Introduction

Methamphetamine (METH) abuse is a global health concern and is associated with a wide range of adverse neurological conditions [8,28]. Experimental and clinical evidence indicates that chronic METH exposure leads to oxidative stress, neuroinflammation, and severe neuropathological complications in affected individuals [3,27]. Consequently, investigation of METH toxicity extends beyond analytical confirmation to rigorous histological and biochemical analysis associated with stimulant toxicity. Understanding the mechanistic relationship between METH use and certain neuropathological conditions in the brain is essential for interpreting observed pathological findings and improving forensic assessment.

METH’s neurotoxic potential is widely understood, and its common pathophysiology is mainly the result of a cascade of significant oxidative stress and excitotoxicity [7,38]. This well-established concept proposes that METH-induced hyperdopaminergia causes dopamine autooxidation, resulting in a significant rise in reactive oxygen species (ROS) and mitochondrial dysfunction, ultimately resulting in neuronal apoptosis and degeneration of monoaminergic terminals [21,26]. While apoptotic pathways are important for METH-induced damage, they alone may be enough to adequately explain the widespread cellular death observed in severe METH intoxication. The apoptotic model fails to effectively explain this in the absence of a solid understanding of the natural mechanism of cellular death. This poses challenges for developing specific histopathological indicators useful in forensic diagnostics.

Our current study focuses on alternate mechanisms of cell death, with an emphasis on iron-dependent cell death. The new discovery of ferroptosis, an iron-dependent form of regulated cell death characterized by the accumulation of lipid peroxides, provides a transformational perspective on neurodegeneration. Unlike apoptosis, ferroptosis is highly dependent on the inactivation of the selenoenzyme glutathione peroxidase-4 (GPX-4), the primary inhibitor responsible for decreasing harmful lipid hydroperoxides inside cellular membranes [12,13,19]. Ferroptosis exhibits morphologically and biochemically distinct features from conventional apoptosis [35], and is characterized by dysfunction of the cystine/glutamate antiporter system Xc-, particularly its SLC7A11 subunit, which regulates intracellular glutathione synthesis and cellular antioxidant defense. Disruption of this pathway promotes oxidative lipid damage and ferroptotic cell death. Therefore, examining indicators of dysfunction in this pathway may provide new insights into the mechanisms underlying METH-induced neurotoxicity.

Although markers such as GPX-4 and lipid peroxidation have been associated with METH exposure, with GPX-4 playing a particularly important role in protecting neurons from oxidative damage, especially in the context of neurodegenerative diseases such as Parkinson’s disease [10,34,37], definitive evidence confirming ferroptotic cell death in this context remains limited, highlighting a considerable translational gap in our understanding of METH neurotoxicity.

Furthermore, understanding the precise mechanism of cell damage is also critical for identifying potential neuroprotective interventions. Cannabidiol (CBD), a non-psychoactive phytocannabinoid, has demonstrated antioxidant, anti-inflammatory, and neuroprotective effects in models of ischemia and excitotoxicity [5,18,22]. Its ability to mitigate oxidative stress makes it a potential therapeutic agent against METH neurotoxicity. However, its efficacy in inhibiting specified ferroptosis-associated pathways remains to be fully explored.

Therefore, this study was designed to investigate ferroptosis-like oxidative injury in the brain following METH exposure and to assess whether pretreatment with CBD can modulate these effects. To address gaps in human data, we first examined ferroptosis-related molecular markers in post-mortem prefrontal cortex (PFC) tissue from verified METH users, providing a human-relevant molecular profile. Subsequently, we employed a murine METH binge model to experimentally evaluate these molecular changes and the preventive effects of CBD in vivo.

