Gallic and ascorbic acids supplementation alleviate cognitive deficits and neuropathological damage exerted by cadmium chloride in Wistar rats

Cadmium is a highly neurotoxic heavy metal that interferes with DNA repair mechanisms via generation of reactive oxygen species. The potentials of polyphenols and antioxidants as effective protective agents following heavy metal-induced neurotoxicity are emerging. We therefore explored the neuroprotective potentials of gallic and ascorbic acids in CdCl2-induced neurotoxicity. Seventy-two Wistar rats were divided into six groups. Group A received distilled water, B: 3 mg/kg CdCl2, C: 3 mg/kg CdCl2 + 20 mg/kg gallic acid (GA), D: 3 mg/kg CdCl2 + 10 mg/kg ascorbic acid (AA), E: 20 mg/kg GA and F: 10 mg/kg AA orally for 21 days. Depression, anxiety, locomotion, learning and memory were assessed using a battery of tests. Neuronal structure and myelin expression were assessed with histological staining and immunofluorescence. The Morris Water Maze test revealed significant increase in escape latency in CdCl2 group relative to rats concurrently treated with GA or AA. Similarly, time spent in the target quadrant was reduced significantly in CdCl2 group relative to other groups. Concomitant administration of gallic acid led to significant reduction in the durations of immobility and freezing that were elevated in CdCl2 group during forced swim and open field tests respectively. Furthermore, GA and AA restored myelin integrity and neuronal loss observed in the CdCl2 group. We conclude that gallic and ascorbic acids enhance learning and memory, decrease anxiety and depressive-like behavior in CdCl2-induced neurotoxicity with accompanying myelin-protective ability.

www.nature.com/scientificreports/ Morris water maze (MWM) test. The maze was a rounded water pool with concealed spherical hidden escape platform placed about two inches below the water surface 23 . The water was made opaque with powdered non-fat milk. The pool was marked north, south, east and west with the escape platform fixed at a specific location. Each rat was placed into the pool from different start points randomly from the 4 locations and allowed to search for the escape stage. The time spent to locate the escape platform was recorded. If the platform was not found after 120 s, the animals were directed towards it and allowed to stay on it for at least 15 s. Individual animals underwent four tests (corresponding to the four different start locations) per day for five uninterrupted days. The animals were allowed inter-trial rest of up to one hour. This represents the acquisition phase of the test. On the 6 th day, a single probe test was carried out to assess spatial memory. During this phase, the escape platform was removed. The retention of the former position of the platform was measured by evaluating the period the animal spent in the target quadrant and the number of crosses within it. The time spent at the opposite of the target quadrant was also recorded and the test documented manually by two independent viewers blinded to the treatment protocol. The MWM commenced fifteen days after the onset of drug administration (Fig. 1B).
Forced swim test (FST). The forced swim test (FST) is one of the most commonly used tests for depressive-like behavior in rodents 24 . Rats were placed in a container filled with clean water (24 ± 1 °C) and were monitored for six minutes 25 . The onset and duration of immobility were recorded using a timer. This test was performed on experimental day 18 (Fig. 1B).

