Alzheimer’s disease (AD) is the most common neurodegenerative disorder worldwide, affecting over 55 million people, with projections estimating nearly 139 million cases by 2050 (Alzheimer’s Association, 2025). AD is characterized by progressive neuronal loss, synaptic dysfunction and cognitive decline, which lead to substantial functional impairment and dependence (Blanton et al., 2023). Alongside cognitive deficits, chronic pain is a frequent but underrecognized comorbidity in AD, although the reported prevalence varies substantially across studies due to differences in assessment tools, disease stage and data sources (van Kooten et al., 2016; Yao et al., 2025). According to the International Association for the Study of Pain (IASP), pain is defined as an unpleasant sensory and emotional experience associated with or resembling, actual or potential tissue damage (Raja et al., 2020). Chronic pain, lasting 3 months or longer, affects over 30% of the global population and is particularly prevalent among older adults (Cohen et al., 2021). In individuals with AD, pain is often expressed non-verbally through behaviors such as frowning, groaning or agitation, and its intensity may fluctuate across disease stages (Liao et al., 2021; Khalid et al., 2022).

Chronic pain exacerbates behavioral and neuropsychiatric symptoms and has been associated with accelerated cognitive decline, whereas neurodegenerative processes linked to cognitive impairment may, in turn, amplify pain perception (Palesi et al., 2016; Atee et al., 2021; Guerreiro et al., 2022). Consistently, patients with chronic pain are reported to be 2.2 times more likely to develop cognitive impairment than those without pain (Huai et al., 2021; Kumaradev et al., 2021). Moreover, a broad spectrum of pain phenotypes has been described in patients with dementia, including musculoskeletal, osteoarticular and headache-related pain (Lin et al., 2018); however, neuropathic pain remains comparatively less systematically investigated and may be underestimated due to communication difficulties and atypical behavioral manifestations in cognitively impaired individuals (Yao et al., 2025). This clinical complexity highlights the need for translational experimental models capable of interrogating shared pathological pathways between chronic pain and neurodegeneration (Niziński et al., 2026). Taken together, these observations provide the rationale for investigating neuroinflammatory and neurodegenerative mechanisms in preclinical models, rather than attempting to fully reproduce the complex clinical phenotype of Alzheimer’s disease.

The mechanisms underlying the interplay between AD and chronic pain are complex and multifactorial. Overactivation of microglia and neuroinflammation, brain structure changes and disruptions in neural circuits contribute both to neuropathic pain and to the progression of AD pathology (Landrø et al., 2013; Samartin-Veiga et al., 2019). Persistent pain triggers neuroinflammatory cascades mediated by activated glial cells, which in turn exacerbate neuronal excitability, increase pain sensitivity and impair cognitive function (Ji et al., 2018; Vergne-Salle and Bertin, 2021). Among the molecular systems implicated, the endocannabinoid system (ECS) - comprising cannabinoid receptors type 1 and 2 (CB1 and CB2), endogenous ligands and metabolizing enzymes - is significantly altered in both chronic pain and AD (Kanwal et al., 2024).

ECS is widely distributed along pain-processing pathways within the central and peripheral nervous systems, as well as in neuroimmune circuits (Finn et al., 2021). Experimental evidence reveals that cannabinoids exert analgesic actions in a variety of preclinical models of acute and chronic pain of diverse etiologies (Wilson et al., 2008; Cox et al., 2007). These effects appear to result from modulation of nociceptive transmission at multiple levels (peripheral, spinal and supraspinal), either by attenuating ascending pain signals or by enhancing descending inhibitory control (Cichewicz and McCarthy, 2003; Cox et al., 2007). At the peripheral level, cannabinoids have been shown to suppress nociceptive input when administered locally, an effect mediated through both CB1 and CB2 receptors (Potenzieri et al., 2008). Within the brain, activation of CB1 receptors by agonists such as Δ⁹-tetrahydrocannabinol engages descending inhibitory pathways involving key pain-modulatory centers (Calignano et al., 1998). In addition to the direct modulation of pain signaling, cannabinoids may also exert analgesic effects indirectly through their anti-inflammatory properties. CB1 and CB2 receptor activation, as well as stimulation of other cannabinoid-sensitive targets, contributes to the suppression of pro-inflammatory cascades (DeLong et al., 2010; Wen et al., 2023). Selective CB2 receptor agonists have demonstrated analgesic efficacy in experimental models of inflammatory and neuropathic pain by reducing inflammatory signaling (Malan Jr et al., 2003; Wen et al., 2023).