Material and methods

Human brain

Post-mortem PFC tissue was collected from individuals with a confirmed history of METH use (n = 5) and age-matched non-drug-using controls (n = 5) through the Forensic Appraisal Center of Kunming Medical University. Consent for the scientific use of brain tissue was obtained from the legal relatives of all deceased cases. All procedures were conducted in accordance with the ethical principles of the World Medical Association and were approved by the Medical Ethics Committee of Kunming Medical University (Approval No. KMMU2022MEC151; November 10, 2022). A summary of toxicological findings for the METH cases is provided in Table I. In the control group, cardiovascular disease was identified as the definitive cause of death. For biochemical and protein-based analyses, the PFC was promptly dissected and stored at –80°C until use. For histological processing, tissues were fixed in 4% paraformaldehyde (Biosharp Life Sciences, BL539A) and cryoprotected by immersion in stepwise sucrose solutions (10%, 20%, and 30%; Beyotime, ST1670). Coronal sections with a thickness of 10 µm were prepared using a freezing microtome (RWD Life Science, FS800A).

Post-mortem human brain tissue is inherently susceptible to confounding factors, including post-mortem interval, agonal state, cause of death, comorbidities, and potential polysubstance use, all of which may influence oxidative stress indicators and protein expression. Although toxicological findings and cause-of-death data were reported, these variables cannot be entirely excluded.

Iron (Fe) concentration

The total iron content of the tissues was measured using an Iron Assay Kit (Beyotime, S1066S). To extract the supernatant, the samples were homogenized in the assay buffer and centrifuged at 12,000 × g for 10 minutes at 4°C. The total protein content was measured with a BCA assay kit. Next, the reaction mixture was incubated with the Iron Probe and Assay Buffer in the dark at 37°C for 60 minutes. Finally, the absorbance was measured at 593 nm with a microplate reader (BioTek Instruments, EPOCH2). Iron level was normalized to the total protein concentration.

Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining

Cellular apoptosis was evaluated using a commercial TUNEL assay kit (Sigma-Aldrich, 12156792910 or 11684817910). Tissue sections were initially fixed with 4% paraformaldehyde for 10 min, rinsed with phosphate-buffered saline (PBS), and permeabilized using 2× Triton X-100 for 10 min at room temperature. Sections were then incubated with the TUNEL reaction solution for 60 min at 37°C, followed by three PBS washes. Nuclear counterstaining was performed with DAPI. Fluorescence images were acquired using a fluorescence microscope and quantified with ImageJ software (v1.46r).

Western blot

Brain tissues were homogenized on ice in RIPA lysis buffer containing a protease inhibitor cocktail, followed by ultrasonic disruption (Epizyme Biotech, PC101) and incubation for 30 min. Protein concentrations were determined using the Pierce BCA protein assay kit (Epizyme Biotech, ZJ102). Equal quantities of protein (25 µg
per lane) were resolved on 12% SDS–polyacrylamide gels and transferred onto PVDF membranes (Sigma-
Aldrich, IPVH00010) using a wet transfer system.

Membranes were blocked with Tris-buffered saline containing 5% skim milk and incubated overnight at 4°C with primary antibodies against GPX-4 (Proteintech, 66983-1-Ig; 1 : 5000). Following washing with TBST (three times, 5 minutes each), membranes were incubated with appropriate HRP-conjugated secondary antibodies (1 : 5000) for 90 min. Protein bands were visualized using a ChemiDoc MP imaging system (Bio-Rad). b-Actin (Proteintech, 66009-1; 1 : 5000) served as the internal loading control. Densitometric analysis was performed using ImageJ software with background correction, and GPX-4 expression levels were normalized to b-actin within each blot.

Malondialdehyde (MDA) detection

Lipid peroxidation was assessed by measuring MDA concentrations using an MDA assay kit (Beyotime, S0131). Tissue samples were subjected to ultrasonic homogenization in lysis buffer and incubated on ice for 30 min. Total protein levels were quantified using a BCA assay. Samples and standards were then reacted with the MDA working solution at 100°C for 15 min. Absorbance was measured at 532 nm using a microplate reader (BioTek Instruments, EPOCH2), and MDA levels were normalized to protein concentration.

