Chemotherapy is a key method for treating cancer and is effective against various tumor types (Anand et al., 2023). However, it also affects healthy tissues owing to its non-selective nature (Blagosklonny, 2023). Additionally, chemotherapy can lead to cellular injury that leads to oxidative stress and inflammation, resulting in toxicity and various side effects (Jiang et al., 2023). An important, yet unexplored side effect of chemotherapy is chemobrain or chemofog, which involves difficulties in concentration, memory, decision-making, learning, and language during and after chemotherapy (Fleming et al., 2023). Up to 70% of cancer survivors experience chemobrain disease, and in 35% of these cases, the symptoms can last for as long as 5 years (Ren et al., 2019). Multiple regions in the brain, including the hippocampus, are primarily responsible for cognitive function and memory formation (Kolibius et al., 2025). Therefore, changes in protein expression or function in the brain can lead to memory impairment (Ding et al., 2023). Currently, the exact causes and mechanisms of chemobrain remain poorly understood.

A regimen of cyclophosphamide, methotrexate, and fluorouracil (CMF) is frequently used to treat different types of cancers, including breast cancer (Park et al., 2017). Cyclophosphamide, an alkylating agent with multiple functions, functions by alkylating DNA, thereby hindering DNA transcription and RNA translation (Ahlmann and Hempel, 2016). Methotrexate acts as a folate antagonist by blocking several enzymes involved in nucleotide synthesis, including dihydrofolate reductase, which facilitates the conversion of dihydrofolate to tetrahydrofolate (Cronstein and Aune, 2020). Fluorouracil, an antimetabolite of the natural nucleobase uracil, inhibits thymidylate synthase, thereby obstructing DNA and RNA syntheses (Sethy and Kundu, 2021). CMF can cross the blood–brain barrier, offering therapeutic effects against cancer cells in the brain (Corley and Allen, 2021). Koppelmans et al. (2012) examined the neuropsychological performance of breast cancer survivors over two decades after receiving adjuvant CMF chemotherapy. Their findings indicated that these individuals scored lower on neuropsychological tests than the random population controls (Koppelmans et al., 2012). Furthermore, studies have shown that CMF administration reduces hippocampal neurogenesis in rodent models of chemobrain, resulting in impaired cognitive function (Briones and Woods, 2011).

Pioglitazone (PGZ), a derivative of thiazolidinedione, serves as an activator of the peroxisome proliferator-activated receptor (PPAR) (Devchand et al., 2018). It has been sanctioned for use in managing diabetes mellitus linked to insulin resistance (Alam et al., 2019). By activating PPAR agonists, PGZ enhances insulin sensitivity, facilitates glucose uptake, and optimizes lipid metabolism (Alam et al., 2019). Moreover, PGZ has shown potential in treating neurodegenerative diseases, highlighting its promise as a therapeutic option for these conditions (Alhowail A. et al., 2022). A previous study has indicated that PGZ positively affects cognitive impairment by stimulating the cholinergic system and reducing neuroinflammation in rat models of neurodegeneration and chemotherapy (Alsaud et al., 2023; Zhao et al., 2021). Pioglitazone (PGZ) has been demonstrated to mitigate diabetes-associated cognitive impairment in rodent models, primarily through the attenuation of neuroinflammatory processes, as evidenced by reduced expression of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β, along with improved insulin sensitivity (Saunders et al., 2021). Furthermore, recent preclinical investigations have evaluated the synergistic neuroprotective potential of PGZ in combination with doxorubicin in animal models (Alsaud et al., 2023). The underlying mechanisms proposed for these effects include the enhancement of mitochondrial bioenergetics, suppression of oxidative stress, downregulation of neuroinflammatory signaling pathways, and inhibition of apoptotic cascades (Alsaud et al., 2023; Alhowail, 2024).

The primary aim of this study was to evaluate the effects of CMF on memory function and explore the possibility of using PGZ in conjunction with CMF to mitigate any cognitive impairments induced by CMF.

