Abstract
Adolescent nicotine use is particularly concerning due to increased susceptibility to long-term effects and dependence during this critical developmental period. This study investigates the therapeutic effects of thiamine on nicotine withdrawal-induced anxiety, anhedonia, and depression in rats. Adolescent rats received nicotine (2 mg/kg, s.c.) for 21 d, followed by 21 d of withdrawal. Thiamine (25 or 50 mg/kg, i.p.) was administered during exposure and withdrawal. Behavioral assessments were used to evaluate anxiety- and depressive-like symptoms, and biochemical analyses measured oxidative stress markers, serotonin levels, MAO activity, BDNF, and GFAP as indicators of neuroinflammation in the prefrontal cortex. Nicotine withdrawal significantly elevated anxiety-, depression-, and anhedonia-like behaviors, increased oxidative stress, and upregulated MAO-A activity and GFAP expression, indicating neuroinflammatory effects. Notably, thiamine administration during both nicotine exposure and withdrawal effectively alleviated these behavioral impairments, restored serotonin levels, reduced oxidative stress markers, and mitigated the increase in GFAP expression. Additionally, thiamine alone has been shown to alleviate anxiety- and depressive-like behaviors. This study highlights thiamine's potential as a promising intervention for managing psychological distress associated with nicotine withdrawal. Given the high prevalence of adolescent nicotine use and its associated mental health challenges, further research on thiamine's mechanisms and therapeutic potential is warranted to improve treatment strategies during this critical developmental stage.
Significance Statement
Nicotine dependence remains a global health challenge, with withdrawal symptoms posing a significant barrier to cessation efforts, particularly in adolescents. This study explores the neuroprotective potential of thiamine in mitigating nicotine withdrawal in adolescent male rats by modulating key neurochemical pathways. Thiamine treatment was found to attenuate withdrawal-associated behavioral disturbances, while also restoring serotonin metabolism, upregulating brain-derived neurotrophic factor, and reducing oxidative stress and neuroinflammation. These findings highlight thiamine's multifaceted role in alleviating nicotine withdrawal by addressing both neurochemical and inflammatory imbalances. This study suggests that thiamine could serve as an adjunct therapeutic approach for nicotine dependence, with implications for improving cessation outcomes during adolescence—a critical period for brain development and vulnerability to dependence.
Introduction
Adolescent nicotine use remains a significant public health concern due to the heightened risk of addiction and increased vulnerability to the long-term adverse effects of nicotine exposure during this developmental stage (Hossaini et al., 2025b). During this critical stage of development, the central nervous system undergoes significant structural and functional changes (Baum et al., 2022), alongside major modifications in neurotransmitter systems associated with reward processing (Bonke et al., 2023). These neurological changes contribute to adolescent-specific behaviors, including increased novelty-seeking and risk-taking (Kim-Spoon et al., 2019). Nicotine, a psychostimulant with parasympathomimetic effects, shares pharmacological properties with amphetamines, increasing its potential for abuse and addiction (Motaghinejad et al., 2016). Chronic nicotine exposure enhances reward processing mechanisms, contributing to its high addiction potential across various tobacco products (Torabi et al., 2019). Adolescents have a lower sensitivity to nicotine's aversive effects, potentially contributing to its higher use in this population (von Deneen et al., 2022). There is a well-documented link between nicotine addiction and psychiatric disorders, particularly anxiety (Feldner et al., 2007; Jobson et al., 2019) and depression (John et al., 2004; Amiry et al., 2023). Anxiety disorders are significantly more prevalent among individuals with nicotine dependence, affecting ∼22% of this population compared with only 11.1% of nondependent individuals (Grant et al., 2004). Nicotine metabolism generates reactive oxygen species (Jobson et al., 2019), leading to oxidative stress that induces cellular damage and contribute to behavioral disorders, including anxiety, depression, neuroinflammation, and neurotransmitter imbalances (Paz et al., 2007; Ahmadi-Soleimani et al., 2023; Amiry et al., 2023).
While pharmacological treatments for nicotine withdrawal exist, research increasingly seeks safer and more effective alternatives (Yammine et al., 2021). Nutritional psychiatry has gained attention for its investigation into how dietary modifications and specific micronutrient supplementation influence mental health, particularly mood disorders (Botturi et al., 2020). Vitamins, as essential organic compounds, act as cofactors in enzymatic reactions; since mammalian cells are typically unable to synthesize them, they must be obtained through dietary intake (Fattal-Valevski, 2011). Thiamine (vitamin B1) is a water-soluble vitamin crucial for neuronal function and energy metabolism (Butterworth, 2003). Its depletion, which can occur within weeks of inadequate intake, has been linked to behavioral impairments in young individuals (Butterworth, 2003). This study aims to evaluate the effects of thiamine administration on anxiety- and depression-like behaviors resulting from nicotine dependence, particularly during the withdrawal period following nicotine exposure. Additionally, it investigates the impact of concurrent nicotine and thiamine administration on the development of these behavioral alterations in rats.
Materials and Methods
Animals
Eighty adolescent male Sprague Dawley rats were selected for this study to reduce biological variability and eliminate confounding hormonal influences related to the estrous cycle, which can affect behavioral and neurochemical responses during nicotine withdrawal (Becker et al., 2005; Carroll and Anker, 2010). Female rodents often exhibit milder withdrawal symptoms—particularly in measures of anxiety, hyperalgesia, and locomotor activity—which may obscure the detection of treatment effects (Chellian et al., 2021). Male adolescents are widely used in nicotine dependence research due to their consistent and well-characterized withdrawal profiles (O'Dell et al., 2007), providing greater experimental control in mechanistic investigations. Rats were obtained from the animal facility of Khatam Al-Nabieen University at postnatal day 21 (P21) and housed in groups of three to four per open-top plexiglass cage. The rats were kept under standardized environmental conditions, featuring a controlled temperature of 22 ± 2°C, humidity levels maintained between 55 and 65%, and a 12 h light/dark cycle, with lights turning on at 6:00 A.M. The animals received a standard laboratory diet (Javaneh Khorasan) containing 46% nanofibrillated cellulose, 25% neutral detergent fibers, 19% protein, and 10% lipids, with ad libitum access to clean water. All experimental procedures were approved by the Animal Ethics Committee of the author's university (AF, knu.edu.af.rec 23, 10/5/2024) and were conducted in strict accordance with the Guide for the Care and Use of Laboratory Animals (Ahmadi-Noorbakhsh et al., 2021).
