Cannabidiol improves short-term memory in a Streptozotocin-induced animal model of Alzheimer’s disease

Cannabidiol improves short-term memory in a Streptozotocin-induced animal model of Alzheimer’s disease | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Cannabidiol improves short-term memory in a Streptozotocin-induced animal model of Alzheimer’s disease Gabrielle Christine Pereira, Vanessa Alexandre Silva, Beatriz Soares-Silva, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7197783/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Alzheimer's disease (AD) is a progressive neurodegenerative pathology and the leading cause of dementia in the elderly. Early impairments in brain glucose metabolism and insulin signaling pathway may contribute to neurodegeneration, promoting oxidative stress, increased amyloid-beta (Aβ) production, Tau hyperphosphorylation, mitochondrial dysfunction, neuroinflammation, and neuronal loss. The search for novel therapeutic strategies that can prevent, or slow AD progression remains a major challenge. Cannabidiol (CBD), a phytocannabinoid, has been shown to exert neuroprotective, antioxidant, and anti-inflammatory effect in various experimental models. This study aimed to evaluate the potential neuroprotective effect of CBD in a rat model of AD induced by streptozotocin (STZ, 3 mg/kg, i.c.v.). Wistar rats (6–7 months old) received CBD (10 mg/kg, i.p.) for 14 consecutive days. During treatment, behavioral assessments including the open field, novel object recognition, sucrose preference, and spontaneous alternation tasks were performed, alongside monitoring of body weight and liquid consumption. At the end of the protocol, brains were collected for immunohistochemistry, immunofluorescence, and oxidative stress analysis. STZ-treated animals displayed cognitive deficits, weight loss, and increased Aβ deposition in the hippocampus. CBD treatment prevented short-term memory impairment, reduced Aβ accumulation in the CA1 and dentate gyrus, and decreased microglial activation (Iba-1 immunoreactivity). In addition, CBD attenuated oxidative damage in the striatum. These findings suggest that CBD exerts neuroprotective effects in this pharmacological model of AD, supporting its potential as a candidate for further investigation in the context of neurodegenerative diseases. Cannabis phytocannabionoid neuroprotection dementia Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Highlights • Cannabidiol prevented short-term memory deficits in an AD rat model. • CBD reduced Aβ deposition and microglial activation in hippocampal regions. • CBD decreased oxidative stress levels in the striatum of STZ-induced rats. • Findings support CBD’s neuroprotective potential for Alzheimer’s disease. Introduction Approximately 55 million people worldwide are affected by some form of dementia, with Alzheimer's Disease (AD) being the most prevalent. This number is expected to rise to 153 million by 2050 (GBD, 2022). Dementia ranks as the seventh leading cause of death and is a significant source of disability among older adults, with an estimated economic impact of $ 1.3 trillion in 2019 (WHO, 2025). AD is characterised by multifactorial neuropathological changes, including amyloid beta dysfunction, tau protein hyperphosphorylation, neurotransmission imbalances, and neuroinflammation. Currently, there are 143 drugs in development for AD, with 83.2% aimed at modifying the disease, and two monoclonal antibodies recently approved (aducanumab and lecanemab), acting in the Aβ peptide agglomerates (Dhillon, 2021 ; Cummings et al., 2022 ; Reardon, 2023 ). However, many of these drugs, like acetylcholinesterase (AChE) inhibitors and memantine, can cause side effects such as nausea, vomiting, and fatigue (Kaduszkiewicz et al., 2005 ; Mimica and Presečki, 2009). In contrast, natural products may have milder side effects compared to drugs available (Deshpande, Gogia and Singh, 2019), interacting simultaneously with multiple AD targets (Patil et al., 2020 ; Noori et al., 2021 ), as these products contain various bioactive substances that may work synergistically through multiple neuroprotective mechanisms (Chen et al., 2021 ). Recent studies have focused on Cannabis sativa (cannabis, hemp) as a medicinal plant, which contains over 400 active compounds with pharmacological potential, as observed in experimental models involving rodents and humans (Karniol et al., 1974 ; Mechoulam and Carlini, 1978 ; Carlini, 2003 , 2004 ). The two most prevalent substances in the plant are delta-9-tetrahydrocannabinol (Δ9-THC), ranging from 10–30%, and cannabidiol (CBD), ranging from 0.1–5%. CBD, the second most abundant phytocannabinoid found in the resin of the flowers, has received considerable attention in research due to its therapeutic effects on various chronic diseases, including AD, Parkinson's disease, epilepsy, rheumatoid arthritis, anxiety, and depressive disorders (ElSohly et al., 2016 ; Pisanti et al., 2017 ; Patricio et al., 2020 ). CBD has shown promising results by reducing β-amyloid expression, oxidative damage, neuronal injury, and apoptosis while promoting neurogenesis and offering neuroprotective effects against excitotoxicity, also regulating caspase 3 to inhibit neuronal apoptosis (Iuvone et al., 2004 ; Esposito et al., 2006 , 2011 ; Fagherazzi et al., 2012 ; Watt and Karl, 2017 ; Kim et al., 2019 ; Li et al., 2020 ; Cooray, Gupta, and Suphioglu, 2020 ). This study aims to evaluate the therapeutic potential of CBD in AD by assessing its effects on key neuropathological mechanisms, including β-amyloid accumulation, oxidative stress, and neuroinflammation. Given the limitations and adverse effects of current AD treatments, the study investigates CBD as a potential alternative with multi-target neuroprotective properties. By reviewing existing research, it aims to determine CBD’s efficacy and advantages over conventional pharmacological interventions. Materials and Methods Animals Eighty male Wistar rats (6-7 months) were obtained from the CEDEME (Centro de Desenvolvimento de Modelos Experimentais – Unifesp), placed in groups of 3 animals in polypropylene cages under controlled ventilation and temperature (22 ± 2°C), with a 12-hour light/dark cycle (lights on at 7 am) and free access to food and water. The use of animals in research was carried out under Brazilian law (Law No. 11.794). The project was submitted to the Ethics Committee on the Use of Animals (CEUA) under the protocol number 1327200121 of the Federal University of São Paulo. All necessary measures were taken to minimise pain, discomfort, and suffering. Drug preparation To prepare a CBD injection of 10 mg/kg in a volume of 1 mL/kg (for rats), the "Working Solution" consisted of 10 mg of CBD / 1 mL of vehicle (5% DMSO + 2% Tween 80 in 0.9% NaCl). The solution was prepared in a dark environment, as CBD is photoreactive and should not be exposed to light. To prepare a donepezil (DON) injection, the "working solution" consisted of 1 mg of DON/1 mL of vehicle (12.5% propylene glycol in 0.9% NaCl). The treatments were administered via intraperitoneal injections (i.p.). Experimental procedures The study consisted of 19 days of experiment. Animals underwent stereotaxic surgery involving bilateral skull perforation on the 1 st day of the experiment (Paxinos and Watson, 2005). Animals from the STZ group received a 3 mg/kg dose of streptozotocin (STZ) through an intracerebroventricular (i.c.v.) injection, and the CTR group received a control vehicle solution in both ventricles (Uchigata et al., 1982; Hoyer, Müller and Plaschke, 1994; Hosokawa, Dolci and Thorens, 2001; Szkudelski, 2001; Baydas et al., 2003; Salkovic-Petrisic et al., 2006; 2013; Zhou et al., 2013; Grieb, 2016). After three days of observation, the animals were assigned to experimental groups: CTR-CTR: [vehicle (i.c.v) + vehicle (NaCl, DMSO, and Tween 80, i.p., n=13)], STZ-CTR: [3 mg/kg STZ (i.c.v.) + vehicle (NaCl, DMSO, Tween 80, i.p., n=14)], CTR-CBD: [vehicle (i.c.v.) + CBD 10 mg/kg, i.p., n=14)], STZ-CBD: [3 mg/kg STZ (i.c.v.) + CBD 10 mg/kg, i.p., n=13)], CTR-DON [Vehicle (i.c.v.) + 1 mg/kg donepezil, i.p., n=13)], STZ-DON: [3 mg/kg STZ (i.c.v.) + 1 mg/kg donepezil, i.p., n=13]. For 14 days, CTR animals received a vehicle solution via i.p. injection, while others received either cannabidiol (CBD, at 10 mg/kg) (Esposito et al., 2006, 2011; Fagherazzi et al., 2012; da Silva et al., 2014; Watt and Karl, 2017; Peres, 2018) or donepezil (DON) at 1 mg/kg (Jayant, Sharma and Sharma, 2016; Choi et al., 2022; Faldu, Patel and Shah, 2023), starting 72 hours post-surgery (Fig. 1). During treatment, the animals were subjected to the following behavioural tests: olfactory sensitivity test (7th day of treatment), open field test (9th day of treatment), novel object recognition test (10th day of treatment), sucrose preference test (10th day of treatment) and spontaneous alternation test (14th day of treatment) (Fig. 1). Behavioural testing Open field test The experiment took place in a circular wooden apparatus measuring 50 cm in diameter and 40 cm in height, designed to assess locomotor activity and habituate animals for a subsequent object recognition test. During the 5-minute test, animals were placed in the centre, and their behaviours were tracked using Anymaze® (Stoelting, USA), which categorises the field into central and peripheral zones. The assessment focused on the distance covered, average speed, and time spent in each zone evaluated. Novel Object Recognition task This task assesses short-term memory by evaluating the recognition of a new object (de Lima et al., 2005). Conducted in a 50 cm diameter, 40 cm high open circular wooden arena, the test used pairs of identical objects differing in colour, size, and shape. During training, animals interacted with two identical objects. After one hour, they were shown a familiar object in the same location alongside a new object. Object selection was randomised. Each session lasted 5 minutes. The discrimination index was calculated using the formula: [time spent on the new object - time spent on the familiar object] / [time spent on the new + time spent on familiar]. Preference was calculated with: [time spent on the new object] / [time spent on new + time spent on familiar]. Spontaneous alternation test The spontaneous alternation test assesses operational memory, which is often impaired in patients with Alzheimer's disease (AD) due to frontal lobe dysfunction (Hughes, 2004). In this test, animals are placed in a maze with four closed arms and an open central area. After being positioned on the central platform facing arm A, they can explore for 5 minutes. Spatial working memory is evaluated by calculating the percentage of alternation, defined as four consecutive, non-repeated entries into different arms, using the formula: [number of alternations / (total number of entries – 3) * 100]. Sucrose preference test This test evaluates hedonic behaviour, specifically anhedonia, which is a reduction in pleasure often seen in depression (Liu et al., 2018) and Alzheimer's disease (Lyketsos et al., 2011). Two bottles, one with water and one with a 2% sucrose solution, are placed in each cage for 48 hours. During the first 24 hours, animals adapt to the setup. The bottles are then refilled and available for 12 hours, with their positions switched to prevent bias. Low sucrose intake indicates anhedonia. Sucrose preference is calculated as [sucrose consumption/ (sucrose consumption + water consumption)] to find the percentage of total liquid consumed (Huynh et al., 2011). Assessment of liquid consumption The average consumption of water and sucrose solution was assessed and determined once by the weight of the bottles before and after the test phase of the sucrose preference behavioural test. Weight loss Animal weighing was updated throughout the experiment to monitor possible losses in body mass. Weighting took place weekly during the 3 weeks of the experiment (approximately one day before i.c.v. surgery, 3 days after