Open field maze (OFM) test.
The open field was used to assess anxiety and locomotion. The apparatus was a box measuring 72 × 72 cm × 36 cm high. One of the walls was covered with a plexiglass so the rats could be visible in the apparatus. The floor area was separated into 25 squares (20 × 20 cm), defined as 9 central and 16 peripheral squares. At the beginning of the test, each animal was placed in the center of the apparatus and then allowed to freely explore it for 5 min. The parameters recorded were line crossing which is the total number of squares visited (a measure of anxiety and locomotion), rearing (the number of times rat stood on its hind legs), grooming (number of times the animal spent licking or scratching itself while stationary), freezing (the duration with which the animal was completely stationary), center square duration (time spent in the central squares of the box), Stretched-Attend Posture (frequency with which the animal displayed forward elongation of the head and the shoulders followed by retraction to the original position) and defecation (the number of fecal boli). The OFM test was performed twenty (20) days after the onset of drug administration.
The elevated plus maze test. The elevated plus maze test was made of two open arms (50 × 10 cm) crossed at right angles with two opposing closed arms of equal sizes. Each animal was placed at the center of the maze, facing one of the closed arms, and allowed to freely explore the arena for 5 min. The anxiety-related measures taken include the frequency and duration with which the animals visited the open arms and the closed arms.
Forelimb support (hanging wire test). A 2 mm thick metallic wire was tightly attached to a frame to avoid vibration or unwanted displacement of the wire during the test. Individual rat was placed on the 2 mm diameter hanging wire with its forelimb and monitored for a period of 2 min. The period of time it took the animal to stay on the wire before falling was recorded. Rats were tested twice and allowed about 2 h of rest in between. The hanging wire was performed twenty-one days after the commencement of drug administration.
In between testing, all the apparatus were cleaned with 70% ethyl alcohol to eliminate odor cues of previously tested rat. All behavioral tests were carried out by an experimenter blinded to the treatment groups.
Animal sacrifice. Twenty-four hours after the behavioral tests, each rat was deeply anesthetized with sodium pentobarbital (10 mg/100 g body weight) and transcardially perfused with physiological saline, followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4) at 4 °C. Brains were removed and placed in 10% formaldehyde and 30% NBF solution for histopathology and immunohistochemistry respectively.
Haematoxylin and Eosin Staining. The brains were processed routinely for paraffin embedding. 5 μm sections were obtained with rotary microtome and processed for Haematoxylin and Eosin staining as previously documented 26 . Sections were observed with light microscope and photomicrograph taken with a Leica LAS-EZ Microscope installed with Leica software application suite version 3.4.

Percentage of initial body weight (%)
weight on day n − baseline weight Baseline weight × 100%. www.nature.com/scientificreports/ Immunofluorescence. Immunofluorescence was performed by de-paraffinizing the brain sections in xylene and rehydration in graded percentages of alcohol. Antigen retrieval was done by placing the slides in 10 mM citrate buffer at 95-100 °C for 15 min. The slides were thereafter blocked in blocking buffer (2X casein/10% goat serum/0.1% Triton X-100 pH 7.4) and later incubated with the following primary antibodies: anti-NeuN polyclonal antibody (1:500; #MAB377, Millipore ® ), anti-MBP rabbit polyclonal antibody (1:500; #PA5-78397, Invitrogen ® ). The sections were rinsed 3 times (5 min each) in PBS and then incubated with secondary antibodies conjugated with Alexa dyes (AF-488, #A11001, Invitrogen and AF-647, #A21245, Life Technologies) at room temperature for 1 h. Sections were then washed 3 times (5 min each) followed by the addition of 2 drops of mounting media with DAPI (Invitrogen #P36935) before sections were cover slipped. The pixel intensities for MBP and NeuN images were analyzed using ImageJ software.
Statistical analysis. Data were analyzed using a one-way analysis of variance (ANOVA) followed by Tukey's post hoc test for comparison between groups. All tests were used with significance set at p < 0.05. The data were presented as mean ± standard deviation. Data analysis indicated no sex differences in any of the parameters investigated above hence we combined data from males and females experimental rats.