This research explored the influence of the CMF chemotherapy regimen on memory function in rats by employing behavioral assessments such as the Y-maze test, the Novel Object Recognition Test (NORT), and fear conditioning memory tasks. In chemotherapy protocols, it is common to combine multiple chemotherapeutic agents to reduce the possibility of drug resistance and to improve therapeutic outcomes (Anand et al., 2023). Both clinical and experimental studies have previously identified memory impairments as a consequence of chemotherapy (Alhowail and Aldubayan, 2023b). Our previous study in rats demonstrated that the CMF regimen impaired memory, confirming a correlation between the CMF regimen and cognitive dysfunction (Alhowail A. H. et al., 2022). In addition, our previous findings have shown that PGZ has promise in ameliorating DOX-induced neurodegeneration and chemobrain damage, as well as in diabetic and Alzheimer’s disease (Alsaud et al., 2023; Alhowail, 2024; Gao et al., 2017; Seok et al., 2019). Therefore, we hypothesized that PGZ could exert a protective effect against CMF-induced cognitive dysfunction, as assessed using the Y-maze, NORT, and fear conditioning memory tests.

Our data indicated significant toxicity associated with the CMF regimen. In particular, the survival rate decreased by 40% in the CMF group, while the combination of CMF and PGZ reduced the survival rate by 30%. Therefore, this is considered a slight mitigation of CMF toxicity by PGZ. These results raised serious concerns regarding the safety and efficacy of PGZ in reducing CMF toxicity. Additionally, body weight data on the safety of PGZ in relation to CMF toxicity were reviewed. These findings indicate a significant 16% reduction in body weight following CMF treatment. However, the addition of PGZ slightly mitigated this weight reduction; the reduction was 11%. Therefore, it is important to understand the mechanisms underlying this effect. In contrast, body weight increased by approximately 11% in the control group and 7% in the PGZ group, respectively.

Cognitive function was assessed using the Y-maze test, NORT, and fear conditioning memory tasks. These methods evaluate spatial recognition and associative learning. The Y-maze test quantifies exploratory behavior in novel environments, whereas the NORT assesses the recognition memory of novel objects (Kim et al., 2023; Kirby et al., 2018). Fear conditioning assesses the formation of associations between neutral and aversive stimuli (Alhowail and Aldubayan, 2023b). These behavioral paradigms provide a comprehensive assessment of various cognitive domains. Analysis of spatial memory using the Y-maze revealed a significant decrease in arm entries and the time spent in the novel arm in the CMF-treated group. While PGZ co-treatment did not improve cognitive impairment. Similarly, NORT results demonstrated reduced novel object exploration in the CMF-treated group, compared with controls. Moreover, the combination of the CMF and PGZ failed to improve novel object exploration. These data suggest that the CMF treatment may negatively affect exploratory behavior. Moreover, while fear conditioning memory tasks are dependent on the hippocampus, they originate in the amygdala (Kim and Cho, 2020). Our results diverged from previous findings, indicating a statistically significant reduction in freezing time in the CMF group (Alhowail A. H. et al., 2022). However, the addition of PGZ co-treatment did not yield further improvements in freezing time. The CMF-treated groups exhibited shorter freezing times than the control group. Collectively, these findings demonstrate the ineffectiveness of PGZ in mitigating CMF-induced toxicity.

