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. 2025 Jul 11;19:272–289. doi: 10.1016/j.ibneur.2025.07.003

Evaluation of the role of omega-3 fatty acids in nicotine-induced neurotoxicity in pregnant Wistar rats and their pups

Joseph Gbenga Omole a, Quadri Kunle Alabi b,⁎, Mayowa Grace Elemile c, Ayodeji Aturamu d, Modinat Adebukola Adefisayo e, Gbenga Olatunde Omotosho f, Samson Mokolade Ige g, Orji Eunice Ibiye h, Tokunbo Olorunfemi Samuel i, Fisayo Nathaniel Ogunleye j, Peace Kehinde Olamilosoye a, Oladele Abiodun Ayoka a
PMCID: PMC12304721  PMID: 40735301

Abstract

This study investigated maternal and their pup neurobehaviour following nicotine exposure during gestational and postnatal periods and neuroprotective effect of Omega-3 fatty acids. Thus, the study used thirty pregnant female Wistar rats for this study. Groups I and II (nonpregnant and pregnant) were treated with 1 ml/kg/day of normal saline for 42 days; III was treated with 4 mg/kg/day of nicotine for 42 days; IV-VI were co-administered nicotine 4 mg/kg and 100, 300, 600 mg/kg/day of Omega-3 fatty acids respectively for 42 days. Results: The beam walk time of the mother rats in groups III and IV were significantly higher when compared with other groups. Similarly, the beam walks time of the pups of groups III and IV were significantly higher when compared with the pups of the mother rats in other groups. The brain dopamine and serotonin levels of mother rats in groups III, IV and V were significantly higher when compared with other groups. Also, the brain dopamine and serotonin levels of the pups in groups III and IV were significantly higher when compared with the pups in other groups. The reduced glutathione and catalase of mother and pup rats in groups III and IV were significantly lower when compared with other groups. Photomicrographs of cerebellum and hippocampus of the rats treated with nicotine showed scattered arrangement of pyramidal cells with vacuolated neurons. These alterations were significantly reversed with Omega-3 fatty acids following nicotine exposure. Conclusions. Omega-3 fatty acids at 300 and 600 mg/kg ameliorated nicotine-induced neurotoxicity in mother rats and their pups.

Keywords: Nicotine, Omega-3 fatty acids, Neurotoxicity, Oxidative stress, Wistar rats

Introduction

Nicotine has been linked to etiopathologies of different diseases, including neurobehavioral toxicity caused by smoking exposure (Law et al., 2003). Brain cellular loss throughout development has been linked to prenatal exposure to nicotine in previous study (Abdel-Rahman et al., 2005). It is a direct cholinergic agonist at the nicotinic acetylcholine receptor (nAChR) site, leading to behavioral effects and cellular loss (Abdel-Rahman et al., 2005, Betnowitz and Jacob, 2000). Exposure to nicotine early in life causes oxidative damage to nicotinic receptors in the brain and subsequently abnormal behavior in adulthood (Abdel-Rahman et al., 2005). The increase dietary nicotine consumption nevertheless has been reported to increase the risk of various behavioral defect and other neural diseases (Dong et al., 2020, Nielsen et al., 2013). This study suggested that nicotine might have opposite functions with respect to neuroprotection and neurodegeneration. An experimental study revealed that nicotine stops dopaminergic neuron loss in a rodent neural disease of memory and cognitive model (Dong et al., 2020, Liu et al., 2017).

Moreover, nicotine has been associated with a decreased risk for Alzheimer’s disease (Townsend et al., 2004, Echeverria and Zeitlin, 2012, Lombardo and Maskos, 2015, Moreno-Gonzalez et al., 2013). Nicotine positively influences different aspects of cognition, including learning, memory, and attention (Ferrea and Winterer, 2009). It also improves mood, stress regulation, anxiety (Ferrea and Winterer, 2009), Alzheimer’s disease, and Parkinson’s disease. According to cell culture experiments, nicotine has neurotrophic properties, and nicotinic acetylcholine receptors (nAChRs) play significant roles in the control of neurodevelopment and maturation of the central nervous system before parturition. (Levin, 2002, Ferrea and Winterer, 2009).

Omega−3 fatty acids are polyunsaturated fatty acids (PUFAs) with a double bond three atoms away from the terminal methyl group in their chemical structure. They are extensively distributed in nature, are vital constituents of animal lipid metabolism, and perform vital functions in the human diet and in human physiology (Scortletti and Byrne, 2013). Omega-3 fatty acids are eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), both of which are often found in marine oils, and alpha-linolenic acid (ALA), which is found in plant oils. (Scortletti and Byrne, 2013). Alpha-linolenic acid contains walnut, edible seeds, algal oil, clary, sage seed oil, flaxseed oil, hemp oil, sacha Inch oil and Echium oil, while sources of the animal omega-3 fatty acids EPA and DHA include fish, fish oil eggs from chickens fed EPA and DHA, krill oil, squid oil and certain algae. Mammals are unable to produce the essential omega-3 fatty acid ALA but acquire it through the diet. Nevertheless, ALA can be used to form EPA and DHA by generating additional double bonds along its carbon chain (desaturation) and extending it (elongation). ALA is subsequently used to produce EPA, which is subsequently used to produce DHA (Scortletti and Byrne, 2013). The omega-3 polyunsaturated fatty acid (particularly, docosahexaenoic) is uniquely enriched in the brain and retina and is required for proper human brain development and visual functions (Birch et al., 2000, Innis and Friesen, 2008). These findings support the beneficial effects of omega-3 fatty acids in different neurological and neurodegenerative disorders (Dennis et al., 2015). DHA, a principal member of the omega-3 fatty acid family, is highly concentrated in the brain and the outer segments of retinal cones and rods, accounting for approximately half of the total omega-3 fatty acids (Parletta et al., 2013). Several studies have revealed that supplementation with DHA, an omega-3 fatty acids during pregnancy has neuroprotective effects on prenatal stress-induced brain disorders in rats and prevents hypertoxic and ischemic injuries of the rat brain (Wainwright, 2002, Feng et al., 2012, Tuzun et al., 2012). Evidence from animal and human studies has shown that the DHA diet supplement reduced hippocampal oxidized protein level thereby positively activate hippocampal Akit and CaMKII levels in improving the cognitive, memory and learning capacity of the rats exposed to brain stress model (Wu et al., 2008). Omega-3 FAs participates in synaptogenesis, synaptic transmission, learning, and memory processes (Cao et al., 2009). Omega-3 FAs have been reported in previous studies to possess antioxidant (Bazan, 2005, Calon et al., 2004, Hashimoto et al., 2002), anti-inflammatory (Bazan, 2005, Marcheselli et al., 2003) and anti-apoptotic properties (Calon et al., 2005). Hence, this study investigated the impact of omega-3 fatty acids on nicotine-induced neurotoxicity in pregnant Wistar rats and their pups.

