Grouping and treatment

Groups Dose
Group I 300 mg/kg eugenol
Group II 150 mg/kg eugenol
Group III 300 mg/kg eugenol + 100 mg/kg
aluminium chloride
Group IV 150 mg/kg eugenol + 100 mg/kg
aluminium chloride
Group V 100 mg/kg of aluminium chloride
Group VI 2 ml distil water

Brain Trace Elements

At the end of the experiment, brains were dissected (under ketamine anaesthesia, the rats were decapitated and the skull was carefully opened to expose the brain) weighed and homogenized in 0.1M Phosphate buffer (pH 7.4) (1g tissue/ 4ml (Ige et al., 2011). The homogenate were then centrifuged and aliquots of the supernatant were obtained for analysis of brain trace elements.

Neurochemical analysis for (Fe, Mn, Mg. and Al) estimation in the in the tissue (brain) homogenate was conducted using atomic Absorption Spectrophotometer (AAS – AA240FS, Varian) at the Multiuser Laboratory, Department of Chemistry Ahmadu Bello University, Zaria. The analytical method for determining metals in biological tissues as reported by Environmental Monitoring Methods Index, EMMI (1997) was adopted and is summarized below:

Tissue preparation/digestion

Take 1 ml (0.25g) of sample (homogenized tissue; 1g in 4ml of phosphate buffer) into boiling tube and add 2 ml of concentrated HNO3. The sample is heated at100°C for 2 hours and allowed to cool. This is followed by addition 0.3 ml of 30% hydrogen peroxide (H2O2) to the already cooled sample. Heat again at 100°C for 1-2 hours and allow to cool. Filter using whatmann’s filter paper.

Dilution/ Deionization

Make volume (of digested sample) into 20ml using deionized water analyze using an atomic absorption spectrophotometry (AAS)

Neurobehavioral Studies:

This method as described by Morris et al., (1982), as reported by Drapeau et al., (2003), for spatial memory and learning was adopted for this study. Rats were tested in a Morris water maze (180 cm diameter, 60 cm height) filled with water. An escape platform was hidden 2cm below the surface of the water in a fixed location in one of the four quadrants halfway between the wall and the middle of the pool.

Procedure:

Before the commencement of the treatment, the rats were trained in the pool daily for four (4) days. During the training, animals were required to locate the submerged platform by using distal extra-maze cues. They were tested for four trials per day (90 seconds with an inter trial interval of 30 seconds and beginning from different start points that varied randomly each day). Rats were tested after seven

(7), fourteen (14) and twenty one (21) days of treatment. Preparation of tissue for microscopy: The brain was removed and fixed in formol saline and processed for microscopy. Tissues were processed to obtain 5 µm thick paraffin sections, stained with haematoxylin and eosin (Feldman and Wolfe, 2014) as outlined below:

Removal of wax with xylene (dewaxing I and II for 3 minutes) and followed by Hydration with graded alcohol; absolute alcohol for 1 minute; 95% alcohol for 1 minute, 70% alcohol for 2minutes, 50% alcohol for 2 minutes and 30% alcohol 1minute.

Staining: Haematoxylin for 10-20 minutes, distilled water (washing), 35% alcohol for 1 minute. Acid – alcohol for 30 seconds (for differentiation between nucleus and cytoplasm). Followed by Distilled water for 1 minute and Staining in 1 % eosin for 2 minutes.

Dehydration in alcohol (90% alcohol 10 – 15 seconds) followed by absolute Alcohol for 1 minute. Clearing in, Xylene-alcohol for 2 minutes, Pure xylene I for 3 minutes and pure xylene III for 3 minutes. Cleared tissue is mounted In DPX

Statistical analysis: Results obtained were analyzed using statistical software, statistical package for social sciences (IBM SPSS version 21.0, SPSS and Microsoft Office Excel 2007 for charts. Results were expressed as mean ± Standard error of mean (S.E.M) and presence of significant differences among means of the groups were determined using one way analysis of variance (ANOVA) with least significant difference (LSD) post hoc test for significance.

Values were considered significant when p≤0.05.