Despite accumulating evidence on the interplay between cognitive decline and chronic pain, no previous studies, to our knowledge, have systematically evaluated standardized, EU-GMP certified Cannabis sativa L. strains in preclinical models designed to investigate mechanistic interactions between peripheral nerve injury and transient cholinergic dysfunction. The strain employed in this study possesses a well-characterized cannabinoid profile and EU-GMP certification ensures reproducibility and compliance with stringent European pharmaceutical standards. Furthermore, our research group has established its safety through comprehensive acute and chronic toxicology studies, providing a rigorous preclinical basis for further evaluation (Filipiuc et al., 2023; Stanciu et al., 2024; Tudorancea et al., 2025).

In the present study, scopolamine was employed to induce transient cholinergic dysfunction and cognitive impairment, a widely used preclinical model for investigating mechanisms underlying memory deficits (Stanciu et al., 2023; Miravalles et al., 2025). Donepezil was included as a reference cognitive drug; it primarily acts through cholinesterase inhibition, but also exerts neuroprotective and anti-inflammatory effects, supporting its use as a comparator in preclinical models of cognitive deficits (Sheikh and Ammar, 2024). Tramadol served as a standard analgesic to validate the pain model; although it is less commonly used for neuropathic conditions and has known central nervous system effects, observational evidence indicates that its use is not associated with an increased risk of Alzheimer’s disease (Oh et al., 2024), justifying its inclusion as a pharmacological benchmark in this preclinical context, while acknowledging that anti-epileptics or antidepressants are more typical first-line choices for neuropathic pain in elderly populations.

Chronic neuropathic pain often coexists with cognitive decline, worsening functional deficits and reducing quality of life in patients with dementia (Oh et al., 2024). Management of this comorbidity is challenging, as conventional analgesics, including opioids like tramadol, may pose additional risks (Musich et al., 2021). Preclinical evidence suggests that prolonged tramadol exposure can lead to hippocampal atrophy, impair learning and memory (Raj et al., 2019; Hussein et al., 2020), yet human studies remain limited and inconclusive (Taipale et al., 2018). Observational studies frequently overlook critical factors such as lifestyle, competing mortality risks and duration of opioid exposure (Oh et al., 2024), underscoring the need for alternative strategies that alleviate pain without compromising cognition.

To investigate potential therapeutic interventions in a clinically relevant context, we employed a dual in vivo model combining chronic neuropathic pain induced by sciatic nerve ligation with scopolamine-induced cognitive impairment. While scopolamine administration does not recapitulate hallmark pathological features of Alzheimer’s disease, such as amyloid-β deposition or tau hyperphosphorylation, it consistently produces deficits in learning, memory and synaptic plasticity, as well as associated neuroinflammatory responses (Stanciu et al., 2023; Miravalles et al., 2025). This pharmacological model has been extensively validated across multiple species, including rodents (mice and rats) and zebrafish, enabling cross-species evaluation of cognitive function, neurochemical alterations and pharmacological interventions (Déciga-Campos et al., 2025; Chen and Yeong, 2020; Budzynska et al., 2015). While limitations exist regarding the full translational relevance to human Alzheimer’s disease, this model remains a valuable tool for preclinical testing of interventions targeting cholinergic deficits, synaptic dysfunction and neuroinflammatory processes (Déciga-Campos et al., 2025; Miravalles et al., 2025). By combining neuropathic pain with cognitive impairment, this approach closely replicates the clinical scenario in which dementia is accompanied by persistent neuropathic pain, enabling simultaneous assessment of peripheral nociceptive processing and central cognitive deficits. The endocannabinoid system plays a central role in maintaining neuronal homeostasis, modulating pain signaling and limiting neuroinflammatory responses via CB₁ and CB₂ receptors (Simankowicz and Stępniewska, 2025). Cannabinoid-based compounds have therefore attracted attention as multi-target agents capable of providing both neuroprotective and anti-inflammatory effects (Kaur et al., 2016). Although the pharmacodynamics of isolated cannabinoids such as CBD and THC are well-characterized, standardized plant-based formulations like Cannabixir® Medium Flos have been less extensively studied. Our study aimed to bridge this gap by assessing the therapeutic potential of an EU-GMP certified Cannabis sativa L. strain in a complex pathological context.

