Curcumin‐loaded cockle shell‐derived calcium carbonate nanoparticles ameliorates lead‐induced neurotoxicity in rats via attenuation of oxidative stress

Abstract A substantial global health burden is associated with neurotoxicity caused by lead (Pb) exposure and the common mechanism of this toxicity is mainly via oxidative damage. Curcumin has remarkable pharmacological activities but remains clinically constrained due to its poor bioavailability when orally administered. Currently, cockle shell‐derived calcium carbonate nanoparticle (CSCaCO3NP) is gaining more acceptance in nanomedicine as a nanocarrier to various therapeutics. This study aimed at investigating the ameliorative effect of curcumin‐loaded CSCaCO3NP (Cur‐CSCaCO3NP) on lead‐induced neurotoxicity in rats. A total of 36 male Sprague–Dawley rats were randomly assigned into five groups. Each group consists of 6 rats apart from the control group which consists of 12 rats. During the 4 weeks induction phase, all rats received a flat dose of 50 mg/kg of lead while the control group received normal saline. The treatment phase lasted for 4 weeks, and all rats received various doses of treatments as follows: group C (Cur 100) received 100 mg/kg of curcumin, group D (Cur‐CSCaCO3NP 50) received 50 mg/kg of Cur‐CSCaCO3NP, and group E (Cur‐CSCaCO3NP 100) received 100 mg/kg of Cur‐CSCaCO3NP. The motor function test was carried out using the horizontal bar method. The cerebral and cerebellar oxidative biomarker levels were estimated using ELISA and enzyme assay kits. Lead‐administered rats revealed a significant decrease in motor scores and SOD activities with a resultant increase in MDA levels. Furthermore, marked cellular death of the cerebral and cerebellar cortex was observed. Conversely, treatment with Cur‐CSCaCO3NP demonstrated enhanced ameliorative effects when compared with free curcumin treatment by significantly reversing the aforementioned alterations caused by lead. Thus, CSCaCO3NP enhanced the efficacy of curcumin by ameliorating the lead‐induced neurotoxicity via enhanced attenuation of oxidative stress.