RESULTS

Eugenol treatment on brain neurotrace element (Iron) following aluminium chloride-induced neurotoxicity, revealed a significant (p< 0.01) reduction in brain iron levels in rats administered 100mg/kg of AlCl3 when compared to control. Treatment with Eugenol, however resulted in significant (p<0.001) elevated level of iron in rats administered 300 mg/kg (eugenol) + 100 mg/kg (AlCl3) and 150 mg/kg (eugenol) + 100 mg/kg (AlCl3) when compared to the group treated with 100 mg/kg AlCl3This elevation was found to be significant (p<0.001). But when comparison is made with the control, the reduced levels of Iron (Fe) in the brain which was observed in rats administered 300 mg/kg (eugenol) + 100 mg/kg AlCl3 and 150 mg/kg (eugenol) + 100 mg/kg AlCl3, was not significant (p>0.05). The Increase iron levels observed in rats administered 300 mg/kg and 150 mg/kg eugenol were found to be not significant (p>0.05) when compared to control. (Figure 1)

Figure 1: Effect of eugenol on neurotrace brain element (Iron Fe) following administration of aluminium chloride on Wistar rats.

n = 5; mean ± SEM One way ANOVA LSD post hoc test: q, s = p<0.01when compared to the AlCl3 y = p<0.001 when compared with control .Group I and II (Eugenol 300mg/kg and 150mg/kg respectively), Group V = (Aluminium chloride 100 mg/kg), Group VI = (Control 2.0ml/kg)

Figure 2: Shows the effect of Eugenol treatment on brain neurotrace element (Magnesium) following aluminium chloride-induced neurotoxicity. This result shows a significant (p<0.01) reduction in brain levels of magnesium in the AlCl3 treated group when compared to the control. Treatment with eugenol, however, revealed a significant (p<0.05) increase in the level of brain magnesium as observed in Groups III and IV when compared to V. However Groups I and II levels of brain magnesium revealed a non significant (p>0.05) difference when compared to control.

Figure 2: Effect of Eugenol on Neurotrace Brain element (Magnesium Mg) following administration of aluminium chloride on Wistar rats

n = 5; mean ± SEM One way ANOVA LSD post hoc test: q = p<0.05 when compared with the AlCl3 treated group; y= p<0.01; when compared with control group respectively. Groups I and II (Eugenol 300mg/kg; 150mg/kg), Group V (Aluminium chloride 100mg/kg), Group VI (Control 2.0ml/kg)

Figure 3: Shows the effect of Eugenol treatment on brain neurotrace element Manganese (Mn) following aluminium chloride-induced neurotoxicity. This result shows a significant (p<0.01) elevated level of brain Manganese in AlCl3 when compared to the control. Treatment with Eugenol however significantly (p<0.05) reduced the manganese level in Groups III and IV when compared to Group V. Comparison of Groups I and II to Group VI reveals a non-statistical significance (p>0.05) between the brain levels of manganese.

Figure 3: Effect of Eugenol on Neurotrace Brain element (Manganese Mn) following administration of aluminium chloride on Wistar rats.

Figure 4: Shows the level of aluminium in the brain following oral administration of aluminium chloride. The result shows a significant (p<0.01) elevation in brain Al levels when AlCl3 is compared to the control. It will be observed that the administration of Eugenol significantly reduced (p<0.05) the level of aluminium as observed in Groups III and IV when compared to Group V.

Figure 4: Effect of Eugenol on Aluminium Brain element following administration of aluminium chloride on Wistar rats.

n = 5; mean ± SEM One way ANOVA LSD post hoc test: q = p<0.05 when compared to the AlCl3 treated group y = p<0.001 AlCl3 treated group is compared to the control group. Groups I and II (Eugenol 300mg/kg; 150mg/kg), Group V (Aluminium chloride 100mg/kg), Group VI (Control 2.0 ml/kg)

Figure 5: Transfer Latency of Wistar rats on Morris water maze habituation.

Group I= Eugenol (300 mg/kg); Group II = Eugenol (150 mg/kg), Group V = Aluminium chloride (100 mg/kg) Group VI = Control (distilled water 2.0 ml/ kg).

Figure 5: shows training latency time in seconds from day 1 to day 4. On day one rats from all groups had an increased latency time when compared to day 2,3 and 4 where there was reduction in latency time.

Figure 6: Effect of Eugenol on Cognition (Morris water maze) following administration of aluminium chloride.

n = 5; mean ± SEM; Paired sample t-test, a,b,c = p<0.05; p<0.01; p<0.001 when comparison is made between pretreatment, day 7, day 14 and day 21 b = p<0.01; c = p<0.001. One way ANOVA LSD post hoc test, x = p<0.05 when comparison is made with the control group at day-14.