Detection of superoxide dismutase (SOD)

For the assessment of SOD activity, a commercial assay kit (Beyotime, S0101) was used. Protein concentration was measured first using the same method described above. The supernatant was treated with the working solution and incubated at 37°C for 30 min. Absorbance was measured at 450 nm with a microplate reader (described above). Relative SOD activity was determined using the formula given in the kit.

Hematoxylin and eosin (H&E) staining

Routine histopathological evaluation was performed using an H&E staining kit (Beyotime, C0105). Tissue sections were fixed in 4% paraformaldehyde for 15 min, washed with distilled water, stained with hematoxylin for 8 min, and counterstained with eosin for 1 min. Sections were subsequently cleared with xylene, dehydrated through graded ethanol solutions, and mounted using a commercial mounting medium (Sigma-Aldrich, 03989). Digital images were obtained using a pathology slide scanner (Kangfeng Bioinformatics Technology, KF PRO-005-EX).

Animals

Adult male C57BL/6 mice (18-20 g; n = 40) were obtained from the Experimental Animal Center of Kunming Medical University. Animals were housed under controlled temperature and humidity conditions with free access to food and water and maintained on a 12-h reverse light/dark cycle. All experimental procedures followed the National Research Council’s Guide for the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committee of Kunming Medical University (Approval No. KMMUX2025070288).

Animal treatments

Following a one-week acclimatization period, mice received four daily intraperitoneal injections of methamphetamine (2 mg/ml per injection) administered at 4-h intervals. This binge-type dosing regimen has been widely used to model patterns of human METH abuse and to induce neurotoxic effects in rodents. CBD (40 mg/ml) was administered intraperitoneally one hour before each METH injection. Following treatment, animals were euthanized and perfused transcardially with saline. The PFC was isolated, stored at –80°C, and processed for subsequent biochemical and histological analyses. Brain tissues were fixed in 4% paraformaldehyde at 4°C, dehydrated in graded sucrose solutions (10%, 20%, and 30%), and sectioned at a thickness of 10 µm using a freezing microtome.

Establishment of the CPP model

The conditioned place preference test was performed using a two-chamber plexiglass apparatus
(32 × 29 × 39 cm), consisting of visually and tactilely distinct compartments. One chamber had black walls and a round-hole floor, while the other had white walls with a palisade-pattern floor. A removable central partition separated the two sections during conditioning. Mouse activity was recorded using a computerized tracking system (v2.8.9.5, Shanghai Yishu Information Technology Co., Ltd., China).

Experimental protocol of the CPP model

The partition was removed, and mice were allowed to freely explore both chambers for 30 min daily for three consecutive days. On Day 4, preconditioning data were collected by recording the residence time in each chamber for 15 min under sound-insulated conditions. Animals showing a strong baseline preference (> 700 s
or < 200 s in a single chamber) were excluded. For each mouse, the compartment with longer occupancy during the pre-test was defined as the preferred side, and the opposite side was designated as the non-preferred chamber. During conditioning, the partition was lowered to restrict animals to a single compartment. The mice were assigned to the METH and METH + CBD groups, received intraperitoneal drug administration, and were immediately confined to the drug-paired chamber for 30 min. They received saline and were confined to the non-drug chamber. CBD-only mice received CBD drug only, and followed the same chamber assignment. Control animals received saline on all training days. For the CPP test, the partition was removed, and mice were allowed to move freely between chambers for 15 min. Time spent in each compartment was recorded automatically. The CPP score was calculated as the difference between time spent in the drug-paired and non-drug chambers (post-test residence time). Change in preference was represented as the post-test minus pre-test value.

Statistical analysis

Data analysis was performed using GraphPad Prism (version 9; GraphPad Software, USA). Data are expressed as mean ± standard deviation (SD). Sample size (n) represents biological replicates and is indicated in the corresponding figure legends. Technical replicates were averaged prior to analysis. Comparisons between human tissue groups were conducted using an independent samples t-test. Data obtained from animal experiments were analyzed using two-way analysis of variance (ANOVA) without repeated measures, followed by Tukey’s HSD post hoc test for multiple comparisons. Assumptions of normal distribution and homogeneity of variance were evaluated prior to analysis. Where applicable, effect sizes were provided to aid interpretation. A p < 0.05 was considered statistically significant.