Materials and methods

Drugs and chemicals

Omega-3 fatty acids (300 mg/capsule) were purchased from Nature Way. Products Inc., Green Bay, W1 54311, USA; nicotine (11 mg/ml) was purchased from Alchem Europe SA. Via al flume 1,6929. Gravesano. Ketamine hydrochloride (50 mg/10 ml) was purchased from SWISS Parenterals Ltd., 809, Karala GIDC. Dist. Ahmedabad 302220. Gujarat, INDIA. Assay kits for biochemical parameters were purchased from Elabscience, Randox Manual/RX Monza, Cusabio and Abcam.

Animal care

Male and female adult Wistar rats weighing between 140 and 150 g were used for this research. They were obtained from a reputable animal care vendor, Ile-Ife, Nigeria. All the animals were kept healthy at favorable temperature and they were allowed free access to food and water. The rats were kept in the animal facility for 2 weeks before the commencement of the experiment.

Experimental design

Thirty (30) adults female and ten (10) adults male Wistar rats weighing between 140 and 150 g were used for this research. The female rats were randomly distributed into six (6) groups such that each group contained 5 female rats. After the distribution of the female rats, each rat was weighed before the commencement of the study. Male rats were introduced into each of the female rat cage in ratio 2:5 for copulation. The pregnancy was tested by the method of Ochiogu et al., 2006 via checking the vaginal smear grossly by hand and under microscope for the presence of protein coagulate plug which was the confirmatory test for the rat pregnancy. Once the protein coagulate was observed on the vaginal smear of each rat (which was regarded as day 1 of conception), the rat was weighed at four-day intervals to check the progress of the pregnancy. During the course of the experiment, the female rats were administered nicotine and omega-3 fatty acids immediately pregnancy was confirmed, at 21 days of delivery and continued at post-delivery for 21 days.

Grouping of experimental animals

Group I included nonpregnant female Wistar rats. The rats were treated with 1 ml/kg physiological saline (0.9 % NaCl) orally daily for 42 days.

Group II included pregnant female Wistar rats. The rats were treated with 1 ml/kg physiological saline (0.9 % NaCl) orally daily for 42 days.

Group III included pregnant female Wistar rats. The rats were treated with 4 mg/kg nicotine orally daily for 42 days (Neogy et al., 2008). Nicotine was given during (21 days) and after pregnancy (21 days).

Group IV included pregnant female Wistar rats. The rats were orally administered 4 mg/kg nicotine daily and 100 mg/kg omega-3 fatty acids orally daily for 42 days. Both nicotine and omega-3 fatty acids were administered together during (21 days) and after pregnancy (21 days) (Abdel Moneim et al., 2011).

Group V included pregnant female Wistar rats. The rats were treated with both 4 mg/kg nicotine orally and 300 mg/kg omega-3 fatty acids orally daily for 42 days.

Group VI included pregnant female Wistar rats. The rats were treated with both 4 mg/kg nicotine daily and 600 mg/kg omega-3 fatty acids orally daily for 42 days (Singh et al., 2016).

The male pups of all the mother rats in the study design were selected for the study along their mother. Each male pup was assigned according to the group of their mother as follows;

Group II: This comprises of four male pups, belong to the female rats treated with 1 ml/kg physiological saline (0.9 % NaCl) orally daily for 42 days.

Group III: Four male pups, belong to the mother rats treated with 4 mg/kg nicotine orally daily for 42 days.

Group IV: Four male pups, belong to the mother rats treated with both 4 mg/kg nicotine daily and 100 mg/kg omega-3 fatty acids orally daily for 42 days.

Group V: Four male pups, belong to the mother rats treated with both 4 mg/kg nicotine daily and 300 mg/kg omega-3 fatty acids orally daily for 42 days.

Group VI: Four male pups, belong to the mother rats treated with both 4 mg/kg nicotine daily and 600 mg/kg omega-3 fatty acids orally daily for 42 days.

Neurobehavioral evaluations

Neurobehavioral tests were carried out for each rat (mothers and their pups) in each group following the standard protocol as described below, and the results were documented.

Inclined plane test

The inclined plane test was used to evaluate limb motor function in the Wistar rats. This procedure followed the methodology of Abou-Donia et al., 2001. The mother rats and their offspring were placed on a flat plane separately in the horizontal position, with the head facing the side of the board to be raised, which was measured to the nearest 5°. The angle at which the rat began to slip downward was recorded. Three trials were conducted for each exposure. The intertrial period was one hour. The results of the three trials were averaged and recorded.

Forepaw grip test

This method was adopted from Abou-Donia et al., 2001 procedure, where the grip strength of the mother and their pups were measured to ascertain appropriate neuromuscular function of the hand and forearm of the rats. This model was carried out on an 8 mm diameter wood dowel that was raised horizontally so that the body weight of the rat is supported on the wood dowel. The time to release the grip by the animal was recorded (in seconds) by a stopwatch. Three successive trials were carried out to measure forelimb strength only.

Beam-walking test

This procedure was experimented as described by Carter et al., 1999 and Perry et al., 1995. The beam-walking or beam balance test measures fine motor balance and coordination in Wistar rats. The goal of this test was for the rat to walk across a horizontal narrow beam to a safe platform without deviating from normal route.

Step-down latency in the active avoidance test

This procedure followed the methodology of Jarvik and Essmann, 1960, which was modified by Hudspeth et al. (1964). A plain block of wood was placed on one side of the grid floor of a continuous avoidance apparatus, which served as a shock-free zone. Each rat was placed in the shock-free zone, and upon stepping down, it received an electric shock (5 V) through the grid floor. The time taken for each rat to step down was recorded with a recorder.

Biochemical analysis

The animals were sacrificed through ketamine and the brain tissues of all the animals (mothers and pups) were carefully removed, washed, and weighed. The tissues were homogenate and centrifuged to separate the wanted from unwanted substance. The supernatant was obtained for the estimation of serotonin, dopamine, superoxide dismutase (SOD) catalase (CAT), reduced glutathione (GSH), acetylcholinesterase, glucose-6-phosphate dehydrogenase (G6PD), inflammatory marker (interleukin 6 and TNF-alpha) and apoptotic marker (caspase-3) activity.

Evaluation of Serotonin

The serotonin concentration in the brain tissue of each rat was measured following the manufacturer’s protocol (Sandwich-ELISA kit, Elabscience; E-EL-R1140). Wistar rat brain tissue was collected for the estimation of serotonin. At the end of this protocol, the serotonin concentration in the brain tissue homogenate was then calculated by extrapolating the OD of the samples from the standard curve. The concentration of brain serotonin was expressed as ng/ml.