| INTRODUC TI ON
Lead (Pb) is a ubiquitous environmental toxic metal that is used in agriculture and modern industries, which consequently causes numerous harmful health effects to man and it is now an important public health burden (Patrick, 2006a). The continuous usage of lead owing to its beneficial physicochemical properties yet harmful to health is from antiquity to modern days (Ansar et al., 2019;McQuirter et al., 2013). Consequently, continuous human exposure to lead is becoming inevitable leading to a significant global challenge (Carrington et al., 2019;Ming et al., 1997). Several health consequences in adults are linked to occupational exposure to lead which remained a major source of lead toxicity (Bhattacharjee et al., 2018). Lead can interrupt normal biological function by causing inflammations and oxidative stress via several pathways leading to cell degeneration and death (Lakshmi et al., 2013;Mason et al., 2014). Neurotoxins such as lead produces an adverse effect on the nervous system resulting in several neuropathological and neurological disorders (Wani et al., 2015). Lead-induced neurotoxicity and neurological disorders are characterized by cognitive deficit, impaired motor function, attention deficit, dullness, low IQ, hyperactivity, and antisocial problems among others (Vlasak et al., 2019).
Preceding studies on humans and experimental animals have documented several lead-induced neurotoxic insults. For example, a significant decrease in motor scores and SOD activity with a resultant increase in MDA with evidence of lead concentration, which resulted in marked histological alterations in the cerebellar cortex of rats induced with 50 mg/kg of lead, were documented (Abubakar, Muhammad Mailafiya, et al., 2019); also, a study has shown that rats exposed to lead at 7.5 mg/kg body weight for 14 days resulted in significant oxidative alterations and histological degeneration in the rats' cerebral cortex and blood-brain barrier (Singh et al., 2017), documented studies reported encephalopathy as a direct consequence of lead exposure which is characterized by dullness, irritability, headache, attention deficit, loss of memory, hearing loss, etc. in children exposed to lead poisoning (Paul & Gupta, 2018), and another study linked the lead exposure to be the major cause of peripheral nervous system dysfunction in adult while the central nervous system is more prominently affected in children (Bellinger et al., 2018;Bose-O'Reilly et al., 2017;Plumlee et al., 2013).
Noteworthy, in the absence of consistent standard neurotherapeutic drugs in allopathic medicine, herb extracts display therapeutic functions in the treatment of many lead-induced neurotoxicity and other related organ toxicity (Hewlings & Kalman, 2017;Shaikh et al., 2009). Nevertheless, many of these available herbs are insoluble, which limits their absorption and subsequent bioavailability (Marslin et al., 2018;Paul & Gupta, 2018;Sharma et al., 2005).
Among the insoluble herbs, curcumin possessed an old documented medicinal history that mirrored the current field of nanomedicine, drawing numerous attention of researchers due to its wide safety margin and health benefits such as antioxidant, anti-inflammatory, and neurotherapeutic activities (Chirio et al., 2019;Mofazzal Jahromi et al., 2014). In spite of all the commendable curative properties of curcumin, the major drawback of poor bioavailability due to poor aqueous solubility, poor absorption from the intestine, rapid metabolism in the liver, and high degree of elimination in the bile has constrained its clinical applications (Priyadarsini, 2014;Yadav et al., 2012). Hence, searching for a safe and potential delivery system that will overcome such limitations to ensure safe delivery within a biological system thereby enhancing curcumin therapeutic efficacy has become the most fascinating and desired area of research in nanotechnology (Basniwal et al., 2014).
Cockle shells from a natural marine source have recently been used as a nanocarrier for the delivery of various therapeutic agents for chemotherapy and antibacterial purposes (Danmaigoro et al., 2017;Hammadi et al., 2017;Isa et al., 2016). This natural biogenic material is a strong source of abundant calcium carbonate existing in aragonite polymorphic form (Hoque et al., 2014;. Its outstanding potential ability to safely deliver several anticancer and antibacterial agent was demonstrated in previous literatures Hamidu et al., 2019;Isa et al., 2016). To date, no study to the best of our knowledge have yet documented cockle shell-derived calcium carbonate nanoparticles (CSCaCO 3 NP) as effective delivery of antioxidant such as curcumin in vivo.
Exposure to metals such as lead has been reported to be one of the leading causes of cerebral and cerebellar toxicity (Sidhu & Nehru, 2004). Cerebrum is the largest part of the brain responsible for superior brain functions such as motor movement, emotions, learning, and recognition, while the cerebellum is the major structure of the hindbrain responsible for motor coordination and balance (Lazarus et al., 2018;Mahmoud & Sayed, 2016). Both cerebellum and cerebrum are delicate structures that are vulnerable to intoxication resulting in a deficit of cognitive abilities and impaired motor coordination and balance (Bhattacharjee et al., 2018;Patrick, 2006b). A previous study reported the common direct culprit of environmental lead exposure to be via ingestion, particularly in drinking water. (Bhattacharjee et al., 2018) Furthermore, lead exposure even at a low level resulted in several pathological conditions with a great impact on the nervous system of the populations exposed Husain, 2015). Thus, regardless of the higher amount of lead exposure, cumulative dose of lead and vulnerability of the individual are strongly linked to health consequences (Bose-O'Reilly et al., 2017;Kim et al., 2014). Hence, in this study, the choice of oral administration of lead at a dose of 50 mg/kg was adopted in order to mimic the environmental exposure of lead to organisms.
The dose of free curcumin at 100 mg/kg revealed non-toxic effects in rats; in fact, a higher dose of curcumin showed no sign of toxicity, indicating its wide safety margin (Sarada et al., 2015;Zhang et al., 2018). Furthermore, the toxicity evaluation of CSCaCO 3 NP in both rats and dogs indicated that CSCaCO 3 NP have a wide safety margin to the biological system in vivo (Danmaigoro et al., 2018;Jaji, Zakaria, et al., 2017). In addition, several in vitro studies reported the great safety and biocompatibility effect of CSCaCO 3 NP on various cell lines Hamidu et al., 2019;Kamba et al., 2014;.
Previous studies have emphasized the targeted effects of different nanoparticles for curcumin's delivery for the treatment of various heavy metals-induced neurodegenerative diseases (Kakkar & Kaur, 2011;Sandhir et al., 2014). However, the current research may be an added advantage because it stressed not only on the targeted effect mechanism of CSCaCO 3 NP but also the ability of the nanoparticle to enhance the therapeutic effect of curcumin. Therefore, the study generally aimed at evaluating the ameliorative effect of curcumin-loaded cockle shell-derived calcium carbonate nanoparticles (Cur-CSCaCO 3 NP) on lead-induced neurotoxicity in rats via behavioral, biochemical, histological, and histochemical assessments. CA, USA), and superoxide dismutase (SOD) assay kit (E-BC-K020, Elabscience Biotechnology Inc.). All other reagents and chemicals used were of high analytical grade quality and higher purity.

