As research into the ECS advances, its therapeutic potential becomes increasingly apparent. Aging—a primary risk factor for cognitive decline, involves neuroanatomical and neurochemical changes that are mirrored by alterations in the ECS (Legdeur et al. 2018). Recent evidence suggest that interactions between the aging brain and the ECS (Hazzaa, et al., 2021), particularly through its modulatory receptors, could influence brain functions related to motor skills, emotions and cognition (Fogaca et al., 2018; Ruiz de Martín et al., 2022). However, targeting the ECS presents challenges, as CB1 and CB2 receptors play crucial roles in regulating neuronal function, leading to diverse effects on neuronal responses (Kamaruzzaman et al. 2023).

Consistent with earlier preclinical and clinical studies (Stanciu et al. 2024; Alshaarawy and Anthony 2019), which reported little to no impact of Cannabis-active constituents on weight gain, our study also observed only a slight weight loss across all groups throughout the experimental period. There were no significant differences in weekly weight variation among the older animal groups, regardless of the Cannabixir® Medium Flos dose (6.25 mg/kg or 25 mg/kg), suggesting that chronic intermittent treatment did not markedly influence weight dynamics in aged rats (Fig. 2). However, statistically significant differences were observed between adult and aged rats across several time points, particularly between weeks 4 and 6 (e.g., 4.47% difference at week 4, p = 0.047, Fig. 2). These findings indicate that aging has a stronger impact on body weight regulation than cannabinoid treatment per se. The better weight maintenance observed in adult rats may reflect age-related declines in metabolic and physiological efficiency in older animals. Supporting the weight-related findings, the hematological evaluation indicated a generally safe profile of Cannabixir® Medium Flos therapy, with all measured parameters remaining within physiological reference ranges for Sprague–Dawley rats (Fig. 3). However, statistically significant hematological changes were observed in the OLD-25 group (25 mg/kg), including increased neutrophil percentages and decreased lymphocyte proportions, suggesting a mild alteration in immune cell distribution. These shifts may reflect subtle effects on erythropoiesis or immune responses, potentially linked to cannabinoid-mediated changes in metabolism or nutrient absorption. Additionally, significant changes in mean corpuscular volume (MCV) were detected, with OLD-6.25 rats showing lower values and OLD-25 rats higher values compared to controls, although still within normal limits (Fig. 3). These findings highlight the importance of further research to clarify the immunomodulatory effects of cannabinoid-based treatments in aged populations and to better understand their implications for long-term safety and pharmacotherapeutic use.

Chronic, intermittent administration of Cannabixir® Medium Flos in both young adult and naturally aged rats did not result in statistically significant changes in motor coordination, endurance, exploratory behavior, or anxiety-like responses. Performance across all behavioral assays—including the RotaRod, Treadmill Fatigue Test, Activity Cage, and Elevated Plus Maze—performance remained comparable between treated and control groups (Figs. 4 and 5). These findings suggest that, at the tested doses, Cannabixir® Medium Flos does not impair locomotor activity or induce anxiety-related alterations, which supports its safety profile in aged models. While minor, non-significant trends, such as a slight reduction in spontaneous locomotion at higher doses, were observed, they did not reach statistical significance and should be interpreted with caution (Fig. 4E and 4 F). The consistency of these results across multiple behavioral paradigms aligns is consistent with previous studies reporting neutral (Varvel et al. 2001) or dose-dependent anxiolytic effects of cannabinoids, particularly those with low ∆9-THC content (Zuardi et al. 2012; Berger et al. 2022; Martin et al. 2021). Although low doses of ∆9-THC have often been associated with anxiolytic effects—potentially through the modulation of approach behavior and the inhibition of anxiety-related circuits involving the prefrontal cortex and amygdala (Rubino et al. 2007), recent evidence suggests that this is not a universal outcome (Ramis, et al., 2016). Specifically, studies have shown that ∆9-THC can produce anxiogenic effects even at low doses, particularly in female subjects, indicating that sex-related differences in cannabinoid sensitivity may critically influence behavioral responses (Salviato et al., 2021). These findings emphasize the biphasic and context-dependent nature of THC’s effects on anxiety, and the importance of considering sex as a biological variable when evaluating cannabinoid-induced emotional modulation (Sharpe et al. 2020). Overall, these data support the notion that Cannabixir® Medium Flos is behaviorally well-tolerated and reinforces its potential for further investigation as part of a neuroprotective strategy, particularly in aging contexts. Future studies should explore long-term administration, a broader dose range, and incorporate additional neurobehavioral endpoints to fully characterize its efficacy and mechanism of action.