Results

Oxidative neurotoxicity induced by METH in human prefrontal cortex

The PFC was identified in the isolated post-mortem human brain samples. A summary of toxicological findings for the METH cases is provided in Table I. This region demonstrated elevated METH-induced oxidative neurotoxicity. Individuals with a history of chronic METH use exhibited significantly higher total iron levels compared with the control group in the human PFC (Fig. 1A). In addition, the proportion of TUNEL-positive cells, indicative of DNA fragmentation, was significantly increased in the METH group compared to the controls (Fig. 1B). Western blot analysis further demonstrated that the expression of GPX-4 protein levels substantially decreased in the METH users compared to the control group (Fig. 1C). Consistent with increased oxidative stress, MDA levels were significantly higher in those with a history of chronic methamphetamine use than in matched control subjects (Fig. 1D), whereas SOD activity was significantly lower compared to controls (Fig. 1E).

Effects of CBD pretreatment on METH-induced conditioned place preference (CPP) in mice

To study the effect of CBD on neuroprotection in vivo, a mouse METH binge model was established (Fig. 2).
This treatment causes severe METH neurotoxicity in mice, consistent with a previous study [30], and mimics the consumption patterns observed among METH users.

The findings demonstrated that repeated METH injections induced CPP. In the CPP post-test, mice in the METH group demonstrated a clear increase in time spent in the drug-paired compartment compared with the control group, indicating successful induction of the CPP model. In Figure 3, A represents the drug-paired compartment, and B represents the non-drug compartment. Mean CPP scores were higher in the METH group than in the control group, and a shift toward positive preference was observed in most animals. In contrast, the CBD-treated group showed a moderate reduction in chamber preference, while the METH + CBD group remained closer to the baseline level (Fig. 3). Overall, the trend demonstrated higher chamber preference in METH-treated mice. Heatmap tracking visually confirmed these behavioral patterns. METH-exposed mice spent more time in the drug-paired chamber, whereas control and METH + CBD mice exhibited a more balanced distribution of time across compartments (Fig. 3).

CBD provided neuroprotection against METH-induced oxidative neurotoxicity in the prefrontal cortex

Subsequent results revealed that METH treatment resulted in a significantly higher total iron concentration than in controls in the mouse PFC. Co-treatment with CBD substantially reduced iron accumulation, while the CBD group did not differ from controls (Fig. 4A).
Similarly, METH exposure substantially increased the DNA fragmentation, as indicated by TUNEL-positive cells. This effect was significantly attenuated by CBD co-treatment. The CBD-only group showed no significant difference compared to controls (Fig. 4B). A considerable decrease in the GPX-4 protein expression was observed in METH-treated mice compared to controls. METH + CBD co-treatment resulted in a substantial partial recovery of GPX-4 levels. The CBD-only group remained comparable to the control group (Fig. 4C). Consistent with oxidative stress, METH significantly elevated MDA levels in the mouse PFC. The increase was significantly reduced by CBD co-treatment, whereas the CBD-only group showed no significant difference from controls (Fig. 4D). In addition, decreased SOD activity was seen compared to the control group. CBD co-administration substantially reversed the decline. SOD activity in the CBD-only group remained unchanged (Fig. 5A). H&E staining of the mouse PFC revealed marked neuronal damage, including shrinkage and pyknosis, in the METH-treated group compared to controls. The METH + CBD group had significantly less histopathological damage, whereas the CBD-only group showed no difference from controls (Fig. 5B).

Discussion

The neurotoxic effects of METH remain a significant public health concern. However, the specific molecular processes underlying neuronal injury are not fully understood. In the present study, we combined post-mortem human brain analysis with an in vivo mouse model to investigate oxidative injury patterns associated with METH exposure. Our findings demonstrate a consistent biochemical profile characterized by iron accumulation, increased lipid peroxidation, reduced GPX-4 expression, and impaired antioxidant defense, which together are consistent with a ferroptosis-like oxidative injury signature in the brain PFC.