Cannabixir® Medium Flos administration was well tolerated, with no significant changes in body weight or serum biochemical parameters, indicating systemic safety and metabolic stability. These observations are consistent with previous reports that cannabinoids, particularly THC and CBD, can modulate multiple signaling pathways without inducing hepatotoxicity or renal dysfunction, even under chronic exposure (Stanciu et al., 2024; Tudorancea et al., 2025). Furthermore, cannabinoids may exert hepatoprotective and renoprotective effects through CB₁- and CB₂-mediated antioxidant and anti-inflammatory mechanisms (Pan et al., 2009; Mecha et al., 2012; Carmona-Hidalgo et al., 2021). CB₁ receptors, predominantly expressed in the brain but also present in hepatocytes and hepatic stellate cells, together with CB₂ receptors, primarily localized in immune cells such as Kupffer and hepatic stellate cells, contribute to maintaining cellular homeostasis and limiting oxidative stress (Louvet et al., 2011; Mukhopadhyay et al., 2011). Mild alterations observed in the tramadol-treated group align with previously reported hepatocellular stress associated with opioid metabolism (Barbosa et al., 2020), supporting the hypothesis that Cannabixir® Medium Flos co-administration may mitigate systemic adverse effects under long-term treatment conditions.

Behavioral nociceptive testing using the Tail Flick, Hot Plate and Randall–Selitto assays revealed a marked and time-dependent analgesic effect of Cannabixir® Medium Flos. Obvious increases in response latencies were observed as early as 30 min post-administration, becoming highly significant at 120 min. Importantly, the CanDTG group exhibited prominently longer latencies than the tramadol-treated group, indicating a more potent and sustained analgesic action. These results strongly support the efficacy of Cannabixir® Medium Flos in modulating nociceptive responses, consistent with cannabinoid-mediated CB1 central inhibition and CB2 peripheral anti-inflammatory mechanisms (Smith and Martin, 1992; Starowicz and Finn, 2017). In contrast, mechanical sensitivity assessed via the Randall–Selitto test showed only limited changes following treatment. The significant decrease in mechanical thresholds at T0 compared to the post-operative baseline confirmed the successful induction of neuropathic pain, establishing a reference for mechanical hypersensitivity. Subsequent time points did not demonstrate further significant alterations, suggesting that cannabinoid compound primarily modulated thermal nociceptive responses rather than mechanical thresholds. This differential effect aligns with prior evidence that cannabinoids preferentially attenuate thermal and chemical nociception, while mechanical nociceptive pathways may be less sensitive to cannabinoid-mediated modulation (Starowicz and Finn, 2017; Bouchet and Ingram, 2020). Collectively, these results indicate that Cannabixir® Medium Flos exhibits stimulus-specific and time-dependent analgesic properties, particularly effective against thermal nociceptive stimuli. The enhanced analgesic effect observed in concurrent therapy, especially in the CanDTG group, further suggests potential synergistic interactions with donepezil and tramadol. However, these findings should be interpreted with caution, as the study was limited by sample size and the use of a single animal model. Further research is warranted to confirm these effects and to investigate their underlying mechanisms across different types and chronicity of pain, pain-processing regions represents an important limitation and that future studies will extend the investigation to cortico-striatal and limbic circuits.

Formal cognitive testing was not conducted to minimize animal stress, in accordance with the 3Rs principles (Replacement, Reduction, Refinement), particularly as multiple nociceptive assays were already included. The pharmacokinetic characteristics of this Cannabis sativa L. strain, previously described by our group (Filipiuc et al., 2023), support the absence of central psychoactive effects at the administered doses. Psychoactive THC remains undetectable in serum at oral doses up to 600 mg/kg and is largely replaced by its acidic precursor tetrahydrocannabinolic acid-A (THCA-A), which exhibits markedly reduced blood–brain barrier penetration and a CB1 receptor affinity at least 62-fold lower than THC. At the doses used in this study, THCA-A primarily mediates peripheral effects through transient receptor potential channels and cyclooxygenase 1/2 modulation, without activating central CB1 receptors. Importantly, the scopolamine-induced cognitive impairment model has been extensively validated by both our research team and the broader literature, demonstrating consistent deficits in hippocampal-dependent learning and memory, synaptic plasticity, and associated neuroinflammatory responses (Poceviciute et al., 2025; Stanciu et al., 2023; Bhuvanendran et al., 2018). These findings support its suitability as a preclinical tool for assessing cognitive impairment in the context of neuropathic pain.