Assessment of the reduced glutathione (GSH) level

GSH was quantified following the protocol reported by Beutler et al. (1963). In brief, 1 ml of the supernatant was added to 0.5 ml of 10 mM Ellman’s reagent. Thereafter, 2 ml of phosphate buffer was added, and the resulting yellow precipitate was read as an absorbance against a blank. Serially diluted standard concentrations were analyzed similarly, and GSH was determined in mg/100 g of the tissue.

Determination of superoxide dismutase (SOD) activity

SOD activity was examined using the method reported by Misra and Fridovich (1972). Two hundred microliters of the supernatant were mixed with Tris–HCl buffer, 300 μL of 2 mM pyrogallol and 30 mM EDTA. Changes in absorbance were recorded for 3 min to obtained SOD activity.

Catalase activity

The brain homogenate of catalase was measured using the Aebi (1984) laboratory method. A total of 2000 μL of phosphate buffer and 200 μL of H2O2 were added to 40 μL of the above brain supernatant. The disappearance of hydrogen peroxide (H2O2) was monitored for 3–4 min at 30 s intervals using a spectrophotometer to obtained catalase activity.

Glucose-6-phosphate dehydrogenase concentration

This enzyme was analyzed according to the laboratory protocol of Kornberg et al. (1955) according to the manufacturer’s instructions (Randox Manual/RX Monza, G-6-PDH). This colorimetric method used a cuvette with a 1 cm light path, a temperature of approximately 37°C, and a wavelength of 340 nm. The concentrations of enzyme in the homogenates were determined by the standard concentration at U/g Hb.

Evaluation of acetylcholinesterase

This activity was assayed by a laboratory protocol according to the manufacturer’s instructions (Abcam; acetylcholinesterase assay kit, colorimetric). Then, 0.6 ml of the assay solution was added to buffer in a vial containing DTNB to make a 20X DTNB stock solution. The solution was incubated at 37°C. A total of 1.0 % of the sample was added to the stock solution, 0.6 ml of ddH2O was added to the vial of acetylthiocholine, and 100 µl of ddH2O with 0.1 % BSA was added to the vial of acetylcholinesterase standard to make a 50 units/ml acetylcholinesterase reaction. The concentration of acetylcholinesterase was obtained and plotted.

Evaluation of Dopamine

This was assayed by a laboratory protocol according to the manufacturer’s instructions (Abcam; Dopamine kit, colorimetric). All the reagents, samples and standards were prepared. The plate was washed 2 times with 1X wash solution before standard, sample and control wells were added. Then, 50 µl of biotin-conjugated antibody working solution was added to each well, and the plate was covered with a plate sealer. All the samples were incubated and washed with IX solution after incubation, and SABC working solution was added to each well and incubated. The samples were then washed again, and 90 µl of TMB substrate was added to each well and incubated in the dark within 15–20 min. The shades of blue were observed in the 3–4 wells at the end of the incubation, and 50 µl of stop solution was added to each well. The wavelength was read at 450 nm within 20 min.

Evaluation of Caspase-3

This was assayed by a laboratory protocol according to the manufacturer’s instructions (Cusabio; caspase-3 assay kit). The prepared reagents, working standards, and samples (brain homogenates) were prepared as described in the protocol manual. Then, 100 μl of standard or sample was added to each well, and the plates were covered with the adhesive strips provided. At the end of the protocol, the optical density of each well was determined within 5 min, and a microplate reader set to 450 nm was used to determine the caspase-3 concentration in the samples.

Evaluation of tumor necrosis factor Alpha and IL-6

The levels of TNF-α and IL-6 in the homogenate from each rat were measured following the manufacturer’s protocol (Sandwich-ELISA kit, Elabscience). Rat brain homogenates were obtained for the estimation of TNF-α and IL-6 levels. Then, 100 μL of brain homogenate was added to each well with diluted TNF-α and IL-6 detection antibodies. At the end of the protocol, the concentrations of TNF-α and IL-6 were expressed as pg/ml.

Histological processes

The brain tissue biopsies (hippocampus and cerebellum) of the rats were fixed in 10 % formalin for staining using hematoxylin-eosin. The processed tissues were observed under microscope and photomicrographs were captured with a camera at 400x magnification. Changes in each subfield (CA1, CA2 and CA3) of the hippocampus were observed under a light microscope and the neuron cells were counted and quantified by image pro software.

Statistical analysis

The data represented in mean ± standard deviation (SD) and one–way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. The results were considered significant when p ˂ 0.05. GraphPad 5.03 (GraphPad Software Inc., CA, USA).

Results

Neurobehavioral evaluation of mother rats and their pups following exposure to nicotine and Omega-3 fatty acids

The beam scores of Mothers’ rats and their pups following nicotine and Omega-3 fatty acid exposure

The beam scores of the maternal rats in groups III and IV (9.40 ± 0.400 and 9.00 ± 0.71, respectively) were not significantly different from those in groups I, II, V and VI (8.60 ± 0.51; 8.80 ± 0.49; 8.60 ± 0.51 and 8.40 ± 0.60, respectively). Similarly, the beam scores of pup rats in groups III and IV (6.60 ± 0.68 and 7.00 ± 0.71, respectively) were not significantly different from those of mothers in groups II, V and VI (7.40 ± 0.51; 8.00 ± 0.45 and 7.40 ± 0.51, respectively) (Fig. 1A-B).

Fig. 1.

Fig. 1

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The Beam score and walk time of the mother and pup following maternal exposure to nicotine and Omega-3 fatty acid, Each value represents Mean ± SD (n = 5). * = significantly different from Group 1 (p < 0.05); µ = significantly different from Group 2 (p < 0.05); α = significantly different from Group 5 (p < 0.05); # = significantly different from Group 6 (p < 0.05).

Times of mothers’ rats and their pups following nicotine and omega-3 fatty acid exposure to beam walk

The beam walk times of the mothers in groups III and IV (9.00 ± 0.32 and 8.00 ± 0.32, respectively) were significantly greater (p = 0.0001) than those in groups I, II, V and VI (6.00 ± 0.45, 6.40 ± 0.51, 5.80 ± 0.37 and 6.00 ± 0.32, respectively). Similarly, the beam walking times of the pups in groups III and IV (17.20 ± 0.97 and 15.60 ± 0.81, respectively) were significantly greater (p = 0.0001) than those of the pups in groups II, V and VI (6.80 ± 0.58; 9.60 ± 0.51 and 8.20 ± 0.66) (Fig. 1C-D).