| Synthesis of CSCaCO 3 NP and Cur-CSCaCO 3 NP
The preparation, synthesis, loading processes, and physicochemical characterizations of CSCaCO 3 NP and Cur-CSCaCO 3 NP as well as the in vitro kinetic release were previously described in the work of  Noteworthily, based on the protocol and procedure of the author's previous study, the best formulation of Cur-CSCaCO 3 NP that gives the best loading content and good encapsulation efficiency was selected for curcumin delivery.

| Experimental design
Following 1 week of acclimatization, the rats were randomly assigned into five groups (A, B, C, D, and E), comprising six rats each; except for control group A, which consists of 12 rats. The groups were as

| Dose preparation of lead and Cur-CSCaCO 3 NP
The lead solution was prepared by dissolving 1 g of lead acetate in 50 ml of deionized water to form a stock solution of 20 mg/ml of lead acetate concentration. Each rat in all the groups with the exception of the rats from control group A was given a dose of 50 mg/kg three times based on their body weight (Ayuba & Ekanem, 2017;Owolabi et al., 2012;Sidhu & Nehru, 2004).
The Cur-CSCaCO 3 NP was weighed to get the exact number of milligrams per kg needed for the rats (i.e., 100 and 50 mg/kg) from which a stock solution was made by dissolving it in 50 ml of deionized water. Each rat (in groups D and E) was given a dose of 50 and 100 mg/ kg three times a week, respectively, based on the body weight.

| Weekly body weight measurement and physical observations
During the period of the experiment, normal physical activities of the rats were observed: daily water intake, voluntary feed intake, fecal output, weight loss, and gain were observed. Starting from week 0 to week 8, weight gain and weight loss were recorded at 1-week intervals for each rat. This was done to monitor the trend of body weight throughout the period of the experiment. The recorded body weights within the study period were subjected to statistical analysis using two-way ANOVA.

| Motor activity test
To monitor the trend of the rat's ability of motor coordination, their forelimb grip balance was tested starting from week 0 to week 8 using a horizontal bar method. The rats were transported into the training room 1 h before the start of the experiment for the rats to adjust to their new environment. The entire motor activity test was done between 9 am and 1 pm.

| Horizontal bar method (HBM)
The method measures forelimb strength and coordination. The rat's ability to grip the bar using their phalanges was assessed weekly.
This method involves the use of 38-cm-long and 2-mm-diameter metal bar, suspended horizontally above 49 cm height with end-toend supports of a laboratory clamp, and a padded surface to ensure rat's soft landing when falling. Each rat was held by the tail, carefully placed at the central point of the metal bar, and allowed its forepaws to grasp the bar, and the tail was released immediately after grasping at the same time the stopwatch was started to measure the time. The translation of the time into scores in this study was done F I G U R E 1 Schematic diagram of the experimental design showing complete periodic activities of the experimental rats. Note: Lead-treated groups (LTG), curcumin 100 mg/kg (cur 100), curcumin-loaded cockle shell-derived calcium carbonate nanoparticles at the dose of 50 and 100 mg/kg, respectively (Cur-CSCaCO 3 NP 50 and 100), enzyme-linked immunosorbent assay (ELISA), lead (lead).