surgery, after 7 days of treatment and after 14 days of treatment). Immunohistochemistry (IHC) Following these tests, on the 19th day of treatment, the animals were euthanised by perfusion, which consisted of accessing the abdominal cavity, exposing the diaphragm muscle, which was sectioned to access the heart. Subsequently, a cannula was inserted into the left ventricle, and a small incision was made in the right atrium. A volume of 200 ml of 0.9% saline solution was injected with a peristaltic pump. Then, 200 ml of 4% paraformaldehyde solution in 0.1 M phosphate buffer, pH 7.4, was injected. After perfusion, the brains were collected by craniotomy and kept in 4% paraformaldehyde for 24 hours. The following day, the brains were removed from the solution and were cryoprotected in 30% sucrose in the refrigerator until immunohistochemistry and immunofluorescence were analysed. The brains were removed from the cryoprotection in 30% sucrose on the day of the sectioning. They were frozen in Tissue-Tek® cryostat inclusion medium (Sakura, Japan) and 4% gelatine in 0.1 M PBS inside a plastic mould. Samples were coronally sectioned at 50 μm using a Leica CM 1950 cryostat. Brain sections were washed and incubated with non-fat milk, then treated with anti-beta-amyloid (1-42) primary antibody (1:500, #bs-0107R Bioss) and secondary antibody (1:1000, #AP132B Millipore). They were processed with the avidin-biotin complex (ABC Kit, Vector Labs) and 0.05% 3,3' diaminobenzidine (DAB) for staining. After sufficient staining, sections were mounted on gelatinised slides and dehydrated through a series of alcohols. Images of the areas of interest were captured, and quantifications were performed using relative optical densitometry. The sections were analysed using a Zeiss Axio Imager M2 microscope with Apotome (Carl Zeiss Microscopy, Germany). Contours were drawn to map the hippocampal region using the Stereo Investigator program (MBF Biosciences, USA). Cell counting was performed to quantify the number of beta-amyloid biomarkers in the hippocampal formation. Immunofluorescence analysis Brain sections were washed in PBS and incubated with normal serum to prevent non-specific binding [3% normal donkey serum (Jackson ImmunoResearch Laboratories, West Grove, PA) in PBS] for two hours. Primary antibodies, anti-GFAP (1:500, #53989282, eBioscience) and anti-Iba1 (1:500, #01919741, FUJIFILM Wako), were added for 48h at 4 ℃ with 0.1% Triton X-100, followed by AF 594 goat-anti-rabbit IgG secondary antibody (1:200, #A11012, Invitrogen) for two hours at room temperature. The sections were mounted on gelatinised slides and cover-slipped with VECTASHIELD antifade mounting medium with DAPI (Vector Laboratories, #H1200). Oxidative stress analysis To assess oxidative stress parameters, another group of animals was previously anaesthetised with isoflurane for euthanasia by decapitation using a guillotine. The brains were stored in a freezer at -80 °C for subsequent measurements of the total oxidant and antioxidant status of the samples. Brains were homogenised in a buffer and centrifuged at 12.000 rpm for 15 minutes at 4 ºC. The supernatant was collected and stored at -20ºC. For Total Antioxidant Status (TAS), 5 µL of the sample was pipetted in duplicate with 5 µL of Trolox and 5 µL of PBS. Then, 200 µL of Reagent 1 was added, and the first absorbance reading was taken at 444 nm. After adding 10 µL of Reagent 2, the solution was incubated for 5 minutes in the dark before the second reading at 444 nm (Erel, 2004). For Total Oxidant Status (TOS), 25 µL of the sample was treated similarly with hydrogen peroxide and PBS, followed by 160 µL of Reagent 1. After the first reading at 560 nm, 8 µL of Reagent 2 was added, and the solution was incubated for 5 minutes before the second reading at 560 nm (Erel, 2005). Data analysis A two-way Analysis of Variance (ANOVA) was conducted for all comparisons between cohorts, followed by Tukey’s post hoc test to identify interaction effects. Outlier values were adjusted through winsorization to the nearest valid value. It is a statistical technique used to limit extreme values in a dataset to reduce the effect of outliers. Instead of removing outliers, it replaces them with the closest value. This helps stabilise variance and improve the robustness of statistical analyses. Data met the assumptions of normality, linearity, and homogeneity. Statistical significance was determined at a p-value of less than 0.05, with results presented as the difference between means (MD). Error bars represented the standard deviation of the mean (SD), and differences were demonstrated using asterisks (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). All analyses and graphs were created using IBM® SPSS Statistics Program (Version 29) (IBM, USA), GraphPad Prism (Version 10), and Microsoft Excel. Results CBD reverses short-term memory deficits in STZ-treated animals that were not able to discriminate a novel object. The analysis of object exploration in novel object recognition showed group x object interaction [F (5,148) = 2.36; p = 0.0426]. Animals in the CTR-CTR (Mean difference = 15.03; p = 0.0003), CTR-CBD (Mean difference = 15.44; p = 0.0001), CTR-DON (Mean difference = 8.57; p = 0.0347), STZ-CBD (Mean difference = 12.07; p = 0.0032) and STZ-DON (Mean difference = 16.76; p < 0.0001) groups explored the novel object for longer compared to the familiar object. Animals in the STZ-DON group explored the new object for longer compared to the CTR-DON (Mean difference = 11.63; p = 0.0493) and STZ-CTR groups (Mean difference = 12.00; p = 0.033) (Fig. 2A). For the object discrimination index, a group x treatment interaction was also observed [F (2,74) = 3.94; p = 0.0235]. Animals in the STZ-CTR group did not discriminate against objects compared to the CTR-CTR (Mean difference = 0.48; p < 0.0001), STZ-CBD (Mean difference = 0.30; p = 0.022) and STZ-DON groups (Mean difference = 0.35; p = 0.0074) (Fig. 2B). STZ-treated animals showed reduced alternation frequency compared to the CTR-CTR group In the analysis of spontaneous alternation, a treatment effect was observed [F (1,74) = 13,28; p = 0,0005]. Animals in the STZ-CTR group had a lower frequency of alternation compared to the CTR-CTR (Mean difference = 15,24; p = 0,0014) (Fig. 3). 3.3. Animals treated with STZ-CBD travelled a greater distance and at a higher speed compared to the animals treated with STZ-CTR and STZ-DON. The analysis of the total distance travelled in the open field revealed a significant treatment effect [F (2,74) = 4.45; p = 0.0149]. Animals treated with STZ-CBD travelled a greater distance compared to CTR-CBD (Mean difference = 2.63; p = 0.0484), STZ-CTR (Mean difference = 4.63; p = 0.0021) or STZ-DON groups (Mean difference = 3.81; p = 0.0154) (Table 1). Regarding average speed, a significant treatment effect was also observed [F (2,74) = 4.90; p = 0.0100]. Animals treated with STZ-CBD moved at a higher speed than animals in the CTR-CBD (Mean difference = 0.008; p = 0.0487), STZ-CTR (Mean difference = 0.014; p = 0.004), and STZ-DON (Mean difference = 0.012; p = 0.0191) groups (Table 1). For distance travelled in the central zone, a significant group effect [F (1,74) = 12.39; p = 0.0007] and treatment effect [F (2,74) = 5.67; p = 0.0051] were found. Animals in the STZ-CBD group spent more time in the central zone compared to animals in the CTR-CBD group (Mean difference = 43.43; p = 0.0019). Additionally, animals in the STZ-CTR group spent more time in the central zone compared to animals in the STZ-DON group (Mean difference = 39.39; p = 0.0128) (Table 1). Animals from the STZ-DON group showed increased sucrose preference compared to other STZ-treated groups Analysis of the sucrose preference test revealed a significant group x treatment interaction [F (2,74) = 6.17; p = 0.0033]. Animals in the CTR-CBD group exhibited a higher preference for sucrose compared to the animals of the STZ-CBD group (Mean difference = 8.00; p = 0.0033). Furthermore, animals in the STZ-DON group showed greater sucrose preference than animals in the STZ-CTR (Mean difference = 8.36; p = 0.0062) and STZ-CBD groups (Mean difference = 9.15; p = 0.0030) (Fig. 4). Animals from the STZ-CBD group showed increased consumption of liquids compared to the cohorts Analysis of mean liquid consumption revealed a significant group x treatment interaction [F (5,148) = 14.86; p < 0.0001]. Animals in the STZ-CBD group consumed more water compared to the CTR-CBD (Mean difference = 45.18; p = 0.0383) and STZ-DON (Mean difference = 44.85; p = 0.0471) groups (Table 2). In terms of sucrose consumption, animals in the STZ-CBD group consumed significantly more than animals in the CTR-CBD (Mean difference = 175.37; p < 0.0001), STZ-CTR (Mean difference = 90.45; p < 0.0001) and STZ-DON (Mean difference = 161.80; P < 0.0001) groups. Additionally, animals in the STZ-CTR group also consumed more sucrose compared to animals in the CTR-CTR (Mean difference = 93.40; p < 0.0001) and STZ-DON (Mean difference = 71.40; p < 0.0001) groups. Animals in the CTR-CTR (Mean difference = 106.20; p < 0.0001), CTR-CBD (Mean difference = 117.60; p < 0.0001), CTR-DON (Mean difference = 92.15; p < 0.0001), STZ-CTR (Mean difference = 185.90; p < 0.0001), STZ-CBD (Mean difference = 247.80; p < 0.0001) and STZ-DON (Mean difference = 130.80; p < 0.0001) groups consumed more sucrose compared to water intake (Table 2). Animals in the STZ groups exhibited greater weight loss compared to the CTR groups In the evaluation of body mass, a significant group x weighing period was observed [F (15,222) = 13.81; p < 0.0001]. Animals in the CTR-CBD group showed a reduction in body mass from the first to the second weighing (Mean difference = 6.42; p < 0.0001), third (Mean difference = 13.71; p = 0.0249) and fourth weight (Mean difference = 6.93; p = 0.0016), as well as the second to the fourth weighting (Mean difference = 11.29; P = 0.0293). In the CTR-DON group, a decrease was observed between the first and second weighting (Mean difference = 5.76; p = 0.0196). The STZ-CTR group exhibited significant weight loss from the first and second (Mean difference = 21.43; p < 0.0001), third (Mean difference = 54.57; p < 0.0001) and fourth weight (Mean difference = 51.86; p < 0.0001) and from the second to the third (Mean difference = 33.14; p = 0.0005) and fourth weightings (Mean difference = 30.43; p = 0.0080). Similarly, STZ-CBD group lost body mass from the first to the second (Mean difference = 22.46; p < 0.0001), third (Mean difference = 77.31; p < 0.0001) and fourth weight (Mean difference = 66.54; p < 0.0001), as well as from the second to the third (Mean difference = 31.66; p < 0.0001) and fourth weightings (Mean difference = 19.51; p = 0.0009). In the STZ-DON group, a reduction was observed from the first to the second (Mean difference = 18.62; p < 0.0001), third (Mean difference = 48.31; p = 0.0013) and fourth weightings (Mean difference = 40.00; p = 0.0043), and from the second to the third (Mean difference = 29.69; p = 0.0088) and fourth weightings (Mean difference = 21.38; p = 0.0450) (Table 3). Furthermore, animals in the STZ-CTR (Mean difference = 54.57; p < 0.001), STZ-CBD (Mean difference = 77.30; p < 0.001) and STZ-DON (Mean difference = 48.30; p = 0.001) groups showed a significant loss in body mass from the third weighing compared to the first. A similar trend was observed in the fourth weighing in the STZ-CTR group (Mean difference = 51.85; p < 0.001), STZ-CBD (Mean difference = 66.53; p < 0.001) and STZ-DON (Mean difference = 40.00; p = 0.008) (Table 3). Treatment with CBD and DON reduced immunoreactivity of amyloid- b (Aβ) in hippocampal regions (Dentate gyrus and CA1) In the analysis of Aβ immunoreactivity in the dentate gyrus region of the hippocampus, a significant group x treatment interaction was observed [F (2,83) = 6.059; p = 0.0035]. Animals in the STZ-CBD group exhibited higher Aβ density compared to animals in the CTR-CBD (Mean difference = 0.027; p = 0.001) and STZ-DON (Mean difference = 0.029; p = 0.0027). Conversely, animals in the CTR-CTR (Mean difference = 0.046; p < 0.0001), STZ-CBD (Mean difference = 0.022; p = 0.03) and STZ-DON (Mean difference = 0.051; p< 0.0001) groups exhibited lower Aβ density when compared to the STZ-CTR group (Fig. 5). In the CA1 region, a significant group x treatment interaction was also observed [F (2,82) = 7.018; p = 0.0015]. Animals in the STZ-CBD group showed higher Aβ density compared to the animals in the CTR-CBD (Mean difference = 0.028; p < 0.0001) and STZ-DON (Mean difference = 0.021; p = 0.0098) groups. Furthermore, animals in the CTR-CTR (Mean difference = 0.050; p < 0.0001), STZ-CBD (Mean difference = 0.024; p = 0.0028) and STZ-DON (Mean difference = 0.046; p < 0.0001) groups presented significantly lower Aβ density compared to the STZ-CTR group (Fig. 6). In the CA3 region, a group x treatment interaction was also identified [F (2,86) = 5.15; p = 0.007]. Animals in the STZ-CBD groups exhibited higher Aβ density when compared to the CTR-CBD (Mean difference = 0.038; p < 0.0001) and STZ-DON (Mean difference = 0.025; p = 0.0022) groups. Additionally, animals in the CTR-CTR (Mean difference = 0.048; p < 0.0001) and STZ-DON (Mean difference = 0.037; p < 0.0001) groups presented reduced Aβ density compared to the STZ-CTR group (Fig. 7). Microglia and astrocytes were increased in the STZ-CTR group, and CBD reduced IBA1 cell number in the hippocampus In the analysis of Iba1 immunoreactivity in the hippocampus region, a significant group x treatment interaction was observed [F (2,24) = 5.75; P = 0.0091]. Animals in the STZ-CBD group exhibited a higher Iba1+ cell population compared to the CTR-CBD group (Mean difference = 271.835; p = 0.032). Conversely, animals in the CTR-CTR (Mean difference = 691.269; p < 0.0001), STZ-CBD (Mean difference = 351.488; p = 0.0194) and STZ-DON (Mean difference = 336.966; p = 0.0255) groups showed reduced Iba1 expression compared to the STZ-CTR group (Fig. 8). Regarding GFAP immunoreactivity in the hippocampus, a group x treatment interaction was also observed [F (2,24) = 3.34; p = 0.05]. Animals in the CTR-CTR group displayed a significantly lower GFAP+ cell population compared to the STZ-CTR group (Mean difference = 683.572; P = 0.0069) (Fig. 8). STZ-CBD animals presented a lower oxidative stress index (OSI) compared to the STZ-CTR group in the striatum In the striatum, a significant group x treatment interaction was observed [F (2,36) = 8.36; p = 0.001]. Animals in the CTR-CTR (Mean difference = 36.93; p = 0.0029), STZ-CBD (Mean difference = 57.26; p < 0.0001) and STZ-DON (Mean difference = 58.48; p < 0.0001) groups exhibited lower oxidative stress indices compared to the STZ-CTR group (Fig. 9A). In the hippocampus, a significant group effect was found [F (1,36) = 13.73; p = 0.0007]. Animals in the CTR-CTR group presented higher oxidative stress indices compared to the STZ-CTR group (Mean difference = 18.06; p = 0.0186). Additionally, animals in the STZ-CBD group presented lower oxidative stress indices compared to the CTR-CBD (Mean difference = 17.57; p = 0.0217) (Fig. 9B). Discussion The results of this study indicate that subchronic treatment with CBD at a dose of 10 mg/kg in rats, using the STZ-induced model of AD, effectively prevented short-term memory deficits. Additionally, it increased fluid intake and locomotor activity, resulting in hyperactivity. These effects were associated with reduced immunostaining for Aβ and IBA1, as well as a decrease oxidative stress index in the striatum. The novel object recognition task is commonly used to assess learning and memory in rats, enabling the evaluation of short- or long-term memory depending on the interval between training and testing (Akkerman, Blokland and Prickaerts, 2014; Lueptow, 2017). In our study, we focused on short-term memory using a one-hour interval. As expected, STZ-injected animals failed to discriminate between novel and familiar objects, indicating a memory deficit (Chen et al., 2013; Grieb, 2016; Andrade et al., 2023). Consistent with previous studies, treatment with CBD and Donepezil attenuated this memory impairment (Cheng et al., 2014; Coles et al., 2020; de Bruin et al., 2011; Guo et al., 2015). In the study by Fagherazzi et al. (2012), treatment with CBD (10 mg/kg) for 14 days showed a better neuroprotective effect in the novel object recognition task compared to a lower dose (5 mg/kg) in a rat model of neurodegeneration induced by iron overdose, effectively preventing deficit in the memory consolidation phase. De Paula Faria et al. (2022) reported that treatment with CBD (20 mg/kg) for 7 days prevented short- and long-term memory deficits in STZ-induced animals. These findings suggest that CBD, when administered in higher and subchronic doses, exerts a neuroprotective effect against memory deficits in models of neurodegeneration. The spontaneous alternation test is used to evaluate the spatial working memory in rodents and is particularly sensitive to changes in the hippocampus and other memory-related brain regions (Lalonde, 2002; Deacon and Rawlins, 2006). In this task, rodents are expected to explore a novel arm of the apparatus, alternating their choices within the maze (Hughes, 2004). In our study, STZ-injected animals treated with vehicle displayed a reduced alternation rate, indicating a working memory deficit. On the other hand, treatment with CBD significantly improved performance in this task, suggesting its potential to mitigate STZ-induced memory impairment. The open field test is widely used to assess rodent behavioural patterns, including anxiety-like behaviour, stereotypy, locomotion, and overall health status (Prut and Belzung, 2003; Kraeuter, Guest, and Sarnyai, 2019). However, when evaluating anxious behaviour, it is important to note that this is an individual test in which the animal is isolated from its social group, a factor that can influence anxiety levels. Moreover, the open arena is larger compared to the animal's cage, and this environmental contrast may elicit distinct behavioural responses under potentially stressful conditions. In our results, animals in the STZ group spent more time in the central zone compared to the animals in the CTR group. Interpretations of this finding may vary in the literature, depending on the treatment applied, the animal model used, and the specific experimental protocol adopted. When comparing our findings with a previous study conducted by our group, which utilised the same protocol and animal model but involved different pharmacological treatments, we observed similar outcomes (Alexandre-Silva et al., 2024). Animals from the STZ-CBD group that spent more time in the centre of the open field test exhibited hyperlocomotion, as indicated by increased average speed, an outcome that some researchers interpret as a potential indicator of anxiety. However, previous studies have suggested that CBD may exert anxiolytic effects (Crippa et al., 2011; Blessing et al., 2015; García-Gutiérrez et al., 2020; de Faria et al., 2020). Further analyses are needed to elucidate the effects of STZ and CBD on anxiety-like behaviours, including the use of traditional evaluation paradigms and detailed assessments of specific ethological behaviours such as exploratory activity, grooming, feeding patterns, and fluid intake (Kelley, 2001). Comorbidities such as depression and anxiety are common among AD patients (Botto et al., 2022). The sucrose preference test is a well-established method for assessing anhedonia, a core symptom of depression (Liu et al., 2018; Höflich et al., 2019). Apathy and depression are classified as Behavioural and Psychological Symptoms of Dementia (BPSD) and are particularly prevalent among older adults (Deardorff and Grossberg, 2019). Moreover, research indicates that anhedonia tends to increase with the severity and progression of AD (Turner and Husain, 2022). Lopez et al. (2003) reported that 61% of the patients evaluated exhibited anhedonia, with the prevalence of symptoms increasing alongside disease progression: 50.5% in mild cases, 65.0% in moderate cases, and 72.0% in severe cases. In cases of mild to moderate dementia, anhedonia was associated with sleep disturbances, depressed mood, and feelings of hopelessness, whereas in severe dementia, it was primarily linked to hopelessness. Similarly, Saz et al. (2009) found that 46.8% of AD patients presented anhedonia, with the prevalence rising from 35.6% in mild to moderate AD to 80.0% in severe stages. When discussing animal models, healthy animals typically exhibit a higher preference for sucrose solution, as it is perceived as pleasurable. A decreased preference may indicate anhedonia (Liu et al., 2018). Pooladgar et al. (2022) suggest that donepezil may exert antidepressant effects through interactions with the sigma-1 receptors, nicotinic acetylcholine receptors, and neurotransmitters such as dopamine and serotonin (Ramakrishnan et al., 2014; Papp et al., 2016; Walczak-Nowicka and Herbet, 2021). In our study, the control and STZ-DON groups exhibited a higher preference for sucrose compared to the STZ-CTR and STZ-CBD groups. However, we cannot conclude that STZ-CTR and STZ-CBD animals are experiencing anhedonia, as their sucrose consumption remained high at 87.9% and 86.8%. Additionally, these groups also showed increased water and sucrose consumption, with donepezil appearing to reverse this trend. Strekalova et al. (2006) found that anhedonic and non-anhedonic mice exhibited different fluid intake levels during and after stress. This increase may be linked to polydipsia, a potential indicator of diabetes mellitus caused by prolonged stress or compulsive behaviour (Schoenecker, Heller, and Freimanis, 2000). The STZ-CBD group showed signs of polydipsia and increased locomotion in the open field test, suggesting repetitive and compulsive behaviour. Additionally, Sofia and Knobloch (1976) studied the effects of CBD, CBN (cannabinol), THC, and d-amphetamine sulphate on food, sucrose, and water consumption in rats, finding a greater preference for calorie-dense sweets in the cohorts. Furthermore, a study by Spindle et al. (2020) revealed that dry mouth was reported in 6.6% of patients taking CBD 100 mg orally, 16.1% with vapour, and 35.9% with 100 mg of CBD alongside 3.7 mg of THC, suggesting that cannabinoids can significantly influence feeding behaviour and preferences. In terms of weight, animals in the STZ groups exhibited a greater loss of body mass compared to those in the CTR groups, beginning from the third weighing, which occurred two weeks after the STZ microinjection. The literature has previously reported a loss of body mass associated with the use of STZ as a model for AD, suggesting that the damage caused by STZ is significant enough to reduce food consumption and hinder the animals from regaining weight (Chen et al., 2013; Paidi et al., 2015; Zappa Villar et al., 2018; Yuliani, Lobentanzer, and Klein, 2021; Silva et al., 2023). In our histological analyses, we aimed to confirm the results from our behavioural tests. We found that the deposition of the Aβ peptide was elevated in the hippocampal regions of CA1, CA3, and the dentate gyrus in the STZ-CTR group, consistent with previous studies indicating this characteristic in this animal model (Ahn et al., 2020; Andrade et al., 2023). Our proposed treatment with CBD at a dosage of 10 mg/kg significantly reduced deposition in the CA3 region and the dentate gyrus. This effect was also observed with the treatment using donepezil, supporting findings in the literature (Esposito et al., 2007; Ye et al., 2015; Takada-Takatori et al., 2019; Chen et al., 2023). These results are consistent with the outcomes of the novel object recognition and alternation tasks, which demonstrated that CBD treatment effectively prevented short-term and spatial working memory deficits in the animals. Similar findings were observed in Iba1 analyses using immunofluorescence. Chronic glial activation, characterised by an increase in reactive microglial