Drugs and experimental procedure
(−)Nicotine base and thiamine were obtained from Sigma-Aldrich and diluted in normal saline (0.9% sodium chloride) for administration and were injected into a final volume of 1 ml. Rats received subcutaneous (s.c.) nicotine injections at a dose of 2 mg/kg every 12 h (6:00 A.M. and 6:00 P.M.). This model is widely utilized in preclinical studies to simulate nicotine dependence, as it allows for the controlled evaluation of key behavioral adaptations such as tolerance, reinforcement, and withdrawal symptoms—core features that underlie nicotine addiction and are essential targets for the development of therapeutic interventions (Chellian et al., 2021). The study consisted of eight experimental groups (n = 10 per group), detailed as follows: The experimental design consisted of eight groups of adolescent rats (n = 10 per group), each receiving specific treatment regimens across two distinct phases—P21–42 (nicotine exposure period) and P42–63 (postexposure period). Group 1 (Vehicle–Vehicle): Served as the control group and received saline injections throughout the entire experimental period (P21–63). Group 2 (Nicotine–Vehicle): Received nicotine (2 mg/kg, s.c.) twice daily during the nicotine exposure phase (P21–42), followed by saline during the postexposure period (P42–63). This group was used to assess the behavioral effects of nicotine administration and spontaneous withdrawal. Groups 3 and 4 (Nicotine–Thiamine): Received nicotine (2 mg/kg, s.c.) during the exposure phase (P21–42), followed by thiamine administration at either 25 mg/kg (Group 3) and 50 mg/kg (i.p.; Group 4), during the postexposure phase (P42–63), to evaluate the potential of thiamine as a postwithdrawal intervention. Groups 5 and 6 (Nicotine + Thiamine–Vehicle): Administered both nicotine (2 mg/kg, s.c.) and thiamine (25 mg/kg for Group 5 and 50 mg/kg for Group 6, i.p.) simultaneously during P21–42, followed by saline injections from P42–63. These groups were included to assess the potential protective effects of thiamine when coadministered with nicotine during exposure. Group 7 (Thiamine–Vehicle): Received thiamine alone (50 mg/kg, i.p.) from P21 to P42, followed by saline from P42 to P63, to evaluate the independent effects of thiamine. Group 8 (Vehicle–Thiamine): Administered saline during the exposure phase (P21–42), followed by thiamine (50 mg/kg, i.p.) during the postexposure phase (P42–63), to determine the efficacy of delayed thiamine treatment. The selected thiamine dosages were informed by previous research demonstrating its neurophysiological benefits (Kim et al., 1990; Karabulut et al., 2022). Furthermore, clinical data indicate that doses ranging from 25 to 50 mg/kg are safely used in human infants and children for the treatment of thiamine deficiency (Smith et al., 2021). The schematic schedule of the experiments is presented in Figure 1.
A detailed timeline outlining the key phases and experimental events in the study. P, postnatal day; OFT, open field test; EPM, elevated plus maze; FST, forced swimming test; SPT, sucrose preference test; MDA, malondialdehyde; TT, total thiol; SOD, superoxide dismutase; CAT, catalase; MAO-A, monoamine oxidase A; BDNF, brain-derived neurotrophic factor; GFAP, glial fibrillary acidic protein.
Behavioral assessments
During the initial week following nicotine cessation, rats typically do not exhibit significant changes in anxiety-like behavior (Chellian et al., 2021). Therefore, in this study, to evaluate the behavioral consequences of nicotine withdrawal in adolescent rats, a battery of validated behavioral tests—including the open field test (OFT), elevated plus maze (EPM), forced swimming test (FST; Popovych et al., 2019), and sucrose preference test (SPT)—was performed on Day 21 following the final nicotine injection. This time point was selected to capture the intermediate withdrawal phase when emotional and cognitive disturbances are typically evident. Before each behavioral test, rats were allowed to acclimate to the testing environment for at least 30 min to reduce environmental stress and increase data reliability. Illumination levels were standardized according to task requirements: 150 lux for the EPM to encourage open-arm exploration, and 40 lux for the OFT to support natural exploratory behavior without inducing anxiety. To avoid potential confounding effects, animals that had completed behavioral testing were housed separately from those awaiting evaluation. Furthermore, all testing apparatuses were sanitized between trials using a 10% ethanol solution to eliminate olfactory cues from previous subjects, ensuring the consistency and validity of behavioral measurements.
Open field test
The OFT is a widely used behavioral assay for assessing anxiety-like behaviors and exploring the neurobiological mechanisms underlying anxiety. It is also employed to evaluate the efficacy of potential anxiolytic compounds (Kraeuter et al., 2019). In this test, an animal is placed in a large, open arena, and its movements are monitored. The test is based on the conflict between the animal's innate fear of open spaces and its curiosity to explore. Time spent in the center of the arena is often interpreted as an indicator of reduced anxiety. More time spent in the center typically suggests lower anxiety levels (Sturman et al., 2018). In this study, the OFT was employed to evaluate anxiety-like behavior. The test was conducted in a square arena measuring 100 × 100 × 40 cm, constructed from opaque, nonreflective material to minimize external visual distractions. Each rat was gently placed in the center of the arena and allowed to explore freely for 5 min. A 5 min duration allows for the collection of reliable data while minimizing the potential for habituation effects, where animals may become accustomed to the environment and alter their behavior over time (Delprato et al., 2017). This time is also long enough to capture the initial anxiety response to a novel environment, which is critical for evaluating the effects of pharmacological interventions or genetic modifications on anxiety and exploratory behavior (Mesiakaris et al., 2024). During the session, behavioral parameters—including the time spent in the central versus peripheral zones—were recorded using an automated video tracking system. The data were subsequently analyzed to assess levels of and anxiety-like behavior.