Results
CdCl 2 administration led to reduction in body weight. We observed a gradual increase in body weight across all the groups in the first nine days of this study. However, by day 10, 11 and 14 there was a decline in body weight gain in the CdCl 2 + AA, CdCl 2 + GA and CdCl 2 groups respectively. The decrease in body weight gain in these groups was initially not significant but by day 16 till the end of the experiment this decrease in weight had become statistically significant relative to the GA alone and AA alone groups (Fig. 2).
Gallic acid improved the learning pattern of rats following impairment by CdCl 2 . During the acquisition trial of the Morris water maze we assessed the learning of the rats as the time it took the rat to find and climb onto the escape platform (escape latency). In all the experimental groups, the escape latency diminished over the 5-day training period (Fig. 3A). Repeated-measures one-way ANOVA showed a significant reduction in escape latency with training (F (6.031, 63.63) = 2.514) p = 0.0316. On the 6th day when the escape platform had been removed we observed the CdCl 2 group had a significant decrease in time spent at the target quadrant when compared to the other groups. Remarkably, the time spent in the target quadrant by the CdCl 2 + GA group was similar to control, which was significantly (p = 0.029) increased when compared to the other groups (Fig. 3B). On the contrary, the CdCl 2 group spent a significantly (p = 0.0001) increased time in the opposite quadrant while the control and the CdCl 2 + GA group had similar significantly (p = 0.041) reduced duration in the opposite quadrant (Fig. 3C).
The depressive-like behavior exhibited in the CdCl 2 group was ameliorated following concurrent administration of gallic acid. During FST, we observed that the onset of immobility was significantly higher in the CdCl 2 + GA and GA alone groups relative to other groups. Similarly, the duration of immobility was significantly increased in the GA alone group, while the CdCl 2 + GA and CdCl 2 + AA groups had significantly lower duration of immobility relative to GA alone and control groups. Overall, our results showed that the concurrent administration of GA or AA with CdCl 2 increased struggling behavior in the experimental rats (Fig. 4).
Since we observed that CdCl 2 + GA and CdCl 2 + AA groups had increased escape or struggling-directed behavior (as depicted by significant decreased duration of immobility) compared to the CdCl 2 alone group we subsequently sought to explore their locomotor activity since most antidepressants are known to reduce locomotion. We consequently investigated their actions on locomotion using the open field maze (OFM). We used the OFM as a positive control to further analyze the effect seen in the FST. Our results indicated similarity Values are presented as mean ± Standard Deviation n = 12/group; a indicates significant difference at p < 0.05 compared with control; b Indicates significant difference at p < 0.05 compared with CdCl 2 (one-way ANOVA; n = 12). GA gallic acid, AA ascorbic acid. www.nature.com/scientificreports/ in line crossing across all the groups except in the AA only group where number of lines crossed was significantly reduced. Interestingly, we also observed a significant reduction in freezing time in the CdCl 2 + GA and CdCl 2 + AA groups relative to the CdCl 2 alone group. In addition, stretched attend posture, number of fecal boli, peripheral square and grooming duration were similar across all the experimental groups (Fig. 5).
In the elevated plus Maze test, One-way analysis of variance (ANOVA) revealed a significant decrease in the frequency of entry into the open arms in the CdCl 2 + AA group when compared with the control (p = 0.0014) and CdCl 2 alone (p = 0.0006) groups. In addition, we observed a significant decrease in the frequency of entry into the close arms in all groups administered CdCl 2 , whether alone or concurrently with GA or AA. However, the time spent in the open and closed arms of the maze were similar across all the groups (Fig. 6). CdCl 2 did not alter hanging latency on the forelimb suspension test. The mean hanging latency was 71.45 ± 11.52, 72.45 ± 13.19, 80.35 ± 11.92, 84.05 ± 11.18, 107.4 ± 5.026 and 105.0 ± 6.407 in the control, CdCl 2 alone, CdCl 2 + GA, CdCl 2 + AA, GA alone and AA alone groups respectively. Our results indicate similarity in mean hanging latency across all groups (Fig. 7).
Gallic acid rescued myelin disruption and reduced astrocytic activation following CdCl 2-induced neurotoxicity. We further investigated the effects of CdCl 2 on integrity of myelin sheaths using anti-MBP antibodies and Luxol Fast Blue (LFB) staining. Anti-MBP immunofluorescence staining showed a reduction in staining intensity in the cortex of CdCl 2 and CdCl 2 + AA rats (Fig. 8A). We observed a significant decrease in expression of neuronal nuclei (NeuN) protein across all groups relative to control (Fig. 8B).Quantitative examination of staining intensity indicated a significant reduction (p < 0.05) in the CdCl 2 and CdCl2 + AA rats compared with control and CdCl 2 + GA rats (Fig. 8C). Furthermore, we observed that LFB revealed the white matter tracts appeared thinner with vacuolations and stained pale in rats administered with CdCl 2 (arrows, Fig. 9). Conversely, deeply stained myelinated fibers were detected traversing through the white matter in control and CdCl 2 + GA groups (Fig. 9). Similarly, the CdCl 2 group had less intensely stained, disorganized cerebellar white matter with significant decrease in number of positive MBP cells. Likewise, our results showed that cells positive for GFAP appear to be enlarged and activated in the CdCl 2 rats (Fig. 10A). In addition, quantification of the expression of GFAP revealed a significant elevation in the CdCl 2 group relative to the other test groups (Fig. 10B).