and astroglial cells, is commonly observed in neurodegenerative diseases. Studies have shown heightened immunoreactivity of these cells in the STZ animal model (Bassani et al., 2017; Luo et al., 2019; Liu et al., 2022), with CBD treatment showing a reduction in this immunoreactivity (Esposito et al., 2007; Schiavon et al., 2014; Li et al., 2022). Our histological results revealed that STZ-treated animals had increased IBA1 microglia and GFAP astrocytes in the hippocampus, whereas CBD treatment significantly reduced the number of IBA1 reactive cells. An important factor contributing to the increase in these biomarkers is the rise in reactive oxygen species. The oxidative stress index (OSI) revealed that animals in the STZ-CBD and STZ-DON groups exhibited lower oxidative stress levels in the striatum compared to the STZ-CTR group, corroborating the literature (Cassol et al., 2010; Valvassori et al., 2011). According to Ferbinteanu (2016), the hippocampus and striatum can function cooperatively in memory formation, with significant contributions from different areas to task performance and learning. The findings of this study suggest that subchronic treatment with CBD at a dose of 10 mg/kg can effectively mitigate memory deficits associated with Alzheimer’s disease in the STZ-induced rat model. The behavioural tests indicated improvements in short-term and spatial working memory, as well as hyperactivity-related behaviours. CBD demonstrated neuroprotective properties, supported by decreased oxidative stress and alterations in neuroinflammatory markers. Additionally, the study highlights the prevalence of anhedonia in Alzheimer’s patients, underscoring the need for continued exploration of behavioural and psychological symptoms associated with the disease. Comorbidities such as anxiety and depression warrant further investigation, particularly in terms of how they impact memory and overall quality of life in affected individuals. Overall, these results emphasise the potential of CBD as a therapeutic agent in combating cognitive impairments and psychological symptoms associated with Alzheimer’s disease. Future studies should further explore the underlying mechanisms and long-term effects of CBD treatment, analyse feed intake by weighing food during each cage change to determine if the results support the hyperphagia observed in AD and its correlation with increased fluid consumption, and also investigate patterns of increased appetite related to CBD treatment. Limitations This study focused on male subjects, despite global data indicating that women are more affected by AD. This limitation is acknowledged in the animal model of AD induced by STZ, as Biasibetti et al. (2017) found that females are generally more resistant to STZ-induced damage. Declarations CRediT authorship contribution statement Conceptualization: GCP, RHL, VCA, JRS, EG, and AMR; data curation: GCP, VAS, BSS, LOT, PSCL, ACCS, EPSJ, RHL, and EG; writing original draft preparation: GCP, EG, and AMR; Review and editing: GCP, VCA, JRS, EG, and AMR; Supervision: EG, and AMR; Funding acquisition: AMR. All authors have read and agreed to the publication of the manuscript. Acknowledgments This research was supported by the São Paulo Research Foundation (FAPESP, grant #20/12053-5, #21/12409-7, #20/09015-4, #22/02320-1, and #22/09866-0), the Coordination for the Improvement of Higher Education Personnel (CAPES) – Finance Code 001, and the National Council of Technological and Scientific Development (CNPq #425694/2016-0, #303325/2017-8, #408377/2021-6 and #310403/2021-9). Declaration of Competing Interest The authors declare that they have no competing interests. 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Intracerebroventricular streptozotocin induces impaired Barnes maze spatial memory and reduces astrocyte branching in the CA1 and CA3 hippocampal regions. J Neural Transm (Vienna) , 125 (12), 1787-1803. https://doi.org/10.1007/s00702-018-1928-7 Zhou, S., Yu, G., Chi, L., Zhu, J., Zhang, W., Zhang, Y., & Zhang, L. (2013). Neuroprotective effects of edaravone on cognitive deficit, oxidative stress and tau hyperphosphorylation induced by intracerebroventricular streptozotocin in rats. Neurotoxicology , 38 , 136-145. https://doi.org/10.1016/j.neuro.2013.07.007 Tables Tables 1 to 3 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1.docx Table2.docx Table3.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7197783","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":489994641,"identity":"eb2289d1-8825-4e03-86ac-d49ba9e07d39","order_by":0,"name":"Gabrielle Christine Pereira","email":"","orcid":"","institution":"Federal University of São Paulo","correspondingAuthor":false,"prefix":"","firstName":"Gabrielle","middleName":"Christine","lastName":"Pereira","suffix":""},{"id":489994646,"identity":"4e053e6c-a70e-4ff6-a083-43570681dd14","order_by":1,"name":"Vanessa Alexandre Silva","email":"","orcid":"","institution":"Federal University of 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Paulo","correspondingAuthor":true,"prefix":"","firstName":"Alessandra","middleName":"Mussi","lastName":"Ribeiro","suffix":""}],"badges":[],"createdAt":"2025-07-23 15:08:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7197783/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7197783/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87500716,"identity":"e5a3bc8c-50b6-4bf1-92c9-6e41540de49e","added_by":"auto","created_at":"2025-07-24 13:51:41","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":73897,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental design. After STZ or vehicle injection during the surgical procedure, animals were given 72h to recuperate. Then, treatment with CBD (10 mg/kg), DON (1 mg/kg) or vehicle solution (i.p. via) lasted for 14 days. The following tests were performed: olfactory discrimination test (Day 7), open field test (Day 9), novel object recognition test (Day 10), sucrose preference test (Day 10) and spontaneous alternation test (Day 14). On day 15, animals were euthanized for tissue collection.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7197783/v1/e363a8eaf1526f01f760a2ab.jpg"},{"id":87501768,"identity":"bb2e88bf-6e9e-4b3c-9ae8-d8be7ea72711","added_by":"auto","created_at":"2025-07-24 13:59:41","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":80487,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of repeated CBD (10 mg/kg) administration in rats subjected to the STZ-induced AD animal model. (A) Exploration time of the familiar and novel objects in the test session. (B) Object discrimination index. The results indicated that animals injected with STZ and treated with vehicle were unable to distinguish between the familiar and the novel objects, suggesting a memory deficit. However, CBD administration ameliorated this impairment (*p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001 compared between groups). Data were analyzed using a two-way ANOVA followed by Tukey’s post hoc test. Error bars represent the standard deviation of the mean (SD).\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7197783/v1/09aaabbce0ed5d5e42422df4.jpg"},{"id":87500720,"identity":"9af044f6-515b-4485-aa63-d65fb8961029","added_by":"auto","created_at":"2025-07-24 13:51:41","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":35142,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of repeated CBD (10 mg/kg) administration in rats subjected to the STZ-induced AD animal model.\u003cstrong\u003e \u003c/strong\u003eThe results indicated that animals injected with STZ and treated with vehicle exhibited a reduced alternation frequency, suggesting a working memory deficit. However, CBD administration was able to ameliorate this deficit. Statistical significance was considered at *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001. Data were analysed using two-way ANOVA. Error bars represent the standard deviation of the mean (SD).\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7197783/v1/0896902b2ba23a1f80e45ce3.jpg"},{"id":87501770,"identity":"3e7ebddd-b4fb-4a6c-9cf6-809ce9dde94d","added_by":"auto","created_at":"2025-07-24 13:59:41","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":36713,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of repeated CBD (10 mg/kg) administration in rats subjected to the STZ-induced AD animal model.\u003cstrong\u003e \u003c/strong\u003eThe results indicated that all cohorts exhibited a preference for sucrose over water consumption. Statistical significance was considered at *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001. The data were analysed using a Two-way ANOVA. Error bars represented the standard deviation of the mean (SD).\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7197783/v1/4fe6da1ec4e6948c3750a312.jpg"},{"id":87500730,"identity":"19704b25-3c1c-4834-9020-085dd98d1468","added_by":"auto","created_at":"2025-07-24 13:51:41","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":142611,"visible":true,"origin":"","legend":"\u003cp\u003eAmyloid-b peptide immunoreactivity in rats submitted to the STZ-induced AD model treated with CBD (10 mg/kg), DON (1 mg/kg), or vehicle solution. (A-F) Representative microscopy images of coronal sections in the dentate gyrus cells. Images taken under the 10x objective (scale bar 50 µm). (G) Bar graph representing Aβ peptide density in the hippocampus. Two-way ANOVA with Tukey’s post hoc test. Post hoc effect of treatment demonstrated by asterisks, (*p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001). Error bars represented the standard deviation of the mean (SD).\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7197783/v1/af5498108c8872ebeedbfe6e.jpg"},{"id":87500722,"identity":"809c4379-e79a-4760-be47-87c1b9640643","added_by":"auto","created_at":"2025-07-24 13:51:41","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":125075,"visible":true,"origin":"","legend":"\u003cp\u003eAmyloid-b peptide immunoreactivity in rats submitted to the STZ-induced AD model treated with CBD (10 mg/kg), DON (1 mg/kg), or vehicle solution. (A-F) Representative microscopy images of coronal sections of the Aβ peptide density in CA1 cells. Images taken under the 10x objective (scale bar 50 µm). (G) Bar graph representing Aβ peptide density in the hippocampus. Two-way ANOVA with Tukey’s post hoc test. Post hoc effect of treatment demonstrated by asterisks, (*p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001). Error bars represented the standard deviation of the mean (SD).\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7197783/v1/ddb180ac0ce970b8e14ddef1.jpg"},{"id":87502136,"identity":"34afeea7-0665-4dee-bf38-23fd3dd00f3d","added_by":"auto","created_at":"2025-07-24 14:07:41","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":116802,"visible":true,"origin":"","legend":"\u003cp\u003eAmyloid-b peptide immunoreactivity in rats submitted to the STZ-induced AD model treated with CBD (10 mg/kg), DON (1 mg/kg), or vehicle solution. Representative microscopy images of coronal sections in the density of CA3 cells. (A-F) Images taken under the 10x objective (scale bar 50 µm). (G) Bar graph representing Aβ peptide density in the hippocampus. Two-way ANOVA with Tukey’s post hoc test. Post hoc effect of treatment demonstrated by asterisks, (*p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001). Error bars represented the standard deviation of the mean (SD).\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7197783/v1/d92d23a0051172085f4b8bf0.jpg"},{"id":87500728,"identity":"1120d462-da58-482b-aadb-ac2afb2b6db9","added_by":"auto","created_at":"2025-07-24 13:51:41","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":143267,"visible":true,"origin":"","legend":"\u003cp\u003e(A – L) Representative confocal microscopy images of immunofluorescence staining in the CA1 region of the hippocampus. (M) IBA1+ microglia (in red) and (N) GFAP+ astrocytes (in green). Sample size: CTR-CTR (n = 5); CTR-CBD (n = 5); CTR-DON (n = 5); STZ-CTR (n = 5); STZ-CBD (n = 5); S TZ-DON (n = 5). Error bar reported in the standard deviation of the mean; two-way ANOVA with Tukey’s post hoc test (*p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001). Error bars represented the standard deviation of the mean (SD).