Elevated plus maze
Following the OFT, the anxiolytic effects of thiamine were assessed using the EPM, a well-established method for evaluating anxiety-like behavior and locomotor activity. The EPM consists of a plus-shaped platform elevated 50 cm above the ground, featuring two open arms and two enclosed arms with 40 cm high walls to provide security. Each rat was individually placed in the central area of the maze under dim lighting, allowing free access to any of the arms. The animal's behavior was recorded over 5 min, with particular emphasis on the time spent in each arm. This test leverages the natural conflict in rats between their instinctual aversion to open spaces and their exploratory drive. Anxiety levels were inferred by comparing the time spent in the open versus the closed arms, as rats with lower anxiety levels are more likely to explore the open arms, whereas those with higher anxiety levels tend to remain predominantly in the closed arms (Acikgoz et al., 2022). In this study, the EPM apparatus was constructed from opaque gray wood to minimize external visual cues and enhance the animals’ sense of security during testing. The maze consisted of two open arms and two closed arms arranged in a plus-shaped configuration and elevated above the floor to provoke an approach-avoidance conflict. Each rat was placed at the center of the maze facing one of the open arms and was allowed to explore freely for 5 min. Behavioral parameters, including the time spent in the open and closed arms, were carefully recorded and analyzed. These measurements provided valuable insights into anxiety-like behaviors and were used to evaluate the potential anxiolytic effects of thiamine administration.
Forced swimming test
In the FST, rats are placed in a water-filled container from which they cannot escape, and their behavior is monitored to assess depressive-like states. The test records the time spent in various behavioral states, including active behaviors such as swimming and struggling, as well as immobility, which is interpreted as a marker of behavioral despair, analogous to depressive symptoms in humans (Pumpaisalchai et al., 2005). In this study, the FST was employed to assess depression-like behavior in rats. Each animal was individually placed in a transparent glass cylinder (height, 50 cm; diameter, 20 cm) filled with water at a depth of 30 cm and maintained at a temperature of 24 ± 2°C, ensuring that the rats were unable to touch the bottom and were required to swim. The test duration was 5 min, during which each rat was gently introduced into the water and its behavior was continuously recorded using a video camera for subsequent analysis. Behavioral responses were categorized into three distinct types: struggling (vigorous activity involving forelimb and hindlimb movements, including attempts to escape by climbing the walls), swimming (active horizontal movement throughout the container without climbing attempts), and immobility (minimal movement, with only slight motions necessary to keep the head above water). These behavioral parameters were later quantified to evaluate the antidepressant-like effects of thiamine.
Sucrose preference test
The SPT is a widely used behavioral assay to assess anhedonia, a core symptom of depression characterized by the inability to experience pleasure. This test evaluates rats’ relative preference for a 1–2% sucrose solution compared with water as an indicator of reward sensitivity (Liu et al., 2018). Rats typically exhibit a preference for sweet, palatable solutions, as sucrose consumption is associated with the activation of reward pathways. A decreased preference for sucrose is therefore considered a sign of anhedonia (Scheggi et al., 2018). This phenomenon is frequently observed in individuals with substance use disorders, major depressive disorder, and various neuropsychiatric conditions, making the SPT a valuable tool for studying the neurobiological mechanisms underlying these disorders in rodents models (Pizzagalli, 2014). In this study, the SPT was conducted to evaluate anhedonia-like behavior, a core symptom of depression. All rats underwent a 4 d adaptation protocol prior to testing. During the first 2 d (Days 1 and 2), animals were provided with two identical bottles containing pure water to minimize any innate side preference. On Day 3, a habituation phase was implemented in which both bottles were filled with a 1% sucrose solution, allowing the rats to become familiar with the sweet taste. On Day 4, following a 12 h period of food and water deprivation, each rat was presented with two bottles: one containing 1% sucrose solution and the other containing pure water. After 12 h of access, the volumes consumed from each bottle were measured to determine sucrose preference. The sucrose preference index was calculated as the ratio of sucrose solution intake to the total fluid intake, providing a quantitative measure of reward sensitivity. Sucrose preference was calculated using the following formula: sucrose preference percentage (%) = sucrose solution consumption / (sucrose solution consumption + water consumption) × 100% (He et al., 2020).
Euthanasia
Following the behavioral experiments, the rats were killed using a 95% CO2 method, employing a step-fill technique in which CO2 was introduced into the euthanasia chamber at a rate of 30% of the chamber volume per minute (Begni et al., 2016). After killing, the brains were carefully extracted, and the prefrontal cortex (PFC) was dissected from the surrounding cortical tissue and promptly frozen for subsequent biochemical analyses. These analyses included measurements of serotonin levels, monoamine oxidase A (MAO-A) activity, brain-derived neurotrophic factor (BDNF), glial fibrillary acidic protein (GFAP), malondialdehyde (MDA), total thiol (TT) content, superoxide dismutase (SOD) activity, and catalase activity.
Biochemical measurements
Measurement of serotonin, MAO-A activity, BDNF, and GFAP
Serotonin concentrations were measured using an ELISA kit from My BioSource (MBS713292). MAO-A activity was determined with a separate ELISA kit from the same supplier (MBS721413). BDNF levels were quantified using a rat-specific ELISA kit from CUSABIO (CSB-E04504r). Similarly, GFAP concentrations were assessed with a CUSABIO ELISA kit designed for rat samples (CSB-E08602r). All experimental procedures were conducted in accordance with the manufacturer's guidelines to ensure the accuracy and reliability of the results. Quantitative analysis involved measuring absorbance with a BioTek microplate reader, and the results were compared against a standard curve established under consistent experimental conditions to ensure validity.