We administered a CMF treatment to introduce cognitive impairment known to occur after this chemotherapy treatment. This study is the first to use CMF medications to predict induced cognitive impairment in combination with pioglitazone. Previous findings revealed that CMF reduces neurogenesis and increases neuroinflammation, inducing cognitive dysfunction (Alhowai et al., 2022; Murillo et al., 2023). Previous studies also revealed that pioglitazone improves neuronal function and reduces neuroinflammation in neurodegenerative diseases (Alzheimer’s and Parkinson’s) as well as diabetes (Saunders et al., 2021; Zamanian et al., 2023). Therefore, we hypothesized that pioglitazone could improve neuronal circuits, reflected in behavioral function. Unfortunately, after conducting the experiment, we did not see much improvement. However, there was a slight mitigation of CMF toxicity, shown by improved survival rate (30% mortality versus 40% in CMF only) and reduced weight loss (11% versus 16%). Furthermore, behavioral assessment results showed no effect in the Y-maze by combining pioglitazone with CMF, whereas slight mitigation was detected in the NOR and fear conditioning memory tests. Therefore, pioglitazone was unable to reverse the effects on neurons after CMF treatment. Our interpretation of these results is that CMF may cause irreversible damage to neurons. Further study is required to investigate the mechanisms of CMF-induced cognitive impairment to better understand its cellular effects and allow for the development of neurotherapies.

Chemotherapy is recognized for inducing oxidative stress, which is characterized by compromised mitochondrial function and elevated levels of reactive oxygen species (ROS) (Zhang et al., 2019). Chemotherapeutic agents such as doxorubicin and cisplatin primarily exert cytotoxic effects by increasing intracellular ROS (Ongnok et al., 2020). Similarly, cyclophosphamide, methotrexate, and fluorouracil (CMF) can promote ROS generation either directly or by impairing mitochondrial function (Sahu et al., 2021). In the present study, CMF treatment significantly impaired mitochondrial complex I activity and elevated ROS levels, indicating the induction of oxidative stress. However, co-treatment with pioglitazone (PGZ) did not mitigate the mitochondrial dysfunction, as evidenced by reducing complex I activity by reducing the oxidized NADH to NAD⁺ that is essential for energy production by formation of adenosine triphosphate (ATP), and failed to rescue the increase in ROS levels compared to the control group, suggesting that PGZ was ineffective in reducing CMF-induced oxidative stress.

Elevated concentrations of pro-inflammatory cytokines such as TNF-α and IL-1β in the brain are indicative of neuroinflammation, a pathological condition that can impair mitochondrial function and trigger oxidative stress (Bhol et al., 2024). In this study, CMF treatment modestly increased levels of TNF-α and IL-1β, consistent with the presence of neuroinflammation. Concurrently, impaired mitochondrial function and elevated ROS levels further supported the occurrence of oxidative stress. These pathological changes are believed to contribute to the cognitive deficits observed following CMF therapy. Although this study hypothesized that PGZ could alleviate CMF-induced cognitive impairment by reducing oxidative damage and neuroinflammation, the results showed that co-treatment with PGZ was ineffective in attenuating the oxidative stress and neuroinflammatory response.

There are a number of strengths and limitations associated with this study. The purpose of this study is to investigate the effects of CMF on cognitive impairment. Additionally, this is the first study to investigate the combination of PGZ as a potential protective benefit with CMF. The use of a homogenous rat population (including rats of the same strain, age, and weight, as well as those that have been shown to be cancer-free) to isolate the impact of CMF and so minimize confounding variables is one of the strengths of this study. Additionally, to ensure consistency with clinical practice, the researchers used medications prescribed by hospitals and obtained from commercial sources. Moreover, our laboratory’s current technical and resource constraints have prevented us from conducting thorough histological analyses to corroborate our functional and behavioral findings. Future investigations should include histological examinations of neuronal architecture, cellular integrity, and any potential pathological changes in these brain regions to validate and build upon our results.

This study investigated the effects of CMF on neurotoxicity and memory dysfunction in rats by assessing survival rates, body weight, and neuronal function. This was achieved using the Y-maze, NORT, and fear conditioning memory behavioral tasks. This study also evaluated the protective potential of PGZ co-administration in mitigating cognitive impairment. Overall, our findings suggest that PGZ was ineffective in mitigating CMF-induced neurotoxicity, as reflected in cognitive function, oxidative stress, and neuroinflammation. Our findings highlight the need for further studies to explore further mechanistic pathways for alleviating chemotherapy-related cognitive impairments in cancer survivors.