\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7197783/v1/66d2508af3dc5a2e2181dff6.jpg"},{"id":87502134,"identity":"1d55e6be-cd1a-4e24-b98a-affaf614f9b8","added_by":"auto","created_at":"2025-07-24 14:07:41","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":66427,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of repeated CBD (10 mg/kg) administration in rats subjected to the STZ-induced AD animal model on oxidative stress assays in the different brain areas.\u003cstrong\u003e (A) \u003c/strong\u003eOxidative Stress Index (OSI) in striatum, and (B) OSI in hippocampus regions. Sample size: CTR-CTR (n = 7); CTR-CBD (n = 7); CTR-DON (n = 7); STZ-CTR (n = 7); STZ-CBD (n = 7); STZ-DON (n = 7). Error bar reported in the standard deviation of the mean; two-way ANOVA with Tukey’s post hoc test, *p \u0026lt; 0.05. Error bars represented the standard deviation of the mean (SD).\u003c/p\u003e","description":"","filename":"9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7197783/v1/fdec69089841deed4b4c50d1.jpg"},{"id":88196270,"identity":"fff6bdbf-e352-44bf-86c2-da85a6fc60ab","added_by":"auto","created_at":"2025-08-03 17:01:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2030988,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7197783/v1/e0e4d3e3-efdf-4c03-b38e-be1dd485cd05.pdf"},{"id":87502133,"identity":"f0cef2e2-d743-484e-9168-411414384795","added_by":"auto","created_at":"2025-07-24 14:07:41","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":31591,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7197783/v1/7b6dbcc9fc42a484e0c2e332.docx"},{"id":87500717,"identity":"b6262257-3ef7-49ee-9f04-27e8e6e574ef","added_by":"auto","created_at":"2025-07-24 13:51:41","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":31427,"visible":true,"origin":"","legend":"","description":"","filename":"Table2.docx","url":"https://assets-eu.researchsquare.com/files/rs-7197783/v1/946e3dd813eaa75897ad2376.docx"},{"id":87500724,"identity":"6e9f7677-6b3e-4724-ab7a-e867360448cf","added_by":"auto","created_at":"2025-07-24 13:51:41","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":31742,"visible":true,"origin":"","legend":"","description":"","filename":"Table3.docx","url":"https://assets-eu.researchsquare.com/files/rs-7197783/v1/82ece288d24b9e66db9fffb7.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Cannabidiol improves short-term memory in a Streptozotocin-induced animal model of Alzheimer’s disease","fulltext":[{"header":"Highlights","content":"\u003cp\u003e\u0026bull; Cannabidiol prevented short-term memory deficits in an AD rat model.\u003c/p\u003e\u003cp\u003e\u0026bull; CBD reduced Aβ deposition and microglial activation in hippocampal regions.\u003c/p\u003e\u003cp\u003e\u0026bull; CBD decreased oxidative stress levels in the striatum of STZ-induced rats.\u003c/p\u003e\u003cp\u003e\u0026bull; Findings support CBD\u0026rsquo;s neuroprotective potential for Alzheimer\u0026rsquo;s disease.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eApproximately 55\u0026nbsp;million people worldwide are affected by some form of dementia, with Alzheimer's Disease (AD) being the most prevalent. This number is expected to rise to 153\u0026nbsp;million by 2050 (GBD, 2022). Dementia ranks as the seventh leading cause of death and is a significant source of disability among older adults, with an estimated economic impact of \u003cspan\u003e$\u003c/span\u003e1.3 trillion in 2019 (WHO, 2025).\u003c/p\u003e\u003cp\u003eAD is characterised by multifactorial neuropathological changes, including amyloid beta dysfunction, tau protein hyperphosphorylation, neurotransmission imbalances, and neuroinflammation. Currently, there are 143 drugs in development for AD, with 83.2% aimed at modifying the disease, and two monoclonal antibodies recently approved (aducanumab and lecanemab), acting in the Aβ peptide agglomerates (Dhillon, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Cummings et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Reardon, \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, many of these drugs, like acetylcholinesterase (AChE) inhibitors and memantine, can cause side effects such as nausea, vomiting, and fatigue (Kaduszkiewicz et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Mimica and Presečki, 2009).\u003c/p\u003e\u003cp\u003eIn contrast, natural products may have milder side effects compared to drugs available (Deshpande, Gogia and Singh, 2019), interacting simultaneously with multiple AD targets (Patil et al., \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Noori et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), as these products contain various bioactive substances that may work synergistically through multiple neuroprotective mechanisms (Chen et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eRecent studies have focused on \u003cem\u003eCannabis sativa\u003c/em\u003e (cannabis, hemp) as a medicinal plant, which contains over 400 active compounds with pharmacological potential, as observed in experimental models involving rodents and humans (Karniol et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e1974\u003c/span\u003e; Mechoulam and Carlini, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e1978\u003c/span\u003e; Carlini, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2003\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). The two most prevalent substances in the plant are delta-9-tetrahydrocannabinol (Δ9-THC), ranging from 10\u0026ndash;30%, and cannabidiol (CBD), ranging from 0.1\u0026ndash;5%. CBD, the second most abundant phytocannabinoid found in the resin of the flowers, has received considerable attention in research due to its therapeutic effects on various chronic diseases, including AD, Parkinson's disease, epilepsy, rheumatoid arthritis, anxiety, and depressive disorders (ElSohly et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Pisanti et al., \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Patricio et al., \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCBD has shown promising results by reducing β-amyloid expression, oxidative damage, neuronal injury, and apoptosis while promoting neurogenesis and offering neuroprotective effects against excitotoxicity, also regulating caspase 3 to inhibit neuronal apoptosis (Iuvone et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Esposito et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2006\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Fagherazzi et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Watt and Karl, \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Kim et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Cooray, Gupta, and Suphioglu, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThis study aims to evaluate the therapeutic potential of CBD in AD by assessing its effects on key neuropathological mechanisms, including β-amyloid accumulation, oxidative stress, and neuroinflammation. Given the limitations and adverse effects of current AD treatments, the study investigates CBD as a potential alternative with multi-target neuroprotective properties. By reviewing existing research, it aims to determine CBD\u0026rsquo;s efficacy and advantages over conventional pharmacological interventions.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eAnimals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEighty male Wistar rats (6-7 months) were obtained from the CEDEME (Centro de Desenvolvimento de Modelos Experimentais – Unifesp), placed in groups of 3 animals in polypropylene cages under controlled ventilation and temperature (22 ± 2°C), with a 12-hour light/dark cycle (lights on at 7 am) and free access to food and water. The use of animals in research was carried out under Brazilian law (Law No. 11.794). The project was submitted to the Ethics Committee on the Use of Animals (CEUA) under the protocol number 1327200121 of the Federal University of São Paulo. All necessary measures were taken to minimise pain, discomfort, and suffering.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDrug preparation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo prepare a CBD injection of 10 mg/kg in a volume of 1 mL/kg (for rats), the \"Working Solution\" consisted of 10 mg of CBD / 1 mL of vehicle (5% DMSO + 2% Tween 80 in 0.9% NaCl). The solution was prepared in a dark environment, as CBD is photoreactive and should not be exposed to light. To prepare a donepezil (DON) injection, the \"working solution\" consisted of 1 mg of DON/1 mL of vehicle (12.5% propylene glycol in 0.9% NaCl). The treatments were administered via intraperitoneal injections (i.p.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperimental procedures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study consisted of 19 days of experiment. Animals underwent stereotaxic surgery involving bilateral skull perforation on the 1\u003csup\u003est\u003c/sup\u003e day of the experiment (Paxinos and Watson, 2005). Animals from the STZ group received a 3 mg/kg dose of streptozotocin (STZ) through an intracerebroventricular (i.c.v.) injection, and the CTR group received a control vehicle solution in both ventricles (Uchigata et al., 1982; Hoyer, Müller and Plaschke, 1994; Hosokawa, Dolci and Thorens, 2001; Szkudelski, 2001; Baydas et al., 2003; Salkovic-Petrisic et al., 2006; 2013; Zhou et al., 2013; Grieb, 2016).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAfter three days of observation, the animals were assigned to experimental groups: CTR-CTR: [vehicle (i.c.v) + vehicle (NaCl, DMSO, and Tween 80, i.p., n=13)], STZ-CTR: [3\u0026nbsp;mg/kg STZ (i.c.v.) + vehicle (NaCl, DMSO, Tween 80, i.p., n=14)], CTR-CBD: [vehicle (i.c.v.) + CBD 10 mg/kg, i.p., n=14)], STZ-CBD: [3 mg/kg STZ (i.c.v.) + CBD 10 mg/kg, i.p., n=13)], CTR-DON [Vehicle (i.c.v.) + 1 mg/kg donepezil, i.p., n=13)], STZ-DON: [3 mg/kg STZ (i.c.v.)\u0026nbsp;+ 1 mg/kg donepezil, i.p., n=13]. For 14 days, CTR animals received a vehicle solution via i.p. injection, while others received either cannabidiol (CBD, at 10 mg/kg) (Esposito\u0026nbsp;et al., 2006, 2011; Fagherazzi\u0026nbsp;et al., 2012; da Silva\u0026nbsp;et al., 2014; Watt\u0026nbsp;and Karl, 2017; Peres, 2018) or donepezil (DON) at 1 mg/kg (Jayant, Sharma and Sharma, 2016; Choi\u0026nbsp;et al., 2022; Faldu, Patel and Shah, 2023), starting 72 hours post-surgery (Fig. 1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDuring treatment, the animals were subjected to the following behavioural tests: olfactory sensitivity test (7th day of treatment), open field test (9th day of treatment), novel object recognition test (10th day of treatment), sucrose preference test (10th day of treatment) and spontaneous alternation test (14th day of treatment) (Fig. 1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBehavioural testing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOpen field test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experiment took place in a circular wooden apparatus measuring 50 cm in diameter and 40 cm in height, designed to assess locomotor activity and habituate animals for a subsequent object recognition test. During the 5-minute test, animals were placed in the centre, and their behaviours were tracked using Anymaze® (Stoelting, USA), which categorises the field into central and peripheral zones. The assessment focused on the distance covered, average speed, and time spent in each zone evaluated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNovel Object Recognition task\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis task assesses short-term memory by evaluating the recognition of a new object (de Lima et al., 2005). Conducted in a 50 cm diameter, 40 cm high open circular wooden arena, the test used pairs of identical objects differing in colour, size, and shape. During training, animals interacted with two identical objects. After one hour, they were shown a familiar object in the same location alongside a new object. Object selection was randomised. Each session lasted 5 minutes. The discrimination index was calculated using the formula: [time spent on the new object - time spent on the familiar object] / [time spent on the new + time spent on familiar]. Preference was calculated with: [time spent on the new object] / [time spent on new + time spent on familiar].