TA B L E 1
Horizontal bar method scoring system (Deacon, 2013)

Falls Time (seconds) Scores
Falling between 1-5 1 Falling between 6-10 2 Falling between 11-20 3 Falling between 21-30 4 Falling after 30 5 Without falling -5 Note: If the experimental animal grasps the bar properly and moves from end to end of the bar without falling, then a maximum score (5) was allotted. All rats underwent the test in three different attempts with brief resting intervals to obtain the best score and prevent error.
in accordance with the intense description by the previous work Deacon (2013). The scoring procedure is shown in Table 1.
The horizontal bar method in this study was used to assess the effect of Cur-CSCaCO 3 NP on the motor coordination initiated by the cerebrum of lead-induced rats. However, to avoid possible confounding factors that may arise when investigating the effect of Cur-CSCaCO 3 NP in the brain of lead-induced Sprague-Dawley rats for the first time, this research confirmed the use of male instead of female gender. This is because female cyclical hormonal changes usually affect their mood swings, thus, sex hormones such as prolactin, progesterone, and estrogen may arbitrarily influence feeding habit, emotion, motor behavior, and cognitive function during the experiments as stated in previous literatures Frye, 2010).

| Sample collection
At the end of the experimental period (8 weeks), all the rats were euthanized, and the brain was harvested, washed thrice in ice-cold saline, and weighed. The brain tissues were separated into two; one portion was stored at −80°C for SOD and ELISA assays and the other portion was preserved in 10% buffered formalin for histological and histochemical analyses. The brain tissue stored at −8°C for MDA and SOD assays was allowed to thaw. Cerebellum and cerebrum were isolated and then homogenized with ice-cold phosphate-buffered saline (PBS) (0.01 M, pH = 7.4) in a volume of 20 times the weight of the tissue to prepare 10% cerebral and cerebellar homogenates at the ratio of 9:1.
The homogenates were centrifuged at 5000 × g for 5 min at 4°C and the final aliquot of the supernatant was separated and kept at −80°C.

| Protein estimation
The total protein concentration of the cerebrum and cerebral tissues was measured using the bicinchoninic acid assay (BCA assay).

| ELISA and SOD activity analyses
Malondialdehyde (MDA) level was detected from the rats' cerebellum and cerebrum homogenates base on the simple principle of competitive ELISA using the MDA ELISA kit (E-EL-006, Elabscience).

| Hematoxylin and eosin (H&E)
The fixed tissues were processed for histological evaluation as earlier described by Danmaigoro et al. (2018) Briefly, the brain tissues were trimmed and dehydrated in ascending concentration of alcohol, cleared in xylene, and further embedded in paraffin wax.
Furthermore, the tissue was trimmed and sectioned to approximately 5 μm thick in size. The sectioned brain tissues were stained using the techniques for standard Harris's hematoxylin and eosin for normal histology and histopathological studies and examined under the light microscope. The degree of tissue injury, necrosis, and inflammatory responses were analyzed.

| Histochemical analysis
The sectioned ribbon brain tissues (cerebellum and cerebrum) were stained using toluidine blue as a special stain. The following reagents were used to prepare the stain; colophonium (resin) 10 g, 95% alcohol 100 ml, toluidine blue 0.1 g, Distilled water 100 ml, and 10% solution of aniline in 95% alcohol. The section tissues were totally immersed in xylene, absolute alcohol, and 95% alcohol. After which they were dipped in alcoholic colophonium solution for 3-5 min and rinsed in two changes of 95% alcohol (3 min each), then stained with toluidine blue (30 s), followed by differentiation in aniline-alcohol and cleared in xylene (two changes) again and, finally, mounted in synthetic resin.