Incline plane test of mothers’ rats and their pups following nicotine and omega-3 fatty acid exposure

There was no significant difference across groups I to VI (63.80 ± 2.01; 59.20 ± 3.26; 56.80 ± 1.93; 57.40 ± 2.04; 63.40 ± 1.83 and 65.00 ± 2.01, respectively). Similarly, the inclination planes of the pups in groups II, III, IV, V and VI (68.80 ± 2.85, 65.40 ± 2.27, 67.20 ± 2.92, 68.20 ± 2.94 and 71.00 ± 3.30, respectively) were not significantly different from one another (Fig. 2A-B).

Fig. 2.

Fig. 2

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The incline plane and grip time of the mother and Pup Following Maternal Exposure to Nicotine and Omega-3 Fatty Acid, Each value represents Mean ± SD (n = 5). * = significantly different from Group 1 (p < 0.05); µ = significantly different from Group 2 (p < 0.05); β = significantly different from Group 4 (p < 0.05); α = significantly different from Group 5 (p < 0.05); # = significantly different from Group 6 (p < 0.05).

Grip Time of mothers’ rats and their pups following nicotine and omega-3 fatty acid exposure

The grip times of the maternal rats in groups I, III, and IV (11.40 ± 0.40, 6.80 ± 0.73 and 9.60 ± 0.40, respectively) were significantly lower (p = 0.0001) than those in groups I, V and VI (13.60 ± 0.24, 12.60 ± 0.24 and 13.20 ± 0.37, respectively). However, the grip time of group II (11.40 ± 0.40) was significantly greater (p = 0.0002) than that of groups III and IV (6.80 ± 0.73 and 9.60 ± 0.40, respectively). The grip time of group IV (9.60 ± 0.40) was significantly greater than that of group III (6.80 ± 0.73). Similarly, the grip times of the pups in groups III and IV (4.60 ± 0.51 and 5.20 ± 0.37) were significantly lower (p = 0.0004) than those of the pups in groups II, V and VI (8.40 ± 0.68; 8.00 ± 0.71 and 8.60 ± 0.93, respectively). However, there was no significant difference in grip time between groups V and VI (8.00 ± 0.71 and 8.60 ± 0.93, respectively) and between group II (8.40 ± 0.68) and group II (8.40 ± 0.68) pups (Fig. 2C-D).

Evaluation of the active avoidance response of mothers’ rats and pups following exposure to nicotine and omega-3 fatty acids

The percentage responses of the maternal rats in groups III and IV (63.00 ± 5.15 and 69.20 ± 6.53, respectively) were significantly lower (p = 0.0001) than those in groups I, II, V and VI (96.00 ± 6.96; 92.60 ± 5.15; 92.20 ± 2.45 and 94.40 ± 3.04, respectively). Similarly, the percentage response of the pup rats in group III was significantly lower (60.00 ± 4.47) than that of the pups in groups II, IV, V and VI (76.80 ± 2.96; 68.80 ± 1.46; 71.60 ± 2.94 and 75.60 ± 2.32, respectively) (Fig. 3A-B).

Fig. 3.

Fig. 3

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The active avoidance and passive avoidance responses of the mother and Pup Following Maternal Exposure to Nicotine and Omega-3 Fatty Acid, Each value represents Mean ± SD (n = 5). * = significantly different from Group 1 (p < 0.05); µ = significantly different from Group 2 (p < 0.05); β = significantly different from Group 4 (p < 0.05); α = significantly different from Group 5 (p < 0.05); # = significantly different from Group 6 (p < 0.05).

Evaluation of the Passive Avoidance Response of Mothers and Pups following Exposure to Nicotine and Omega-3 Fatty Acids

The step-down latency of the maternal rats in groups III and IV (10.40 ± 0.51 and 13.40 ± 1.08, respectively) was significantly lower (p = 0.0010) than that of the maternal rats in groups I, II, V and VI (15.40 ± 1.29; 15.40 ± 1.33; 16.40 ± 1.08 and 17.60 ± 0.75, respectively) (Fig. 3C).

Pup rats in group III had significantly lower (p = 0.0001) step-down latency (4.40 ± 0.51) than did pups in groups II, IV, V and VI (10.20 ± 0.80; 9.20 ± 0.37; 9.60 ± 0.51 and 10.80 ± 0.37, respectively). However, the step-down latency of pups in group IV (9.20 ± 0.37) was significantly lower than that in group VI (10.80 ± 0.37) (Fig. 3D).

Brain Dopamine (mg/dl) in mothers’ rats and their pups following exposure to nicotine and omega-3 fatty acid

The brain dopamine levels of the maternal rats in groups III, IV and V (24.12 ± 1.74, 18.43 ± 1.64 and 13.97 ± 1.57, respectively) were significantly greater (p = 0.0001) than those in groups I, II and VI (5.37 ± 0.83, 5.13 ± 0.75 and 6.38 ± 1.20, respectively). However, groups IV and V (18.43 ± 1.64 and 5.13 ± 0.75, respectively) had significantly lower (p = 0.0034) dopamine levels than did group III (24.12 ± 1.74). Similarly, the brain dopamine concentrations of the pup rats in groups III and IV (16.90 ± 0.86 and 10.17 ± 0.62, respectively) were significantly greater (p = 0.0001) than those of the pups in groups II, V and VI (3.34 ± 0.36; 7.28 ± 0.33 and 4.58 ± 0.82, respectively). There was significantly less brain dopamine in the pups in group IV (10.17 ± 0.62) than in those in group III (16.90 ± 0.86) (Fig. 4A-B).

Fig. 4.

Fig. 4

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The brain dopamine and serotonin level of the mother and Pup Following Maternal Exposure to Nicotine and Omega-3 Fatty Acid, Each value represents Mean ± SD (n = 5). * = significantly different from Group 1 (p < 0.05); µ = significantly different from Group 2 (p < 0.05); β = significantly different from Group 4 (p < 0.05); α = significantly different from Group 5 (p < 0.05); # = significantly different from Group 6 (p < 0.05).

Brain Serotonin (ng/ml) in mothers’ rats and their pups following exposure to nicotine and omega-3 fatty acid

The serotonin levels of the maternal rats in groups III, IV and V (35.58 ± 0.97, 39.45 ± 1.47 and 24.78 ± 1.51, respectively) were significantly greater (p = 0.0001) than those in groups I, II and VI (15.92 ± 1.42, 16.70 ± 1.57 and 20.29 ± 0.78, respectively). However, group V (24.78 ± 1.51) had significantly lower (p = 0.0001) serotonin levels than groups III and IV (35.58 ± 0.97 and 39.45 ± 1.47, respectively). Similarly, the Pup rats in groups III and IV had significantly greater (p = 0.0001) serotonin levels than did the pups in groups II, V and VI (13.43 ± 0.89; 14.53 ± 1.98 and 18.73 ± 0.78, respectively). There was significantly greater brain serotonin in the pups of group VI (18.73 ± 0.78) than in those of group II (13.43 ± 0.89) (Fig. 4C-D).