In parallel, our results highlight that Cannabixir® Medium Flos may offer neuroprotective benefits in naturally aged rodents, potentially through modulation of the ECS. Histological examination of the hippocampus—a region rich in endocannabinoids and CB1 receptors (Glass et al. 1997a, b), revealed significant improvements in the number of neuronal layers in the treatment groups (OLD-6.25 and OLD-25), resembling those of ADULT-Con rats. Additionally, there was a reduction in cells with hyperchromatic and elongated nuclei in the DG region compared to the OLD-Con group (Fig. 6). These observations indicate that this Cannabis strain may positively influence neurogenesis by enhancing neuronal growth and function, likely mediated through ECS modulation. The hippocampus, including its subregions such as DG, CA1, CA2, and CA3, is crucial for cognitive functions and is strongly associated with the onset and progression of age-related neurodegenerative diseases (Giannos and Prokopidis 2022). The ECS, comprising CB1 and CB2 receptors, plays a key role in regulating neurogenesis, synaptic plasticity, and neuroinflammation. Activation of the ECS has been shown to promote neuronal survival and reduce inflammation, processes that are essential in combating the detrimental effects of aging on the brain (Rahimi and Askari, 2023). The structural and functional changes observed in the aging hippocampus (Figs. 7 and 8) are linked to several underlying mechanisms, including neuronal loss, impaired neurogenesis, neuroinflammation, and oxidative stress (Bettio et al. 2017; Giannos and Prokopidis 2022; Hamezah et al. 2017). These mechanisms are interrelated and often trigger cascades of pathological changes as aging progresses. For instance, hippocampal atrophy often accompanies a decline in neurogenesis, which is further compounded by neuroinflammatory responses specific to aging (Bettio et al. 2017). While we hypothesize that THCA-A and CBGA from Cannabixir® Medium Flos could play a role in the observed neuroprotective effects, a comprehensive quantitative analysis of the cannabinoid profile of the product is essential to confirm this hypothesis. The literature on these two compounds is still limited, but some existing studies support this possibility. THCA-A, the acidic precursor to THC and a non-psychotropic cannabinoid, has been suggested to have neuroprotective properties, potentially due to its ability to downregulate pro-inflammatory mediators commonly associated with aging (Nadal et al. 2017). This effect occurs via activation of the peroxisome proliferator-activated receptor gamma (PPAR-gamma) and the CB1 cannabinoid receptor (Nadal et al. 2017; Palomares et al. 2020). In addition, CBGA recently demonstrated an anti-inflammatory effect in an acute nephropathy mouse model by suppressing mRNA expression of the cytokines evaluated and a neuroprotective effect in Parkinson’s disease models (Suzuki et al. 2023). Cannabigerol (CBG), the decarboxylated version of CBGA, exerts an antioxidant effect by activating CB2 receptors and inhibits the translocation of nuclear factor-κB (NF-κB), while also modulating the mitogen-activated protein kinase (MAPK) pathway (Carone et al. 2024). These combined actions ultimately lead to the inhibition of cell death. Additionally, CBG has demonstrated an anti-inflammatory effect through downregulation of cytokine activity both at the peripheral and central level (Gugliandolo et al. 2018). Another major compound present in our product is CBD. Schiavon et al. showed that low doses of CBD, between 3 mg/kg and 30 mg/kg led to the improvement of neuronal proliferation (Schiavon et al., 2016). Furthermore, CBD has shown protective effects on the brain that have been linked to its ability to facilitate the survival and differentiation of newborn neurons of the DG (Luján et al., 2019). Based on the current data, we can suggest that this compound shows potential at the neuronal level. However, the mechanisms underlying its influence require further rigorous investigation and robust statistical analysis to be fully understood.

Using GFAP and CD68 immunolabeling as markers of astrocyte and microglia activation, we observed significant immunohistochemical alterations in the hippocampus of treated aged rats compared to the control groups (Fig. 7). While these markers are commonly employed to indicate glial cell activation, they do not provide direct evidence of functional alterations within these cell types. Therefore, although our findings suggest glial activation, further research is necessary to explore the functional consequences of these changes in astrocyte and microglial activity. Focusing on GFAP, a significant increase in astrocyte immunolabeling was noted in the OLD-25 group when compared to both the OLD-Con (p = 0.03) and ADULT-Con (p = 0.03) groups. The highest average number of GFAP-positive cells was observed in the OLD-25 group (535.50 ± 45.96), while the lowest was recorded in the OLD-Con group (180.50 ± 50.50) (Fig. 7). This suggests an activation of astrocytes in response to treatment, although direct functional changes in astrocyte activity remain unproven. Moreover, we observed a reduction in the number of CD68-marked macrophages in the dentate gyrus and Ammon’s horn of the treated aged rats, closely resembling the histological profile of young adult rats. In contrast, the OLD-Con group exhibited astrocytes appearing as cellular debris or intensely stained with variable sizes, and the number of CD68-positive macrophages was more than three times higher than in the treated groups. The increased number of astrocytes in the treated groups may exert a neuroprotective role by modulating neuroinflammation (Miguel-Hidalgo 2023). Astrocytes can influence the release of pro-inflammatory cytokines, as observed in our study, where the OLD-Con animals displayed higher levels of pro-inflammatory cytokines such as TNF-α (Fig. 9). This modulation by astrocytes helps in promoting the clearance of inflammatory mediators, thereby creating a more favorable microenvironment for neuronal survival (Sofroniew and Vinters 2010). Astrocytes also play a crucial role in regulating extracellular glutamate levels. Activation of the ECS can modulate glutamate uptake and release by astrocytes, preventing excitotoxicity and subsequent neuronal damage (Navarrete and Araque 2008). Cannabinoid receptor activation may induce astrocyte hypertrophy, leading to enhanced astrocytic coverage of synapses and improved synaptic stability, thereby conferring neuroprotection (López et al. 2018; Glass et al., 1997).

