Human and animal populations interact with their environment via food, air and water on a daily basis, as such exposes them to toxic substances, such as chemicals and heavy metals, capable of causing harm  or even death (Wade et al., 2002; Burger et al., 2013). Heavy metals are natural constituents of the earth crust, their biochemical balance are easily altered by human needs for improving quality of live and well-being (Das  et al., 2014). Once present in the environmenteven in trace amount can pose a serious problem for all organism and prolong exposure creating a deleterious health effect in humans, since they cannot be degraded or destroyed (Chen and Chen, 2001; Sedbrook, 2016). Lead is one of the common toxic heavy metals due to the ease in mining and refining. It is used in building construction, making of water pipes, lead-acid batteries, bullets and shot, weights, as part of solders, pewter’s fusible alloys, as well as radiation shield (Duah et al., 2012). The wide use of lead had turned lead poisoning into an ever present environmental and health challenge, hence the increased blood-lead level (Ahmed et al., 2013). Lead exposure affect both central and peripheral nervous system resulting to nerve cell degeneration and demyelination (Sanders et al., 2009; Abeer, 2012; Assi et al., 2016). 

The cerebrum is rostral most part of the brain responsible for higher brain function, such as motor movements, perception of stimuli, emotions, problem solving and recognition (Singh, 2002; Owolabi et al., 2014). The cerebrum is vulnerable to damage from a variety of sources such as developmental defects, degenerative diseases, infectious processes, trauma and tumors (Klementiev et al., 2007).  Heavy metals exposure which lead happen to be one, have been reported to be one of the leading cause of cerebral injuries (Korogi et al., 2011; Fonfria et al., 2005; Wagner et al., 2010; Owoeye and Farombi, 2015).

Pharmacotherapy and psychoactive drugs in the last two decades have gain recognitions, due to its efficacy in the management of neurological related disorders. However, several studies have revealed that such relieve are temporal with manifestation of various side effects (Handa, 1995; Mireille et al., 2017). Traditional medical practice has gained interest in the world over due to the wide spread usage of medicinal plants and its consumption, especially in developing countries (Ashafa and Olunu, 2011; Sujith et al., 2012).

Phoenix dactylifera L. (date palm) and its various parts are widely used in folk medicine for the treatment of various ailment and disorders, such as memory disturbance, fever, inflammation, paralysis, and even nervous disorders (Nadkarni, 1976; Elgindi et al., 2015; Alhaider et al., 2017). Several researchers have documented on the rich nutritional value, high dietary fibre and essential mineral of date palm, such as phosphorus, iron, potassium and a significant amount of calcium and vitamins (Mohamed and Al-Okbi, 2004; Usama et al., 2009; Yusuf et al., 2017). Several studies on extracts of date palm have indicated the presence of antioxidant properties (Mansouri et al., 2005; Al-Qarawi et al., 2008; Agbon et al., 2016); these antioxidant activities are attributed to a wide range of phenolic and flavonoid compounds and some Vitamin in date palm (Vayalil, 2012; Benmeddourt et al., 2013).  

The aim of this study was to histologically and histochemically assess the therapeutic  effect  of  ethanol fruit extract of P. dactylifera (EFPD) against lead acetate-induced cerebral alterations in Wistar rats.

Plant collection and identification

Dried P. dactylifera (date palm) fruits were obtained at a local market (Samaru) in Zaria, Nigeria and was authenticated and given a Voucher Specimen Number of 7130, at the Herbarium Unit of the Department of Botany, Faculty of Life Sciences, Ahmadu Bello University, Zaria, Kaduna State, Nigeria.

Extract preparation and phytochemistry

Extraction of P. dactylifera fruit and phytochemical screening were conducted in the Department of Pharmacognosy and Drug Development, Faculty of Pharmaceutical Sciences, Ahmadu Bello University, Zaria. The method of maceration as reported by Agbon et al. (2013) for the preparation of ethanol fruit extract of P. dactylifera was adopted. The method of Trease and Evans (2002) as reported by Oni et al. (2015) was adopted for photochemical screening.