Brain acetylcholinesterase (pg/ml) in mothers’ rats and their pups following nicotine and omega-3 fatty acid exposure

The acetylcholinesterase activities of groups III and IV (15.92 ± 1.21 and 16.84 ± 1.47, respectively) were significantly lower (p = 0.0001) than those of groups I, II, V and VI (46.03 ± 0.75; 42.09 ± 2.11; 46.17 ± 1.27 and 45.05 ± 1.89, respectively). Similarly, Pup rats in groups III and IV had significantly lower (p = 0.0001) acetylcholinesterase levels (23.12 ± 1.15 and 24.25 ± 1.75) than did pups in groups II, V and VI (42.19 ± 0.88; 38.63 ± 1.97 and 42.64 ± 1.23) (Fig. 5A-B).

Fig. 5.

Fig. 5

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The brain acetylcholinesterase and glucose 6 phosphate dehydrogenase activities of the mother and Pup Following Maternal Exposure to Nicotine and Omega-3 Fatty Acid, Each value represents Mean ± SD (n = 5). * = significantly different from Group 1 (p < 0.05); µ = significantly different from Group 2 (p < 0.05); α = significantly different from Group 5 (p < 0.05); # = significantly different from Group 6 (p < 0.05).

Brain glucose-6 phosphate dehydrogenase (U/g Hb) in mothers’ rats and their pups following nicotine and omega-3 fatty acid exposure

The glucose-6 phosphate dehydrogenase levels of the maternal rats in groups III and IV (3.50 ± 0.31 and 4.85 ± 0.42, respectively) were significantly lower (p = 0.0001) than those in groups I and VI (7.86 ± 0.92 and 8.64 ± 0.77, respectively). However, groups II and V had significantly greater (p = 0.0004) levels of glucose-6 phosphate dehydrogenase (6.61 ± 0.58 and 6.79 ± 0.48) than did group III (3.50 ± 0.31) (Fig. 5C). Similarly, Pup rats in groups III and IV had significantly lower (p = 0.0007) glucose-6 phosphate dehydrogenase levels (3.30 ± 0.45 and 3.89 ± 0.85, respectively) than did pups in groups II and VI (8.39 ± 1.27 and 8.39 ± 0.27, respectively). There was a significant increase in the glucose-6 phosphate dehydrogenase activity of pups in group V (6.02 ± 0.88) compared with that in group III (3.30 ± 0.45) (Fig. 5D).

Brain Caspase-3 (U/mg protein) in mother rats and pregnant plants following nicotine and omega-3 fatty acid exposure

The caspase-3 levels of the maternal rats in groups III and IV (9.00 ± 0.62 and 9.62 ± 0.82, respectively) were significantly greater (p = 0.0001) than those in groups I, II and VI (4.11 ± 0.84; 4.71 ± 0.63 and 2.99 ± 0.41, respectively). However, caspase-3 was significantly lower in group V (5.51 ± 0.72) than in group III (9.00 ± 0.62) (Fig. 6A). Similarly, compared with the pups in groups II, V and VI, the Pup rats in groups III and IV had significantly greater (p = 0.0005) levels of caspase-3 (7.79 ± 0.67 and 6.23 ± 0.48) (3.41 ± 0.79; 3.98 ± 0.59 and 3.34 ± 0.63) (Fig. 6B).

Fig. 6.

Fig. 6

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The brain caspase-3 activity of the mother and Pup Following Maternal Exposure to Nicotine and Omega-3 Fatty Acid, Each value represents Mean ± SD (n = 5). * = significantly different from Group 1 (p < 0.05); µ = significantly different from Group 2 (p < 0.05); α = significantly different from Group 5 (p < 0.05); # = significantly different from Group 6 (p < 0.05).

Brain TNF-α (pg/ml) in mothers’ rats and their pups following nicotine and omega-3 fatty acid exposure

The tumor necrosis alpha values of the maternal rats in groups III and IV (17.48 ± 2.13 and 16.00 ± 2.21, respectively) were significantly greater (p = 0.0001) than those in groups I, II, V and VI (5.82 ± 0.76, 9.05 ± 0.69, 8.88 ± 0.90 and 5.82 ± 0.63, respectively). Similarly, Pup rats in groups III, IV and V had higher (p = 0.0041) tumor necrosis-alpha (8.32 ± 0.72; 8.62 ± 1.24 and 6.29 ± 0.73) than did the pups in group II (3.59 ± 0.48). The TNF-α was significantly higher (p = 0.0131) in the groups III and IV pup rats (8.32 ± 0.72 and 8.62 ± 1.24, respectively) when compared with the group VI (4.18 ± 0.68). However, no significant difference in tumor necrosis-alpha between group II (3.59 ± 0.48) and group VI (4.18 ± 0.68) (Fig. 7A-B).

Fig. 7.

Fig. 7

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The brain tumor necrosis alpha and interleukin-6 level of the mother and Pup rats Following Maternal Exposure to Nicotine and Omega-3 Fatty Acid, Each value represents Mean ± SD (n = 5). * = significantly different from Group 1 (p < 0.05); µ = significantly different from Group 2 (p < 0.05); α = significantly different from Group 5 (p < 0.05); # = significantly different from Group 6 (p < 0.05).

Brain interleukin-6 (pg/ml) in Mothers’ Rats and Their Pups Following Nicotine and Omega-3 Fatty Acid Exposure

The interleukin-6 levels of the maternal rats in groups II, III, IV, and V (10.78 ± 0.72, 22.07 ± 1.18, 19.52 ± 1.84 and 11.70 ± 0.96, respectively) were significantly greater (p = 0.0001) than those in groups I and VI (7.47 ± 1.15 and 7.80 ± 0.88, respectively). However, groups II and V had significantly lower (p = 0.0001) interleukin-6 (10.78 ± 0.72 and 11.70 ± 0.96) levels than groups III and IV (22.07 ± 1.18 and 19.52 ± 1.84, respectively). Similarly, the Pup rats in groups III and VI had significantly greater (p = 0.0003) interleukin-6 (7.79 ± 0.67 and 6.23 ± 0.48, respectively) levels than did the pups in groups II, V and VI (3.41 ± 0.79; 3.98 ± 0.59 and 3.34 ± 0.63, respectively (3.41 ± 0.79), compared with groups II and V (3.98 ± 0.59 and 3.34 ± 0.63) (Fig. 7C-D).