The attenuation of METH‑induced CPP by CBD co‑administration adds behavioral relevance to our biochemical observations. While the neurotoxic binge model demonstrated that CBD reduces markers of oxidative injury, the CPP experiment extends this finding to the motivational domain of addiction [1,39]. CBD may disrupt the neuroplasticity that underlies the associative learning of drug reward, according to the significant decrease in place preference [4]. There are two possible non-exclusive mechanisms for this. One possibility is that CBD prevents METH-induced synaptic maladaptation in reward circuits such as the medial PFC and nucleus accumbens, which are essential for memory consolidation, whereas another is that CBD decreases the acute reinforcing or stimulating effects of METH through modulation of dopamine signaling, resulting in a weaker unconditioned stimulus [17]. Our findings do not differentiate between these possibilities but provide behavioral evidence consistent with neurochemical protection.

Our findings suggest that prolonged METH consumption in humans and a binge regimen in mice consistently induce a biochemical signature characterized by GPX-4 depletion, increased iron accumulation and MDA levels, and decreased SOD activity. These results are consistent with previous studies [16,19,33]. The convergence of human and animal data is a significant strength of this work. Both post-mortem human samples and the mouse model demonstrated similar biochemical changes, such as increased iron and MDA levels, decreased GPX-4 expression, and decreased SOD activity. This cross-validation improves the biological relevance of the findings, with human data representing practical drug exposure in humans and the animal model allowing for controlled mechanistic inquiry. Furthermore, we also observed that CBD pretreatment partially attenuated this ferroptosis-like cascade and the associated histopathological impairment, consistent with the study by Shen et al. [33]. However, because CBD was administered only as a pretreatment, our experimental design models are a prophylactic rather than a therapeutic intervention.

Historically, the dopaminergic terminal toxicity model has been used to study METH-induced brain damage. METH stimulates dopamine release and autoxidation, creating reactive oxygen species (ROS) that damage presynaptic terminals, resulting in dopamine and transporter depletion over time [3,7]. This paradigm, while fundamental, was mostly neuron-centric. Over the last decade, the focus has shifted substantially to include comprehensive oxidative stress and non-apoptotic cell death mechanisms. Our findings of extensive lipid peroxidation and GPX-4 loss in the PFC support this change, transforming the narrative from selective terminal damage to a more diffuse, ferroptosis-like cellular loss impacting whole neuronal populations.

The nature of cell death in METH intoxication remains unclear. Early studies strongly suggested apoptosis, citing DNA fragmentation and caspase-3 activation [6,16,24]. However, the distinctive biochemical combination we observed – GPX-4 downregulation, iron overload, and lipid peroxidation – is consistent with the characterization of ferroptosis described by Dixon et al. [9]. Observations that were previously unclear under a strictly apoptotic paradigm, such as the insufficient efficacy of caspase inhibitors in completely stopping METH-induced cell death, may be explained by the involvement of additional cell-death pathways [11]. The TUNEL-positive DNA fragmentation observed in our study and reported by others may represent a parallel event in a PANoptosis-like process or a consequence of ferroptotic signaling [29], rather than evidence of apoptosis alone. These findings provide a possible link between previously proposed mechanisms of METH-induced cell death. Studies that manipulate GPX-4 provide evidence connecting METH to ferroptosis. METH directly depletes GPX-4 in vitro, and its genetic overexpression has been reported to confer substantial resistance to METH toxicity, according to previous research by Imam et al. [20]. Our results of GPX-4 depletion in human post-mortem tissue support this preclinical observation. However, we did not conduct functional rescue experiments using ferroptosis-specific inhibitors such as ferrostatin-1 or liproxstatin-1, which would be necessary to more definitively establish the involvement of ferroptosis in METH-induced cell death.