Experimental animals

Twenty Wistar rats (male and female; 100 to 180 g) were obtained from Animal House of the Department of Human Anatomy, Faculty Basic Medical Sciences, College of Health Sciences, Ahmadu Bello University, Zaria and housed in new wired cages in the same animal house were rats acclimatized for two weeks prior to the commencement of the experiment. The rats were separated into five groups; one control and four treatment groups. The rats were housed under standard laboratory condition, light and dark cycles of 12 h and were provided with standard rodent pellet diet and water ad libitum. The treatment groups were administered, in addition to feed and water, lead acetate/EFPD/DMSA for a period of five weeks. The rats were weighed before and after the experiment and weight changes were computed and analysed.    

Drug

Lead acetate (Analytical) manufactured by British Drug Houses (BDH) Laboratory Chemicals Division, Poole, England, was obtained and used as neurotoxin for the experiment.

Dimercaptosuccinic acid (DMSA, Analytical) manufactured by Best of Chemical (BOC) Sciences, New York, USA was obtained and used for the experiment as standard chelating drug.

Experimental procedure

Twenty Wistar rats were grouped into five groups (A to E) of four rats each. Group A (control) was administered distilled water (2 ml/kg), while groups B to E were treatment groups. Cerebral damage was induced in rats by the administration of lead acetate (120 mg/kg; 20% LD₅₀) (Sujatha et al., 2011) as reported by Yusuf et al. (2017). Groups B, C, D and E were administered lead acetate (120 mg/kg) for a period of 3 weeks, after which groups C and D were administered ethanol fruit extract of P. dactylifera (500 and 1,000 mg/kg, respectively, that is, 10 and 20% of LD₅₀ oral in rats (Agbon et  al.,  2017)  and  group  E  was  administered  DMSA  (10 mg/kg) (Chen et al., 1999) for an additional period of 2 weeks. All administrations were via oral route, once daily.

Histological and histochemical studies

At the end of the experiment, rats were euthanized and brain organs were harvested. Harvested brain organs were fixed in Bouin’s fluid and tissues processed using routine histological techniques, stained with Haematoxylin and Eosin (H&E) and histochemical stains (Cresly Fast Violet [CFV] for Nissl substance and Bielschowsky for neurites/nerve fibres) for light microscopic examination.

Data analysis

Results obtained were analysed using the statistical software, Statistical Package for Social Sciences (IBM SPSS version 20.0) and results were expressed as mean ± standard error of mean (SEM). Presence of significant difference among mean of the groups was determined using one-way analysis of variance (ANOVA) with Tukey post hoc test. Paired sample t-test was employed for the comparison of means as appropriate. Values were considered significant when p Ë‚ 0.05.

Phytochemical analysis

Phytochemical analysis of ethanol fruit extract of P. dactylifera (EFPD) produced positive reaction for secondary metabolite and negative for some shown in Table 1.

Physical observation

During the period of administration, physical activities of the rats were observed. Rats in the control group were observed to exhibit normal physical activities, such as movement and playfulness, whereas rats in the treatment groups exhibited aggressiveness and decreased activity, especially in lead acetate-treated group.

The weight of the rats in all groups were observed to have increased (p>0.05), except in the lead acetate-treated group (p<0.05) when initial and final weights were compared. However, there was no significant difference (p>0.05) in weight change (difference in initial and final weights) when treated groups were compared with the control (Table 2).

Histological and histochemical examination

Histological examination of sections of cerebral cortex of rats stained with routine (H&E) histological and histochemical (CFV and Bielschowsky) stains revealed the following.

The cerebral sections of rats in the control group revealed normal histoarchitecture of the cerebral cortex with distinctive appearance of cortical neurones arranged in six layers (I to VI). Morphology of the ganglionic layer (Layer V) revealed dense distribution of large pyramidal cells; ganglion or Betz cells and glial cells. Histochemical (CFV) staining for Nissl substance revealed normal appearance of distinct intensely stained pyramidal cells and Bielschowsky revealed normal neuronal fibres (Plate 1A).