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSpontaneous alternation test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe spontaneous alternation test assesses operational memory, which is often impaired in patients with Alzheimer's disease (AD) due to frontal lobe dysfunction (Hughes, 2004). In this test, animals are placed in a maze with four closed arms and an open central area. After being positioned on the central platform facing arm A, they can explore for 5 minutes. Spatial working memory is evaluated by calculating the percentage of alternation, defined as four consecutive, non-repeated entries into different arms, using the formula: [number of alternations / (total number of entries – 3) * 100].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSucrose preference test\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis test evaluates hedonic behaviour, specifically anhedonia, which is a reduction in pleasure often seen in depression (Liu et al., 2018) and Alzheimer's disease (Lyketsos et al., 2011). Two bottles, one with water and one with a 2% sucrose solution, are placed in each cage for 48 hours. During the first 24 hours, animals adapt to the setup. The bottles are then refilled and available for 12 hours, with their positions switched to prevent bias. Low sucrose intake indicates anhedonia. Sucrose preference is calculated as [sucrose consumption/ (sucrose consumption + water consumption)] to find the percentage of total liquid consumed (Huynh et al., 2011).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssessment of liquid consumption\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe average consumption of water and sucrose solution was assessed and determined once by the weight of the bottles before and after the test phase of the sucrose preference behavioural test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWeight loss\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnimal weighing was updated throughout the experiment to monitor possible losses in body mass. Weighting took place weekly during the 3 weeks of the experiment (approximately one day before i.c.v. surgery, 3 days after surgery, after 7 days of treatment and after 14 days of treatment).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemistry (IHC)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing these tests, on the 19th day of treatment, the animals were euthanised by perfusion, which consisted of accessing the abdominal cavity, exposing the diaphragm muscle, which was sectioned to access the heart. Subsequently, a cannula was inserted into the left ventricle, and a small incision was made in the right atrium. A volume of 200 ml of 0.9% saline solution was injected with a peristaltic pump. Then, 200 ml of 4% paraformaldehyde solution in 0.1 M phosphate buffer, pH 7.4, was injected.\u003c/p\u003e\n\u003cp\u003eAfter perfusion, the brains were collected by craniotomy and kept in 4% paraformaldehyde for 24 hours. The following day, the brains were removed from the solution and were cryoprotected in 30% sucrose in the refrigerator until immunohistochemistry and immunofluorescence were analysed. The brains were removed from the cryoprotection in 30% sucrose on the day of the sectioning. They were frozen in Tissue-Tek® cryostat inclusion medium (Sakura, Japan) and 4% gelatine in 0.1 M PBS inside a plastic mould. Samples were coronally sectioned at 50 μm using a Leica CM 1950 cryostat.\u003c/p\u003e\n\u003cp\u003eBrain sections were washed and incubated with non-fat milk, then treated with anti-beta-amyloid (1-42) primary antibody (1:500, #bs-0107R Bioss) and secondary antibody (1:1000, #AP132B Millipore). They were processed with the avidin-biotin complex (ABC Kit, Vector Labs) and 0.05% 3,3' diaminobenzidine (DAB) for staining. After sufficient staining, sections were mounted on gelatinised slides and dehydrated through a series of alcohols. Images of the areas of interest were captured, and quantifications were performed using relative optical densitometry.\u003c/p\u003e\n\u003cp\u003eThe sections were analysed using a Zeiss Axio Imager M2 microscope with Apotome (Carl Zeiss Microscopy, Germany). Contours were drawn to map the hippocampal region using the Stereo Investigator program (MBF Biosciences, USA). Cell counting was performed to quantify the number of beta-amyloid biomarkers in the hippocampal formation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBrain sections were washed in PBS and incubated with normal serum to prevent non-specific binding [3% normal donkey serum (Jackson ImmunoResearch Laboratories, West Grove, PA) in PBS] for two hours. Primary antibodies, anti-GFAP (1:500, #53989282, eBioscience) and anti-Iba1 (1:500, #01919741, FUJIFILM Wako), were added for 48h at 4 ℃ with 0.1% Triton X-100, followed by AF 594 goat-anti-rabbit IgG secondary antibody (1:200, #A11012, Invitrogen) for two hours at room temperature. The sections were mounted on gelatinised slides and cover-slipped with VECTASHIELD antifade mounting medium with DAPI (Vector Laboratories, #H1200).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOxidative stress analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess oxidative stress parameters, another group of animals was previously anaesthetised with isoflurane for euthanasia by decapitation using a guillotine. The brains were stored in a freezer at -80 °C for subsequent measurements of the total oxidant and antioxidant status of the samples.\u003c/p\u003e\n\u003cp\u003eBrains were homogenised in a buffer and centrifuged at 12.000 rpm for 15 minutes at 4 ºC. The supernatant was collected and stored at -20ºC. For Total Antioxidant Status (TAS), 5 µL of the sample was pipetted in duplicate with 5 µL of Trolox and 5 µL of PBS. Then, 200 µL of Reagent 1 was added, and the first absorbance reading was taken at 444 nm. After adding 10 µL of Reagent 2, the solution was incubated for 5 minutes in the dark before the second reading at 444 nm (Erel, 2004). For Total Oxidant Status (TOS), 25 µL of the sample was treated similarly with hydrogen peroxide and PBS, followed by 160 µL of Reagent 1. After the first reading at 560 nm, 8 µL of Reagent 2 was added, and the solution was incubated for 5 minutes before the second reading at 560 nm (Erel, 2005).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA two-way Analysis of Variance (ANOVA) was conducted for all comparisons between cohorts, followed by Tukey’s post hoc test to identify interaction effects. Outlier values were adjusted through \u003cem\u003ewinsorization\u003c/em\u003e to the nearest valid value. It is a statistical technique used to limit extreme values in a dataset to reduce the effect of outliers. Instead of removing outliers, it replaces them with the closest value. This helps stabilise variance and improve the robustness of statistical analyses.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Data met the assumptions of normality, linearity, and homogeneity. Statistical significance was determined at a p-value of less than 0.05, with results presented as the difference between means (MD). Error bars represented the standard deviation of the mean (SD), and differences were demonstrated using asterisks (*p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001). All analyses and graphs were created using IBM® SPSS Statistics Program (Version 29) (IBM, USA), GraphPad Prism (Version 10), and Microsoft Excel.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eCBD reverses short-term memory deficits in STZ-treated animals that were not able to discriminate a novel object.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe analysis of object exploration in novel object recognition showed group x object interaction [F (5,148) = 2.36; p = 0.0426]. Animals in the CTR-CTR (Mean difference = 15.03; p = 0.0003), CTR-CBD (Mean difference = 15.44; p = 0.0001), CTR-DON (Mean difference = 8.57; p = 0.0347), STZ-CBD (Mean difference = 12.07; p = 0.0032) and STZ-DON (Mean difference = 16.76; p \u0026lt; 0.0001) groups explored the novel object for longer compared to the familiar object. Animals in the STZ-DON group explored the new object for longer compared to the CTR-DON (Mean difference = 11.63; p = 0.0493) and STZ-CTR groups (Mean difference = 12.00; p = 0.033) (Fig. 2A).\u003c/p\u003e\n\u003cp\u003eFor the object discrimination index, a group x treatment interaction was also observed [F (2,74) = 3.94; p = 0.0235]. Animals in the STZ-CTR group did not discriminate against objects compared to the CTR-CTR (Mean difference = 0.48; p \u0026lt; 0.0001), STZ-CBD (Mean difference = 0.30; p = 0.022) and STZ-DON groups (Mean difference = 0.35; p = 0.0074) (Fig. 2B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSTZ-treated animals showed reduced alternation frequency compared to the CTR-CTR group\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the analysis of spontaneous alternation, a treatment effect was observed [F (1,74) = 13,28; p = 0,0005]. Animals in the STZ-CTR group had a lower frequency of alternation compared to the CTR-CTR (Mean difference = 15,24; p = 0,0014) (Fig. 3).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.3. Animals treated with STZ-CBD travelled a greater distance and at a higher speed compared to the animals treated with STZ-CTR and STZ-DON.\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe analysis of the total distance travelled in the open field revealed a significant treatment effect [F (2,74) = 4.45; p = 0.0149]. Animals treated with STZ-CBD travelled a greater distance compared to CTR-CBD (Mean difference = 2.63; p = 0.0484), STZ-CTR (Mean difference = 4.63; p = 0.0021) or STZ-DON groups (Mean difference = 3.81; p = 0.0154) (Table 1).\u003c/p\u003e\n\u003cp\u003eRegarding average speed, a significant treatment effect was also observed [F (2,74) = 4.90; p = 0.0100]. Animals treated with STZ-CBD moved at a higher speed than animals in the CTR-CBD (Mean difference = 0.008; p = 0.0487), STZ-CTR (Mean difference = 0.014; p = 0.004), and STZ-DON (Mean difference = 0.012; p = 0.0191) groups (Table 1).