Group I = Eugenol (300 mg/kg); Group II = Eugenol (150 mg/kg), Group V = Aluminium chloride (100 mg/kg) Group VI = Control (distilled water 2.0 ml/ kg).

Figure 6: Shows a significant (p<0.05) increase in latency time on Day 14 in the group treated with AlCl3 when compared to pre-treatment and this increase in latency time is significant (p<0.001) when compared to the control (Grp VI) on day 14. Administration of eugenol however was able to reduce latency time this reduction was significant (p<0.05; p<0.001) in Group III and IV when pre-treatment is compared to day 21 .

A B

G

P

P

C

G

P

PV

E

PV

P

G

G

P

PV G

D

G

P

PV

F

PV

G

P

H

P G PV

I J

G

P

P G

K L

P

P

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O

Figure 7 : Shows the micrograph of the section of the cerebral cortex (Layer III and V). A and B shows the histological features of the cerebral cortex of the control rat. C and D shows cerebral cortex (layer III and V) of Group V that was administered 100mg/kg aluminium chloride with perineuronal vacoulations (PV). E and F shows cerebral cortex (Layer III and V) of rats administered 300 mg/kg of eugenol and 100mg/kg aluminium chloride showing mild perineuronal vacoulations. G and H shows the cerebral cortex of rats (Layer III and V) administered 150mg/kg eugenol and 100mg/kg aluminium chloride showing very mild perineuronal vacoulations when compared to the group administered 100mg/kg of aluminium chloride only, I and J shows the cerebral cortex of rats (Layer III and V) administered 300mg/kg of eugenol showing normal histology of the cortex when compared to the control group, L and M shows the cerebral cortex of rats (Layer III and V) administered 150mg/kg eugenol showing a normal histology of the cerebral cortex when compared to the control (Pyramidal cell P, Glial cell G, Oligodendrocyte, O, Perineuronal vacoulations PV).

DISCUSSION

Oral administration of aluminium chloride resulted in extensive neuronal vacuolation and necrosis (neuro-degeneration) of the cerebral cortex of wistar rats (Buraimoh et al., 2012). These degenerative changes could occur in the following ways such as suppression of neuronal energy production (especially mitochondrial energy production) and greatly enhances excitotoxic sensitivity of neurons (Henneberry, 1989; Nicholls and Budd, 1998; Beal et al., 1993).

Aluminium is also known to inhibit or suppress cellular energy-producing enzymes, including mitochondrial electron transport enzymes (Blaylock and Ridgeland, 2004). The clinical importance of neuronal energy suppression by aluminium lies in the fact that mitochondrial energy suppression is intimately connected as an early event to neurodegenerative diseases such as Alzheimer’s dementia and Parkinson’s disease (Meltzer et al., 1996; Schapira et al., 1998). Hence neuronal energy suppression is one of the bases for cellular degeneration within the central nervous system (Gibson et al., 1999).

The main mechanism of aluminium toxicity involves the disruption of the homeostasis of metals, such as magnesium (Mg), calcium (Ca), and iron (Fe) manganese. The physical and chemical properties of aluminium allow it to effectively mimic these metals in their respective biological functions and trigger biochemical anomalies.

Aluminium has been shown to replace Mg and bind to phosphate groups on the cell membrane (Kawahara and Kato, 2011).

Oral exposure to aluminium results in accumulation within the cerebral cortex, cerebellum and hippocampus of the brain and thus affect some essential elements (Fe, Zn, Cu, Mn, and Mg) contents at varying levels (Kruck et al, 2004). Previous studies have correlated neurological disorders to the accumulation of aluminium chloride in the brain of Wistar rats (Mahmoud and Marwa, 2017; Sies and Jones, 2007)

Manganese is an essential mineral for maintaining brain function, manganese toxicity in humans is associated with Parkinsonian-like symptoms such as ataxia and altered balance may develop (Watts, 1990). Exposure to aluminium has been shown to induce changes in the cerebral, cerebellar and hippocampal levels of neurotrace elements (Mahmoud and Marwa, 2017).

In this study exposure to aluminium resulted in increased levels of manganese and this increase was higher than the control group. Increase in the levels of manganese within the brain also act as a prooxidant and hence a toxicant to the brain (elevated amounts) which is deleterious to neurons within the brain. However, administration of eugenol was able to lower brain manganese levels close to normal as observed in Group III and IV.