Brain superoxide dismutase (µ/mg protein) in maternal rats and their pups following exposure to nicotine and omega-3 fatty acids

The superoxide dismutase activity of the maternal rats in groups III and IV (3.86 ± 0.65 and 3.10 ± 0.51, respectively) was significantly lower (p = 0.0001) than that of the rats in groups I, II, V and VI (8.36 ± 0.88; 7.09 ± 0.44; 6.16 ± 0.47 and 7.95 ± 0.83, respectively). Similarly, the Pup rats in group III had significantly lower (p = 0.0357) superoxide dismutase (4.11 ± 0.13) levels than did the pups in groups II, V and VI (7.57 ± 0.97; 6.98 ± 0.72 and 6.56 ± 0.93, respectively). However, there was no significant difference in superoxide dismutase activity in the pups of group IV (5.30 ± 0.13) compared with those of groups II, V and VI (7.09 ± 0.44, 6.16 ± 0.47 and 7.95 ± 0.83, respectively) (Fig. 8A-B).

Fig. 8.

Fig. 8

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The brain antioxidant activities of the mother and Pup rats Following Maternal Exposure to Nicotine and Omega-3 Fatty Acid, Each value represents Mean ± SD (n = 5). * = significantly different from Group 1 (p < 0.05); µ = significantly different from Group 2 (p < 0.05); β = significantly different from Group 4 (p < 0.05); α = significantly different from Group 5 (p < 0.05); # = significantly different from Group 6 (p < 0.05).

Brain Catalase (µM/mg protein) in mother rats and their pups following nicotine and omega-3 fatty acid exposure

The catalase activity of the mother rats in group III (3.73 ± 0.20) was significantly lower (p = 0.0001) than that of the rats in groups I, II, IV, V and VI (7.03 ± 0.48; 5.69 ± 0.42; 6.31 ± 0.64; 7.01 ± 0.46 and 10.94 ± 0.95, respectively). However, group VI had significantly greater (p = 0.0001) catalase (10.94 ± 0.95) than groups I, II, IV and V (7.03 ± 0.48; 5.69 ± 0.42; 6.31 ± 0.64 and 7.01 ± 0.46, respectively). Similarly, Pup rats in groups III and IV had significantly lower (p = 0.0001) catalase (3.73 ± 0.32 and 3.92 ± 0.67, respectively) levels than did pups in groups II, V and VI (7.36 ± 0.95, 8.38 ± 0.66 and 9.62 ± 0.90, respectively) (Fig. 8C-D).

Glutathione (µ/mg protein) in the brains of mothers’ rats and their pups following nicotine and omega-3 fatty acid exposure

The levels of reduced glutathione in the maternal rats in groups III and IV (7.01 ± 0.46 and 10.94 ± 0.95, respectively) were significantly lower (p = 0.0001) than those in groups I, II, V and VI (11.63 ± 0.70; 11.41 ± 0.84; 10.55 ± 0.50 and 13.07 ± 1.21, respectively). However, group VI had significantly more reduced glutathione (13.07 ± 1.21) than did group V (10.55 ± 0.50). Similarly, compared with the pups in groups II, V, and VI, the pups in groups III and IV had significantly lower (p = 0.0019) levels of glutathione (9.20 ± 0.79 and 7.81 ± 1.28, respectively) (15.35 ± 1.74, 12.43 ± 0.81, and 13.56 ± 0.87, respectively) (Fig. 8E-F).

Histopathological studies

Photomicrograph of the CA1 region of the hippocampus of a female Wistar rat showed normal arrangement of the pyramidal neuronal cells across the groups except for group 3, which showed a scattered arrangement of the cells. The CA2 region of the hippocampus of group 3 showed poor arrangement of the pyramid cells across the groups. The CA3 region of the hippocampus showed poor arrangement of pyramidal cells in groups 3 and 4 compared with the other groups. The quantitative analysis of the cells in the hippocampus indicated significant distorted (p < 0.05) of the cells in the subfield CA1, CA2 and CA3 of the groups exposed to nicotine. However, following treatment with omega-3 fatty acid, the reduction of the neuronal cells was prevented, particularly, in the groups received higher doses of omega-3 fatty acids (Fig. 9, Fig. 10, Fig. 11, Fig. 12, Fig. 13).

Fig. 9.

Fig. 9

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Photomicrographs of Hippocampi of the Mother Rats Following Exposure to Nicotine and Omega-3 Fatty Acid, Photomicrograph of the CA1 region of the hippocampus of a female Wistar rat. The normal arrangement of the pyramidal cells across the groups was very intact except for group 3, which showed a scattered arrangement of the cells. The blue arrows indicate vacuolated neurons stained with H&E; × 400. Fig. 9. Each value represents Mean ± SD (n = 5). * = significantly different from Group 1 (p < 0.05); µ = significantly different from Group 2 (p < 0.05); α = significantly different from Group 5 (p < 0.05); # = significantly different from Group 6 (p < 0.05) Fig. 9.

Fig. 10.

Fig. 10

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Photomicrographs of Hippocampi of the Mother Rats Following Exposure to Nicotine and Omega-3 Fatty Acid, Photomicrograph of the CA2 region of the hippocampus of a female Wistar rat. Only group 3 showed poor arrangement of the pyramid cells across the groups. The arrows also represent vacuolated neurons. H&E staining; x400.Fig. 10. Each value represents Mean ± SD (n = 5). * = significantly different from Group 1 (p < 0.05); µ = significantly different from Group 2 (p < 0.05); α = significantly different from Group 5 (p < 0.05); # = significantly different from Group 6 (p < 0.05) Fig. 10.

Fig. 11.

Fig. 11

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Photomicrographs of Hippocampi of the Mother Rats Following Exposure to Nicotine and Omega-3 Fatty Acid, Photomicrograph of the CA3 region of the hippocampus of a female Wistar rat: There was poor arrangement of pyramidal cells in Groups 3 and 4 compared with the other groups. The blue arrow shows vacuolated neurons. H&E staining; x400.Fig. 11. Each value represents Mean ± SD (n = 5). * = significantly different from Group 1 (p < 0.05); µ = significantly different from Group 2 (p < 0.05); α = significantly different from Group 5 (p < 0.05); # = significantly different from Group 6 (p < 0.05) Fig. 11.

Fig. 12.

Fig. 12

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Photomicrographs of Hippocampi of the Pup Rats Following Maternal Exposure to Nicotine and Omega-3 Fatty Acids, Photomicrograph of the CA1 region of the hippocampus of Pup Wistar rats: the micrograph shows good arrangement of the pyramidal cells across all the groups. The blue arrow shows vacuolated neurons. H&E staining; x400.Fig. 12. Each value represents Mean ± SD (n = 5). µ = significantly different from Group 2 (p < 0.05); α = significantly different from Group 5 (p < 0.05); # = significantly different from Group 6 (p < 0.05) Fig. 12.

Fig. 13.