Quantification of MDA, SOD, TT, and CAT
MDA levels, as an indicator of lipid peroxidation, were measured using the thiobarbituric acid reactive substances (TBARS) method. Briefly, 0.5 ml of homogenized tissue supernatant was mixed with 2.5 ml of 20% trichloroacetic acid (TCA) and 1 ml of 0.67% thiobarbituric acid (TBA; Sigma-Aldrich). The mixture was heated at 95°C for 30 min in a water bath, then cooled to room temperature, and centrifuged at 3,000 rpm for 10 min. The absorbance of the resulting supernatant was measured at 532 nm. The concentration of MDA was calculated and expressed as nmol/mg tissue (Ohkawa et al., 1979; Akbari et al., 2023).
Total thiol groups were measured using Ellman's reagent [5,5′-dithiobis-(2-nitrobenzoic acid), DTNB; Sigma-Aldrich]. In brief, 50 µl of tissue homogenate was mixed with 1 ml of Tris-EDTA buffer, pH 8.6, and 50 µl of 10 mM DTNB. The mixture was incubated at room temperature for 15 min, and the absorbance was recorded at 412 nm. The concentration of thiol groups was calculated using a molar extinction coefficient of 13,600 M−1cm−1 and expressed as µmol/mg protein (Ellman, 1959).
SOD activity was quantified using a commercial colorimetric assay kit (e.g., Cayman Chemical, catalog #706002), following the manufacturer's protocol. This assay is based on the enzymatic production of superoxide radicals, which react with a tetrazolium salt (WST-1) to form a colored formazan dye measurable at 450 nm. SOD in the sample inhibits the formation of this dye by dismutating superoxide radicals. One unit of SOD activity is defined as the amount of enzyme that causes a 50% inhibition of the color development. Absorbance was measured and SOD activity was calculated using the provided standard curve.
CAT activity was determined spectrophotometrically by measuring the decomposition of hydrogen peroxide (H2O2) according to the method of Aebi (1984). Briefly, 50 µl of tissue homogenate was added to 2.95 ml of 30 mM H2O2 (Sigma-Aldrich) prepared in 50 mM phosphate buffer, pH 7.0. The decrease in absorbance was recorded at 240 nm over a period of 1 min. CAT activity was expressed as µmol H2O2 decomposed per minute per mg protein (Aebi, 1984; Akbari et al., 2023). All absorbance measurements were performed using a UV–Vis spectrophotometer (Model: UV-1600, Shimadzu).
Statistical analyses
The data were analyzed using GraphPad Prism software (version 8.4.3). Statistical evaluation was carried out through one-way ANOVA, followed by Tukey's post hoc test for multiple comparisons. The results are presented as the mean ± standard error of the mean, with statistical significance defined at α = 0.05, representing a 95% confidence interval.
Results
Thiamine mitigates anxiety-like behaviors associated with nicotine withdrawal
The OFT revealed that nicotine withdrawal induced anxiety-like behaviors, as indicated by a significant decrease in time spent in the central area of the arena (F(7,54) = 44.59, p < 0.001; Fig. 2A) and a corresponding increase in exploratory activity within the peripheral zones (F(7,54) = 21.23, p < 0.001; Fig. 2B). Thiamine administration at 50 mg/kg during both nicotine exposure and withdrawal significantly increased central area exploration compared with the nicotine-vehicle group (p < 0.001; Fig. 2A). Additionally, thiamine at 50 mg/kg alone during P42–63 significantly increased central time compared with the vehicle-vehicle group (p < 0.05; Fig. 2A). Similarly, thiamine at 25 mg/kg during nicotine withdrawal and at 50 mg/kg during both exposure and withdrawal significantly reduced peripheral exploration compared with the nicotine-vehicle group (p < 0.05, p < 0.01, and p < 0.001, respectively; Fig. 2B).
Effects of thiamine on anxiety-related behaviors in the open field test (OFT). A, Time spent in the central area of the arena; (B) time spent in the peripheral area. Data are expressed as mean ± SEM (n = 10). Significant differences: *p < 0.05 and ***p < 0.001 compared with the vehicle–vehicle group; +p < 0.05, ++p < 0.01, and +++p < 0.001 compared with the nicotine–vehicle group.
Findings from the EPM supported the OFT results, with nicotine withdrawal significantly decreasing time in the open arms (F(7,54) = 35.72, p < 0.001; Fig. 3A) and increasing time in the closed arms (F(7,54) = 31.68, p < 0.001; Fig. 3B) compared with the vehicle-vehicle group. Notably, thiamine at 50 mg/kg during both nicotine exposure and withdrawal significantly increased time spent in the open arms (p < 0.01; Fig. 3A) and reduced time in the closed arms (p < 0.01 and p < 0.001, respectively; Fig. 3B) relative to the nicotine-vehicle group. However, thiamine at 50 mg/kg alone did not significantly alter open or closed arm time compared with the vehicle-vehicle group.
Effects of thiamine on anxiety-related behaviors in the elevated plus maze (EPM). A, Time spent in the open arms; (B) time spent in the closed arms. Data are shown as mean ± SEM (n = 10). Significant differences: *p < 0.05 and ***p < 0.001 compared with the vehicle–vehicle group; +p < 0.05, ++p < 0.01, and +++p < 0.001 compared with the nicotine–vehicle group.
Thiamine mitigates depression-like behaviors associated with nicotine withdrawal
In the FST, nicotine withdrawal significantly increased depressive-like behaviors, as evidenced by a reduction in struggling time (F(7,54) = 41.16, p < 0.001; Fig. 4A), an increase in immobility (F(7,54) = 35.42, p < 0.001; Fig. 4B), and a decrease in swimming time (F(7,54) = 47.32, p < 0.001; Fig. 4C) compared with the vehicle-vehicle group. Thiamine administration at 25 mg/kg during nicotine withdrawal significantly reduced immobility time (p < 0.01; Fig. 4B). Additionally, thiamine at 50 mg/kg, both during nicotine exposure and withdrawal, significantly increased struggling (p < 0.01 and p < 0.001, respectively; Fig. 4A) and swimming times (p < 0.01 and p < 0.001, respectively; Fig. 4C), while reducing immobility (p < 0.01 and p < 0.001, respectively; Fig. 4B) compared with the nicotine-vehicle group. However, thiamine at 50 mg/kg alone did not produce significant behavioral changes in the FST.
