psychiatric consequences. This study evaluated the neuroprotective potential of vitamin B-complex against

METH-induced cerebellar and cerebral toxicity in adolescent male Wistar rats. Fifty-eight rats weighing 115–

128 grams were used, with 28 employed for toxicity testing and 30 randomized into six experimental groups (n

= 5). Group A (Negative Control) and received feed and distilled water only. Group B received 8 mg/kg of

methamphetamine. Group C and D received 50 mg/kg and 100mg/kg of vitamin B-complex respectively.

Group E received a co-administration of 8 mg/kg methamphetamine and 50 mg/kg of vitamin B-complex,

while Group F received a co-administration of 8 mg/kg of methamphetamine and 100 mg/kg of vitamin B-

complex. Treatments were administered orally for 28 days. Neurobehavioral evaluations (Morris water maze

and hanging wire test) were conducted during days 24–28 to capture sub-chronic functional outcomes. At

termination, animals were anesthetized, brains harvested, and tissues processed for biochemical and

neurotoxicity, supporting its potential as an adjunctive therapeutic strategy for substance-related neurological

disorders.

Methamphetamine (METH), commonly known as “ice,” “crystal,” or locally as Mkpurummiri in South-East

Nigeria, is a highly addictive psychostimulant and a major global public health concern (1, 2). Structurally

related to amphetamine, which is used clinically in attention-deficit hyperactivity disorder (ADHD) and

narcolepsy, METH is largely abused recreationally due to its potent, long-lasting euphoric effects that are

stronger and cheaper than cocaine (3, 4). It can be ingested orally, snorted, injected, or smoked, with smoking

being the most common route (4). Rising use continues to impose devastating effects on individuals, families,

linked with paranoia, hallucinations, delusions, and psychosis, while withdrawal features depression, anxiety,

hypersomnolence, and agitation. Some symptoms resolve quickly, but cognitive and affective deficits often

persist, correlating with ongoing neuronal damage (5, 7).

Mechanistically, METH primarily damages dopaminergic and serotonergic terminals rather than cell bodies.

Excessive dopamine release drives oxidative stress, lipid peroxidation, mitochondrial dysfunction, and

excitotoxicity, while hyperthermia worsens injury (5, 8). These processes impair executive function and motor

coordination, with meta-analyses confirming deficits in working memory, processing speed, and impulse

control (1).

Another critical dimension of METH abuse is nutritional compromise. Substance use disorders often cause

deficiencies in essential vitamins, especially B vitamins, which are vital for energy metabolism and

apoptosis, gliosis, and oxidative damage in models of METH exposure and diabetes-related neurodegeneration

(10, 13).

Given the central role of the cerebrum in higher cognitive functions and the cerebellum in motor control and

coordination, and their known vulnerability to oxidative and excitotoxic injury (14), investigating the

protective role of vitamin B complex against methamphetamine-induced neurotoxicity in these brain regions is

highly justified. This is particularly relevant in adolescence, a critical stage of brain maturation and heightened

susceptibility to substance-induced damage (3).

Therefore, this study evaluates the neuroprotective effects of vitamin B complex against METH-induced

cerebellar toxicity in adolescent male Wistar rats. By targeting oxidative stress, neurotransmitter imbalance,

and energy deficits, B-complex supplementation may offer a safe and accessible strategy to mitigate METH-

Nigeria. The animals were acclimatized for two weeks under standard laboratory conditions, housed in well

ventilated cages at room temperature with a 12-hour light/dark cycle, and maintained on standard rat chow

(Agro Feed Mill, Nigeria Ltd.) and distilled water ad libitum. The use of animals at this age and weight

corresponds to the adolescent stage of rat development, which is marked by rapid brain maturation and

increased vulnerability to neurotoxic insults, making them appropriate for this study. All experimental

procedures complied with the National Institute of Health Guide for the Care and Use of Laboratory Animals

(15). Twenty-eight rats were assigned to toxicity testing, while fifty-six were used for the main experimental

Methamphetamine (METH) was obtained through the National Drug Law Enforcement Agency (NDLEA),

while vitamin B-complex was purchased from a licensed pharmaceutical supplier. METH was reconstituted in

distilled water following Madden et al.’s method (16), and vitamin B-complex prepared according to the

32.5 mg/kg, while vitamin B-complex showed no mortality up to 5000 mg/kg, indicating a wide margin of

safety.

Thirty (30) of the acclimatized adolescent male Wistar rats were randomly assigned into six groups (A–F) of 5

rats each. Group A (Negative Control) received distilled water. Group B received 8 mg/kg of

methamphetamine. Group C and D received 50 mg/kg and 100mg/kg of vitamin B-complex respectively.