| Statistical analysis
All analyses were conducted using GraphPad Prism (GraphPad Prism software, Inc, Version 6.01, San Diego, California, USA) and SPSS. Differences in p values <0.05 were statistically significant for the purpose of comparison. The data obtained were presented as mean ± standard error of the mean (SEM). Data obtained from the horizontal bar method (HBM) and weekly body weight (WBW) were analyzed using repeated measures followed by Tukey's post hoc test. The data obtained from the effect of lead on various parameters were conducted using Student's unpaired samples t-test while the data obtained from histology, ELISA, and SOD analysis were all analyzed by one-way ANOVA followed by Tukey's post hoc test.

| Physical observations
At the early weeks of lead induction, the rats showed no pronounced physical evidence of toxicity. Subsequently, decreased feed and water intake with minimal gross evidence of toxicity (i.e., rough fur and slight body weakness) were observed among all the lead-treated groups of rats in the subsequent weeks of lead induction. However, treatments with Cur-CSCaCO 3 NP remarkably improved the eating habit of the rats and reduced their body weakness.

| Ameliorative effects of Cur-CSCaCO 3 NP on the motor score of rat motor functions
Repeated measures showed a statistically significant interaction between the effect of treatment and the weeks of treatment [F (24, 160) = 3.780, p < 0.0001] in the motor activities score of the rats. Tukey's post hoc test showed a statistically significant decrease (p < 0.05) in the motor score of the rats for their ability to maintain a forelimb grip balance on weeks 3, 4, and 5 by the Cur 100, Cur-CSCaCO 3 NP 50, and Cur-CSCaCO 3 NP 100 groups when compared to the control group of rats. Subsequently, a similar trend was also observed on week 6 of the test where a statistically significant decrease (p < 0.05) in the motor score of the rats for their ability to maintain a forelimb grip balance by rats of Cur 100 and Cur-CSCaCO 3 NP 50 groups when compared to the control group. Conversely, a statistically significant increase (p < 0.05) was observed in the motor score of the rats on week 6 by the control and Cur-CSCaCO 3 NP 100 groups when compared to the Cur 100 group of rats. Furthermore, a statistically significant decrease was observed (p = 0.0367) in the motor score of the rats for their ability to maintain a forelimb grip balance on week 7 by the Cur 100 when compared to the control group of rats. No statistically significant differences were observed in all the groups when compared to the control group on week 8 as shown in Figure 3.
F I G U R E 2 Effect of lead on the motor score of rats after 4 weeks of induction. Values were presented as mean ± SEM, n = 6. *p < 0.05 versus control group.

| Effect of lead on body weight of rats
To evaluate the effect of oral administration of lead on body weight,

| Effect of Cur-CSCaCO 3 NP on body weight of rats
To

| Effects of lead on the weight of organs
Unpaired sample t-tests were conducted to compare the weight of the cerebrum and cerebellum of rats treated with lead (50 mg/ kg) and that of the control group. There was a significant F I G U R E 3 Effect of cur-CSCaCO 3 NP and curcumin on the motor score of rats exposed to lead. Values were presented as mean ± SEM, n = 6. *p < 0.05 versus control, #p < 0.05 versus Cur 100.

F I G U R E 4 Effect of lead on the rats' body weight after 4 weeks of induction.
Values were presented as mean ± SEM, n = 6. *p < 0.05 versus control group.

| Effects of Cur-CSCaCO 3 NP on the weight of organs
As shown in Figure 7, one-way ANOVA revealed statistically significant difference in the weights of the cerebellum and cerebrum

| Effect of lead on SOD activities
The activity of SOD in the cerebellum and cerebrum of the lead-

| Effect of Cur-CSCaCO 3 NP on SOD activities
As shown in Figure 9,

| Effect of Cur-CSCaCO 3 NP on MDA level
As shown in Figure 11, based on the ELISA results, the one-way ANOVA revealed statistically significant differences in MDA

| Histological examination of the cerebral cortex using H&E stain after lead induction
The histological section in Figure 12a showed the normal histological structure of the cerebral cortex and normal neuronal cell distributions and cellular morphology of the cerebral cortex in the control group of rats. However, the section in Figure 12b