\u003c/p\u003e\n\u003cp\u003eFor distance travelled in the central zone, a significant group effect [F (1,74) = 12.39; p = 0.0007] and treatment effect [F (2,74) = 5.67; p = 0.0051] were found. Animals in the STZ-CBD group spent more time in the central zone compared to animals in the CTR-CBD group (Mean difference = 43.43; p = 0.0019). Additionally, animals in the STZ-CTR group spent more time in the central zone compared to animals in the STZ-DON group (Mean difference = 39.39; p = 0.0128) (Table 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimals from the STZ-DON group showed increased sucrose preference compared to other STZ-treated groups\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnalysis of the sucrose preference test revealed a significant group x treatment interaction [F (2,74) = 6.17; p = 0.0033]. Animals in the CTR-CBD group exhibited a higher preference for sucrose compared to the animals of the STZ-CBD group (Mean difference = 8.00; p = 0.0033). Furthermore, animals in the STZ-DON group showed greater sucrose preference than animals in the STZ-CTR (Mean difference = 8.36; p = 0.0062) and STZ-CBD groups (Mean difference = 9.15; p = 0.0030) (Fig. 4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimals from the STZ-CBD group showed increased consumption of liquids compared to the cohorts\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnalysis of mean liquid consumption revealed a significant group x treatment interaction [F (5,148) = 14.86; p \u0026lt; 0.0001]. Animals in the STZ-CBD group consumed more water compared to the CTR-CBD (Mean difference = 45.18; p = 0.0383) and STZ-DON (Mean difference = 44.85; p = 0.0471) groups (Table 2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn terms of sucrose consumption, animals in the STZ-CBD group consumed significantly more than animals in the CTR-CBD (Mean difference = 175.37; p \u0026lt; 0.0001), STZ-CTR (Mean difference = 90.45; p \u0026lt; 0.0001) and STZ-DON (Mean difference = 161.80; P \u0026lt; 0.0001) groups. Additionally, animals in the STZ-CTR group also consumed more sucrose compared to animals in the CTR-CTR (Mean difference = 93.40; p \u0026lt; 0.0001) and STZ-DON (Mean difference = 71.40; p \u0026lt; 0.0001) groups. Animals in the CTR-CTR (Mean difference = 106.20; p \u0026lt; 0.0001), CTR-CBD (Mean difference = 117.60; p \u0026lt; 0.0001), CTR-DON (Mean difference = 92.15; p \u0026lt; 0.0001), STZ-CTR (Mean difference = 185.90; p \u0026lt; 0.0001), STZ-CBD (Mean difference = 247.80; p \u0026lt; 0.0001) and STZ-DON (Mean difference = 130.80; p \u0026lt; 0.0001) groups consumed more sucrose compared to water intake (Table 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimals in the STZ groups exhibited greater weight loss compared to the CTR groups\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the evaluation of body mass, a significant group x weighing period was observed [F (15,222) = 13.81; p \u0026lt; 0.0001]. Animals in the CTR-CBD group showed a reduction in body mass from the first to the second weighing (Mean difference = 6.42; p \u0026lt; 0.0001), third (Mean difference = 13.71; p = 0.0249) and fourth weight (Mean difference = 6.93; p = 0.0016), as well as the second to the fourth weighting (Mean difference = 11.29; P = 0.0293). In the CTR-DON group, a decrease was observed between the first and second weighting (Mean difference = 5.76; p = 0.0196). The STZ-CTR group exhibited significant weight loss from the first and second (Mean difference = 21.43; p \u0026lt; 0.0001), third (Mean difference = 54.57; p \u0026lt; 0.0001) and fourth weight (Mean difference = 51.86; p \u0026lt; 0.0001) and from the second to the third (Mean difference = 33.14; p = 0.0005) and fourth weightings (Mean difference = 30.43; p = 0.0080). Similarly, STZ-CBD group lost body mass from the first to the second (Mean difference = 22.46; p \u0026lt; 0.0001), third (Mean difference = 77.31; p \u0026lt; 0.0001) and fourth weight (Mean difference = 66.54; p \u0026lt; 0.0001), as well as from the second to the third (Mean difference = 31.66; p \u0026lt; 0.0001) and fourth weightings (Mean difference = 19.51; p = 0.0009). In the STZ-DON group, a reduction was observed from the first to the second (Mean difference = 18.62; p \u0026lt; 0.0001), third (Mean difference = 48.31; p = 0.0013) and fourth weightings (Mean difference = 40.00; p = 0.0043), and from the second to the third (Mean difference = 29.69; p = 0.0088) and fourth weightings (Mean difference = 21.38; p = 0.0450) (Table 3).\u003c/p\u003e\n\u003cp\u003eFurthermore, animals in the STZ-CTR (Mean difference = 54.57; p \u0026lt; 0.001), STZ-CBD (Mean difference = 77.30; p \u0026lt; 0.001) and STZ-DON (Mean difference = 48.30; p = 0.001) groups showed a significant loss in body mass from the third weighing compared to the first. A similar trend was observed in the fourth weighing in the STZ-CTR group (Mean difference = 51.85; p \u0026lt; 0.001), STZ-CBD (Mean difference = 66.53; p \u0026lt; 0.001) and STZ-DON (Mean difference = 40.00; p = 0.008) (Table 3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTreatment with CBD and DON reduced immunoreactivity of amyloid-\u003c/strong\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;(A\u0026beta;) in hippocampal regions (Dentate gyrus and CA1)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the analysis of A\u0026beta; immunoreactivity in the dentate gyrus region of the hippocampus, a significant group x treatment interaction was observed [F (2,83) = 6.059; p = 0.0035]. Animals in the STZ-CBD group exhibited higher A\u0026beta; density compared to animals in the CTR-CBD (Mean difference = 0.027; p = 0.001) and STZ-DON (Mean difference = 0.029; p = 0.0027). Conversely, animals in the CTR-CTR (Mean difference = 0.046; p \u0026lt; 0.0001), STZ-CBD (Mean difference = 0.022; p = 0.03) and STZ-DON (Mean difference = 0.051; p\u0026lt; 0.0001) groups exhibited lower A\u0026beta; density when compared to the STZ-CTR group (Fig. 5).\u003c/p\u003e\n\u003cp\u003eIn the CA1 region, a significant group x treatment interaction was also observed [F (2,82) = 7.018; p = 0.0015]. Animals in the STZ-CBD group showed higher A\u0026beta; density compared to the animals in the CTR-CBD (Mean difference = 0.028; p \u0026lt; 0.0001) and STZ-DON (Mean difference = 0.021; p = 0.0098) groups. Furthermore, animals in the CTR-CTR (Mean difference = 0.050; p \u0026lt; 0.0001), STZ-CBD (Mean difference = 0.024; p = 0.0028) and STZ-DON (Mean difference = 0.046; p \u0026lt; 0.0001) groups presented significantly lower A\u0026beta; density compared to the STZ-CTR group (Fig. 6).\u003c/p\u003e\n\u003cp\u003eIn the CA3 region, a group x treatment interaction was also identified [F (2,86) = 5.15; p = 0.007]. Animals in the STZ-CBD groups exhibited higher A\u0026beta; density when compared to the CTR-CBD (Mean difference = 0.038; p \u0026lt; 0.0001) and STZ-DON (Mean difference = 0.025; p = 0.0022) groups. Additionally, animals in the CTR-CTR (Mean difference = 0.048; p \u0026lt; 0.0001) and STZ-DON (Mean difference = 0.037; p \u0026lt; 0.0001) groups presented reduced A\u0026beta; density compared to the STZ-CTR group (Fig. 7).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMicroglia and astrocytes were increased in the STZ-CTR group, and CBD reduced IBA1 cell number in the hippocampus\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the analysis of Iba1 immunoreactivity in the hippocampus region, a significant group x treatment interaction was observed [F (2,24) = 5.75; P = 0.0091]. Animals in the STZ-CBD group exhibited a higher Iba1+ cell population compared to the CTR-CBD group (Mean difference = 271.835; p = 0.032). Conversely, animals in the CTR-CTR (Mean difference = 691.269; p \u0026lt; 0.0001), STZ-CBD (Mean difference = 351.488; p = 0.0194) and STZ-DON (Mean difference = 336.966; p = 0.0255) groups showed reduced Iba1 expression compared to the STZ-CTR group (Fig. 8).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRegarding GFAP immunoreactivity in the hippocampus, a group x treatment interaction was also observed [F (2,24) = 3.34; p = 0.05]. Animals in the CTR-CTR group displayed a significantly lower GFAP+ cell population compared to the STZ-CTR group (Mean difference = 683.572; P = 0.0069) (Fig. 8).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSTZ-CBD animals presented a lower oxidative stress index (OSI) compared to the STZ-CTR group in the striatum\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the striatum, a significant group x treatment interaction was observed [F (2,36) = 8.36; p = 0.001]. Animals in the CTR-CTR (Mean difference = 36.93; p = 0.0029), STZ-CBD (Mean difference = 57.26; p \u0026lt; 0.0001) and STZ-DON (Mean difference = 58.48; p \u0026lt; 0.0001) groups exhibited lower oxidative stress indices compared to the STZ-CTR group (Fig. 9A).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the hippocampus, a significant group effect was found [F (1,36) = 13.73; p = 0.0007]. Animals in the CTR-CTR group presented higher oxidative stress indices compared to the STZ-CTR group (Mean difference = 18.06; p = 0.0186). Additionally, animals in the STZ-CBD group presented lower oxidative stress indices compared to the CTR-CBD (Mean difference = 17.57; p = 0.0217) (Fig. 9B).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe results of this study indicate that subchronic treatment with CBD at a dose of 10 mg/kg in rats, using the STZ-induced model of AD, effectively prevented short-term memory deficits. Additionally, it increased fluid intake and locomotor activity, resulting in hyperactivity. These effects were associated with reduced immunostaining for Aβ and IBA1, as well as a decrease oxidative stress index in the striatum.\u003c/p\u003e\n\u003cp\u003eThe novel object recognition task is commonly used to assess learning and memory in rats, enabling the evaluation of short- or long-term memory depending on the interval between training and testing (Akkerman, Blokland and Prickaerts, 2014; Lueptow, 2017). In our study, we focused on short-term memory using a one-hour interval. As expected, STZ-injected animals failed to discriminate between novel and familiar objects, indicating a memory deficit (Chen et al., 2013; Grieb, 2016; Andrade et al., 2023). Consistent with previous studies, treatment with CBD and Donepezil attenuated this memory impairment (Cheng et al., 2014; Coles et al., 2020; de Bruin et al., 2011; Guo et al., 2015).\u003c/p\u003e\n\u003cp\u003eIn the study by Fagherazzi et al. (2012), treatment with CBD (10 mg/kg) for 14 days showed a better neuroprotective effect in the novel object recognition task compared to a lower dose (5 mg/kg) in a rat model of neurodegeneration induced by iron overdose, effectively preventing deficit in the memory consolidation phase. De Paula Faria et al. (2022) reported that treatment with CBD (20 mg/kg) for 7 days prevented short- and long-term memory deficits in STZ-induced animals. These findings suggest that CBD, when administered in higher and subchronic doses, exerts a neuroprotective effect against memory deficits in models of neurodegeneration.\u003c/p\u003e\n\u003cp\u003eThe spontaneous alternation test is used to evaluate the spatial working memory in rodents and is particularly sensitive to changes in the hippocampus and other memory-related brain regions (Lalonde, 2002; Deacon and Rawlins, 2006). In this task, rodents are expected to explore a novel arm of the apparatus, alternating their choices within the maze (Hughes, 2004). In our study, STZ-injected animals treated with vehicle displayed a reduced alternation rate, indicating a working memory deficit. On the other hand, treatment with CBD significantly improved performance in this task, suggesting its potential to mitigate STZ-induced memory impairment.\u003c/p\u003e\n\u003cp\u003eThe open field test is widely used to assess rodent behavioural patterns, including anxiety-like behaviour, stereotypy, locomotion, and overall health status (Prut and Belzung, 2003; Kraeuter, Guest, and Sarnyai, 2019). However, when evaluating anxious behaviour, it is important to note that this is an individual test in which the animal is isolated from its social group, a factor that can influence anxiety levels. Moreover, the open arena is larger compared to the animal's cage, and this environmental contrast may elicit distinct behavioural responses under potentially stressful conditions. In our results, animals in the STZ group spent more time in the central zone compared to the animals in the CTR group. Interpretations of this finding may vary in the literature, depending on the treatment applied, the animal model used, and the specific experimental protocol adopted. When comparing our findings with a previous study conducted by our group, which utilised the same protocol and animal model but involved different pharmacological treatments, we observed similar outcomes (Alexandre-Silva et al., 2024). Animals from the STZ-CBD group that spent more time in the centre of the open field test exhibited hyperlocomotion, as indicated by increased average speed, an outcome that some researchers interpret as a potential indicator of anxiety. However, previous studies have suggested that CBD may exert anxiolytic effects (Crippa et al., 2011; Blessing et al., 2015; García-Gutiérrez et al., 2020; de Faria et al., 2020). Further analyses are needed to elucidate the effects of STZ and CBD on anxiety-like behaviours, including the use of traditional evaluation paradigms and detailed assessments of specific ethological behaviours such as exploratory activity, grooming, feeding patterns, and fluid intake (Kelley, 2001).