The immunohistochemical analyses focused on the hippocampus, a brain region critically involved in learning, memory and synaptic plasticity, which are prominently affected in the scopolamine-induced cognitive impairment model (Miravalles et al., 2025). A panel of well-established markers was selected to comprehensively assess neuronal health and neuroinflammatory responses: NeuN for neuronal integrity, GFAP and Iba1 for astrocytic and microglial activation, Caspase-3 for apoptosis, and IL-6 and CD-163 for neuroinflammation. This targeted approach allowed the evaluation of key cellular processes relevant to cognitive function and neuropathic pain while maintaining a manageable scope for analysis. In the hippocampus, treatment with the cannabinoid compound, either alone or in combination with donepezil or tramadol, reduced GFAP and Iba1 expression, indicating attenuation of astrocytic and microglial activation. Notably, the combination of Cannabixir® Medium Flos with donepezil (CanDG group) produced an even greater reduction in GFAP expression, suggesting that the cannabinoid formulation may potentiate the anti-inflammatory effects of donepezil. These findings are consistent with previous studies showing that cannabinoids can suppress neuroinflammation by downregulating proinflammatory cytokines such as IL-6 and TNF-α (Puffenbarger et al., 2000; Nagarkatti et al., 2009). High astrocytic densities observed in the SG and TG groups reflect a pronounced neuroinflammatory response, in line with reports linking opioid exposure to oxidative stress and glial activation (Park et al., 2024; Abdelmoez, 2024). Several studies also support the role of GFAP upregulation in opioid dependence and tolerance (Torkzadeh-Mahani et al., 2019; Marie-Claire et al., 2004), where chronic drug-induced astrogliosis is considered an innate immune response to neurotoxicity and brain injury, potentially affecting synaptogenesis, neurogenesis, apoptosis and/or necrosis (Hussein et al., 2020). In our study, the increased GFAP expression aligns with signs of glial proliferation and hypertrophy.

Caspase-3 and IL-6 immunoreactivity patterns in the hippocampus indicate that scopolamine induces both apoptotic and inflammatory responses, consistent with its well-documented neurotoxic and cholinergic-blocking effects (Cheon et al., 2021). Tramadol produced a similar but less pronounced increase in Caspase-3 expression, aligning with reports of opioid-related oxidative stress and neuronal vulnerability (Abdelmoez, 2024). In contrast, treatment with donepezil or Cannabixir® Medium Flos markedly reduced apoptotic activity, with the greatest suppression observed in the combined Cannabixir®–donepezil group. This synergistic effect suggests that the cannabinoid formulation enhances donepezil’s neuroprotective efficacy, possibly through modulation of CB1/CB2 receptor-mediated pathways that inhibit caspase activation and promote neuronal survival. Likewise, the significant reduction in IL-6 levels following Cannabixir®–donepezil co-administration supports the anti-inflammatory potential of this therapy. Previous studies have shown that cannabinoids downregulate pro-inflammatory cytokines and microglial activation in models of neurodegeneration (Lisboa et al., 2016; Martín-Moreno et al., 2011), while donepezil exerts indirect anti-inflammatory effects through cholinergic enhancement (Cui et al., 2019). The nearly 90% suppression of IL-6 observed here is consistent with these findings and reinforces the hypothesis that concurrent targeting of cholinergic and endocannabinoid systems can effectively mitigate neuroinflammatory signaling.