Effects of thiamine on depressive-like behaviors in the forced swim test. A, Time spent struggling; (B) time spent immobile; (C) time spent swimming. Data are represented as mean ± SEM (n = 10). Significant differences: ***p < 0.001 compared with the vehicle–vehicle group; +p < 0.05, ++p < 0.01, and +++p < 0.001 compared with the nicotine–vehicle group.
Thiamine mitigates anhedonia-like behaviors associated with nicotine withdrawal
The SPT results indicated that nicotine withdrawal induced anhedonia-like behaviors, as reflected by a significant decrease in sucrose preference compared with the vehicle-vehicle group (F(7,54) = 40.36, p < 0.001; Fig. 5). Notably, thiamine treatment at 50 mg/kg, administered both during nicotine exposure and throughout withdrawal, significantly increased sucrose preference relative to the nicotine-vehicle group (p < 0.01 and p < 0.001, respectively; Fig. 5). Additionally, thiamine administration alone at 50 mg/kg during P42–63 significantly enhanced sucrose preference compared with the vehicle-vehicle group (p < 0.05; Fig. 5).
Effects of thiamine on anhedonia-like behaviors in the sucrose preference test (SPT). Data are shown as mean ± SEM (n = 10). Significant differences: *p < 0.05 and ***p < 0.001 compared with the vehicle–vehicle group; +p < 0.01 and ++p < 0.001 compared with the nicotine–vehicle group.
Thiamine modulates oxidative stress parameters associated with nicotine withdrawal
Biochemical analyses revealed that nicotine withdrawal significantly disrupted oxidative stress homeostasis in the PFC. This disruption was characterized by a marked increase in MDA levels (F(7,54) = 46.25, p < 0.001; Fig. 6A), alongside decreased thiol concentrations (F(7,54) = 33.15, p < 0.001; Fig. 6B) and reduced enzymatic activities of SOD (F(7,54) = 28.75, p < 0.001; Fig. 6C) and CAT (F(7,54) = 33.27, p < 0.001; Fig. 6D) compared with the vehicle-vehicle group. Notably, coadministration of thiamine at 25 mg/kg significantly decreased MDA levels (p < 0.05; Fig. 6A). Furthermore, thiamine treatment at 50 mg/kg during both nicotine exposure and withdrawal led to significant reductions in MDA levels (p < 0.05 and p < 0.001; Fig. 6A) and increases in thiol concentrations (p < 0.05 and p < 0.005; Fig. 6B) and SOD activity (p < 0.01 and p < 0.001; Fig. 6C). Similarly, CAT activity was significantly elevated compared with the nicotine-vehicle group (p < 0.01 and p < 0.001; Fig. 6D). Additionally, thiamine administration at 50 mg/kg alone from P42–63 significantly reduced MDA levels while enhancing thiol concentrations and SOD and CAT activities compared with the vehicle-vehicle group (p < 0.05; Fig. 6A–D).
Effects of thiamine on oxidative stress markers in the prefrontal cortex. A, Malondialdehyde (MDA) levels; (B) total thiol (TT) levels; (C) superoxide dismutase (SOD) activity; (D) catalase (CAT) activity. Data are presented as mean ± SEM (n = 10). Significant differences: *p < 0.05 and ***p < 0.001 compared with the vehicle–vehicle group; +p < 0.05, ++p < 0.01, and +++p < 0.001 compared with the nicotine–vehicle group.
Thiamine ameliorates neurochemical alterations associated with nicotine withdrawal
Nicotine withdrawal was associated with a significant decrease in serotonin levels (F(7,54) = 99.65, p < 0.001; Fig. 7A), along with a pronounced increase in MAO-A activity (F(7,54) = 129.65, p < 0.001; Fig. 7B). Additionally, there was a marked reduction in BDNF levels (F(7,54) = 43.95, p < 0.001; Fig. 7C) and an increase in GFAP expression (F(7,54) = 10.65, p < 0.001; Fig. 7D) compared with the vehicle-vehicle group. Administration of thiamine at 25 mg/kg during nicotine exposure significantly increased serotonin levels (p < 0.05; Fig. 7A). Furthermore, thiamine treatment at 50 mg/kg during both nicotine exposure and withdrawal effectively elevated serotonin levels (p < 0.01 and p < 0.001, respectively; Fig. 7A), decreased MAO-A activity (p < 0.01 and p < 0.001, respectively; Fig. 7B), restored BDNF levels (p < 0.05 and p < 0.001, respectively; Fig. 7C), and reduced GFAP expression compared with the nicotine-vehicle group (p < 0.05 and p < 0.001, respectively; Fig. 7D). Notably, thiamine administration at 50 mg/kg alone from P42 to 63 resulted in a significant increase in serotonin levels compared with the vehicle-vehicle group (p < 0.05; Fig. 7A).
Effects of thiamine on cortical levels of neurotransmitters and biomarkers. A, Serotonin levels; (B) monoamine oxidase-A (MAO-A) activity; (C) brain-derived neurotrophic factor (BDNF) levels; (D) glial fibrillary acidic protein (GFAP). Data are expressed as mean ± SEM (n = 10). Significant differences: *p < 0.05 and ***p < 0.001 compared with the vehicle–vehicle group; +p < 0.05, ++p < 0.01, and +++p < 0.001 compared with the nicotine–vehicle group.