Group E received a co-administration of 8 mg/kg methamphetamine and 50 mg/kg of vitamin B-complex,

while Group F received a co-administration of 8 mg/kg of methamphetamine and 100 mg/kg of vitamin B-

complex. Administration was carried out orally using an orogastric cannula at the designated doses for each

experimental group.

Morris Water Maze test involved pre-training with a visible platform followed by hidden platform trials in

opaque water to assess spatial learning and memory (20).

Twenty-four hours after the final treatment (day 29), the animals were fasted overnight and anesthetized with

ketamine hydrochloride (40 mg/kg, intraperitoneally). Blood samples were obtained via ocular puncture and

transferred into plain, sterilized glass tubes without anticoagulant for biochemical assays. The blood was

centrifuged using a laboratory ultracentrifuge (New Life model), and the resulting serum was separated and

stored under refrigeration until analysis.

While still under deep anesthesia, the animals were humanely sacrificed in accordance with institutional ethical

guidelines. The skull was carefully opened, and the brain was excised. The cerebellum and cerebrum were

dissected, rinsed in cold normal saline to remove blood residues, blotted dry, and weighed. Portions designated

saline, centrifuged at 3000 rpm for 20 minutes, and the supernatant stored at 2 °C until analysis, while samples

(21) with absorbance set at 530 nm. Results were expressed as nmol MDA/h/g tissue.

Reduced Glutathione (GSH) was determined using Ellman’s method with DTNB; absorbance at 412 nm and

expressed as μmol/g tissue (22).

Superoxide Dismutase (SOD) was measured by monitoring NBT reduction; absorbance at 560 nm and

expressed as μmol/min/mg protein (23).

Fixed portions of the brain tissues (cerebrum and cerebellum) were processed using standard histological

techniques. Briefly, samples were dehydrated in ascending grades of ethanol (50%, 70%, 95%, and absolute),

cleared in xylene, and infiltrated with molten paraffin wax at 75 °C before embedding in paraffin blocks at 58–

Values are expressed as Mean ± SEM (n = 5). Statistical significance was determined using one-way ANOVA;

Table 5 shows the effects of methamphetamine and vitamin B-complex on oxidative stress biomarkers in the

cerebrum and cerebellum of Wistar rats. Methamphetamine significantly increased malondialdehyde (MDA)

levels compared with the control, while co-treatment with vitamin B-complex attenuated this effect.

Superoxide dismutase (SOD) activity was significantly altered across groups, whereas reduced glutathione

(GSH) concentrations did not differ significantly among the treatment groups.

Table 5: Effect of Methamphetamine and Vitamin B-Complex on Oxidative Stress Markers

Values are expressed as mean ± SEM (n = 5). One-way ANOVA was used to compare groups; p ≤ 0.05 was

considered statistically significant.

H&E-stained cerebellar sections revealed normal cortical architecture in the control group (A), showing well-

defined molecular (A), Purkinje (C), and granular layers (B) with intact Purkinje cells and clear nuclei. The

Cresyl violet–stained cerebellar sections showed clear differences across groups. The control group (A)

displayed normal trilaminar architecture with well-outlined Purkinje cells (red arrows) and intense Nissl

staining. The methamphetamine-treated group (B) exhibited severe neuronal degeneration with pyknotic and

karyorrhectic Purkinje cells (black arrows), disorganized layers, and reduced staining intensity. Groups C and

D (Vitamin B complex only) maintained normal cerebellar structure and deeply stained Purkinje cells (red

arrows), though slight scattering of the Purkinje layer was observed in group D. Co-treated groups (E and F)

showed partial to near-complete recovery of neuronal organization, with mild to pronounced aggregation of

H&E-stained sections of the cerebrum showed normal cortical organization in the control group (A), with

organized cortical structure, with group F exhibiting the greatest degree of preservation.

Cresyl violet–stained sections of the cerebrum revealed normal neuronal architecture in the control group (A),

characterized by distinct pyramidal neurons with well-defined nuclei and cytoplasm (red arrows). The

methamphetamine-treated group (B) showed scanty degeneration of pyramidal neurons (red arrows) within the

cortical region. Groups C and D (Vitamin B complex only) displayed intensely stained pyramidal neurons and

dendrites with deeply stained Nissl substance, indicating essentially normal neurons (red arrows). In the co-

treated groups (E and F), pyramidal neurons appeared aggregated with increased color intensity (red arrows),