Magnesium (Mg) is known to play an important role in supporting brain plasticity, this primes the brain for maximal learning, memory and cognitive function. Increasing brain magnesium levels have been shown to restore critical brain Plasticity and thus improves cognition (Slutsky et al., 2010)

In this study, decreased Mg brain levels as observed in aluminium treated group. This is in tandem with the study of Slutsky et al., 2010. Eugenol was able to reverse the reduction in the Mg levels that were induced by aluminium resulting in an increase in Mg levels when compared to the control group. The groups administered eugenol only (Groups I and II) showed elevated brain Mg levels when compared to the control (Group VI). Eugenol’s ability to increase brain Mg levels might be responsible for its cognitive improving properties. In a Eugenol the salvaged groups (Group III and IV) was able to elevate magnesium close to Group VI.

Iron deficiency is not perceived as a life-threatening disorder. But lowered levels of Iron (Fe) has resulted in impaired behaviors including learning (Youdim, 2008).

Results from this study revealed reduced brain iron levels in Group V when compared to Group VI. Also, groups treated with eugenol (III and IV) showed an increase in Fe levels when compared to the aluminium treated group. Rats that received eugenol showed increased levels of Fe When compared to the control group. Reduced Fe levels in rat brains (Group V) might be responsible for cognitive deficits elicited by rats which might result in a defective dopaminergic interaction with the opiate system and cholinergic neurotransmission.

Elevated levels of aluminium in the brain have been associated with neurological diseases such as Alzheimer’s or Parkinsonism (Exley, 2004), which has been attributed to the accumulation of such metals in the brain of affected individuals (Walton, 2012).

Oral exposure to aluminium results in accumulation within the hippocampus of the brain and thus affect essential trace elements (Fe, Zn, Cu, Mn, and Mg) contents in the hippocampus at varying levels (Sies and Jones, 2007). Previous studies have correlated neurological disorders to the accumulation of aluminium chloride in the brain of Wistar rats (Mahmoud and Marwa, 2017). Aluminium has been revealed to affect the homeostasis of brain neurotrace elements which are essential for brain function.

Morris water maze is one of the most widely used tasks in behavioural neuroscience for studying the psychological process and neural mechanisms of spatial learning and memory (Brandies et al., 1989; He et al., 2011). Learning and memory of rats is reflected by escape latency compared to the performance at pre-treatment session.

Increased latency as observed with aluminium treatment at day-7, 14 and 21 is an indication of learning and memory impairment. Memory forms can be classified as declarative or explicit (ability to recall past events deliberately) and are hippocampus dependant; and non-declarative or procedural (implicit), defined by unconsciously performed skills (motor or cognitive) that are mainly dependent on the straitum and cerebellum (Packard and McGaugh, 199). Eugenol treatment showed a decrease in latency time when compared to the aluminium treated group, and administration of Eugenol especially at day-21 of treatment had a neuroprotective effect on aluminium intoxication by decreased latency. Zhibin et al. (2013) also reported that Eugenol can increase learning and memory, using MWM to assess learning and memory.

In this study, light microscopic examination of histological (Haematoxylin and Eosin H&E) sections routinely stained histological sections of the Cerebral cortex –layer III and V were conducted as shown in Figure 7. Neurodegeneration is a process involved in both neuropathological conditions and brain ageing (Kumar and Khanum, 2012). Histoarchitectural distortion of neural tissue manifesting as neuronal degenerative changes are indicative of neurotoxicity in the central nervous system (Nahla et al., 2011; Kalantariapour et al., 2012). Degenerative changes are observed as cortical neuronal shrinkage, perineuronal vacuolations, loss of pyramidal neurone process in sections of the brain studied regions of aluminium-treated rat compared to the control, indicates treatment (aluminium) related neurotoxicity and result obtained from the histological study is in agreement with the studies carried out by buraimoh et al., (2012). However treatment with eugenol was able to protect the histological features of the cerebrum and this is in agreement with the study carried out by Mahmoud and Marwa, 2017.

CONCLUSION

The present study concludes that Eugenol has the ability to protect and enhance brain function by restoring brain neurotrace elements (Iron, Magnesium and Manganese), improving cognitive deficits and preserving histoarchitecture of the cerebral cortex from histoarchitectural changes induced by aluminium.

ACKNOWLEDGMENT

This is to acknowledge Mr Peter Akpulu, chief technologist at the Department of Anatomy Ahmadu Bello University, Zaria Nigeria.

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