Discussion
Although nicotine dependence and withdrawal are known to be associated with behavioral and neurochemical disturbances in both humans and animals (Haidary et al., 2024; Hossaini et al., 2025a), nicotine continues to be widely consumed by adolescents (Beckmann et al., 2024). Importantly, low doses of nicotine are insufficient to elicit spontaneous withdrawal symptoms such as anxiety-like behavior. These observations suggest that the extent of nicotine dependence in rats is closely linked to both the administered dose and the duration of exposure (Chellian et al., 2021). High-dose nicotine exposure during adolescence leads to significant and enduring changes in behavioral responses to addictive substances in adulthood (Slotkin et al., 2014; Renda et al., 2020). This exposure is associated with neurobehavioral alterations including heightened anxiety-like behaviors and disruptions in reward processing (Slawecki et al., 2003). Moreover, adolescent nicotine administration results in pronounced neurochemical changes, including reduced serotonin levels (Slotkin et al., 2014), impaired serotonin receptor function, and dysregulated cell signaling mediated by adenylyl cyclase (Xu et al., 2002). Structural brain changes have also been reported, including reductions in total gray matter (GM) volume, region-specific GM loss in the medial prefrontal cortex (mPFC), and altered GM volume covariation between the mPFC and other brain regions (Chen et al., 2025). Studies highlight that the developing brain is particularly susceptible to the neurotoxic effects of nicotine, resulting in long-lasting alterations to neurotransmitter systems that can increase the risk of emotional dysregulation and behavioral disorders later in life (Slotkin et al., 2014).
In this study, we focused on nicotine dependence rather than addiction, as the model was designed to evaluate behavioral and neurochemical changes related to withdrawal, such as anxiety, depression, and anhedonia. To effectively model nicotine dependence and subsequent withdrawal in adolescent rats, we administered subcutaneous nicotine at a dose of 2 mg/kg. Although this dose may be considered relatively high for rodent models, multiple studies have demonstrated that such dosing is necessary to reliably induce robust and measurable nicotine withdrawal symptoms (Amiry et al., 2023; Beheshti et al., 2025; Hossaini et al., 2025b). Importantly, rodent models typically require higher nicotine doses than humans due to interspecies differences in nicotine metabolism, receptor sensitivity, and pharmacokinetics. For instance, the plasma half-life of nicotine in rats is significantly shorter (∼1 h) than that in humans (2 h) (Chellian et al., 2021), necessitating higher dosing to achieve comparable receptor occupancy and plasma nicotine concentrations. Moreover, lower nicotine doses in adolescent and adult rats often fail to produce consistent spontaneous withdrawal symptoms, including anxiety-like behavior, hyperalgesia, and cognitive deficits (Chellian et al., 2021). With regard to translational relevance, it is important to recognize that nicotine withdrawal in humans occurs within a complex biopsychosocial context (Bruijnzeel, 2017; Chellian et al., 2022). Continued tobacco or e-cigarette use is not driven by withdrawal symptoms alone but also shaped by a dynamic interplay of biological factors (e.g., neuroadaptations; Bruijnzeel, 2012), psychological influences (e.g., stress, mood disorders, and habit formation; Pergadia et al., 2014; Bruijnzeel, 2017), and interconnectedness of factors (Torres et al., 2013; Isuru and Rajasuriya, 2019).
The findings demonstrate that nicotine withdrawal induces anxiety-like behaviors, as observed in the OFT and EPM. Specifically, nicotine withdrawal significantly increased anxiety-like behaviors, as observed in both the OFT and EPM. In the OFT, the significant reduction in time spent in the central area by the nicotine-vehicle group corroborates the established understanding that withdrawal from nicotine can lead to heightened anxiety levels in rats. This behavior aligns with previous research indicating that anxiety is often manifested through increased avoidance of open, exposed areas (Prut and Belzung, 2003; Sudakov et al., 2013; Hossaini et al., 2025a), reflecting the animals’ instinctive responses to perceived threats in their environment. Similarly, the EPM results showed decreased open-arm exploration and increased time in the closed arms, reinforcing the anxiogenic effects of nicotine cessation. These findings align with previous studies reporting that nicotine withdrawal disrupts anxiolytic behavioral patterns (Amiry et al., 2023; Rezaei Moghadam et al., 2023), likely due to alterations in neurotransmitter systems, including serotonergic signaling (Haidary et al., 2024; Hossaini et al., 2025a).
A key finding of this study is the anxiolytic effect of thiamine administration. Thiamine treatment significantly mitigated nicotine withdrawal-induced anxiety, as evidenced by an increase in central area exploration in the OFT and enhanced open arm time in the EPM. The effect was dose dependent, with 50 mg/kg producing the most robust behavioral improvements, in line with previous findings that thiamine influences serotonergic pathways (Mrowicka et al., 2023), which are critical for anxiety regulation (Albert et al., 2014). The anxiolytic effects of thiamine are consistent with existing literature that highlights the essential role of B vitamins, particularly thiamine, in brain health and mood regulation (Jahan-Mihan et al., 2024; Melki et al., 2024). Moreover, thiamine deficiency has been linked to various neurological disorders, emphasizing its importance in maintaining mental well-being (Fattal-Valevski, 2011). Research also indicates that thiamine supplementation may improve cognitive function in patients with neurodegenerative diseases, suggesting a strong connection between thiamine levels and mental health (Iskra and Trufanov, 2023). These findings collectively support the potential of thiamine as a therapeutic agent for managing anxiety related to nicotine withdrawal.
Interestingly, thiamine alone at 50 mg/kg (without nicotine exposure) also increased center time in the OFT, suggesting potential intrinsic anxiolytic properties, indicating that thiamine itself has intrinsic anxiolytic effects independent of nicotine exposure or withdrawal. Additionally, studies have found that better thiamine levels are linked to improved clarity, composure, and energy, highlighting its importance in cognitive and emotional processes (Benton et al., 1997), supporting the idea that thiamine may have intrinsic effects on anxiety. Yet, more in-depth investigations are needed to uncover the exact mechanisms at play.
