Treatment-related cognitive deficits are one of the most common side effects of chemotherapy [1]. Previous studies in several rodent models have reported multiple possible mechanisms contributing to cognitive deficits following chemotherapy treatment with DOX, including inflammation, dysregulation of apoptosis, changes in neurotransmitter levels, glial cell interactions, neurogenesis inhibition, hormonal changes, alterations in synaptic plasticity, and long-term potentiation [3,4]. These mechanisms have been studied in parallel with anxiety and depressive behaviors for years [1,10].

Treatment with DOX frequently results in neurological side effects in animal models, including cognitive deficits [4,7,29,38,39]. Sameha et al. demonstrated that DOX-treated male Wistar rats showed reduced locomotor and exploratory behavior in Open Field and Elevated Plus Maze tasks [39]. In the current study, DOX-treated animals spent significantly less time exploring the open arms and more time in the closed arm of the Elevated Plus Maze, indicating anxious-like behavior. We did not find any difference in the Open Field Test between groups, which contrasts with the findings of Keeney et al., who reported an effect of doxorubicin on locomotor activity using the same test [40]. This discrepancy may be attributed to using different animal models and dosages. The anxiety-like behaviors observed in the Elevated Plus Maze were not confounded by locomotion in the Open Field Test, as there were no significant changes in rat behavior during their performance in the Open Field Test.

DOX-induced depression is often studied together with anxiety-like behaviors. In addition to problems in concentration, memory, and decision-making, individuals with depression experience changes in sleep patterns, changes in appetite, and thoughts of harming themselves [41,42]. Like anxiety, cancer patients may experience depressive symptoms before, during, and a long time after chemotherapy [43]. In addition to this, other studies [10,44] have shown an identical effect in different strains of rats that show depression-like behavior following DOX treatment, indicating that DOX administration can induce depression-like behaviors in rodents irrespective of species and dose administered. Consistent with previous studies, DOX-treated rats in our study exhibited depression-like behavior as manifested by a reduced sucrose preference index in the Sucrose Preference Test (Figure 7), which is indicative of anhedonia [44].

In patients with anxiety, 40% are resistant to the use of serotonin reuptake inhibitors (SSRIs) [45]. Given this substantial proportion of patients who may not benefit from SSRIs, it is important to explore alternative treatment options. In this context, investigating CBD as a potential treatment for anxiety becomes a logical and necessary step to address the needs of those who do not find relief with conventional therapies. CBD is highly recognized as a non-psychotomimetic component of Cannabis sativa, having a broad range of therapeutic potential [24,46,47]. While THC’s mechanism of action depends on CB1 and CB2 receptors, which are the two potent endocannabinoid system receptors, CBD is known to act independently of these receptors [17,48,49,50]. However, the precise mechanism of action for how CBD alters the behavioral changes induced by Chemo-brain has not been verified, to the best of our knowledge.

CBD has proven to be an effective medicinal therapy in several animal and human studies. Cunha et al. (1980) first reported that a CBD dose of 200 mg/day was effective in the treatment of seizures in humans [51]. A double-blinded, placebo-controlled trial was conducted among 120 children and young adults to study the efficacy of CBD against drug-resistant epilepsy in Dravet syndrome [52]. The study found that the use of CBD results in a greater reduction in convulsive seizure frequency than the control [52]. Unlike THC, CBD is non-euphoric in nature as it does not activate central CB1 receptors [53,54]. However, CBD has shown its potent antioxidant and anti-inflammatory role via its interaction with PPARy (Peroxisome Proliferator-Activated Receptor Gamma), TRPV1 (Transient Receptor Potential Vanilloid 1) channels, and 5-HT1A (5-Hydroxytryptamine Receptor 1A) receptors [53,55]. During these interactions, CBD is capable of activating the serotonin 1A receptor and blocking the activation of GPR55 (G-protein-Coupled Receptor 55, nucleoside transporter) and the TRP (Transient Receptor Potential) cation channel [56]. CBD can also interact with voltage-dependent anion-selective channel protein (VDAC1) [57]. Like THC, CBD is also highly lipid soluble. It gets easily distributed in the brain, adipose tissue, and several other organs [56]. The bioavailability of CBD depends upon the route of administration. For instance, CBD taken as oil or via oral route is believed to be most effective due to its lipophilic nature [56]. Over the past several decades, the unique pharmacological features of CBD and differential manipulations in dosing and the route of administration of CBD have provided a much better understanding of the potential of CBD to diminish psychotropic effects.

Interestingly, several other receptors, like 5HT1A, GPR55, and PPARγ, have been identified to be involved in the therapeutic actions of CBD [46,58]. Some studies have also mentioned that CBD modulates neuroprotection by increasing the levels of an endogenous cannabinoid called Anandamide (AEA) by inhibiting the anandamide hydrolysis pathway mediated by Fatty Acid Amide Hydrolase (FAAH) [46,59]. Benefits have been shown in mice with mild traumatic brain injuries, where oral cannabinoids improved social interactions and reduced aggressive behavior [17], and rats with neuropathic pain, where repeated CBD treatment prevented anxiety-like behavior [19,24].

Consistent with these studies, we detected lower anxiety symptoms in CBD-treated rats, as demonstrated by the less time spent in the closed arm of the Elevated Plus Maze and significantly more time exploring the open arm of the maze (Figure 3). Furthermore, CBD-treated animals buried a significantly lower number of marbles in the marble burying task, suggesting that the rats were less engaged in repetitive behavior after CBD treatment (Figure 5). This is an important finding because it shows that CBD may be effective against obsessive-compulsive disorder (OCD), which is one of the core symptoms of anxiety [60].