In line with previous research, our study indicates that different doses of Cannabixir® Medium Flos exert distinct effects on cell proliferation and apoptosis in aged rats. The PCNA marker revealed perivascular clusters of labeled nuclei, with a mean count of 15.50 ± 3.54, which was significantly lower than in the other groups (p < 0.05), indicating reduced cellular proliferation in the OLD-Con group (Fig. 8). Additionally, the low occurrence of caspase-3 positive cells in the OLD-Con group suggests a relatively low level of apoptosis (Fig. 7), although further analysis of cleaved caspase-3 would be necessary to confirm this. These findings are consistent with literature suggesting that cannabinoids can enhance neurogenesis and reduce neuronal apoptosis in aging preclinical models (Schuele et al. 2022; Valeri and Mazzon 2021).

The distribution of PCNA-positive nuclei in the OLD-25 group, which appeared more diffuse, and the low occurrence of caspase-3 positive cells, suggest that increasing the dose does not significantly enhance cell proliferation or apoptosis at this concentration, potentially indicating a plateau effect (Hernandez-Hernandez and Garcia-Fuster 2022; Bilkei-Gorzo et al., 2017)). On the other hand, the observed clusters of PCNA-positive cells around blood vessels and the noticeable reduction in caspase-3 expression in the DG region in the OLD-Con rats suggest that without cannabinoid therapy, there is reduced cell proliferation (Figs. 7 and 8). However, the reduction in caspase-3 expression in this group does not directly indicate increased apoptosis, as further analysis would be needed to assess this relationship more thoroughly. These findings support the role of cannabinoids in mitigating age-related neurodegenerative changes (Martin-Moreno et al. 2012) and emphasize the importance of dose optimization for maximizing therapeutic benefits in aging through cannabinoid administration.

While animal studies provide valuable insights, translating these findings to human contexts requires careful consideration due to species-specific differences and the lack of detailed analyses on cognitive function changes following Cannabis and cannabinoids use. Continued research is necessary to refine dosage strategies, assess the effects of concurrent substance use—especially in individuals with neurodegenerative or chronic pain conditions—and explore modulatory factors such as cognitive reserve and gender differences that may influence how Cannabis and cannabinoids interact with aging.

Although our findings offer important insights, several limitations must be considered when translating our findings to human contexts. One such factor is the exclusive use of male animals in the experiment. Although male rats were chosen to limit variability introduced by hormonal fluctuations, the absence of females in our study may limit the generalizability of the results, given the well-documented sex differences in neurobiology, neuroinflammation, and the effects of cannabinoid treatments (Cooper and Haney 2016). Sex differences are crucial for fully understanding the therapeutic potential of cannabinoids, particularly in age-related conditions. Therefore, future research should include both males and females to compare these sex differences. Behavioral evaluations in animals—such as anxiety, physical performance related to aging frailty, and exploratory behavior—can introduce observer subjectivity, which may affect the accuracy of causality interpretations (McGonigle and Ruggeri 2014). Moreover, significant differences in immune responses, neuroinflammation, and brain structure between rodents and humans can influence research outcomes and limit the direct applicability of findings to human conditions. For example, inflammatory pathways and neurobiological processes can vary substantially between species, impacting the relevance of results obtained from rodent models (Mestas and Hughes 2004). Additionally, species-specific differences in metabolism, genetics, environmental factors, and microbiome play a critical role in determining the pharmacokinetics and pharmacological effects of new drugs. Variations in cannabinoid receptor expression and metabolic enzyme activity between species can affect drug efficacy and safety profiles (Beauchamp et al. 2022; Duffy 2020). These factors underscore the need for cautious interpretation when extrapolating animal models results to human applications. To address these limitations and enhance the translational relevance of our research, future studies should focus on refining dosage strategies and exploring different administration routes. Additionally, investigating the effects of concurrent substance use, particularly in populations with neurodegenerative or chronic pain conditions, will provide a more comprehensive understanding of cannabinoid interactions. Finally, examining factors such as cognitive reserve, gender differences, and individual variability will help elucidate how Cannabis and cannabinoids influence aging and support the development of personalized therapeutic approaches.