This study also found that nicotine withdrawal induces depression-like behaviors, as demonstrated in the FST. Specifically, nicotine withdrawal resulted in a significant decrease in struggling time, an increase in immobility, and a reduction in swimming time, all of which are hallmark indicators of behavioral despair commonly observed in depressive-like states (Yankelevitch-Yahav et al., 2015). Thiamine administration mitigated nicotine withdrawal-induced depression-like behaviors. Thiamine at 25 mg/kg during nicotine withdrawal significantly reduced immobility time, indicating an antidepressant-like effect. More notably, 50 mg/kg of thiamine, administered both concurrently with nicotine and throughout withdrawal, significantly increased struggling and swimming behaviors while reducing immobility, suggesting a stronger therapeutic effect at higher doses. These findings suggest that thiamine may counteract the behavioral despair induced by nicotine withdrawal, potentially through its neuroprotective and neuromodulatory properties. The results indicated that nicotine withdrawal induces anhedonia-like behaviors, as evidenced by a significant decrease in sucrose preference in the SPT. This finding is consistent with existing literature that underscores the impact of substance withdrawal on mood and reward processing, highlighting how nicotine cessation can lead to negative emotional states (Alkhlaif et al., 2017; Murdaugh et al., 2024). Importantly, thiamine administration at 50 mg/kg, significantly attenuated anhedonia-like behaviors, as reflected by an increase in sucrose preference. This effect was observed both when thiamine was administered concurrently with nicotine and throughout the withdrawal period, suggesting a protective role against withdrawal-induced reward deficits. Moreover, thiamine at 50 mg/kg alone (without nicotine exposure) also significantly enhanced sucrose preference, indicating that its effects may extend beyond nicotine withdrawal to more generalized reward-enhancing properties. These results support the hypothesis that thiamine is essential for emotional regulation and mood stabilization (Mrowicka et al., 2023). Nonetheless, additional research is essential to elucidate the precise mechanisms underlying these effects.
The PFC plays a pivotal role in the regulation of anxiety and depression by modulating emotional responses and executive functions (Fitzgerald et al., 2019; Kenwood et al., 2022). Dysregulation of PFC activity has been consistently associated with the pathophysiology of anxiety disorders and major depressive disorder (Shiba et al., 2016).
Notably, nicotine exposure has been shown to induce considerable oxidative stress in the PFC, as evidenced by increased MDA levels, decreased thiol concentrations, and reduced enzymatic activities of SOD and CAT. These findings align with previous studies indicating that nicotine withdrawal triggers excessive oxidative stress, leading to neuronal damage, neuroinflammation, and impaired cognitive and affective functions (Haidary et al., 2024; Hossaini et al., 2025a). Given the critical role of oxidative stress in neuropsychiatric disorders, such as anxiety, depression, and cognitive impairments, the observed biochemical alterations further support the notion that oxidative damage contributes to the behavioral disturbances associated with nicotine withdrawal (Fedoce et al., 2018; Dash et al., 2024). Importantly, thiamine administration mitigated nicotine withdrawal-induced oxidative stress. Thiamine at 25 mg/kg significantly reduced MDA levels and increased thiol concentrations, suggesting an initial protective effect against lipid peroxidation and oxidative imbalance. More notably, thiamine at 50 mg/kg, administered both during nicotine exposure and withdrawal, further enhanced antioxidant defenses by significantly decreasing MDA levels while simultaneously increasing thiol concentrations and restoring the activities of SOD and CAT. These findings indicate that higher doses of thiamine offer greater neuroprotective benefits, potentially by enhancing endogenous antioxidant defense mechanisms. The mechanisms underlying thiamine's protective effects against oxidative stress may involve its role in mitochondrial function, redox homeostasis, and neuroprotection. Thiamine is a critical cofactor for several enzymes involved in energy metabolism and oxidative phosphorylation, and its deficiency has been linked to increased oxidative stress and neurodegeneration (Mrowicka et al., 2023). By enhancing mitochondrial function and reducing oxidative damage, thiamine may help prevent neuronal injury and dysfunction associated with nicotine withdrawal. Additionally, thiamine's ability to increase SOD and CAT activity suggests that it strengthens endogenous antioxidant defenses, further reducing oxidative damage and promoting neuronal resilience. The antioxidant effects of thiamine align with existing literature that underscores its role as an antioxidant agent. Additionally, thiamine has been shown to reduce oxidative damage in various experimental settings (Lukienko et al., 2000), including diabetes and liver disease, where it alleviates oxidative stress by modulating metabolic pathways (Kohda et al., 2019). Interestingly, thiamine at 50 mg/kg alone (without nicotine exposure) also significantly improved oxidative stress markers compared with the vehicle group, indicating that its effects are not limited to withdrawal-induced oxidative damage but may also provide baseline neuroprotective benefits. This suggests that thiamine supplementation could serve as a potential strategy for maintaining oxidative balance and preventing neurodegenerative processes, even in the absence of external stressors.
The present study also reveals that nicotine withdrawal induces significant neurochemical alterations, including a reduction in serotonin levels, an increase in MAO-A activity, and decreased BDNF levels. These findings suggest that nicotine withdrawal is associated with serotonergic dysregulation and neurotrophic impairment which may contribute to the behavioral and affective disturbances commonly observed during withdrawal. These observed neurochemical alterations are consistent with the dysregulation of serotonergic pathways during nicotine withdrawal. This dysregulation is particularly concerning, as serotonin plays a critical role in mood regulation, and its depletion is frequently associated with anxiety and depressive-like symptoms (Amiry et al., 2023). The increase in MAO-A activity further exacerbates this effect, as MAO-A degrades serotonin, leading to further reductions in its availability and exacerbating withdrawal-related mood disturbances. These findings align with previous research suggesting that nicotine dependence modulates serotonergic neurotransmission, and withdrawal from chronic nicotine exposure results in serotonin depletion and heightened monoaminergic degradation, contributing to mood instability and anhedonia (Hitsman et al., 2007). Importantly, thiamine administration was found to mitigate these neurochemical disruptions. Thiamine at 25 mg/kg significantly increased serotonin levels, suggesting a partial restoration of serotonergic function. More notably, thiamine at 50 mg/kg, administered both during nicotine exposure and withdrawal, effectively elevated serotonin levels, reduced MAO-A activity, restored BDNF levels, and decreased GFAP expression. These findings highlight the neuroprotective and neuromodulatory properties of thiamine, particularly in the context of nicotine withdrawal-induced neurochemical deficits. The ability of thiamine to increase serotonin levels while simultaneously reducing MAO-A activity suggests a potential regulatory effect on monoaminergic neurotransmission. Thiamine has been implicated in neurotransmitter synthesis and mitochondrial function, both of which play crucial roles in maintaining serotonergic homeostasis (Mrowicka et al., 2023). By reducing MAO-A activity, thiamine may help preserve serotonin availability, counteracting the serotonergic depletion associated with nicotine withdrawal. Additionally, the restoration of BDNF levels suggests that thiamine supports neuroplasticity and neuronal survival, as BDNF is essential for synaptic plasticity, mood regulation, and cognitive function (Lu et al., 2014; Begni et al., 2016).