Fig. 13

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Photomicrographs of Hippocampi of the Pup Rats Following Maternal Exposure to Nicotine and Omega-3 Fatty Acids, Photomicrograph of the CA2 region of the hippocampus of a Pup Wistar rat. The micrographs show good arrangements of the pyramidal cells across all the groups. The blue arrows show vacuolated neurons. H&E staining; x400.Fig. 13. Each value represents Mean ± SD (n = 5). µ = significantly different from Group 2 (p < 0.05); α = significantly different from Group 5 (p < 0.05); # = significantly different from Group 6 (p < 0.05) Fig. 13.

Photomicrograph of the CA1 region of the hippocampus of Pup Wistar rats shows good arrangement of the pyramidal cells across all the groups. The CA2 region of the hippocampus of the Pup Wistar rats showed good arrangements of the pyramidal cells across all the groups. The CA3 region of the hippocampus of the Pups Wistar rats show good arrangements of the pyramidal cells across all the groups except groups 3 and 4 that show signs of vacuolation in the supporting cells. There was significant different (p < 0.05) across all the cells in the subfield CA1, CA2 and CA3 of the hippocampus of pup following maternal exposure to nicotine. The pup in the groups in which their mother treated with omega-3 fatty acid had significant improvement (p < 0.05) in the number of cells in the hippocampus compared with nicotine group (Fig. 12, Fig. 13, Fig. 14, Fig. 15).

Fig. 14.

Fig. 14

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Photomicrographs of Hippocampi of the Pup Rats Following Maternal Exposure to Nicotine and Omega-3 Fatty Acids, Photomicrograph of the CA3 region of the hippocampus of Pups Wistar rats. The micrographs show good arrangements of the pyramidal cells across all the groups. However, there were signs of vacuolation in the supporting cells in groups 3 and 4 (arrow). H&E, x400.Fig. 14. Each value represents Mean ± SD (n = 5). µ = significantly different from Group 2 (p < 0.05); α = significantly different from Group 5 (p < 0.05); # = significantly different from Group 6 (p < 0.05) Fig. 14.

Fig. 15.

Fig. 15

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Photomicrographs of the cerebellum of mother rats following maternal exposure to nicotine and omega-3 fatty acids. Photomicrograph of the cerebellum of a female Wistar rat. The micrographs show normal histoarchitecture of the cerebellum, with the Purkinje cells (yellow arrows), molecular layer (ML) and granular layer (GL) appearing intact except in group 3 at the junction between the molecular and Purkinje cell layers, which shows some signs of histological distortion. H&E staining, × 400.Fig. 15.

Photomicrograph of the cerebellum of the mother rats showed normal histoarchitecture of the cerebellum, with the Purkinje cells, molecular layer and granular layer appearing intact except in group 3 at the junction between the molecular and Purkinje cell layers, which shows some signs of histological distortion. The cerebellum of Wistar rat pups show normal histoarchitecture of the cerebellum, with the Purkinje cells, molecular layer and granular layer appearing intact except in group 3, in which the Purkinje cells are not distinctively visible (Fig. 16).

Fig. 16.

Fig. 16

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Photomicrographs of the cerebellum of mother rats following maternal exposure to nicotine and omega-3 fatty acids, Photomicrograph of the cerebellum of Wistar rat pups. The micrographs show normal histoarchitecture of the cerebellum, with the Purkinje cells (yellow arrows), molecular layer (ML) and granular layer (GL) appearing intact except in group 3, in which the Purkinje cells are not distinctively visible. H&E staining, × 400.Fig. 16.