The cerebral sections of lead acetate-treated rats revealed histo-architectural degeneration of neurons such as satellitosis, perineuronal vacuolations, necrosis and neuronal cytoplasmic shrinkage. CFV staining revealed chromatolysis, cytoplasmic shrinkage and indistinct staining intensity and Bielschowsky stain revealed pyramidal neuronal degeneration as loss of neuronal fibres when compared with the control (Plates 1B, 2B and 3B).

Examination of the cerebral sections of ethanol fruit extract of P. dactylifera (500 and 1,000 mg/kg)-treated rats, revealed mild cerebral cortex histo-architectural distortions such as perineuronal/cytoplasmic vacuolation and pkynosis; CFV staining showed reduced staining intensity and Bielschowsky staining revealed loss of neuronal fibres when compared with the severe distortions of the lead acetate-treated group (Plates 1C, 1D, 2C and 2D).

The histological features of the DMSA-treated group revealed relatively similar histo-architecture, with mild distortion, when compared with control (Plates 1E, 2E and 3E).

In this study, phytochemical analysis of ethanol fruit extract of P. dactylifera revealed the presence of secondary metabolites such as, flavonoids, saponins, tannins and alkaloids which have been reported to have neuroprotective activities (Chan et al., 2012; Wan Ismail and MohdRadzi, 2013; Hwang et al.,  2015).  This  finding is in agreement with the reported phytochemical constituents in fruit extract of P. dactylifera L. (Faqir et al., 2012; Abiola et al., 2017).

Reduced physical activity observed among the lead acetate-treated rats, reflects treatment-related toxicity. This agrees with the findings on drug-related toxicity and physical activity as reported by Agbon  et  al.  (2014)  and Yusuf et al. (2017). Body weight changes are strong pointers of general health status and drug related toxicity in animals (Mukinda, 2007; Salawu et al., 2009).

Significant weight decrease observed in lead acetate-treated rats is indicative of heavy metal related toxicity. Heavy metals intoxication, have been implicated for poor appetite and malabsorption of nutrients in animal  models (Jadhav et al., 2007; Wadaan, 2009). Finding is in accordance with the report of Yusuf et al. (2017) which observed remarkable decrease in weight in lead-exposed rats. Body weight gain observed in EFPD-treated rats could be attributed to the plant’s wide range of essential nutrients with high caloric value, particularly carbohydrates and lipids (Shaba et al., 2015; Punia, 2016; Megbo et al., 2017). This finding is in agreement with the report of Wahab et al. (2010) and Haouas et al. (2014).

Neuronal degeneration have been identified as one of the major causes of neuronal cell apoptosis (cell death), which could be as a result of disease (pathological) or natural (aging) condition (Mazanetz and Fischer, 2007; Kumar and Khanum, 2012) exerting extrinsic insults or traumatic stresses on the cells (Kumar et al., 2009; Sivanandam and thakur, 2012). Characteristic neuronal histo- and cyto-architectual distortions, such as neuronal shrinkage, perineuronal vacuolation, satellitosis, and indistinct staining intensity, loss of pyramidal neuron processes, necrosis and pkynosis observed in  this  study are indicators of neurodegenerative changes. Satellitosis observed in this study is indicative of treatment related toxicity, a condition marked by an accumulation of neuroglia cells around damaged or necrosed neurones of the central nervous system, often as a prelude to neuronophagia (Manickam et al., 2009). Findings are in consistence with the reports on neurodegenerative changes following heavy metals (lead, mercury, cadmium) exposure. These metals have capacity to induce nervous tissue insult (Amal and Mona, 2009; Fakunle et al., 2013; Wani et al., 2015; Butt et al., 2018) and disrupt the release mechanism of calcium-dependent neurotransmitter and DNA repairs (Hartwig, 1994; Sharma et al., 2014).