Our study also demonstrated significant neuroinflammation during nicotine withdrawal, as evidenced by elevated GFAP expression in the PFC, which may contribute to the disruption of serotonergic signaling, a key neurotransmitter system critically involved in mood regulation (Hossaini et al., 2025b). GFAP is an intermediate filament protein that is released by activated astrocytes within the central nervous system and is widely recognized as a significant biomarker for neuroinflammation and astrocyte activation (Leipp et al., 2024). Research on the association between neuron–astrocyte interactions in depression indicates that astrogliosis, characterized by elevated GFAP levels, is correlated with disrupted monoamine signaling. This specifically implicates serotonin dysfunction as a key factor in mood disturbances (Zhou et al., 2019; Yao et al., 2023). Importantly, thiamine administration led to a reduction in GFAP expression within the PFC, indicating attenuation of astrocyte activation and neuroinflammation. This finding further supports thiamine's neuroprotective properties, suggesting its potential role in preserving neuronal integrity and facilitating functional recovery.
Interestingly, thiamine at 50 mg/kg alone (without nicotine exposure) also led to a significant increase in serotonin levels, suggesting that its neuromodulatory effects extend beyond nicotine withdrawal. This finding implies that thiamine supplementation may enhance serotonergic function even under baseline conditions, potentially offering therapeutic benefits for individuals with mood disorders characterized by serotonergic dysfunction. This is supported by the fact that thiamine is essential for the synthesis of neurotransmitters, including serotonin, which significantly influences anxiety and mood. Research indicates that increased thiamine levels can enhance serotonergic activity, potentially leading to reduced anxiety-like behaviors (Mrowicka et al., 2023). However, more studies are required to clarify the specific mechanisms involved.
While the findings of this study are promising, some limitations should be acknowledged. Firstly, the use of twice daily subcutaneous nicotine injections to induce dependence, while effective in producing withdrawal-related neurobehavioral changes in rodents, does not fully replicate the pharmacokinetic profile of human nicotine consumption. As described by Benowitz (2010), habitual smokers typically experience ∼20 distinct nicotine peaks per day, resulting in rapid and dynamic fluctuations in plasma nicotine levels. In contrast, our dosing regimen generates a more sustained exposure pattern and lacks the high-frequency oscillations characteristic of real-world tobacco or e-cigarette use. Although this protocol is widely accepted in preclinical studies and provides a controlled framework for assessing withdrawal mechanisms (Pandey et al., 2001; Campos et al., 2008; Chellian et al., 2021), this pharmacokinetic discrepancy represents a translational limitation. Future studies should employ more physiologically relevant delivery systems, such as inhalation models or programmable infusions, which may better simulate human nicotine intake patterns and enhance the ecological validity of the findings.
The second limitation of the current study is the reliance solely on GFAP levels to assess neuroinflammation. While GFAP is a well-established marker of astrocyte activation (Agnello et al., 2025; Youn et al., 2025), neuroinflammation is a complex process involving multiple cell types and signaling pathways, including microglial activation (DiSabato et al., 2016) and pro-inflammatory cytokine release (Müller and Di Benedetto, 2025). Therefore, exclusive measurement of GFAP may provide an incomplete picture of the inflammatory response. Future studies should incorporate additional markers, such as Iba1 for microglia, as well as cytokines like IL-1β and TNF-α, which would offer a more comprehensive assessment of neuroinflammatory changes associated with nicotine withdrawal and related interventions (Kumar et al., 2024). Recognizing this limitation is important for contextualizing our findings and guiding more detailed mechanistic investigations.
Additionally, the absence of a positive control, such as varenicline, limits the ability to compare the efficacy of thiamine with established pharmacotherapies for nicotine dependence. Varenicline, a partial agonist of nicotinic acetylcholine receptors, has been shown to reduce withdrawal symptoms and cravings and could serve as a benchmark for evaluating new therapeutic agents. Future studies should incorporate positive control groups to provide a clearer context for assessing thiamine's efficacy relative to standard treatments. While thiamine appears effective in mitigating symptoms of nicotine withdrawal, its precise mechanism of action remains to be fully elucidated. Further investigation into how thiamine modulates serotonin pathways, oxidative stress responses, and neuroinflammatory markers would enhance our understanding of its therapeutic potential. Additionally, studies exploring different dosages, treatment durations, and combinations with other pharmacological agents could help optimize the clinical application of this treatment. Finally, clinical trials will be essential to determine whether thiamine supplementation is effective and safe for managing nicotine withdrawal in human populations.
Conclusion
Thiamine appears to offer a promising therapeutic strategy for mitigating the behavioral and neurochemical alterations associated with nicotine withdrawal. Its effects on anxiety-like, depression-like, and anhedonia-like behaviors, along with its ability to modulate oxidative stress and restore neurochemical balance, highlight its potential as a treatment for nicotine dependence. However, the inclusion of a positive control and further mechanistic studies are necessary to fully evaluate thiamine's efficacy and its place within the broader landscape of treatments for nicotine withdrawal and dependence.
Data Availability
The data generated during this study are available upon request from the corresponding author.
Footnotes
The authors declare no competing financial interests.
We thank the Medical Sciences Research Center of Khatam Al-Nabieen University for their valuable collaboration and for providing the necessary resources to conduct this study.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.
References
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