| Histochemical examination of the cerebral cortex using toluidine blue stain after lead induction
To further confirm the neurodegenerative effect of lead on the cerebrum of rats, a special stain was performed using a toluidine blue stain. The section from rats treated with lead (LTG) shows cellular degenerations with hyperchromatic neuronal cells (Figure 13b) when compared with a cerebral section of rats from the control F I G U R E 7 Effects of Cur-CSCaCO 3 NP and curcumin on the weight of organs in rats exposed to lead. Values were presented as mean ± SEM, n = 6. #p < 0.05 versus Cur 100 group

| Histological examination of the cerebral cortex using H&E stain after Cur-CSCaCO 3 NP treatment
The histological section in Figure 14b showed that the neuronal cells had lost their characteristic shapes and appeared irregular.
The neuronal cells appeared darkly stained with pyknotic nuclei. Some cells are multipolar with vacuolar space around them.

| Histological examination of the cerebellum using H&E stain after lead induction
Sections in Figure 16a  Values were presented as mean ± SEM, n = 3. *p < 0.05 versus control F I G U R E 9 Effect of Cur-CSCaCO 3 NP and curcumin on superoxide dismutase (SOD) activities in the cerebellum and cerebrum of lead-administered rats after 4 weeks of treatment. Values were presented as mean ± SEM, n = 3. #p < 0.05 versus Cur 100.

cells. Further prominent alterations including cellular shrinkage, scattered glia cells, and hyperchromatic Purkinje cells appeared
to be surrounded by vacuolar space (Figure 16b).

| Histochemical examination of the cerebellum using toluidine blue stain after lead induction
The toluidine blue-stained section from rats treated with lead (

| Histochemical examination of the cerebellum using toluidine blue stain after Cur-CSCaCO 3 NP treatment
Histochemical examination of the sections of the cerebellum from rats of Cur 100 showed alteration in the Purkinje cell layer with few degenerated Purkinje cells. However, the molecular and granular layers appeared to be normal (Figure 19b). Furthermore, the cerebellar section from the Cur-CSCaCO 3 NP 50 and Cur-CSCaCO 3 NP 100 showed restoration of the healthy Purkinje cells.
The molecular, Purkinje, and granular layers appeared to be normal as shown in Figure 19c