In parallel, NeuN immunolabeling revealed preserved neuronal integrity in both Cannabixir® Medium Flos-treated and combined therapy groups, whereas the tramadol-treated group exhibited notable neuronal loss, though less severe than scopolamine alone. Comparatively, the CanDG and CanDTG groups displayed the highest neuronal densities, underscoring the superior neuroprotective effect of concurrent Cannabixir® Medium Flos and donepezil treatment. These findings highlight that, while tramadol may partially mitigate pain-related stress, it does not provide the same level of hippocampal protection as Cannabixir® Medium Flos or its combination with donepezil. This outcome corresponds with literature demonstrating that cannabinoids exert neuroprotective effects by limiting excitotoxicity, oxidative damage and apoptosis (Udovin et al., 2020), while donepezil promotes synaptic plasticity and neuronal resilience (Wu et al., 2024). Taken together, these results support a complementary mechanism through which Cannabixir® Medium Flos and donepezil act to preserve hippocampal structure and function by simultaneously suppressing inflammation and apoptosis.

The chronic ligation model produces profound alterations in the sciatic nerve, including demyelination, myelin sheath degeneration, infiltration of inflammatory cells and disorganization of nerve fibers (Al-Qudah and Al-Dwairi, 2016). In our study, these pathological changes were evident across all experimental groups, including animals treated with scopolamine, tramadol, donepezil, as well as those receiving cannabinoid alone or in combination. The extent of nerve damage varied according to the treatment regimen. The SG group exhibited the most severe impairments in axonal regeneration, characterized by marked Caspase-3 and IL-6 overexpression, moderate GFAP levels, comparatively low Iba-1 and CD163 immunoreactivity relative to controls. Conversely, the most favorable regenerative outcomes were consistently observed in the groups treated with cannabinoid compound. Their relative efficacy in promoting sciatic nerve recovery appeared to follow the order: CanDG > CanDTG > CanTG > CanG, consistent with Schwann cell activation and axonal regeneration. Tramadol treatment increased GFAP-positive cells. Similarly, Caspase-3 levels were lowest in these groups, highlighting the anti-apoptotic role of the cannabinoid compound. Inclusion of Cannabixir® Medium Flos, either alone or in combination with donepezil, tramadol or both, further increased Iba1-positive cell density (p < 0.01) compared with scopolamine or groups treated with donepezil or tramadol alone. While GFAP’s function in the central nervous system (CNS) is relatively well established, its role in the peripheral nervous system remains less defined. Some evidence suggests that GFAP expression in Schwann cells contributes to repair mechanisms (Jessen and Mirsky, 2019). Interestingly, GFAP-deficient mice exhibit normal CNS development, with nerve regeneration only modestly delayed compared to wild-type counterparts (Triolo et al., 2006; Berg et al., 2013). Although only a subset of brain structures and markers was examined, the observed modulation of these markers provides evidence supporting the neuroprotective and neuroreparative potential of Cannabixir® Medium Flos. Future studies could expand the analyses to additional brain regions and molecular targets to further elucidate the compound’s mechanisms of action.

The present study highlights the distinct therapeutic profiles of Cannabixir® Medium Flos in a dual model of chronic neuropathic pain accompanied by cognitive impairment. As a single agent, Cannabixir® Medium Flos demonstrated analgesic effects, attenuated neuroinflammatory responses, preserved hippocampal neuronal integrity and promoted peripheral nerve regeneration. Tramadol alone, while partially effective in alleviating pain, showed limited neuroprotective effects, highlighting its potential limitations in addressing the combined pathology of neuropathic pain and cognitive decline.

Combination therapies showed additive trends, with Cannabixir® Medium Flos together with donepezil (CanDG) or with donepezil and tramadol (CanDTG) exhibiting more pronounced effects on glial activation, apoptotic and pro-inflammatory markers, and neuronal and axonal integrity. These findings suggest that targeting cannabinoid-mediated, cholinergic and opioid-related mechanisms may provide a multi-faceted approach to managing pain and neurodegeneration, although further studies are needed to confirm these effects.

Overall, our results provide a preclinical basis for exploring combination strategies that integrate Cannabixir® Medium Flos with conventional agents. Future investigations should clarify the molecular pathways involved, including CB₁/CB₂ receptor signaling, modulation of neuroinflammation and Schwann cell-mediated peripheral nerve repair, as well as assess long-term safety and efficacy across diverse models of neurodegeneration and chronic pain. Such studies will be essential to determine the translational potential of these co-therapies and to guide the development of interventions that may improve functional outcomes and quality of life in patients with dementia and comorbid conditions.