The advantages of ferroptosis over other death mechanisms, however, are strongly contested. According to the study by Liao et al. [25], necroptosis was the predominant cell death under their particular single-high-dose METH model, as demonstrated by the phosphorylation of RIPK1 and the protective effects of necrostatin-1. Fundamental methodological differences are possibly the cause of this seeming inconsistency. While acute, high-dose models may cause distinct, more immediate neuroinflammatory cascades, the binge administration model employed in our study and others [23,41] may produce sustained metabolic and oxidative damage that primarily engages the GPX-4-linked ferroptosis pathway.

Our findings support an increasing amount of research demonstrating CBD’s neuroprotective potential [33,40]. The primary issue is determining its mechanism of action. One possibility, consistent with our findings of reduced MDA levels and restored SOD activity, is that CBD acts as a direct antioxidant, reducing oxidative stress and enhancing endogenous antioxidant defenses, potentially via the Nrf2 pathway [2]. In contrast, another study focused on receptor-mediated processes. Gray et al. [15] found that CBD’s protection against METH-induced hyperthermia and microglial activation was lost in TRPV1 knockout mice. Furthermore, recent research by Grabon et al. [14] found that CBD’s anti-inflammatory actions in a METH model were mediated via CB2 receptors on microglia. Our findings do not address these mechanisms, but they add a po-
tential dimension to the effects of CBD pretreatment by showing attenuation of a ferroptosis-like biochemical signature. Although our study demonstrates that CBD pretreatment attenuated these METH-associated changes, the effects of post-METH CBD administration are also relevant to its potential therapeutic application. Preclinical research shows that CBD exposure following METH treatment may attenuate oxidative damage, apoptosis, and behavioral relapse even when given after exposure [34,42]. Studies that apply CBD (e.g., 1-10 mg/kg i.p. or 1-10 µM in vitro) 30-60 minutes after METH exposure (4-40 mg/kg) have reported reductions in LDH release and Ca²⁺ overload, and improved cell viability. These studies also reported reductions in ROS and lipid peroxidation and, in some models, reduced TUNEL positivity [32,33]. In contrast to pretreatment’s preventative emphasis, this post-METH efficacy has also been reported in withdrawal models, with attenuation of conditioned place preference reinstatement and self-administration [36]. Proposed mechanisms include restoration of dopamine homeostasis, anti-inflammatory effects, and TRPV1 signaling [31]. Future studies must clarify whether CBD attenuates ferroptosis-related changes through direct effects on lipid peroxidation or through upstream regulation of TRPV1/CB2 signaling, potentially leading to GPX-4 stabilization and altered iron homeostasis.

Conclusions

Our study integrates post-mortem human brain data with an in vivo mouse model to characterize molecular and biochemical alterations associated with METH exposure and METH-induced neurotoxicity. The findings demonstrate a consistent pattern of iron accumulation, increased lipid peroxidation, reduced glutathione peroxidase-4 (GPX-4) expression, and impaired antioxidant defenses in the PFC, consistent with a ferroptosis-like oxidative injury profile. In addition, CBD pretreatment attenuated these alterations and modulated behavioral responses in the experimental model, suggesting a potential protective effect of CBD under preventive conditions.

Limitations

Despite the findings of this study, a few key shortcomings remain. The small sample size (n = 5) per group) limits statistical power, and the post-mortem brain tissue is subject to inherent confounding factors that can influence the biochemical measurements. The current findings support an association between METH-associated neurotoxicity and ferroptosis-related changes, but there is no definitive evidence of ferroptotic cell death. Ferroptosis should be further validated by using ferroptosis-specific inhibitors such as ferrostatin-1 or liproxstatin-1 and assessing additional ferroptosis markers such as ACSL4 and SLC7A11/xCT in future studies. Further mechanistic studies are required to determine the therapeutic relevance of CBD, particularly in post-exposure models. Overall, this study provides a foundation for future investigations targeting oxidative injury pathways in METH-related brain damage.

Funding

This work was supported by National Natural Science Foundation (82260336, 82371898 and 82560335) Yunnan Fundamental Research Kunming Medical University Project (202301AY070001-026).

Disclosures

Approval of the Bioethics Committee was not re-
quired.

The authors report no conflict of interest.