| DISCUSS ION
Previous studies on rats and humans have shown that exposure to lead could induce a series of pathological alterations resulting in serious health implications, manifesting different pernicious effects on multiple organs, particularly the brain (Abubakar,  (Husain, 2015), and loss of integrity of blood-brain barrier (Beata et al., 2007). In this study, lead induction in rats revealed marked loss of brain and body weights. In addition, poor performance performance (Adolph & Franchak, 2017;Deacon, 2013;Ivens & Machemerf, 1998). On the account of this, the motor functions of rats in the control group improved by the subsequent weeks in this study, owing to their excellent learning skills, which contributed to their strong motor coordination. However, a progressively significant decrease in motor function and poor learning process was observed in rats administered with lead. This is in agreement with the previous work of Nehru and Sidhu (2002), who reported poor learning performance in measurement of motor coordination skills in lead-exposed rats, and thus, concluded that lead exposure produces behavioral and motor coordination disturbances which are associated with dopaminergic and cholinergic neurotransmission in the CNS. In addition, previous studies also reported a decrease in cognitive and motor functions in rats exposed to lead (Azzaoui et al., 2009;Luthman et al., 1992;Mason et al., 2014;Sabbar et al., 2018). Conversely, the present study There are reports on the loss of body weights with resultant organ damage in animals continuously exposed to heavy metals such as lead (Alwaleedi, 2016;Amjad et al., 2013;Kabeer et al., 2019).
In the present study, the observed decrease in the brain and body weights of lead-administered rats were due to the toxic effect manifestation of lead exposure in the rats. The significant reduction in body weight markedly increased with the duration of oral lead administration, which explained the progressive body weight reduction observed in later weeks (week 4) of this study. Thus, the weight loss observed might be associated with the lead ability to interrupt the absorption and metabolism feed nutrients, which is impactful to health as reported earlier by Alwaleedi (2016). Body weight reduction in lead-induced toxicity was reported in previous literatures (Abdel Moneim et al., 2011;Khan et al., 2008;Varnai et al., 2004).
However, treatment with curcumin and Cur-CSCaCO 3 NP showed body weight gain in the animals and stabilized their diet condition, although body weight gain was more obvious in rats treated with Cur-CSCaCO 3 NP. This is because Cur-CSCaCO 3 NP demonstrated an enhanced efficacy on the lead-induced rats, and thus, stabilizing their body weights and improved the rats' condition as well as their diet condition. This is in accordance with previous literatures which documented improved diet conditions and an increase in body weight after treatment with both free curcumin and curcuminloaded nanoparticles (Abdel et al., 2022;Husain, 2015). Another possible reason could be due to CSCaCO 3 NP consisting of calcium, which may actually contribute to the increase in bone density that may consequently constitute to increase in rats' weight since body weight is directly associated with bone mineral density as re- One of the major consequences of lead-induced toxicity is oxidative stress which reflects an imbalance of oxidative status due to continuous free radicals liberation and insufficient antioxidant activity to detoxify the resulting damage (Offor et al., 2017). Thus, lead can cause oxidative damage via two mechanisms operating simultaneously, which are overproduction of ROS and depletion of antioxidant reserves . These liberated ROS causes damage to cells by acting directly on lipid membranes resulting in lipid peroxidation (Wani et al., 2015). In this study, lead administration significantly decreases SOD activities and increases the MDA levels in the rats' serum, as well as cerebral and cerebellar tissue ho-  (Singh et al., 2017), (Barbara et al., 2017;Flora et al., 2013;Huang et al., 2017;Maithilikarpagaselvi et al., 2016;Motterlini et al., 2000;Sandhir et al., 2014;Tiwari et al., 2013;Yadav et al., 2012).
The nervous system among other biological systems is the most susceptible to lead toxic insults (Mahmoud & Sayed, 2016).
The mechanism of lead intoxication in the brain results from its efficient ability to cross the blood-brain barrier and initiate various pathological alterations to membrane-bound enzymes responsible for maintaining redox homeostasis, thus, causing oxidative stress which consequently leads to cell damage .
Neuronal degeneration has been linked to signs of heavy metalinduced neuronal death (Bhattacharjee et al., 2018). Toluidine blue stain is an important special stain for the brain tissue specifically Nissl bodies, nerve cells, and glia (Sridharan & Shankar, 2012). The basic thiazine metachromatic dye has a high affinity for acidic tissue components (nucleic acid blue and polysaccharides purple).
It improves and enhances the sharpness of histological images (Sridharan & Shankar, 2012). In this study, the histological and histochemical analyses of the cerebral cortex from lead-induced rats spaces in the cerebellum of lead-exposed rats (Husain, 2015;Saleh & Meligy, 2018;Sidhu & Nehru, 2004;Yusuf et al., 2017). Brain tissue injury may be reversible (healing) or irreversible (permanent cell death) (Chongtham & Agrawal, 2016). However, this study clearly demonstrated the potential benefits of Cur-CSCaCO 3 NP against lead-induced toxicity. Correspondingly, administration with Cur-CSCaCO 3 NP regardless of the dose has shown a better amelioration pattern when compared to the free curcumin treatment owing to its antioxidant property. In the same manner, curcumin-loaded lipid nanoparticles revealed a higher ameliorative effect than free curcumin in reversing the pathological alterations induced by aluminum chloride in mice brain section (Kakkar & Kaur, 2011).

| CON CLUS ION
This study has shown that lead induction in rats resulted in oxidative stress, decreased body weight, and deficit in motor functions.
In addition, lead induction in rats for 4 weeks was enough to affect Further studies on the mechanism of how Cur-CSCaCO 3 NP crosses the blood-brain barrier to execute its therapeutic effects should be studied. This might provide an additional clue on the mechanism behind the antioxidant effect of Cur-CSCaCO 3 NP.

ACK N OWLED G M ENT
The authors acknowledge the financial support from the Universiti Putra Malaysia (Grant number GP-IPS 9663600).