We also evaluated CBD for its potential to ameliorate impaired object recognition memory following DOX. In our rats, DOX administration resulted in impaired object recognition memory, as evidenced by the reduced novelty preference following DOX treatment in the Novel Object Recognition test. CBD-treated animals showed higher novelty preference than the DOX-treated group (p = 0.019), suggesting improved object recognition memory after CBD treatment (Figure 6). However, the DOX + CBD group showed no significant differences in object recognition compared to DOX. Oral treatment of CBD alone improves neuroprotection in our rats, but further studies are needed to prove the potential of CBD to improve object recognition memory following DOX treatment.

Immune response mediated by resident glial cells in the nervous system maintains neuronal health [61]. Any changes in these resident cells due to pathological conditions can lead to neuroinflammation, causing learning and memory dysfunction [62]. Neuroinflammation involves the activation of astroglia and microglial cells, leading to the release of proinflammatory cytokines. Increased levels of proinflammatory cytokines, including Tnf and Il1b, have been observed in patients following chemotherapy [40]. DOX-induced neuroinflammation has been widely studied in several animal models, including different strains of rats, such as Wistar, Sprague Dawley, and athymic nude rats [10,63,64,65]. Studies of the downstream effects of cannabinoid compounds in glial cells provide a basis for the study of the anti-inflammatory properties of CBD. For example, immune system cells express cannabinoid receptors, as evidenced by increased Cnr1 mRNA expression [66] and increased Cnr2 receptor expression in response to activated microglia and astrocytes [66]. Moreover, CBD could reduce the expression of cytokines, including Tnf, in a mouse model of hypoxic–ischemic brain damage, indicating its ability to reduce neuroinflammation [67].

Pro-inflammatory cytokines trigger cascades of pathways that may lead to glial cell activation, resulting in cognitive deficits [3,4]. Their expression by immune cells is highly capable of activating NF-κB transcriptional activity via inhibiting inhibitors of κB kinase and DNA binding domains of NF-κB [68]. We conducted gene expression analyses from the freshly dissected prefrontal cortex and hippocampal tissues of DOX- and CBD-treated rats to corroborate these findings. In our study, mRNA expression of the proinflammatory cytokines Tnf, Nfkb1, and Il1b were significantly increased after exposure to DOX alone but not in rats treated with DOX and CBD. Consistent with this, we found elevated hippocampal Il6 mRNA levels after DOX treatment. Ongnok et al. also found a similar increase in mRNA expression of Il6 following DOX in their model [11].

Conversely, we did not detect any significant differences in cortical mRNA levels after DOX treatment. DOX-induced activation of proinflammatory genes may occur in a brain-region-specific manner. CBD is capable of inhibiting iNOS (inducible Nitric Oxide Synthase) expression via the activation of the PPARγ pathway and increases anti-inflammatory cytokines [68]. In our study, the expression of anti-inflammatory genes, including Il10 and Tgfb1, were all significantly decreased in DOX-challenged rats, while CBD treatment showed the opposite effects (Figure 8 and Figure 9).

CBD treatment reduced mRNA expression levels of Aif1 and Gfap, indicating that CBD prevents glial cell activation. This implies that CBD might be a promising agent that can prevent neuroinflammation from the prolonged activation of microglia and astrocytes or potentially prevent cognitive deficits during chemotherapy treatment.

Our study revealed that neuroinflammation is vital in DOX-induced cognitive deficits, particularly hippocampal damage. Thus, our results substantially support previous reports of the neuroprotective benefits of CBD [17,28,44,46,48]. Neuroinflammation caused by DOX may contribute to the impairment of neurological functions. We found that the reduced expression of neuroinflammatory-associated mRNA corresponded with reductions in anxiety and depression-like behaviors in chemotherapy-treated animals given CBD. Consistent with the behavioral effects observed in the different treatment groups, our results showed increased mRNA expression of pro-inflammatory genes (Tnf, Nfkb1, and Il1b), reduced mRNA expression of anti-inflammatory genes (Il10 and Tgfb1), as well as a marked increase in the gene expression of Aif1 and Gfap in the prefrontal cortex as well as the hippocampus of DOX-treated animals, which was reduced by oral treatment with CBD.

Some limitations of our study should be addressed in the future to draw definitive conclusions about the efficacy of CBD in Chemo-brain. We recognize that a larger sample size may have enabled us to detect some more subtle differences in behavior or mRNA expression than our current data allow. However, our sample was sufficient to detect differences between treatment groups due to the effect sizes observed and yielded an overall power greater than or equal to 0.8 for all statistical tests performed.

Our preliminary study aimed to explore the effects of DOX specifically in a context relevant to breast cancer research. Therefore, we focused on female animals because DOX is most used in the treatment of triple-negative breast cancer [69]. Consequently, we did not assess sex differences in this initial phase. Other cancers where DOX is used include ovarian cancer, leukemia, and certain types of lymphoma, but breast cancer remains one of the most common applications. We acknowledge that future studies should consider including both male and female subjects to investigate any potential sex-based differences in response to DOX and CBD.

Additionally, DOX can influence appetite, as we show in Figure 2. This may impact the interpretation of our SPT results. Due to this potential confounder, follow-up studies are needed that incorporate additional measures to separate mood effects from appetite changes, such as including behavioral assays to evaluate mood or using food intake assessments alongside the SPT.

Our study does not account for tumor-induced animal models of cancer, as all experiments were performed using normal rats. Additionally, our study does not provide information regarding the expression of CBD receptors, which should be addressed in future experiments. Based on our findings that CBD treatment altered the neuroinflammatory mediators in normal rat hippocampi and prefrontal cortices, it would also be interesting to elucidate the neuroprotective effects of CBD under different dosing conditions.