In light microscopy, rough endoplasmic reticulum and free ribosomes appear as basophilic granular areas (Nissl bodies) with Cresy Fast Violet staining. Neuronal degeneration has been related to reduction in Nissl substances (Akassoglou et al., 2004) involving degradation of β-tubulin, an important component of the neuronal cytoskeleton, and these effects  are  evoked  by N-methyl D-aspartate receptor (NMDAR) function (Xu et al., 2012). Findings in these studies are in accordance with the reports of Ajibade et al. (2012) and Agbon et al. (2014) with loss of Nissl substance in cerebellar neurones and shrinkage of the nucleus following chemically induced toxicity. The brain among other tissues and organ with high lipid content is the most sensitive target of heavy metal intoxication. Lead neurotoxic effect results from its ability to cross the blood brain barrier readily and influence alteration of certain membrane bound enzymes responsible for protecting the biological systems against reactive oxygen species (ROS) and oxidative stress (Xu et al., 2008; Yun et al., 2011; Fakunle et al., 2013; Ibegbu et al., 2013). Naqi (2015) and Owolabi et al. (2017) reported cortical cerebellar histo-architectural distortions on exposure to lead in rats. Bielschowsky staining technique demonstrates neuronal processes of the central nervous system. Loss of neuronal processes observed in the lead-treated rats is indicative of treatment related toxicity. Heavy metals have been reported to disrupt micro-skeletal like structures in the brain cells (Leong et al., 2001).

Neuronal injury may result in reversible or irreversible cell damage or cell death (Kumar et al., 2006). Mild cortical cerebral histo-architectural distortion in rats treated with DMSA and EFPD (500 and 1000 mg/kg) after exposure to lead acetate, was observed when compared with the severe histo-architectural distortions observed in the lead acetate-treated group.

In this study, histo-architectural features of DMSA (succimer)-treated group were comparable with the control. Thus, indicates the therapeutic activity of succimer. Succimer is an established metal ion chelating agent recommended for the treatment of heavy metal toxicity, such as lead (Kalia and Flora, 2005; Lowry, 2010).

Natural agents with antioxidant properties are beneficial in attenuating drug-induced oxidative stress in biological systems (Musa et al., 2012; Bauchi et al 2016). P. dactylifera antioxidant activities are attributed to a wide range  of phenolic  phytochemical  constituents   (Vayalil, 2012; Benmeddourt et al., 2013). Flavonoid, a polyphenolic compound, found in P. dactylifera fruits have been reported to have strong ROS scavenging and metal ions chelating activities; an antioxidant that shields neurones from lethal damage and has ability to suppress neuroinflammation (Pujari, 2011; Rice, 2001; Komaki et al., 2015). Findings are in line with the reported ameliorative potentials of P. dactylifera fruit extract; Agbon et al. (2017) reported ameliorative activity of ethanol fruit extract against inorganic mercury induced cerebral and cerebellar alterations in Wistar rats and Yusuf et al. (2017) reported neuroprotective activity on lead acetate-induced toxic effects in cerebellum. Thus, indicating the therapeutic potentials of EFPD on heavy metals induced cortical cerebral alterations.

In light of the observed results of the present study, it could be concluded that ethanol fruit extract of P. dactylifera possess therapeutic potentials against lead acetate-induced cortical cerebral alterations in Wistar rats. The therapeutic property of the extract, comparable to the standard drug, DMSA, could be attributed to the antioxidant properties of its constituent phytochemicals, such as flavonoid.

The authors have not declared any conflict of interests. 

The authors thank the Ahmadu Bello University, Zaria, Kaduna State, Nigeria for providing an enabling environment to conduct this study and appreciate the following research assistants: Ivang, Andrew, Usman, M. Ibe, and Makena Wusa for their technical support and the Department of Human Anatomy Department, Faculty of Basic Medical sciences, College of Health Sciences, Ahmadu Bello University, Zaria for providing the facilities to conduct this study.