CdCl 2 -induced Purkinje cell degeneration was ameliorated by gallic and ascorbic acids. In
Wistar rats, the cerebellum is made up of molecular, Purkinje and granular cell layers (Fig. 11). Our results showed a disruption with lack of clear organization of the Purkinje cell layer in the CdCl 2 group. Additionally, the remaining Purkinje cells appeared disorganized with distorted cytoplasms. We further investigated this apparent neuronal loss, by carrying out Purkinje cell counts in the different treatment groups. Our results revealed a significant decrease in entire Purkinje cell number in the CdCl 2 group (9% decrease in total number; p = 0.032). However, the total number of Purkinje neurons were similar in the control and groups administered gallic acid.

Discussion
Cadmium has been reported by several studies to induce behavioral deficits such as memory impairment, anxiety and depression [27][28][29] . It does this primarily through the generation of reactive oxygen species (ROS) 9 . Gallic acid is a phenolic acid with antioxidant activity which has been reported over the past years to be effective against nervous system disorders such as depression, anxiety, ischaemia, Alzheimer's and Parkinson's diseases 14 . Oral supplementation with ascorbic acid has also been used to mitigate heavy metal toxicity as well as protect against cadmium induced toxicity 18 . The present study was carried out to evaluate the effects of gallic acid on cadmiuminduced neurotoxicity with focus on neuro-behavioral deficits such as memory loss, anxiety, depression and loss of motor coordination; and compare its effects with that of ascorbic acid, a common antioxidant.
The decrease in body weight in the CdCl 2 group reported in this study is in consonance with the reports of El-Demerdash et al. 30 and Prabu et al. 31 . This observed weight loss may be as a result of the association of CdCl 2 administration with disruption in glucose regulation resulting in impaired protein, carbohydrate and lipid metabolism as previously highlighted 32 . Conversely, Young et al. 33 reported an increase in body weight following CdCl 2 administration. This variability in results may be due to the different exposure routes and dosage of CdCl 2 in the studies. While in the previous study, CdCl 2 was administered in water, our study utilized oral gavage to ensure that the full dose of CdCl 2 was delivered directly to the stomach. However, the association of CdCl 2 administration with weight alteration is outside the aim of this study.
The Morris water maze in rodents is a test for spatial learning and memory that relies on external or extramaze cues to locate a submerged escape platform 34 . It tests for hippocampal dependent learning, which includes acquisition and long-term memory and plays an important role in the validation of cognitive studies in rodent models 35 . In the present study, learning occurred across all the groups but there was a significant decrease in time to locate the escape platform in the CdCl 2 alone group when compared with the control which indicates that CdCl 2 caused a learning deficit. In the probe phase, CdCl 2 alone group showed a significant reduction in the time spent in the target quadrant when compared with the other experimental groups which is indicative of an impairment in spatial memory. It is anticipated that rats would use the visual stimuli from the cues within the testing room to form a "spatial orientation map" in their brain leading to a decrease in escape latency over the course of the test. However, this was not so in the CdCl 2 only, this corroborates the findings of Halder et al. 28 that cadmium causes a decrease in retention memory. Remarkably, CdCl 2 rats treated with gallic and ascorbic acid spent similar time at the target quadrant as the control group in the probe trial of the MWM test, indicating a recovery in memory retention. Although CdCl 2 caused a significant impairment in memory retention, spatial www.nature.com/scientificreports/ learning was still evident during the acquisition phase of the test as the animals spent reduced time in finding the escape platform with increasing number of trials. Interestingly, a study by Wang