\u003c/p\u003e\n\u003cp\u003eComorbidities such as depression and anxiety are common among AD patients (Botto et al., 2022). The sucrose preference test is a well-established method for assessing anhedonia, a core symptom of depression (Liu et al., 2018; Höflich et al., 2019). Apathy and depression are classified as Behavioural and Psychological Symptoms of Dementia (BPSD) and are particularly prevalent among older adults (Deardorff and Grossberg, 2019). Moreover, research indicates that anhedonia tends to increase with the severity and progression of AD (Turner and Husain, 2022). Lopez et al. (2003) reported that 61% of the patients evaluated exhibited anhedonia, with the prevalence of symptoms increasing alongside disease progression: 50.5% in mild cases, 65.0% in moderate cases, and 72.0% in severe cases. In cases of mild to moderate dementia, anhedonia was associated with sleep disturbances, depressed mood, and feelings of hopelessness, whereas in severe dementia, it was primarily linked to hopelessness. Similarly, Saz et al. (2009) found that 46.8% of AD patients presented anhedonia, with the prevalence rising from 35.6% in mild to moderate AD to 80.0% in severe stages.\u003c/p\u003e\n\u003cp\u003eWhen discussing animal models, healthy animals typically exhibit a higher preference for sucrose solution, as it is perceived as pleasurable. A decreased preference may indicate anhedonia (Liu et al., 2018). Pooladgar et al. (2022) suggest that donepezil may exert antidepressant effects through interactions with the sigma-1 receptors, nicotinic acetylcholine receptors, and neurotransmitters such as dopamine and serotonin (Ramakrishnan et al., 2014; Papp et al., 2016; Walczak-Nowicka and Herbet, 2021). In our study, the control and STZ-DON groups exhibited a higher preference for sucrose compared to the STZ-CTR and STZ-CBD groups. However, we cannot conclude that STZ-CTR and STZ-CBD animals are experiencing anhedonia, as their sucrose consumption remained high at 87.9% and 86.8%. Additionally, these groups also showed increased water and sucrose consumption, with donepezil appearing to reverse this trend. Strekalova et al. (2006) found that anhedonic and non-anhedonic mice exhibited different fluid intake levels during and after stress. This increase may be linked to polydipsia, a potential indicator of diabetes mellitus caused by prolonged stress or compulsive behaviour (Schoenecker, Heller, and Freimanis, 2000). The STZ-CBD group showed signs of polydipsia and increased locomotion in the open field test, suggesting repetitive and compulsive behaviour. Additionally, Sofia and Knobloch (1976) studied the effects of CBD, CBN (cannabinol), THC, and d-amphetamine sulphate on food, sucrose, and water consumption in rats, finding a greater preference for calorie-dense sweets in the cohorts. Furthermore, a study by Spindle et al. (2020) revealed that dry mouth was reported in 6.6% of patients taking CBD 100 mg orally, 16.1% with vapour, and 35.9% with 100 mg of CBD alongside 3.7 mg of THC, suggesting that cannabinoids can significantly influence feeding behaviour and preferences.\u003c/p\u003e\n\u003cp\u003eIn terms of weight, animals in the STZ groups exhibited a greater loss of body mass compared to those in the CTR groups, beginning from the third weighing, which occurred two weeks after the STZ microinjection. The literature has previously reported a loss of body mass associated with the use of STZ as a model for AD, suggesting that the damage caused by STZ is significant enough to reduce food consumption and hinder the animals from regaining weight (Chen et al., 2013; Paidi et al., 2015; Zappa Villar et al., 2018; Yuliani, Lobentanzer, and Klein, 2021; Silva et al., 2023).\u003c/p\u003e\n\u003cp\u003eIn our histological analyses, we aimed to confirm the results from our behavioural tests. We found that the deposition of the Aβ peptide was elevated in the hippocampal regions of CA1, CA3, and the dentate gyrus in the STZ-CTR group, consistent with previous studies indicating this characteristic in this animal model (Ahn et al., 2020; Andrade et al., 2023). Our proposed treatment with CBD at a dosage of 10 mg/kg significantly reduced deposition in the CA3 region and the dentate gyrus. This effect was also observed with the treatment using donepezil, supporting findings in the literature (Esposito et al., 2007; Ye et al., 2015; Takada-Takatori et al., 2019; Chen et al., 2023). These results are consistent with the outcomes of the novel object recognition and alternation tasks, which demonstrated that CBD treatment effectively prevented short-term and spatial working memory deficits in the animals. Similar findings were observed in Iba1 analyses using immunofluorescence. Chronic glial activation, characterised by an increase in reactive microglial and astroglial cells, is commonly observed in neurodegenerative diseases. Studies have shown heightened immunoreactivity of these cells in the STZ animal model (Bassani et al., 2017; Luo et al., 2019; Liu et al., 2022), with CBD treatment showing a reduction in this immunoreactivity (Esposito et al., 2007; Schiavon et al., 2014; Li et al., 2022). Our histological results revealed that STZ-treated animals had increased IBA1 microglia and GFAP astrocytes in the hippocampus, whereas CBD treatment significantly reduced the number of IBA1 reactive cells.\u003c/p\u003e\n\u003cp\u003eAn important factor contributing to the increase in these biomarkers is the rise in reactive oxygen species. The oxidative stress index (OSI) revealed that animals in the STZ-CBD and STZ-DON groups exhibited lower oxidative stress levels in the striatum compared to the STZ-CTR group, corroborating the literature (Cassol et al., 2010; Valvassori et al., 2011). According to Ferbinteanu (2016), the hippocampus and striatum can function cooperatively in memory formation, with significant contributions from different areas to task performance and learning. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe findings of this study suggest that subchronic treatment with CBD at a dose of 10 mg/kg can effectively mitigate memory deficits associated with Alzheimer’s disease in the STZ-induced rat model. The behavioural tests indicated improvements in short-term and spatial working memory, as well as hyperactivity-related behaviours. CBD demonstrated neuroprotective properties, supported by decreased oxidative stress and alterations in neuroinflammatory markers. Additionally, the study highlights the prevalence of anhedonia in Alzheimer’s patients, underscoring the need for continued exploration of behavioural and psychological symptoms associated with the disease. Comorbidities such as anxiety and depression warrant further investigation, particularly in terms of how they impact memory and overall quality of life in affected individuals. Overall, these results emphasise the potential of CBD as a therapeutic agent in combating cognitive impairments and psychological symptoms associated with Alzheimer’s disease. Future studies should further explore the underlying mechanisms and long-term effects of CBD treatment, analyse feed intake by weighing food during each cage change to determine if the results support the hyperphagia observed in AD and its correlation with increased fluid consumption, and also investigate patterns of increased appetite related to CBD treatment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLimitations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study focused on male subjects, despite global data indicating that women are more affected by AD. This limitation is acknowledged in the animal model of AD induced by STZ, as Biasibetti et al. (2017) found that females are generally more resistant to STZ-induced damage.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: GCP, RHL, VCA, JRS, EG, and AMR; data curation: GCP, VAS, BSS, LOT, PSCL, ACCS, EPSJ, RHL, and EG; writing original draft preparation: GCP, EG, and AMR; Review and editing: GCP, VCA, JRS, EG, and AMR; Supervision: EG, and AMR; Funding acquisition: AMR. All authors have read and agreed to the publication of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the São Paulo Research Foundation (FAPESP, grant #20/12053-5, #21/12409-7, #20/09015-4, #22/02320-1, and #22/09866-0), the Coordination for the Improvement of Higher Education Personnel (CAPES) – Finance Code 001, and the National Council of Technological and Scientific Development (CNPq #425694/2016-0, #303325/2017-8, #408377/2021-6 and #310403/2021-9).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAhn, Y., Seo, J., Park, J., Won, J., Yeo, H. 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Neuroprotective effects of edaravone on cognitive deficit, oxidative stress and tau hyperphosphorylation induced by intracerebroventricular streptozotocin in rats. \u003cem\u003eNeurotoxicology\u003c/em\u003e,\u003cem\u003e 38\u003c/em\u003e, 136-145. https://doi.org/10.1016/j.neuro.2013.07.007 \u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 3 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Cannabis, phytocannabionoid, neuroprotection, dementia","lastPublishedDoi":"10.21203/rs.3.rs-7197783/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7197783/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAlzheimer's disease (AD) is a progressive neurodegenerative pathology and the leading cause of dementia in the elderly. Early impairments in brain glucose metabolism and insulin signaling pathway may contribute to neurodegeneration, promoting oxidative stress, increased amyloid-beta (Aβ) production, Tau hyperphosphorylation, mitochondrial dysfunction, neuroinflammation, and neuronal loss. The search for novel therapeutic strategies that can prevent, or slow AD progression remains a major challenge. Cannabidiol (CBD), a phytocannabinoid, has been shown to exert neuroprotective, antioxidant, and anti-inflammatory effect in various experimental models. This study aimed to evaluate the potential neuroprotective effect of CBD in a rat model of AD induced by streptozotocin (STZ, 3 mg/kg, i.c.v.). Wistar rats (6\u0026ndash;7 months old) received CBD (10 mg/kg, i.p.) for 14 consecutive days. During treatment, behavioral assessments including the open field, novel object recognition, sucrose preference, and spontaneous alternation tasks were performed, alongside monitoring of body weight and liquid consumption. At the end of the protocol, brains were collected for immunohistochemistry, immunofluorescence, and oxidative stress analysis. STZ-treated animals displayed cognitive deficits, weight loss, and increased Aβ deposition in the hippocampus. CBD treatment prevented short-term memory impairment, reduced Aβ accumulation in the CA1 and dentate gyrus, and decreased microglial activation (Iba-1 immunoreactivity). In addition, CBD attenuated oxidative damage in the striatum. These findings suggest that CBD exerts neuroprotective effects in this pharmacological model of AD, supporting its potential as a candidate for further investigation in the context of neurodegenerative diseases.\u003c/p\u003e","manuscriptTitle":"Cannabidiol improves short-term memory in a Streptozotocin-induced animal model of Alzheimer’s disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-24 13:51:36","doi":"10.21203/rs.3.rs-7197783/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"57751629-579b-4088-a98b-713023feea2b","owner":[],"postedDate":"July 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-03T16:53:09+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-24 13:51:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7197783","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7197783","identity":"rs-7197783","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}