Discussion

Omega-3 FAs showed adequate neuroprotective in pregnant Wistar rats and their male pups following maternal exposure to nicotine during and after pregnancy periods. Testing for neurobehavioral deficits, the beam walk time, forepaw grip strength, inclined plane and active avoidance showed alteration pattern in the male pups from nicotine-exposed mothers on postnatal day 21. These neurobehavioral deficits have been demonstrated initially by the nicotine exposed mother rats during gestational and postnatal periods of the study. These neurobehavioral anomalies demonstrate treatment-related sensorimotor deficits that may be justified by the histopathological alterations present in the cerebellum and hippocampus (Abdel-Rahman et al., 2005). Since significant alterations exist in the cerebellum, cerebellar neuronal and glial inadequacies might cause beam walking deficits. These neuropathological alterations might be linked to nicotine-induced oxidative stress (Guan et al., 2003). Moreover, nicotine agonists have been shown to promote synaptic regulation in developing cerebellar Purkinje neuronal cells (Kawa, 2002). Neurobehavioral abnormality has been reported in previous study (Adriani et al., 2003) to involved nicotine exposure during peri-adolescence in rats. However, the neurobehavioral deficits in the beam walk time, beam walk score, forepaw grip strength and inclination plane caused by nicotine-induced oxidative damage were reversed following treatment with omega-3 FAs as a neuroprotective supplement. There was a significant increase in dopamine and serotonin in the brains of nicotine-treated rats, and these changes were reversed by coadministration of omega-3 FAs. This finding is in agreement with Song et al. (2009) and Taepavarapruk and Song (2010), who reported ameliorative effects of omega-3 FAs against olfactory bulbectomy and IL-1 beta induced neurobehavioral deficits in rats. In the present study, the activities of the enzymes, acetylcholinesterase and glucose-6-phosphate dehydrogenase (G6PD) were significantly reduced in nicotine-treated rats compared to control and omega-3 FAs rats, which might be attributed to the oxidative stress caused by nicotine on the neuronal activity. The nicotine-induced reduction in acetylcholinesterase and G6PD activities has been shown to be protected following cotreatment with omega-3 FAs, indicating the neuroprotective and antioxidant efficacy of omega-3 FAs (Ross et al., 2007). In the present study, elevated levels of caspase 3, TNF-alpha and IL-6, were linked to neuroinflammation in nicotine-treated maternal rats and their pups, revealing ongoing inflammation. Inflammation in the brain could be advantageous in maintaining its homeostasis in response to infection. However, overwhelming of inflammatory process during undergoing infection on brain cells over the protecting mechanism of the neuronal tissue resulting in damaging of brain cells that are vital for normal functions of the brain activity. The undergoing brain inflammation involves microglia, which is the protecting mechanism of the brain cells (Aloisi, 2001). When triggered, these cells synthesize pro- and anti-inflammatory cytokines. However, as result of continuous production of proinflammatory cytokines, these molecules become neurotoxic, leading to neuronal injury involved in numerous brain pathologies (Blais and Rivest, 2003, Layé, 2010, Solito and Sastre, 2012). This inflammatory action of nicotine on the neuronal cells readily linked with brain oxidative injury. The anti-inflammatory effect of omega-3 FAs was demonstrated in this study by the significant reduction in the elevated levels of TNF-alpha and IL-6 induced by nicotine. Numerous epidemiologic and observational studies have revealed that subjects with greater blood omega-FA levels have decreased proinflammatory cytokine production (Ferrucci et al., 2006, Kiecolt-Glaser et al., 2011, Farzaneh-Far et al., 2009, Alfano et al., 2012). In addition, a diet rich in EPA decreased the synthesis of the proinflammatory cytokine interleukin (IL)-1b and ameliorated synaptic plasticity impairment in the hippocampus of old rats (Martin et al., 2002, Lynch et al., 2007). In contrast, low dietary intake of omega-3 FAs has detrimental effects on the brain, particularly during the perinatal period of brain development. For example, dietary omega-3 FA deficiency beginning on the first day of gestation reduces the DHA level, changes the microglial phenotype and motility, and increases the expression of the brain proinflammatory cytokines IL-6 and TNF-alpha in the pup brains of mice and rats (McNamara et al., 2010). Caspase 3 was significantly lower in omega-3FA-treated mothers and pup rats than in nicotine-treated mothers and pup rats. This demonstrated the antiapoptotic efficacy of omega-3 FAs against the neurotoxic effect of nicotine. This indicated that omega-3 FAs prevented apoptosis in protected rats. This finding is supported by a previous study (Suphioglu et al., 2010), which demonstrated a direct association between DHA treatment and caspase-3 protein levels. In the present study, the activities of SOD and CAT and the amount of reduced glutathione were significantly reduced in nicotine-treated rats, suggesting membrane injury, which may be due to free radical production. SOD and CAT oppose radicals such as superoxide and hydrogen peroxide, respectively. Glutathione peroxidase has a scavenging effect against peroxyl, alkxyl and alkyl radicals that can form from oxidized membrane compounds and uses GSH as a substrate (Das et al., 2009). The present study corroborates the earlier work of Jain and Flora (2012), who discovered that increasing the concentration of lipid peroxidation in nicotine-treated rats is linked to reducing the activities of CAT and SOD. In the present study, nicotine-induced decreases in the levels of CAT and SOD and a decrease in the level of glutathione were protected following coadministration of nicotine and omega-3 FAs, suggesting that nicotine has a neuroprotective effect. The nicotine-induced reduction in acetylcholinesterase and G6PD activities has been shown to be protected following cotreatment with nicotine and omega-3 FAs, showing its neuroprotective efficacy. Histopathological findings in this study revealed a significant reduction in the number of surviving Purkinje neuronal cells in the cerebellum and CA1, CA2 and CA3 subfield of the hippocampus in pups on PND 21 from mothers exposed to nicotine on gestational days (GDs) 1–21. These findings indicated that these Purkinje cells in the cerebellum and subfields of the hippocampus are sensitive to the deleterious effects of nicotine. The cerebellum is a well-organized structure in which neuronal purkinje signals are the major outputs of the cerebellar cortex (Altman, 1972a, Altman, 1972c, Altman, 1972b), and it is one of the first brain structures to transform. The hippocampus is a temporal lobe brain structure peculiarly linked to memory, emotion, and neurogenesis. It appears to be sensitive to the effects of drugs abuse (Kutlu et al., 2016). Nicotine exposure is particularly damaging to the antenatally developing hippocampus, as cholinergic activity modulates relocation and constituents of nascent hippocampal circuitry (Lauder and Schambra, (1999). In the subfield of the hippocampus, prenatal nicotine exposure is linked to an increase in dying neuron density, evidence by the reduction in the hippocampus neuronal cells quantification in various subfields of the histophotomicrographs. These decreased in neuron cells counts observed in the nicotine exposed rats is due to cellular injury. This histopathological study was in conformity with the previous study which revealed reduction in the size of cells in the hippocampal at CA3 region following maternal exposure to nicotine during the gestational period (Abdel-Rahman et al., 2005). In the present study, the groups treated with omega-3 FAs, particularly, the groups that received highest doses showed improvement in number of cells in the subfields of hippocampus. Thus, cotreatment of rats with omega-3 FAs reversed brain injury in the cerebellum and hippocampus compared to that in rats exposed to nicotine only.

Conclusion

In conclusion, the results of the present study indicated that omega-3 fatty acids (FAs) at 300 and 600 mg/kg/body weight ameliorate nicotine-induced brain oxidative stress through direct scavenging of reactive oxygen species (ROS) and prevention of compromise brain inflammation. Cotreatment with nicotine and omega-3 FAs minimized the effects of nicotine-induced biochemical and histopathological alterations. However, 600 mg/kg of omega-3 FAs was more effective at ameliorating these effects. This study revealed that omega-3 fatty acids could be a good supplement for both expectant and nursing mothers during the antenatal and postnatal periods and could support cognitive improvement and prevent total neurodegeneration during the developmental stages of the offspring. Therefore, further studies on the impact of omega-3 fatty acids on the prevention of nicotine or any other environmentally toxic agents during pregnancy and during the developmental stages of offspring are needed.

Authors contributions

J.G. Q.K. and O.A., intellectualized the knowledge, performed the experiment, prepared the data, done data analysis and wrote the draft of the manuscript. M.G., A.A., M.A., G.O., S.M., O.E., T.O., F.N.O., P.K., corrected and reviewed the results, and contributing to writing and editing of the final version of the manuscript.

CRediT authorship contribution statement

Ogunleye Fisayo N.: Software, Resources. Tokunbo Olorunfemi Samuel: Visualization, Software, Resources. Orji Eunice Ibiye: Resources, Methodology, Formal analysis. Samson Mokolade Ige: Visualization, Software, Data curation. Gbenga Olatunde Omotosho: Visualization, Resources, Methodology. Modinat Adebukola Adefisayo: Resources, Project administration, Investigation. Ayodeji Aturamu: Validation, Resources, Funding acquisition. Mayowa Grace Elemile: Software, Resources. Alabi Quadri: Writing – review & editing, Visualization, Validation, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Oladele Abiodun Ayoka: Writing – review & editing, Validation, Supervision, Conceptualization. Joseph Gbenga Omole: Writing – original draft, Resources, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization. Peace Kehinde Olamilosoye: Visualization, Software, Resources, Data curation.

Consent to participate

Not Applicable.

Consent to publish

Not Applicable.

Ethical standard disclosure

The experimental animals used in this study complied with the ARRIVE guidelines (Kilkenny et al., 2010) and were tested according to the National Academy of Sciences Guide for the Care and Use of Laboratory Animals (National Research Council Committee for the Guide, Care and Use of Laboratory Animals, 2011). The study was approved by the Health Research Ethics Committee (HREC) of the Institute of Public Health, College of Health Sciences, Obafemi Awolowo University, Ile-Ife, Osun, State, Nigeria, with approval no: IPH/OAU/12/1925.

Funding

This research was funded solely by the authors.

Conflict of Interest

The authors of this manuscript declare no conflicts of interest.

Acknowledgement

Not Applicable.

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