et al. 36 aligns with this finding that cadmium-treated mice exhibit a deficit in this spatial memory and did not show improvements even after nine weeks of the cessation of cadmium. Increased cadmium levels have also been linked to the presence of depressive-like behavior 29 . The forced swim test is a widely used test to evaluate depressive disorders 24 . The increase in the duration of immobility that is seen in the cadmium alone group is indicative of anxiety as earlier documented 25,37 . Remarkably, gallic acid and ascorbic acid treated groups reduced significantly the duration of immobility indicating their anti-depressive like potential in cadmium-induced toxicity.  www.nature.com/scientificreports/ The fear-related behaviors ranging from locomotion, freezing duration, exploratory activity and emotional state examined in our study revealed a dramatic decline in anxiety-like behaviors following administration of gallic and ascorbic acids. This outcome further supports that ascorbic acid promotes anxiolytic effects in diverse animal models as previously documented in zebrafish 38 , mice 39 and rats 40 . Although, the exact mechanisms of anxiolytic activity of these compounds remains to be decisively established, reports have suggested anti-oxidant related decrease of brain oxidative stress and NMDA antagonism 41,42 . On the other hand, some authors have associated this activity to their ability in reducing circulating levels of cortisol, a stress hormone 43 .
The hanging wire test is a test of muscle strength 44 . Cadmium has been previously linked with motor activity impairment 45 . Our study nevertheless showed no significant difference in hanging latency across all the groups. This incongruence in hanging latency across the groups in this study may be due to the shorter duration of administration of the metal in our study compared to earlier reports 14,45 that had a 28-day of administration of cadmium.
Demyelination is due to several pathologies in the brain such as inflammation, viral infection, focal compression, metabolic derangements and ischaemia 46 . Accumulation of cadmium has been linked to loss of myelin and reduced expression of myelin basic protein (MBP) in the cerebral and cerebellar cortices of rats 47 . Our study showed a reduced myelin expression in the cadmium treated group which was restored in both the ascorbic and gallic acid treated groups. The loss of cerebral neurons observed in LFB and demyelination in both MBP and LFB may be responsible for the impaired memory that was observed in the Morris water maze test. This study suggests that the demyelination could be due to an inflammatory process.
Astrocytes are cells that produce intermediate filaments such as glial fibrillary acidic protein (GFAP). Astrocytic activation (or astrocytosis) results from almost all insults or injury to the brain. The increased GFAP expression could also indicate an ongoing inflammation in cadmium toxicity leading to astrogliosis 48 . This supports the claim of astrocytic involvement in heavy metal neurotoxicity 49 . This astrogliosis was however reversed in the groups treated with gallic acid and ascorbic acid. Plant metabolites such as soybean supplementation in diet has also been shown to reduce arterial and cardiac injuries induced by cadmium 50 . Hence we propose that gallic acid being a secondary polyphenol with antioxidant potential could be beneficial against damages caused by cadmium chloride-induced experimental neurotoxicity and behavioral alterations. These effects can both be linked to their antioxidant activities.

Conclusion
Cadmium chloride caused a deficit in learning and memory with accompanying evidences of anxiety and depressive-like behavior which are indicative of neurotoxicity. Concurrent administration of gallic acid and ascorbic acid ameliorate the neurotoxic effects induced by cadmium. Although gallic acid showed a better neuroprotective effects over ascorbic acid; these compounds should be explored in population that are environmentally or occupationally exposed to toxic levels of cadmium.

Data availability
The supporting data of this study are available from the corresponding author upon reasonable request.